| April/May 2011 Volume 35 Biomarkers for Chronic Obstructive … · 2016-04-25 · The development of drug-resistant bacteria is the inevitable result of natural selection, but imprudent

Also in this issue :

Community acquired pneumonia Pg. 14

Predictive markers in thoracic tumours Pg. 18

Immunodiagnostic assays and influenza Pg. 32

Fully automated HER2 FISH test Pg.44

HIV/CD4 dual test Pg.46

QC strains for antimicrobial susceptibility testing Pg.48

Biomarkers for Chronic Obstructive Pulmonary Disease Pg.10

Weekly news updates on www.cli-online.com | April/May 2011 | Volume 35

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ContentsFRONT COVER

FEATURES [10-12] Biomarkers for Chronic Obstructive Pulmonary Disease

[14-17] Community Acquired Pneumonia: the CAPNETZ study

[18-20] Predictive markers for anti-angiogenic therapy in thoracic tumours

[22-27] Uncommon invasive mould infections

[28-30] Laboratory detection of respiratory viruses in older adults

[32-34] Immunodiagnostic assays and influenza

[36-37] Molecular diagnostics: the changing culture of medical microbiology

[38-40] Detection of antigen - specific B cells by ELISPOT

REGULARS [6] Editor’s letter

[7-8] News in brief

[42-43] Scientific literature review

[44-48] Product news

[49] Book reviews

[50] Industry news

Chronic Obstructive Pulmonary Disease (COPD) is a smoking-related multicomponent condi-tion that is characterised by partially reversible airflow obstruction. There is an urgent need for biomarkers to support patient stratification, predict outcome, facilitate the development of new therapies and ultimately guide therapy. The three year observational study ‘Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points’ (ECLIPSE) aims to identify and develop new biomarkers for COPD.

Also in this issue :

Community acquired pneumonia Pg. 14

Predictive markers in thoracic tumours Pg. 18

Immunodiagnostic assays and infl uenza Pg. 32

Fully automated HER2 FISH test Pg.44

HIV/CD4 dual test

Pg.46

QC strains for antimicrobial susceptibility testing Pg.48

Biomarkers for Chronic Obstructive Pulmonary Disease Pg.10

Weekly news updates on www.cli-online.com | April/May 2011 | Volume 35

8763CLI April/Mei 2011_P1-13.indd 1 21/04/11 12:26

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World-wide infec-tious diseases cur-rently account for over ten million deaths annually, and the emergence and dissemination of resistant strains of

bacteria, such as multidrug-resistant Staphylococcus aureus and Myco-

bacterium tuberculosis, as well as extensively drug-resistant tuber-culosis, are already compounding the problem. Now the NDM-1 gene, which confers resistance to the potent carbapenem antibiotics used against multi-resistant strains of gram-negative bacilli, has been found in several Enterobacteriaceae including the ubiquitous Escherichia

coli. So far one in ten of these strains is resistant to all known antibiotics; the effect on global health could thus be catastrophic.Reflecting the very urgent need for solutions, the theme of this year’s World Health Day was “Antimicro-bial Resistance: no action today, no cure tomorrow”. To act prudently it is necessary to elucidate the rea-

sons why we are facing such a grave problem in the first place. The development of drug-resistant bacteria is the inevitable result of natural selection, but imprudent use has accelerated their evolution. Over-liberal medical prescription of unnecessary antibiotics without prior diagnostic testing, unregu-lated sources of drugs enabling “self-prescription”, as well as pre-mature cessation of treatment, all fuel resistance. The vast quantity of antimicrobials routinely used in industrialised agriculture also aug-ments the problem.Coupled with this is the dearth of new antibiotics in the pipeline. It is commonly but unfairly assumed that pharmaceutical companies prefer to direct their research and devel-opment efforts towards potentially more lucrative drugs for the long-term treatment of chronic diseases. Yet the arrival of the genomic era fifteen years ago (and the sequencing of Haemophilus influenzae) precipi-tated huge efforts by pharmaceutical companies in the drive to discover new classes of antibiotics; unfortu-nately very few new drugs emerged. The unrealistic demands of regula-tory bodies no doubt exacerbated the problem, since a clear advantage over existing drugs is demanded. Alterna-tives which may not have been supe-rior at the time approval was sought may be vital for the treatment of resistant strains. A good example is vancomycin; this drug may not be as effective as methicillin for treatment of susceptible S. aureus infections, and would not have been approved under current regulations, but it is vital for the treatment of MRSA.Clearly then, there is need for con-certed and immediate action by governments and regulatory bodies, health professionals, industry and the general public. Without this, accord-ing to Dr Margaret Chan, WHO Director General, “the world is head-ing towards a post-antibiotic era, in which many common infections will no longer have a cure and, once again, will kill unabated”.

The need for new antimicrobials: a race against time

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New prostate cancer test gives more accurate diagnosis

A new PSA test to screen for prostate can-cer more accurately has been shown in a large multi-centre clinical trial to identify men with prostate cancer, particularly the aggressive form of the disease, and to sub-stantially reduce the number of false posi-tives compared to the two currently avail-able commercial PSA tests, according to newly published research from Northwest-ern University, USA. Elevated levels of PSA can indicate prostate cancer but can also be caused by prostate inflammation or enlargement, or other conditions. The only currently approved screening tests for prostate cancer result in a high number of false positives and lead to unnecessary biopsies and possible over-detection and over-treatment of indolent cancer, which never would have caused suffering or death. The new test is more specific and accurate than the currently available blood tests for early prostate can-cer detection according to lead investiga-tor William Catalona, M.D., director of the clinical prostate cancer programme at the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. The test will have applications in the detection of more life-threatening prostate cancers and reduce unnecessary biopsies in men 50 years of age and older.The study, which will be published in the May issue of the Journal of Urology, fol-lowed 900 patients from 10 sites. The results showed that the new screening test, a simple blood test named the Pro-PSA test, is particularly useful for patients with a normal prostate exam whose PSA is 2 to 10 ng/mL, a range considered as the diag-nostic gray zone because most men with higher levels have prostate cancer and most men with lower levels do not.The Pro-PSA test measures a more spe-cific, truncated PSA subform, namely (-2)Pro-PSA. Even greater accuracy can be achieved when the results are analysed with a mathematical formula that provides an overall Prostate Health Index. (The for-mula divides the Pro-PSA level by that of

the free-PSA. Then the quotient of the two is multiplied by the square root of the total PSA). The logic behind this formula is that the higher the Pro-PSA and the total PSA and the lower the free-PSA, the more likely the patient has aggressive prostate cancer.The new Pro-PSA test was recently approved for commercial use in Europe, and the FDA is currently reviewing data from the study, which was conducted in collaboration with Beckman Coulter, Inc. www.northwestern.edu/newscenter/

results show benefits in using acute kidney injury criteria in the diagnosis of cirrhosis

Especially in end stage cirrhosis, lLiver dis-ease is associated with a high mortality due to renal failure, . Defining more sensitive tests that help to identify patients at risk of renal failure or death earlier is critical to enable physicians to intervene and ensure the patient has the best possible outcome. The first clinical study investigating the use of the AKIN criteria (Acute Kidney Injury Network) in cirrhosis has shown significant benefits that have the potential to change future diagnosis, according to results from a Spanish study presented recently at the International Liver Congress. As screen-ing and differential diagnosis are becom-ing increasingly important in relation to managing health service provision, if these results are confirmed in larger studies, the AKIN criteria have the potential to replace current screening and diagnosis criteria in hospitalised cirrhotic patients.This prospective study aimed to assess the value of the AKIN criteria in predict-ing outcomes in hospitalised cirrhotic patients. Out of 300 patients admitted to hospital for complications of cirrhosis, 88 (29%) developed renal failure according to the AKIN criteria. Three-month sur-vival of these patients was 38%, compared with 87% for patients who did not develop renal failure (p< 0.01). Renal failure in cirrhosis is currently defined as serum creatinine greater than 1.5 mg/dL. According to the study inves-tigators this definition has two shortcom-ings: firstly it represents a very low glo-merular filtration rate (GFR), and secondly

it may not detect significant changes in GFR because it does not take into account variations in creatinine values. In contrast, the AKIN criteria are much more sensi-tive since they consider renal failure as an increase in serum creatinine greater than or equal to a 0.3mL/dL (≥50% increase) com-pared to baseline within 48 hours. Further-more, when the AKIN criteria were com-bined with the current definition of renal failure, patients meeting the AKIN criteria in whom serum creatinine reached a peak value of >1.5 mg/dL (n=60) had a signifi-cantly lower survival compared to patients with a peak value ≤ 1.5 mg/dL (n=28) (29% vs 58%, respectively; p=0.026). Out of the 300 patients, 30 patients had serum cre-atinine >1.5 mg/dL but did not meet the AKIN criteria. However, three-month sur-vival of these patients was 80%.http://tinyurl.com/6k8hfdj

Big size multi-touch display turned into a microscope

Researchers at the Institute for Molecu-lar Medicine Finland (FIMM), in col-laboration with the Finnish company Multitouch Ltd, have created a hand and finger gesture controlled microscope. The method is a combination of two technolo-gies: web-based virtual microscopy and a giant-size multitouch display. The result is an entirely new way of performing microscopy: by touching a table, or even wall-sized screen, the user can navigate and zoom within a microscope sample in the same way as with a conventional microscope. Using the touch control it is possible to move from the natural size of the sample to a 1000-fold magnification, at which cells and even subcellular details can be seen. The giant size, minimum 46” screen is similar to but bigger than iPad. Biological samples are digitised using a microscopy scanner and stored on an image server. Samples displayed on the screen are then continuously read from the server over the internet and the size of a single sample can be up to 200 gigabytes. The developers think that the method will revolutionise microscopy teaching: a group of students can stand around the display together with the teacher and examine the

– April/May 20117NEwS IN BrIEf

same sample. The multitouch microscope can recognise the hands of multiple users at the same time.Web-based virtual microscopy – the WebMicroscope – was developed a few years ago by researchers at the universities of Helsinki and Tampere, Finland and has been well received among students. The multitouch microscope builds upon this technology and makes it even more useful for teaching. At scien-tific meetings this technology is excellent in a situation where a group of users need to simultaneously view a microscopy sam-ple, for example when a consensus needs to be reached concern-ing a new disease entity or a rare case.http://tinyurl.com/6zbbypq

New test detects early-stage, asbestos-related pulmonary cancer

Researchers at NYU Langone Medical Center, USA have investigated a novel protein test to detect early-stage, asbestos-related pulmo-nary cancer. The test can accurately identify pro-teins secreted from can-cerous tumours caused by asbestos exposure. The study was presented recently at the 2011 Annual Meeting Ameri-

can Association for Cancer Research. In a blinded test performed under the sponsorship of the US National Cancer Institute’s Early Detection Research Network Biomarker Discovery Lab, researchers detected 15 of 19 cases of stage 1 or stage 2 malignant pleural mesothelioma. The study shows the test is approximately 80 percent sensitive in identifying disease. In addition, the specificity of the test was 100 percent. Malignant pleural mesothelioma is an aggressive, asbestos-related pulmonary cancer that develops in the lining of the lungs. Each year, the disease causes an estimated 15,000 to 20,000 deaths worldwide. It can be fatal within 14 months following diagnosis because of the advanced stage that is typically found.

The goal of a new diagnostic test is to find the cancer early enough to effectively treat it. The research team used the Multiplex SOMA-mer Assay by SomaLogic, Inc. to examine 170 blood samples from 90 patients diagnosed with malignant mesothelioma and 80 par-ticipants who were previously exposed to asbestos. The technol-ogy uses SOMAmers, chemically modified single-stranded DNA molecules to bind specifically to target proteins, and so to identify and quantify biomarkers. The test measures 19 protein biomark-ers for malignant pleural mesothelioma and is able to find and quantify the small amount of proteins secreted by tumour cells. Ongoing studies are refining the test and validating the results in other patient blood samples. http://tinyurl.com/3bskw9r

researchers link herpes to Alzheimer’s diseaseLaboratories at the University of New Mexico (UNM), Brown University, and House Ear Insti-tute (HEI), USA have developed a new technique to observe herpes sim-plex virus type 1 (HSV1) infections growing inside cells. HSV1, the cause of

the common cold sore, persists in a latent form inside nerve cells. Re-activation and growth of HSV1 infections contribute to cogni-tive decline associated with Alzheimer’s disease.Tagging herpes virus inside cells with green fluorescent protein, scientists used live confocal imaging to watch HSV1 particles emerge from infected cells. Newly produced viral particles exit the cell nucleus and then bud into cellular membranes contain-ing amyloid precursor protein (APP). Electron microscopy at HEI detailed the ultrastructural relationship between HSV1 par-ticles and APP. This interaction between viral particles and cellular APP results in changes in cellular architecture and the distribution of APP, the major component of senile plaques found in the brains of Alzheimer’s disease patients. Results from this study indi-cate that most intracellular HSV1 particles undergo frequent, dynamic interplay with APP, which facilitates viral transport while interfering with normal APP transport and distribution. This dynamic interaction reveals a mechanism by which HSV1 infection leads to Alzheimer’s disease.In developed countries approximately 20 percent of children are infected with HSV1 prior to the age of five. By the second and third decades of life, as much as 60 percent of the population is infected, and late-in-life infection rate reaches 85 percent.Symptoms of primary HSV1 infection include painful blisters of the mouth, lips or eyes. After infection, HSV1 persists in nerve cells by becoming latent. Upon re-awakening, new viral particles are made in the neuron and then travel back out to re-infect the mucous membrane. Many infected people experi-ence sporadic episodes of viral outbreaks as the well-known recurrent cold sore.The researchers recommend people to treat a cold sore as quickly as possible to minimise the amount of time the virus is actively traveling through a person’s nervous system. The faster a cold sore is treated, the faster the HSV1 returns to a dormant stage.http://tinyurl.com/6gatpd

NEwS IN BrIEf – April/May 2011 8


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Chronic obstructive pulmonary disease (COPD) is a major cause of morbid-ity and mortality worldwide and is esti-mated to become the third leading cause of death in 2020. It is a multicomponent condition that is characterised by par-tially reversible airflow obstruction, typi-cally caused by the inhalation of cigarette smoke and other aerotoxins. The airflow obstruction results from emphysema and airways disease and may be punc-tuated by infective exacerbations. How-ever there are also systemic manifesta-tions including loss of lean body mass, fatigue, depression and increased risk of cardiovascular disease. Forced expira-tory volume in one second (FEV1) is the most widely accepted measure of disease severity but this only reflects one aspect of the disease and is not predictive of dis-ease progression. More sensitive meth-ods of assessing COPD severity and progression are needed.

what makes a good biomarker?It is important to consider the require-ments of a good biomarker. This is ide-ally a protein that can easily be assayed i.e. should be present in the blood, urine, sputum or breath condensate. A biomarker needs to be reliable and robust but in addition its level should reflect disease status and response to therapy. The ideal biomarker would allow evaluation of the disease activity and severity, prognosis and progression of the condition. It may also be useful as an intermediate surrogate end-point for drug development.

The ECLIPSE studyThe Evaluation of COPD Longitudi-nally to Identify Predictive Surrogate End-points study (ECLIPSE, ClinicalTri-als.gov identifier NCT00292552, www.ECLIPSE-copd.com) has recruited 2164 COPD patients and 245 healthy controls, who have been followed for three years. Objectives were to a) define clinically rel-evant COPD sub-types with the Global Initiative for Chronic Obstructive Disease (GOLD) stage II-IV (FEV1 <80% pre-dicted); b) identify and define parameters that predict disease progression; c) meas-ure known biomarkers in blood, urine, sputum and breath condensate and d) use genetic analysis, proteomics, RNA tran-scriptomics and metabolomics to identify novel biomarkers. The end-points include evaluation of lung physiology, imaging and health outcome [1].

Exacerbations of COPDAn exacerbation of COPD is defined as a sustained worsening of the patient’s con-dition, from the stable state and beyond normal day-to-day variation. Exacer-bations can be acute in onset and may warrant additional treatment such as corticosteroids, antibiotics or both. Exac-erbations reduce quality of life and appear to accelerate the decline in lung function that characterises COPD. An analysis of the ECLIPSE cohort showed that the single most reliable predictor of exac-erbations in an individual is a history of previous exacerbations. The ‘exacerbator’ phenotype is largely independent of lung function and stable over the three year

study [2]. It is thus important to define the factors that render some individuals susceptible to exacerbations of COPD.

Lung-derived biomarkers of COPDSurfactant Protein D (SP-D)Comprising surfactant protein (SP) A, B, C and D, pulmonary surfactant is a lipopro-tein complex formed in the lung by type II pneumocytes. SP-A and -D are part of the innate immunity system and possess a carbohydrate-binding domain able to bind to bacteria, viruses, DNA and neutrophils. This leads to enhanced phagocytosis and clearance of microorganisms from the lungs. SP-B and C are hydrophobic pro-teins involved in the biophysical function of the surfactant. Serum SP-D levels were higher in individuals with COPD in the ECLIPSE cohort compared to smoking and non-smoking controls with normal lung function. There was no difference in SP-D levels in different components of the COPD phenotype, including GOLD stage of airflow obstruction (based on FEV1), chronic bronchitis or emphysema. The SP-D level was associated with more fre-quent exacerbations and there was a weak correlation between serum SP-D and age and body mass index [3].

Clara Cell secretory protein-16 (CC-16)Clara cell secretory protein 16 (CC-16) is a member of the secretoglobulin family of small secreted dimeric proteins. Produced by Clara cells in the bronchioles and small and large airways, CC-16 plays a role in immunity and protection against oxidative stress and carcinogenesis. It is increased by exposure to smoke, ozone and other lung irritants. A pilot study using ECLIPSE sam-ples demonstrated that the levels of serum CC-16 were stable over three months in individuals with COPD. Data from the ECLIPSE cohort showed that CC-16 was lower in COPD patients than in smokers and former smokers. However, the level of CC-16 was not associated with the severity of COPD as defined by FEV1. Neverthe-less, high levels of CC-16 correlated with reversible airflow obstruction in former smokers with COPD [4].

The development of biomarkers for Chronic Obstructive Pulmonary Disease: the ECLIPSE studyChronic Obstructive Pulmonary Disease (COPD) is a smoking-related multicomponent condition that is characterised by partially reversible airflow obstruction. There is an urgent need for biomarkers to support patient stratification, predict outcome, aid the development of new therapies and ultimately to guide therapy. We review here biomarker identification resulting from the three year observational study “Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points” (ECLIPSE) whose aim is the development of new biomarkers for COPD.

by Dr A. Duvoix, Dr B. Miller, Dr R. Tal-Singer and Prof. D. A. Lomas

– April/May 2011 Biomarkers10r

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CCL18/PArCPulmonary and Activation-Regulated Chemokine (CCL18/PARC) is mainly expressed in the lungs by monocytes, macrophages and dendritic cells. Its expression reflects fibrotic activity in individuals with pulmonary fibrosis. It is also increased in childhood acute lym-phoblastic leukaemia and acute coronary syndromes. CCL18/PARC levels were found to be raised in individuals with COPD from the ECLIPSE cohort when compared to both smokers and non-smoker controls with normal lung func-tion. It was not associated with the sever-ity of the disease (as defined by FEV1) but was associated with mortality and cardiovascular disease. CCL18/PARC levels were stable over a year and are likely to represent a biomarker of the risk of cardiovascular disease in individuals with COPD [5].

Systemic biomarkers of COPDProteins that are not directly associated with lungs or airways can also be useful biomarkers if they relate to the condition. COPD patients may have evidence of systemic inflammation and thus inflam-matory proteins can be considered as potential biomarkers.

fibrinogenFibrinogen is a soluble plasma glycopro-tein that is synthesised in the liver and is converted by thrombin into fibrin during blood coagulation. Fibrinogen is released into the blood in response to stimulation by interleukin 6 (IL-6). Raised levels of fibrin-ogen are associated with an increased risk of vascular disease [6]. Previous work has shown that levels of fibrinogen are raised in COPD, independent of the contribu-tion from cardiovascular disease [7]; lev-els of circulating fibrinogen are associated with the severity of airflow obstruction in COPD with higher levels being associated with an increased risk of exacerbations and of death from COPD [8].

IL-6Synthesised by airway epithelium, mac-rophages and other cells at the site of inflammation in response to environmental stress such as smoking, IL-6 has both pro- and anti-inflammatory proprieties (through TNFα and IL-1 inhibition, and activation of IL-1ra and IL-10). Plasma IL-6 levels were increased during exacerbations of COPD in proportion to the fibrinogen increase [9]. Serum and sputum levels of IL-6 were also more likely to increase when the patient experiences frequent exacerbations of

COPD [10]. Circulating IL-6 levels were sig-nificantly higher in individuals with COPD when compared to controls [11].

CrPC-reactive protein (CRP) is synthesised in the liver as a response to inflammation and the production of IL-6. CRP binds to phos-phocholine, expres and activates the comple-ment system via the C1Q complex. COPD patients have raised levels of circulating CRP compared to controls [8, 11-13]. The levels were significantly higher in patients who were declining rapidly clinically [10].

Therapeutic intervention and biomarkers of COPDSP-D/CCL-18 – SteroidsPrednisolone is a steroid that is widely used to treat exacerbations of COPD. A six week treatment with a tapering dose of oral prednisolone has been shown to reduce SP-D levels and CCL18/PARC in individuals with COPD. There was no effect on serum fibrinogen, IL-1b, IL-8, IL-6, MPO or MMP-9. Inhaled corticoster-oids also reduced serum SP-D and CC-16 levels. Thus, these lung-derived biomarkers may be useful in the development of anti-inflammatory therapies in individuals with COPD [3, 5].

– April/May 201111

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fibrinogen – p38 MAP kinasep38 MAP kinase is involved in the pathway that links environmen-tal stress to gene expression. It stabilises mRNA and so increases the expression of inflammatory molecules such at TNF-α and IL-6. The level of p38 MAP kinase is elevated in individuals with COPD and inhibitors of p38 MAP kinase have anti-inflammatory effects in mice. The administration of an oral p38 MAP kinase inhibitor significantly reduced plasma fibrinogen levels after 12 weeks of treatment in individuals with COPD. It is therefore hypothesised that this therapy may have a role in reducing the systemic inflam-mation in individuals with COPD, thereby reducing the risk of cardiovascular disease, exacerbations and mortality that are asso-ciated with elevated fibrinogen levels [14]. This will need to be evaluated in further clinical trials.

ConclusionECLIPSE is a three year observational study designed to identify new biomarkers for COPD. So far, we have shown that lung derived proteins such as SP-D, CC-16, CCL18/PARC and some systemic proteins such as fibrinogen, IL-6 and CRP are potentially useful

biomarkers for COPD. In particular, serum SP-D, CC-16 and CCL18/PARC levels are associated with the disease status, serum SP-D was related to the risk of exacerbations and CCL18/PARC to the risk of cardiovascular disease. Moreover, increased levels of proteins linked to systemic inflammation such as fibrinogen and CRP are associated with increasing mortality in individuals with COPD. The response to therapeutic intervention is a characteristic of a good biomarker and indeed the levels of SP-D, CC-16 and CCL18/PARC vary in response to treatment with inhaled or oral steroids whilst fibrinogen levels are reduced upon treatment with a p38 MAP kinase inhibitor.

In conclusion, in addition to clinical measurements, a panel of blood biomarkers may be useful in defining the risk of progres-sion and outcome in COPD and so improve the care of COPD patients.

references1. Vestbo J et al. Evaluation of COPD Longitudinally to Identify Predic-

tive Surrogate End-points (ECLIPSE). Eur Respir J 2008; 31(4): 869-73.2. Hurst JR et al. Susceptibility to exacerbation in chronic obstructive

pulmonary disease. N Engl J Med 2010; 363(12): 1128-38.3. Lomas DA et al. Serum surfactant protein D is steroid sensitive and

associated with exacerbations of COPD. Eur Respir J 2009; 34(1): 95-102.

4. Lomas D A et al. Evaluation of serum CC-16 as a biomarker for COPD in the ECLIPSE cohort. Thorax 2008; 63(12): 1058-63.

5. Sin DD et al. Serum PARC/CCL-18 Concentrations and Health Out-comes in Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 2011; doi:10.1164/rccm.201008-1220OC.

6. Danesh J et al. Plasma fibrinogen level and the risk of major cardio-vascular diseases and nonvascular mortality: an individual participant meta-analysis. JAMA 2005; 294(14): p. 1799-809.

7. Gan W Q et al. Association between chronic obstructive pulmonary disease and systemic inflammation: a systematic review and a meta-analysis. Thorax 2004; 59(7): 574-80.

8. Garcia-Rio F et al. Systemic inflammation in chronic obstructive pul-monary disease: a population-based study. Respir Res 2010; 11: 63.

9. Wedzicha J A et al. Acute exacerbations of chronic obstructive pulmo-nary disease are accompanied by elevations of plasma fibrinogen and serum IL-6 levels. Thromb Haemost 2000; 84(2): 210-5.

10. Higashimoto Y et al. Serum biomarkers as predictors of lung function decline in chronic obstructive pulmonary disease. Respir Med 2009; 103(8): 1231-8.

11. Tanni SE et al. Smoking status and tumor necrosis factor-alpha medi-ated systemic inflammation in COPD patients. J Inflamm (Lond) 2010; 7: 29.

12. Quint JK et al. Serum IP-10 as a biomarker of human rhinovirus infec-tion at exacerbation of COPD. Chest 2010; 137(4): 812-22.

13. Yanbaeva DG et al. IL6 and CRP haplotypes are associated with COPD risk and systemic inflammation: a case-control study. BMC Med Genet 2009; 10: 23.

14. Lomas DA et al. An oral inhibitor of p38 MAP kinase reduces plasma fibrinogen in COPD patients. J Clin Pharmacol 2011 (in press).

The authorsAnnelyse Duvoix1, Bruce Miller2, Ruth Tal-Singer2, David A. Lomas1*1Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Hills Road, Cambridge, UK2GlaxoSmithKline, King of Prussia, PA, USA*Corresponding author:Prof. David A. Lomase-mail: [email protected]

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Community-acquired pneumonia (CAP) is the most common potentially fatal infectious disease in the western industrialised coun-tries. Clinical chemistry is routinely used for the diagnosis of infection and the follow-up of the disease; the laboratory parameters commonly measured are the leukocyte count (WBC) and C-reactive protein (CRP) and, in some hospitals, procalcitonin (PCT). In recent years biomarkers have been inten-sively studied in CAP not only for accurate diagnosis but also for determining microbio-logical aetiology, the severity of disease, prog-nosis and treatment decisions. By analysing the data from the prospective study carried out by the German competence network CAPNETZ, we had the unique opportunity of evaluating established and new biomark-ers of CAP in a very large cohort of patients. Exclusion criteria for CAPNETZ were age < 18 years, acquired or therapeutically induced immune deficiency, active tuberculosis or a possible nosocomial genesis of infection (hospitalisation less than four weeks prior to infection). For more information about CAPNETZ see www.capnetz.de. This article gives an overview of our findings, and the implications of these results for every day clinical practice.

Biomarkers and aetiology of CAPSeveral inflammatory markers, such as CRP and WBC, are routinely used in the diagno-sis of pulmonary infections. However, they are non-specific and unhelpful for the dif-ferentiation of bacterial or viral aetiology of pneumonia. PCT is a promising alternative in this respect, since its level rapidly increases in

bacterial infections but remains low in viral diseases. The aim of our study was to inves-tigate whether inflammatory markers meas-ured at admission are helpful in predicting the microbiological aetiology in CAP patients [1].

We enrolled 1337 patients with proven CAP and an intensive microbiological work-up was carried out. At the point of inclusion into the study, samples were taken for the determination of laboratory parameters and microbiology tests. Patients were clas-sified according to microbial diagnosis and the well-established “Confusion, Respiratory rate, Blood pressure, 65 years of age and older clinical score” (CRB-65 score). Patients with proven typical bacterial aetiology showed significantly higher levels of PCT, CRP and WBC than patients with atypical (Myco-plasma, Legionella, Chlamydia) or viral aeti-ology. In the differentiation of S. pneumo-niae CAP from CAP due to atypical or viral aetiology, a PCT cut-off level of 0.1 ng/mL showed an odds ratio of 8.3 whereas a cut-off level of 0.25 ng/mL showed an odds ratio of 3.2 In patients with atypical aetiology, levels of PCT, CRP and WBC were comparable to those in patients with viral aetiology. In con-trast to CRP and WBC there was a marked increase in PCT levels depending on the severity of CAP. The conclusions were that PCT, CRP and WBC values were significantly higher in CAP caused by classical bacterial pathogens compared to atypical bacterial or viral pneumonia, but they did not allow an individual prediction of the aetiology. Due to the large overlap of biomarker values in CAP with typical, atypical and viral aetiology it is

not possible to determine the aetiology of CAP in an individual patient. Thus inflam-matory biomarkers cannot be used in the choice of the appropriate individual antibi-otic therapy, principally because the elevation of inflammatory biomarkers depends not only on the aetiology of the infection but also on the severity of CAP. Thus, high PCT val-ues are indicative not only of a S. pneumoniae aetiology, but also of severe CAP. As a con-sequence, the application of established and validated clinical scoring systems, such as the CRB-65 score, is still needed in practice for the evaluation of CAP severity and the need for therapeutic interventions [2]. Biomarkers and antibiotic therapyEffective antibiotic therapy in CAP leads to a reduction in inflammatory biomarkers, so the longitudinal measurement of inflam-matory biomarkers is recommended for the evaluation of the efficacy of therapy. How-ever, many patients presenting in the emer-gency department have previously been treated as outpatients with antibiotics. (The CAPNETZ study showed that approximately 25% of hospitalised patients with CAP had been pre-treated with antibiotics in an outpa-tient setting). There are only very limited data concerning the influence of such antibiotic pre-treatment (APT) on the levels of inflam-matory parameters. Therefore, one aim of our study was to investigate, in hospitalised CAP patients, the influence of APT not only on the levels of the inflammatory biomarkers WBC, CRP and PCT, but also on the levels of the cardiovascular biomarkers pro-arginin-vaso-pressin (Copeptin) and pro-atrial natriuretic peptide (proANP) [3, 4, 5].

Our first study involved 370 hospitalised patients with CAP [3]. On admission the lev-els of the cardiovascular biomarker Copep-tin and the inflammatory markers PCT were measured (using kits from B.R.A.H.M.S., Germany, now part of Thermo Fisher Sci-entific) and CRP and WBC and the clinical CRB-65 score were determined. Eighty-five patients had had APT and 285 patients had not. Copeptin levels increased with increas-ing severity of CAP in patients without APT but not in patients who had had APT. Patients with APT showed significantly lower levels of

Biomarkers in community acquired pneumonia – lessons from the German competence network CAPNETZThe role of both established and new biomarkers in community-acquired pneumonia (CAP) was evaluated in the large multicentre cohort study conducted by the German competence network CAPNETZ. It was found that new cardiovascular biomarkers are suitable for the evaluation of short- and long-term survival in CAP. The combination of several biomarkers reflecting different pathophysiological pathways has the potential to improve management of CAP in the future.

by Dr Stefan Krüger, Dr Dirk Frechen, Dr Mathias W. Pletz, Dr Gernot Rohde and the CAPNETZ Study Group

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Copeptin and PCT, but not CRP and WBC compared to those without APT. The second study involved 991 hospitalised CAP patients [4]. On admission the levels of PCT, CRP and WBC were measured and the clinical CRB-65 score determined. There were 232 APT patients and 759 patients with no APT. Patients without APT had significantly higher levels of PCT and WBC, but not of CRP, compared to those with APT. Copeptin and PCT thus seem to be more sensitive bio-markers in CAP with more dynamic kinetics and react more promptly to effective therapy than the usual markers of CRP and WBC. As a consequence APT should be regarded as a factor that influences the correct interpreta-tion of cardiovascular (Copeptin, ANP) and inflammatory biomarkers. This is important for making a correct evaluation of biomarker kinetics under antibiotic therapy and for drawing the adequate diagnostic and therapeutic conclusions.

Biomarker and prognosisThe currently established risk scoring sys-tems for CAP are the complicated pneu-monia severity index (PSI) and the com-paratively simple CRB-65 score. However, these scores have been validated for the estimation of short-term mortality only. In patients surviving the acute phase of CAP there is an increased long-term mortality but up till now there has been no predic-tive scoring system for this; there is no biomarker for the estimation of long-term prognosis of CAP.

Short-term mortalityIn the first study we evaluated the prognos-tic value of the inflammatory markers WBC, CRP and PCT for the prediction of short-term mortality at 28 days [6]. We enrolled 1671 patients with proven CAP. The param-eters PCT, CRP, WBC and CRB-65 score were determined on admission. Patients were followed for 28 days. In 70 patients who died during the follow-up period, PCT lev-els on admission predicted the severity and outcome of CAP with a prognostic accu-racy similar to that of the clinical CRB-65 score and higher than that of the established inflammatory markers CRP and WBC. In the second study we analysed the new car-diovascular markers Copeptin and proANP in 589 patients. We found that proANP and Copeptin were at least comparable to the clinical CRB-65 score for the prediction of short-term mortality [7].

Long-term mortalityThe interesting results of these two stud-ies with respect to short-term mortal-ity prompted us to investigate long-term

mortality at 180 days in follow-up studies with larger numbers of patients [8]. In the first study we enrolled 1740 patients with proven CAP. Parameters determined on admission were ProANP, Copeptin, PCT, CRP, WBC and the CRB-65 score. Patients were followed for 180 days. ProANP and Copeptin levels were found to increase with increasing severity of CAP. In patients who died within 28 and 180 days, the initial proANP and Copeptin levels were found to be significantly higher than in survivors. In Receiver Operating Characteristic (ROC) analysis for 28 and 180 days survival respec-tively, the Areas Under the Curves, (AUCS or C-index) for Copeptin (0.84 and 0.78) and proANP (0.81 and 0.81) were superior to those of the CRB-65 score (0.74 and 0.71) and the inflammatory markers PCT, CRP and WBC. In multivariable Cox propor-tional-hazards regression analyses adjusted for comorbidity and pneumonia severity, proANP and Copeptin were found to be independent and the strongest predictors of short- and long-term mortality.

In the meantime other groups were show-ing that other new biomarkers such as pro-endothelin-1 (proET-1) and pro-adrenomedullin (proADM) could be use-ful for the prediction of prognosis in lower respiratory tract infections. However, until now there has been no study that compared all new biomarkers in the same study, so it was unknown which of the new biomarkers could be the best for use in clinical practice or in future biomarker-guided therapeutic intervention studies in CAP. For this rea-son, we wanted to validate the predictive potential of the new biomarkers proADM, proANP, Copeptin and proET-1 compared to the inflammatory biomarkers PCT, CRP, WBC and the clinical severity score CRB-65 for short-term and long-term mortality [9]. (The parameters proADM, proANP, Copep-tin and proET-1 can all be assayed using tests by B.R.A.H.M.S., Germany, now Thermo Fisher Scientific). We enrolled 728 patients with a follow- up of 180 days. In patients who died within 28 and 180 days, the levels of proADM, proANP, Copeptin, proET-1 and PCT, and also the CRB-65 score, were signifi-cantly higher than in survivors. In Cox regres-sion analysis proADM had the best perfor-mance for the prediction of 28 day and 180 day survival. The C-index of proADM for 28 days survival (0.85) was superior to those of proANP (0.81), Copeptin (0.78) and CRB-65 score (0.72) for the prediction of mortality. As for the prediction of mortality at 180 days, the C-index of proADM (0.78) was higher than those of proANP (0.74), Copeptin (0.73), proET-1 (0.76) and PCT, CRP and WBC.


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ProADM was independent of the CRB-65 score and added prognos-tic information for short- and long-term mortality. It was concluded that all the new biomarkers were good predictors of short- and long-term mortality, were superior to the inflammatory markers PCT, CRP and WBC and were at least comparable to the clinical CRB-65 score. Among the new biomarkers proADM showed the best performance so a combination of the CRB-65 score with proADM might be the best predictor of mortality.

How can it be explained that cardiovascular biomarkers are better for the prediction of short- and long-term mortality than inflammatory biomarkers? One explanation is that the inflammatory markers PCT, CRP and WBC are predominantly useful for the diagnosis of infection, whereas the new cardiovascular biomarkers reflect different aspects of CAP. There are various possible mechanisms which could support this. First, all of these new cardiovascular markers are elevated in sep-sis which is one of the main causes of short-term death in the acute phase of CAP. Secondly, Copeptin, proANP, proET-1 and proADM are increased in patients with cardiac failure. The elevation of these biomarkers in CAP might be due to underlying pre-existing cardiac disease or septic cardiomyopathy. We demonstrated that Copeptin, proANP and proADM are independent of pre-existing diagnosis of chronic heart disease or heart failure. As a result, CAP may aggravate underlying and previously unknown cardiovascular or renal disease due to acute inflammatory activation. The fact that proADM seems to be superior to the other cardiovascular markers proANP, Copeptin and proET-1 might be explained by the multiple functions of adre-nomedullin. In contrast to the parameters ANP, arginin-vasopressin and endothelin, which have predominantly cardiovascular actions, adrenomedullin possesses not only cardiovascular activity but also anti-inflammatory and antibacterial functions.

Implications for practical management of CAP Several investigations have shown consistently that, after the acute phase of CAP, long-term mortality is increased compared to age-matched cohorts. After 180 days the mortality rate is double that after 28 days. The highest impact of CAP on long-term mortality is in the first year after the initial CAP episode, but the association with increased long-term mortality could be demonstrated over a period of at least five years. Main causes of death after a CAP epi-sode are cardiovascular diseases and cancer, but chronic lower res-piratory disease, renal failure and infection also play an important role. In many patients who, long-term after the onset of CAP, die of cardiovascular disease, cancer and renal failure, the existence of such underlying pathologies is frequently unknown prior to the occurrence of CAP and even after the initial CAP episode. This sup-ports the traditional hypothesis that, in many cases, especially in the elderly, CAP might be a sentinel event for an underlying life-limiting disease. The new cardiovascular biomarkers might become new and useful additional prognostic markers for short- and long-term risk assessment in CAP. Elevated levels of these new cardio-vascular biomarkers in CAP identify a high risk population. As a consequence increased attention to possible cardiovascular disease, chronic lung disease and cancer and closer medical follow-up may be indicated. Whether this can result in an improved long-term out-come in CAP patients remains to be evaluated in future prospective randomised controlled studies, which include the measurement of the new biomarkers in the treatment algorithm.

ConclusionsThe CAPNETZ study has allowed us to perform biomarker stud-ies in very large cohorts of patients. We found that biomarkers are not helpful in the individual determination of the microbiological aetiology of CAP; in addition, they can be influenced by pre-treat-ment with antibiotics. For the diagnosis of infection inflammatory biomarkers are used. However, the widely used markers CRP and WBC do not correlate well with the severity and prognosis of CAP; for this, PCT is much more useful. There are many methodologi-cally excellent studies that have shown that a PCT-based algorithm can reduce the duration of antibiotic therapy compared to standard management of CAP [10-13]. In lower respiratory tract infections in general, a PCT-guided algorithm can significantly reduce anti-biotic exposure. In CAP, cardiovascular biomarkers have a higher prognostic value than inflammatory biomarkers and have also the possibility of detecting previously unknown cardiovascular-CAP comorbidities, which might lead to improved long-term outcomes in patients identified as high-risk. The pathophysiological mecha-nisms behind an infectious disease like CAP are far too complex to be reflected by a single biomarker as a surrogate marker for diagnosis, therapeutic decision-making or prognosis. However, the combination of several biomarkers reflecting different pathophysi-ological features of the disease, such as PCT for the bacterial/infec-tion aspects and the more multiplex proADM for inflammatory and circulatory aspects might have future potential for improved diagnosis, differential diagnosis and risk assessment in CAP.

references1. Krüger S et al, and the CAPNETZ study group. Inflammatory parameters predict etiologic

patterns but do not allow for individual prediction of aetiology in patients with CAP - Results from the German competence network CAPNETZ. Respiratory Research 2009; 10:65

2. Bauer TT et al, and the CAPNETZ study group. CRB-65 predicts death from community-acquired pneumonia. J Intern Med 2006; 260:93-101.

3. Krüger S et al, and the CAPNETZ study group. Pro-vasopressin (copeptin) in patients with community-acquired pneumonia – influence of antibiotic pre-treatment: results

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from the German competence network CAPNETZ. J Antimicrob Chemother 2009; 64:159-162

4. Krüger S et al, and the CAPNETZ study group. Assessment of inflammatory mark-ers in patients with community-acquired pneumonia – influence of antimicrobial pre-treatment. Results from the German competence network CAPNETZ. Clin Chim Acta 2010; 411:1929-1934

5. Krüger S et al. Influence of antibiotic pre-treatment on pro-atrial natriuretic pep-tide in community-acquired pneumonia. Eur Respir J 2008; 32 (Supplement): 383s

6. Krüger S et al. Pro-atrial natriuretic pep-tide and pro-vasopressin to predict sever-ity and prognosis in community-acquired pneumonia. Results from the German competence network CAPNETZ. Inten-sive Care Med 2007; 33: 2069-2078.

7. Krüger S et al. Procalcitonin predicts patients at low risk of death from com-munity- acquired pneumonia. Eur Resp J 2008; 31:349-355.

8. Krüger S et al, and the CAPNETZ study group. Pro-atrial natriuretic peptide and pro-vasopressin to predict short- and long-term survival in community-acquired pneumonia. Results from the German competence network CAP-NETZ. Thorax 2010; 65: 208-214

9. Krüger S et al, and the CAPNETZ study group. Cardiovascular and inflamma-tory biomarkers to predict short- and long-term survival in community-acquired pneumonia. Results from the German competence network CAP-NETZ. Am J Respir Crit Care Med 2010; 182:1426-1434

10. Burkhardt O et al. A simple procalci-tonin-guided strategy results in safe reductions of antibiotic use in patients with symptoms of acute respiratory tract infections in primary care. Eur Respir J 2010; 36:601-607

11. Christ-Crain M et al. Procalcitonin Guidance of Antibiotic Therapy in Community-acquired Pneumonia: A Randomized Trial. Am J Respir Crit Care Med 2006;174:84–93

12. Christ-Crain M et al. Effect of procal-citonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial. Lancet 2004;363:600–607

13. Schuetz P et al. Effect of procalcitonin-based guidelines vs standard guide-lines on antibiotic use in lower res-piratory tract infections: the ProHOSP randomized controlled trial. JAMA 2009;302:1059-1066

The authorsStefan Krüger1, Dirk Frechen1, Mathias W. Pletz2, Gernot Rohde3 and the CAPNETZ Study Group4

1Medizinische Klinik I, Univer-sitätsklinikum RWTH Aachen, Germany2Medizinische Klinik II, Univer-sitätsklinikum Jena, Germany 3Department of Respiratory Medi-cine, MUMC+, Maastricht, The Netherlands 4T. Bauer, J. Hecht (Berlin), B. Hauptmeier, S. Ewig (Bochum), C.

Schumann (Ulm), T. Schaberg, I. Hering (Rotenburg/Wümme), K. Dalhoff, P. Heyer (Lübeck), M. Pre-diger, K. Zobel (Cottbus), T. Welte, M. Pletz, J. Rademacher (Han-nover), B. Drewelow, J. Majcher-Peszynska (Rostock), N. Suttorp, A. Tessmer (Berlin, Charité), O. Bur-ghuber, G. Rainer (Wien), W. Peter-mann, H. Buschmann, R. Kröning, Y. Aydin (Paderborn), S. Krüger (Aachen), W. Pankow, A. Lies (Neu-kölln), R. Marre (Ulm), G. Rohde (Maastricht), R. Bals (Homburg/

Saar), H. Schütte (Berlin), G. Bar-ten, L. Gosman (Hannover), H. von Baum (Ulm, Med. Microbiology), P. Martus (Berlin), T. Illmann, M. Wallner (Ulm) and all study nurses.Address for correspondence:Priv. Doz. Dr. med. Stefan KrügerMedical Clinic I Medical Faculty, RWTH Aachen University Pauwelsstr. 30, D – 52057 Aachen, Germanytel: +49 241 8035832e-mail: [email protected]


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A broad variety of therapeutic strategies targeting specific receptors or defined signalling pathways are being explored in clinical studies including therapies for thoracic tumours such as non-small cell lung cancer (NSCLC) and malig-nant mesothelioma. One such strategy is based on the widely accepted obser-vation that most solid tumours require the creation of new blood vessels for fur-ther growth and metastasis, which may be achieved by induction of endothelial cell sprouting from the pre-existing vas-culature (angiogenesis). Alternatively tumour vessels may derive from vascu-logenesis, the formation of new blood vessels from circulating endothelial precursor cells, or by co-opting the pre-existing vasculature. These processes are regulated by multiple pro-angiogenic and anti-angiogenic factors that may be produced by tumour and stromal cells. However, currently vascular endothelial growth factor (VEGF) is still considered to be one of the most potent angiogenic and endothelial cell survival factors.

Recently, two large clinical trials (ECOG4599, AVAiL) demonstrated the efficacy of the anti-VEGF antibody beva-cizumab in combination with a platinum-containing chemotherapy for patients with advanced NSCLC of non-squamous histology, resulting in FDA approval for this indication. In both studies, there was a large subset of patients who did not respond, or even experienced tumour progression during therapy. Hence, pre-dictive markers would be helpful in selecting patients who may benefit from this therapy. Moreover, additional novel agents are being tested in clinical trials. The likely future consequence is that cli-nicians will need to decide which agents should be used for which patients.

In contrast to a prognostic factor that is indicative of tumour biology regard-less of the treatment, a predictive marker should provide information on whether the patient is likely to benefit from a given treatment. An anti-angiogenic agent spe-cifically targeting one selected pathway should exert the maximum effect on those tumours that critically depend on the given pathway. The types of investigation for bio-markers for anti-angiogenic therapy most often explored include studies on tumour-derived tissue and assessment of circulat-ing markers that are detected by peripheral blood analyses. A variety of markers may be of potential predictive value indicat-ing either response or resistance to a given therapy. The final goal is the identification of a biomarker that is helpful in clinical decision making. However, the search for biomarkers has just begun. Even the few large clinical trials that included molecu-lar analyses published to date have yielded a confusing variety of potential markers. Some of these markers and the rationale are briefly discussed below.

Markers in tumour biopsiesMost clinical studies investigating the prevalence and prognostic impact of angiogenesis in solid tumours have used tissue samples and assessed microvessel density (MVD) after performing immu-nohistochemistry. Two meta-analyses reported conflicting results on the prog-nostic importance of MVD in various sub-sets of patients, which may be due partly to heterogeneous methodologies. To date, there has been no clinical study reporting on MVD as a predictive marker in NSCLC, but trials are ongoing. In colorectal cancer patients treated with chemotherapy with or without bevacizumab, the pre-therapeutic MVD was not a significant indicator of the benefit of bevacizumab addition in 312

out of a total of 813 patients. Since NSCLC tumours may also co-opt preexisting blood vessels that may respond differently to anti-angiogenic therapy, MVD may be only predictive in subsets of patients.

Alternatively, the vessel density and intra-tumoural blood supply may be estimated using radiological techniques such as con-trast enhanced MRI or positron emission tomography (PET). In addition VEGF expression and VEGFR activation status in endothelial cells may predict the thera-peutic response to anti-VEGF directed therapy. Both high VEGF-A and VEGF-C protein expression have been associated with poor NSCLC survival in various clini-cal studies. To date there are no clinical studies that have correlated VEGF expres-sion with response to anti-VEGF agents. Interestingly, VEGFR expression has been documented in endothelial, stromal and even tumour cells, which may impact on the potential predictive value.

Circulating markersMost data on potential predictive mark-ers for anti-angiogenic therapy have been generated by measuring circulating angio-genic proteins in serum or plasma. In ret-rospective analyses, the predictive value of pre-treatment circulating VEGF levels provided inconsistent results in several studies, including studies on anti-VEGFR inhibitors in NSCLC patients. In the ECOG 4599 study, patients with high pre-treatment VEGF levels were more likely to respond to bevacizumab-containing therapy, but this was not predictive of sur-vival. However a meta-analysis, including four randomised studies with 1816 bev-acizumab-treated solid tumours of types including NSCLC, identified circulating VEGF as a prognostic but not predictive marker. Some studies reported elevations of VEGF and decreases in sVEGFR-2 with anti-VEGF therapy. Interestingly, similar changes were also seen in normal, tumour-free mice treated with sunitinib, indicating that these molecular changes are a systemic response to drug treatment. As in NSCLC patients, only limited data are available on the predictive value of circulating VEGF levels in patients with malignant mesothe-lioma. Results of a phase II study with 108 mesothelioma patients may suggest that

Predictive markers for anti-angiogenic therapy in thoracic tumoursVarious anti-angiogenic agents are being explored as therapeutic strategies for thoracic tumours. Since the regulation of angiogenesis is complex and may vary between individuals it is of utmost importance to select patients who may benefit from a specific therapy appropriately.

by Dr N. Reinmuth, Dr M. Meister, Dr M. Steins, Dr P. A. Schnabel, Dr F. J. F. Herth and Dr M. Thomas.

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anti-VEGF therapy could benefit patients with malignant mesothelioma and baseline VEGF levels at or below the median, how-ever, confirmatory studies are needed.

The predictive value of various cell adhe-sion molecule (CAM) families has also been investigated. During anti-angiogenic therapy, CAMs may be shed into the cir-culation where they may act as markers of activated or damaged endothelial cells. Hence, selected CAMs have been investi-gated as potential markers for monitor-ing anti-angiogenic therapies. For exam-ple, endothelial E-selectin is synthesised by endothelial cells and involved in the recruitment of leukocytes and tumour progression. Elevated expression levels of soluble E-selectin in the peripheral blood have been found in some small series of lung cancer patients. In the ECOG4599 study, E-selectin levels decreased signifi-cantly during treatment, and a significantly longer overall survival in patients treated with chemotherapy and bevacizumab was indicated when the level dropped to less than 5.35 ng/mL.

Another class of important CAMs, the immunoglobulin superfamily includ-ing ICAM-1, seems to be especially

produced in activated endothelial cells. The ECOG4599 study identified low baseline ICAM-levels as a significant predictive fac-tor for improved progression-free survival, but not for overall survival, when beva-cizumab was added to cytotoxic therapy. Further important candidates for potential predictive markers include cadherins such as E-Cadherin and Vascular endothelial (VE) -cadherin as well as the large family of integrins that mediate cell-ECM and cell-cell interactions. Avβ3 integrin is being elucidated for use in molecular imaging of angiogenesis using αvβ3 targeted nano-particles, however its use as a potential biomarker for anti-angiogenic therapy remains speculative. Results from clinical studies are pending.

As well as angiogenesis and co-option of pre-existing blood vessels, it has been pos-tulated that vasculogenesis for the de novo synthesis of new blood vessels by endothe-lial progenitor cells (EPCs) plays an impor-tant role beyond embryonic development. While the incorporation of EPCs has been demonstrated in tumour vascularisa-tion, the significance of this finding is still being debated. In tumour patients includ-ing those with NSCLC, EPCs have been found in varying amounts depending on

the method used to measure them. Since anti-vascular therapy may alter the num-bers of EPCs, this may predict response to therapy. In addition to EPCs, the presence of circulating endothelial cells (CEC) in the peripheral blood, which are shed from the vessel wall, have been considered to be a promising surrogate marker for vascular damage. In healthy individuals, CECs are rarely detected while increased numbers have been reported in a variety of cancer and lymphoma patients.

There is controversy regarding the identi-fication of both EPCs and CECs. Most but not all authors reported using flow cytom-etry techniques to detect these cells, but reported very different numbers of these cells in the peripheral blood. To date, there is no consensus regarding marker expres-sion, washing and centrifugation steps, the appropriate panel of monoclonal antibodies and gating techniques. Moreover, the phe-notype of these cells may change when they are cultured in vitro. Hence, many questions remain to be addressed regarding the origin and characterisation of circulating endothe-lial cells. However, EPC and CEC cell num-bers may be promising predictive markers for anti-angiogenic cancer therapy. This has not been addressed in large clinical studies.


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future directionsIdentification of predictive markers will be crucial for further development of anti-angiogenic therapy strategies. As well as the parameters described above, various other factors may be useful for prediction of anti-angiogenic therapy. For example, many other angiogenic factors contribute to blood vessel for-mation in tumours. Besides VEGF, known as a key-factor for regulating angiogenesis, other growth factors such as PDGF, bFGF and angiopoietins, as well as various CAMs, have also shown to be important in various tumour models. Moreo-ver, vascular remodelling that affects vascular stability may be an important determinant of the response of endothelial cells to anti-vascular therapy. In this process, the interaction of endothelial cells with other stromal cells such as pericytes, vascular smooth muscle cells (VSMCs) and fibroblasts plays a fundamental role.

However, after identifying a predictive marker, further impor-tant challenges remain before such markers can be used in clinical practice. For example, the technology used to measure

various biomarkers has not been standardised. The selection of high-quality reagents, standardised protocols and choice of markers is necessary. Moreover there is no consensus on the interpretation and scoring of the data generated by these methods. For some markers such as microvessel density, stand-ard procedures have been described, and recommendations for the selection of the appropriate clinical trial design for bio-marker identification, as well as discussions of ethical issues, have been published.

To investigate whether a biomarker predicts success for a given treatment, clinical trials should include molecular analyses of patient-derived material. This may necessitate repeated assess-ment of selected markers. Thus, cancer tissue samples should be collected before and, if possible, during or after therapy. It is likely that a combination of markers or a “signature” might prove to be of greater prognostic and predictive value than a single factor. Eventually even though it may may not be possible to characterise every patient, the primary aim is to define sub-groups with an exceptionally improved or decreased response to a selected agent.

Statement of potential conflicts of interestsNiels Reinmuth: Speakers and advisory honoraria (Lilly Germany GmbH; Boehringer Ingelheim Pharma GmbH & Co. KG). Michael Thomas: Speakers and advisory honoraria (AstraZeneca GmbH; Lilly Germany GmbH; Roche Germany GmbH; Boehringer Ingelheim Pharma GmbH & Co. KG). For all other authors: no conflicts of interests.

Selected literature1. Dowlati A et al. Cell adhesion molecules, vascular endothelial growth factor,

and basic fibroblast growth factor in patients with non-small cell lung cancer treated with chemotherapy with or without bevacizumab--an Eastern Coop-erative Oncology Group Study. Clin Cancer Res 2008: 14(5): 1407-1412.

2. Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer. 2008: 8(8): 579-91.

3. Peppercorn J et al. Ethics of mandatory research biopsy for correlative end points within clinical trials in oncology. J Clin Oncol. 2010; 28(15): 2635-40.

4. Reck M et al. Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung can-cer: AVAil. J Clin Oncol 2009: 27(8): 1227-1234.

5. Reinmuth N et al. Current data on predictive markers for anti-angiogenic therapy in thoracic tumours. Eur Respir J. 2010; 36(4): 915-24.

6. Sandler A et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006: 355(24): 2542-2550.

7. Sargent DJ et al. Clinical trial designs for predictive marker validation in can-cer treatment trials. J Clin Oncol 2005: 23(9): 2020-2027.

The authorsNiels Reinmuth1,2, Michael Meister1, Martin Steins2, Philipp A. Schnabel3, Felix JF Herth4, Michael Thomas1,2

Translational Research Unit1, Departments of Thoracic Oncology2, Pneumology & Respiratory Critical Care Medicine4, Thoraxklinik and Institute of Pathology3 University of Heidelberg, 69120 Heidelberg, GermanyCorresponding author:Niels Reinmuth, M.D.Department of Internal Medicine – Thoracic OncologyClinic for Thoracic Diseases / University of HeidelbergAmalienstr. 5D-69126 Heidelberg, GermanyTel + 49 6221-396 1301 e-mail: [email protected]

– April/May 2011 Prognostic markers20

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Clinicians are confronted by a bewilder-ing array of environmental moulds that may occasionally cause devastating dis-ease. Numerical models of fungal diversity calculate a lower limit of approximately 600,000 fungal species [1]. While the num-ber of moulds infecting humans is much more limited, their diversity, rarity, and the variability of both host factors and the circumstance of presentation all limit our ability to make evidence-based decisions based on observations and experience. Excellent reviews have been recently writ-ten on aspergillosis, mucormycosis and fusariosis, to name only a few [2,3,4].

Agents of mucormycosisThe Mucorales, similar to Aspergillus, are environmental moulds predominantly involved in decay. Unlike Fusarium, Scedosporium, Acremonium, Scopulari-opsis or Trichoderma, Mucorales are rarely

encountered in onychomycosis or any other parasitic or commensal relationship with animal hosts. Human infections are incidental and a biological dead-end for the organism. The Mucorales include sev-eral genera involved in sino/respiratory, rhinocerebral, pulmonary and traumatic invasive presentations. In immunocompro-mised patients, the agents of mucormyco-sis may establish an opportunistic infection following inhalation of spores. Infections also commonly originate in the sinuses and nasal turbinates, and in the immuno-suppressed can progress rapidly from these sites. Spores vary with Mucorales species and range in size from 3 -10 microns, with small spores being more likely to reach pulmonary alveoli. Clinically, Rhizopus species are most common, followed by Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea and Rhizomucor [Table 1]. As would be expected, this species

distribution varies considerably with geo-graphic locale and seasonal moisture changes. The predominance of one genus over another must be viewed cautiously. Culture recovery of Mucorales is poor and thus there is a potential for recovery bias depending on which species survive in vitro culture conditions. As with aspergil-losis, immunosuppression is a major risk factor and particularly so for patients with prolonged neutropenia such as haema-topoietic stem cell transplant recipients and heavily treated leukaemia patients. In normal hosts, Mucorales are readily killed by neutrophils and macrophages, and also strongly activate innate immune mecha-nisms such as C3 complement and Toll receptors. Interestingly however, the most common invasive presentation of mucor-mycosis is not in immunocompromised patients, but rather in patients with poorly controlled diabetes (both type I and II) and is frequently associated with ketoacido-sis or high dose corticosteroid treatment. Another condition predisposing to inva-sive disease in immunocompetent hosts is iron overload. Iron overload, particularly when associated with treatment using the bacterially-derived iron chelator desferri-oxamine (Deferral), can result in invasive disease comparable in severity to that seen in the most immunosuppressed host. Fur-thermore, mucormycosis is increasingly reported as a late complication seen in haematopoietic stem cell transplant recipi-ents following recovery from neutropenia suggesting the possibility of nosocomial iron overload as a risk factor.

Diagnosis of mucormycosisPatients with invasive mucormycosis tend to progress rapidly, with angioinvasion, throm-bosis and infarction of involved tissue. Diag-nosis requires a high level of clinical suspi-cion in the appropriate patient population, and is frequently supported by CT findings. These findings are rather non-specific, but are occasionally confirmed by histology or culture methods. Histologically, Mucorales produce large ribbon-like hyphae of irregu-lar diameter, with few septae. Nuclei, when seen, are syncytial and found near the grow-ing hyphal tips. In severely immunocompro-mised patients with rapidly growing lesions the Mucorales may show more septae, and in

Uncommon invasive mould infections: do they share common targets?

Environmental moulds may occasionally cause devastating disease. This article emphasises recent advances in our understanding of the pathogenesis and physiology of Mucorales and attempts to derive lessons that may be generalised to the rarely encountered invasive environmental moulds.

by Dr J.J. Tarrand, Dr E.A. Wagar, Dr G.L. Love and Dr D.P. Kontoyiannis

– April/May 2011 fungal infections22r

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Agents of Mucormycosis Agents of Hyalohyphomycosis

Rhizopus arrhizus (Rhizopus oryzae) Acremonium sp. Microsphaeropsis sp.Rhizopus microsporus Aphanoascus sp. Myriodontium sp.Rhizomucor pusillus Aspergillus sp. Nannizziopsis sp.Rhizopus stolonifer Arthrographis sp. Neocosmospora sp.Cunninghamella bertholletiae Beauveria sp. Paecilomyces sp.Apophysomyces elegans Cephalotheca sp. Penicillium sp.Saksenaea vasiformis Cerinosterus sp. Onychocola sp.Absidia corymbifera Chrysonilia sp. Ovadendron sp.Mucor circinelloides Chrysosporium sp. Pseudallescheria sp.Syncephalastrum racemosum Colletotrichum sp. Scedosporium sp.Cokermyces recurvatus Coprinus sp. Schizophyllum sp.Mortierella wolfii Cylindrocarpon sp. Scopulariopsis sp.

Fusarium sp. Scytalidium sp.Geotrichum sp. Tritirachium sp.Gibberella sp. Trichoderma sp.Gymnascella sp. Tubercularia sp.Lecythophora sp. Verticillium sp.Metarhizium sp. Volutella sp.Microsphaeropsis sp.

Table 1. The species of clinically relevant fungi.

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sparse or low fungal burden samples where only hyphal fragments may be seen, the fungal elements may be mistakenly identified as sep-tate hyphae. In culture colonies grow rapidly, are raised and fluffy, and are readily identified to the genus level. Microscopically, Mucorales are identified by their large irregular hyphae, by the arrangement of their sporangiophores, by the presence and arrangement of rhizoids, and by the presence or absence of a distinct collaret at the base of the sporangium [Figure 1]. On CT of the lung Mucorales tend to produce focal lesions with a variable degree of nodularity. With thrombosis this lesion may show increased contrast uptake at the periphery of the lesion, but this ‘halo’ effect is not always present. Radiographic finding are not specific, particularly in early lesions. The aspergillus galactomannan assay is typically negative, whereas the Beta-D-glucan assay may be positive or negative, and both assays have high false-positive rates. Histological confirmation is limited by sampling error, particularly in small samples collected by fine needle aspiration. Cul-ture yield of Mucorales is typically poor, with less than 20% of his-tologically demonstrated cases recovered from deep biopsy material. Further, contamination of specimens or samples in the laboratory is difficult to rule out. Our findings suggest that Mucorales die rapidly following collection from a deep tissue source. We have seen annexin V and tunnel assay findings suggesting an apoptosis-like process. An extensive search for media factors was unrewarding, however increasing the incubation temperature to 37°C doubled clinical yield [6]. Holding samples on ice prior to culture inoculation prevented mould loss in Aspergillus and may be advisable for all fungal sam-ples [6]. Nonetheless, culture remains problematic. Antibody-based immunohistochemistry and in situ hybridisation should help distin-guish Mucorales from septate moulds in tissue sections. Great hope is held out for PCR assays in mucormycosis, however the low level of DNA-aemia resulting from these durable organisms, combined with the rapid clearance of free DNA, may hamper assay development.

Treatment of mucormycosisMucorales remain difficult to treat; nevertheless, encouraging advances have been made. High doses of amphotericin B, lipo-somal amphotericin B, and amphotericin B lipid complex have all been reported to be useful. While the lipid amphotericin B formulations seem comparable in antifungal efficacy, the lipid formulations are better tolerated; however, no benefit for higher doses could be demonstrated in clinical trials. Aerosolised ampho-tericin B has been associated with success as has the combination

of amphotericin and caspofungin. Most azoles, including vori-conazole, have no activity in mucormycosis, however posacona-zole, at 800mg/day is active. In an open label salvage setting sev-eral retrospective studies have shown response rates >50% with posaconazole.

Adjunctive therapies are also of great interest. Granulocyte colony stimulating factor, GMCSF, and interferon gamma have all been associated with favourable outcomes. As noted above, iron overload and iron import seem to be critical to Mucorales and sufficent to predispose to invasive disease. The recent discovery of the xenosi-derophore import pathway and the fact that newer iron chelators are not imported suggest that iron deprivation may be useful clinically. Indeed the synthetic iron chelators deferiprone and deferasirox have been effective in animal models as single agents and also when used in combination with amphotericin B or other antifungals. Multicentre pilot studies of this approach are underway in mucormycosis.

Agents of hyalohyphomycosisSeveral excellent reviews have been written on Aspergillosis, Fusarium and Scedosporium [7]. Most, if not all, of the predispos-ing factors for Aspergillosis also predispose to the less commonly encountered agents of hyalohyphomycoses: Scedosporium, Acre-monium, Paecilomyces, Trichoderma and Scopulariopsis. The term hyalohyphomycetes has no taxonomic meaning, indicating only a loose grouping of non-pigmented septate moulds. Unlike aspergil-losis, frequently these agents are see in localised infections in immu-nocompetent hosts following trauma, in nail bed infections, or in infections associated with medical devices. However, in immuno-compromised hosts hyalohyphomycetes can occasionally present as deep-seated invasive sino-pulmonary disease and mimic aspergillo-sis clinically. In clinical laboratories they grow rapidly and are relia-bly identified to the genus level. Clinicians must always consider the dilemma of disease attribution. These organisms are common in the environment and may appear incidentally in respiratory samples after being trapped in mucus, or may be contaminants in the labora-tory environment. Again, culture recovery is poor for the true mould infections. Biopsy samples with histological evidence of aspergillosis grew the organism in <5%, and in an untreated animal model with a high tissue mould burden, one gram samples of involved lung could lose recovery completely in 2 hr at room temperature [6,8]. Molecu-lar methods hold out hope for improved diagnosis of rare moulds, but at this time remain largely developmental.

There are over 100 species of Fusarium with approximately 80 well characterised toxins. They are common in the plant rhizosphere and are found as plant pathogens. In addition they are pathogenic, largely through intoxication, to a large variety of animals, with equine toxic alimentary aleukia being a typical example. Fusar-ium can infect immunocompetent hosts causing endophthalmi-tis, onychomycosis, sinusitis, and peritonitis following dialysis. In immunocompromised hosts disease can extend from the sinuses, the nail beds, from catheters or other medical devices, or occa-sionally from the alimentary tract, as well as from inhaled spores. In the immunocompromised setting fusariosis shares many pre-disposing features with aspergillosis – predominantly neutrope-nia. The role of mycotoxins in invasive infection, if any, is not yet demonstrated. Like aspergillosis angioinvasion and thrombosis are prominent and lesions may appear similar on CT, but the ‘halo sign’ is generally absent. However, Fusarium and Scedosporium have the ability to form blastospores in tissue and are recovered well from blood culture. These yeast forms predispose to dis-semination, and in Fusarium cold necrotic gray skin nodules are a

– April/May 2011 fungal infections24

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frequent clinical finding. Differentiation of Fusarium and Scedosporium from Aspergil-lus is important since the former tend to be resistant to amphotericin-B.

Although there are common predisposing factors there are also features unique to the rarely encountered invasive moulds. Scedosporium can produce invasive pul-monary disease clinical indistinguishable from Aspergillus; in addition Scedosporium can present in immunocompetent hosts as an ascending lymphadenitis mimick-ing sporotricosis. Like Fusarium and Acremonium, Scedosporium are com-mon agents of onychomycosis which can become invasive in immunocompromised patients. S. prolificans (teleomorph- Pet-rella) is genetically distinct from S. apio-spermium and appears to be more aggres-sive. Invasive disease is generally similar to aspergillosis except that like Fusarium, Scedosporium tend to produce dissemi-nated skin lesions. The distribution of S. prolificans is relatively narrow being seen in Australia, the west cost of the United States, and on the iberian peninsula. S. prolificans can produce invasive disease following trauma even in immunocompe-tent hosts, and in neutropenic hosts mor-tality approaches 100%. Effective treat-ment regimes have not been identified. The prognosis depends largely on the host immune status. Acremonium species are found worldwide in soil, associated with the rhizosphere, and in rotting mushrooms. They are com-mon plant and insect pathogens and are

only rarely invasive in humans with skin, nail and lung being the prominent portals of entry. They appear largely resistant to current antifungals and treatment failure is > 50%. No optimal regime has been identified. The outcome of severe infec-tions caused by Acremonium depends largely on the recovery of neutrophil count, surgical intervention, and prompt diagnosis and treatment. Scopulariopsis species are widely distrib-uted soil organisms, occasionally associ-ated with invasive disease in immuno-compromised patients. Airborne spores, onychomycosis and trauma are the por-tals of entry. Most antifungals have lim-ited activity against Scopulariopsis species including amphotericin B, echinocandins, itraconazole, voriconazole, and posacona-zole. However, FG3409, a representative of a new antifungal class, shows promising activity. Clinical experience treating deep seated Scopulariopsis infections is limited and no effective regime has been identified. Paecilomyces and Tricoderma are seldom associated with severe invasive infection in immunocompromised patients. Inva-sive cases are associated with pulmonary, skin, or catheter infections. Most species of Paecilomyces are broadly resistant to current antifungals, but posaconazole and voriconazole show excellent MIC levels in vitro. Trichoderma is broadly resistant to most antifungals including posaconazole, however, caspofungin appears useful.

Treatment of hyalohyphomycosisAll of these hyalohyphomycetes remain difficult to treat and our understanding is hampered by their rarity. A potential path to discovery may be along similar lines to those now being applied to Mucorales, where accepted antifungals are being cau-tiously investigated in combination with adjuvant agents. For example, all of these moulds are adapted for soil environ-ments, including their siderophores and the bacterially-derived xenosiderophores used to scavenge iron. Controlling the availability of iron is a major and general host defence among animals. Therefore, it would be interesting to know if the novel synthetic iron chelators desferasirox and deferiprone can substantially deprive these organisms of iron. If so iron chela-tion as an adjunct to therapy warrants further study.

Interestingly, there is emerging evidence that immunosuppressed patients, diabetics, ketoacidotics and iron overload patients

share common features. The most pro-foundly immunosuppressed patients tend to get the most transfusion products, and iron overload has been shown to correlate with disease severity and rate of progres-sion for aspergillosis [9]. Any organism not adapted to the host may be iron-limited and would be expected to behave in a similar manner. Further, it has been demonstrated that diabetic patients, even in the absence of ketoacidosis, have a highly activated hae-meoxidase (HO) pathway, with exaggerated inflammation and generation of free iron in situ and with higher iron levels in the blood stream [10]. Genetic variants of hap-toglobin have been shown to predispose to diabetic complications, are know to release iron more easily and warrant investigation in relation to invasive mycoses [11]. Simi-larly, ketoacidosis decreases the iron bind-ing capacity of transferrin resulting in iron release, as well as affecting several other iron handling pathways [12].

Further, cholesterol may play a role in anti-fungal effectiveness. We have found that Aspergillus is able to import cholesterol and insert it effectively into its plasma mem-brane. In the presence of cholesterol (or human serum) a dose-dependent decrease in anti-ergosterol activity is seen [13]. In vitro amphotericin B and itroaconazole showed a 2 to 64 fold decrease in activity in the presence of 50 to 400 mg/dL cho-lesterol, and surprisingly vorizonazole was unaffected by cholesterol. Whether this mechanism applies more broadly to rare invasive environmental moulds is currently unknown. Animal models are needed. It is interesting to note that those moulds that produce statins can out-compete other moulds in their niche.

The exact mechanism of action of the ergos-terol-depleting drugs is unknown. Recently exciting insights into the mechanism of action of the ergosterol depleting drugs have been published [14,15]. It appears that all of these agents (azoles, allylamines, mor-pholines, and polyenes) show their highest impact at the level of the fungal vacuole func-tion and through the vacuolar H+ ATPase. This enzyme plays a critical role in hydrogen ion gradient formation and is critical to many cell functions. This V-ATPase was shown to be critically dependent on ergosterol. Alka-line conditions and increased calcium ions disrupt this vacuolar ATPase, and, interest-ingly, both alkalinisation and the calcium-active anti-arrhythmia agent amiodarone are sufficent to block fungal growth in vitro. When combined with azoles amiodarone enhanced killing in murine models.

– April/May 2011 fungal infections26

Figure 1. Photomicrographs of Mucorales features: a) Sporangia containing sporangiospores. b) Mucor demonstrating a distinct collarette. c) Rhizopus showing sporangiophore and adjacent rhizoids. d) Broad irregular aseptate hyphae with ribbon like folding. e) Characteristic vascular invasion. f) Poor staining with GMS silver stain.

SummaryThus, recent insights into fun-gal and host physiology are beginning to explain the long observed discrepancy between the good in vitro antifungal activity of the anti-ergosterol agents and their disappointing clinical effectiveness. While it may never be feasible to char-acterise the optimal therapy for each of these rare invasive moulds in clinical trials, it may be possible to evaluate one or more combinations of the adjunct agents outlined above with the current best-practice antifungal regimes similar to what is being done with mucor-mycosis. Hopefully, we will find that these rare moulds have more in common than may at first appear.

references1. Schmit JP, Mueller GM. An estimate of the

lower limit of global fungal diversity. Biodi-vers Canserv 2007; 16:99-111.

2. Dagenais TR, Keller NP. Pathogenesis of Aspergillus fumigatus in invasive aspergil-losis. Clin Microbiol Rev 2009; 22:447-465.

3. Spellberg B, Edwards J, Ibrahim A. Novel perspectives on Mucormyco-sis: Pathophysiology, Presentation, and Management. Clin Microbiol Rev 2005; 18:556-569.

4. Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev 2007; 20:695-704.

5. Kontoyiannis DP et al. Increased culture recovery of Zygomycetes under physi-ologic temperature conditions. Am J Clin Pathol 2007; 127:208-212.

6. Tarrand JJ et al. Aspergillus hyphae in infected tissue: evidence of physiologic adaptation and effect on culture recovery. J Clin Microbiol 2005; 43:382-386.

7. Cortez KJ et al. Infections Caused by Sce-dosporium spp. Clin Microbiol Rev. 2008; 21:157-197.

8. Tarrand JJ et al. Diagnosis of invasive sep-tate mould infections. Am J Clin Pathol 2003: 119:854-858.

9. Kontoyiannis DP et al. Increased bone marrow iron stores is an independent risk factor for invasive aspergillosis in patients with high-risk hematologic malignancies and allogeneic stem cell transplantation. Cancer 2007; 110:1303-1306.

10. Grochot-Przeczek A et al. Heme oxyge-nase-1 in neovascularisation: A diabetic perspective. Thrombosis Haemostasis 2010; 104:424-431.

11. Levy AP et al. Downregulation of the hemoglobin scavenger receptor in indi-viduals with diabetes and Hp 2-2 geno-type. Circ Res 2007; 101:106-110.

12. Fernandez-Real JM et al. Crosstalk between iron metabolism and diabetes: interacting pathways linking glucose and iron metabolism. Diabetes 2002; 51:2348-54.

13. Xiong Q et al. Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors. Antimicrob Agents Chem-other 2005; 49:518-524.

14. Yong-Qiang Z, Rao R. Beyond ergosterol: Linking pH to antifungal mechanisms. Virulence 2010; 1:551-554.

15. Gamarra S, Rao R. Mechanism of the synergistic effect of amiodarone and flu-conazole in Candida albicans. Antimicrob Agents Chemother 2010; 54:1753-1761.

The authorsJ.J Tarrand.1*, E.A. Wagar1, G.L Love.2 and D.P. Kontoyiannis3 1Department of Laboratory Medicine2Quest Diagnostics-Sacramento

and Department of Infectious Diseases Infection Control and Employee Health 3The University of Texas M. D. Anderson Cancer Center *Corresponding author: Jeffrey J. Tarrand, MDDepartment of Laboratory Med-icine, Unit 84, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boule-vard, Houston, TX 77030, USA. e-mail: [email protected]



Respiratory tract infections (RTI) cause con-siderable morbidity and mortality among the elderly population due to the general decline in the function of the respiratory and immune systems with age [1, 2]. Older adults are also more likely to have co-morbid cardiopulmo-nary conditions that increase the risk of severe RTI. Many of these RTIs are viral in origin, with viruses contributing from one-third to up to 80% of acute RTI [3, 4]. Infectious out-breaks commonly occur among older adult communities, such as residents in nursing homes and those using day-care facilities. The recognition of an infectious outbreak can pre-vent further spread of disease and allow early administration of treatment [5].

Diagnosis of viral respiratory tract infectionsThe diagnosis of RTI can be made based upon clinical presentation, radiological and laboratory evidence, and epidemiologi-cal data. While clinical signs and symptoms can suggest a specific infectious cause, they are not always reliable; clinical syndromes with different viral causes overlap, thus they cannot be used to confidently determine the aetiologic agent [1, 6]. Up until recently, reliable tests that were both very rapid and

accurate were unavailable, hindering the laboratory diagnosis of RTI in the elderly. The ability to detect and identify respiratory viruses is important for decreasing the use of antibiotics and unnecessary medical pro-cedures, administering appropriate antiviral treatment when necessary, and instituting infection control measures in hospitalised patients and those in skilled nursing facilities.

The most common causes of severe viral RTI in adults are influenza virus and respir-atory syncytial virus. Other causes include parainfluenzavirus, adenovirus, rhinovirus, coronavirus and human metapneumovirus. Some non-molecular methods for detecting respiratory viruses in the clinical laboratory are rapid antigen testing, direct fluorescent antibody (DFA) testing, viral culture and serological testing. Many of these meth-ods are limited in the number of different viruses they can detect. For example, coro-navirus and rhinovirus infections are not routinely assessed by DFA or rapid anti-gen testing, and they may not be routinely assessed by viral culture depending on the laboratory’s protocol. However, rhinovirus and coronavirus have been known to cause severe and fatal RTI in older adults [7, 8].

Some methods of respiratory virus identifica-tion are slow and cumbersome. Viral culture requires at least 24 hr using shell vial with blind staining, and conventional culture may take one to two weeks for detection of viral replication. Serological testing is highly sen-sitive, but usually reserved for retrospective analysis because paired acute and convales-cent sera are required for accurate diagnosis, particularly in older adults who have already been exposed to many different respiratory viruses in their lifetime. DFA and rapid anti-gen testing are rapid test modalities, but they are insensitive compared to molecular test-ing, particularly in older adults [1, 6]. Nega-tive results must be confirmed by a more sensitive method if it is important to detect the particular virus. Emerging molecular diagnostic techniques now offer the poten-tial to test for a greater range of viruses with increased sensitivity and speed. Multiplex reverse transcription-polymerase chain reac-tion (RT-PCR) for rapid diagnosis of viral RTI is a powerful tool, providing potential clinical and financial benefits for the health-care system [9, 10]. With excellent sensitiv-ity and specificity, multiplex RT-PCR can help make the diagnosis of viral respiratory infections in a timely and clinically relevant manner, allowing for more effective clinical care. Molecular methods can be designed to easily detect a wide range of viral agents, with some molecular platforms capable of detect-ing up to 15 to 20. Some examples include the xTAG RVP (Luminex Corporation, Aus-tin, TX, USA), PLx Multicode Respiratory Viral Panel (Eragen Biosciences, Inc., Madi-son, WI, USA), eSensor Respiratory Virus Panel (GenMarkDx, Carlsbad, CA, USA) and Infiniti RVP Plus (Autogenomics, Vista, CA, USA). With time, technological advances are expected to result in highly accurate diagno-ses requiring minimal hands-on time and providing even faster turnaround time than DFA or rapid antigen tests.

Diagnostic assay performance in the older adult populationProtocols for both traditional and newer diagnostic methods have been defined in diverse adult and paediatric populations. Older adults represent a distinct population for respiratory virus testing. RTIs in these populations usually represent reinfection where there is pre-existing immunity. Older adults experience fewer RTIs than children,

Laboratory detection of respiratory viruses in older adultsHighly sensitive assays for respiratory viruses (RSV) are necessary with older adults to minimise false negative results. Rapid antigen testing is not recommended for these patients, and DFA and culture results should be interpreted with caution as these methods are insensitive. Nucleic acid amplification-based testing, with its superior sensitivity, is the most robust method to detect respiratory viruses in older adult populations.

by Dr R. C. She

– April/May 2011 Virology28r

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Figure 1. The differential sensitivity of direct fluorescent antibody testing for influenza A during the 2009 pandemic is shown by age group. The sensitivity of this method decreases with age, and is significantly less in older adults (> 49 years) than in young children (0-5 years) (p = 0.013). The sensitivity was determined using shell vial culture as the gold standard and testing was performed at ARUP Laboratories, Salt Lake City, UT, USA (data not published).

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and they also shed fewer viral particles. Therefore, the optimal diagnostic approach for older adults with viral RTI differs from younger patients. DFA testing of respira-tory specimens, although relatively insensi-tive, remains a useful test in young children because they tend to shed copious amounts of virus [Figure 1]. This facilitates diagnosis in paediatric patients because DFA is gen-erally quicker and simpler to perform than multiplex RT-PCR. In the older adult popu-lation however, DFA and culture are not very useful [11, 12]. Although convenient, rapid antigen testing for RSV is not recom-mended for testing in older adults due to its insensitivity in this patient population [11]. Rapid antigen detection tests for influenza A and B are variably sensitive and specific. While they may be helpful where resources are limited and other test modalities must be performed off-site, their sensitivity in the older adult population is reported to be only 10-20% [13, 14]. During the peak influenza season, empirical treatment of influenza with antiviral medication in older adult patients may be more cost-effective than perform-ing a rapid test, especially for those who are unvaccinated or at a high risk of complica-tions from influenza infection [15]. Respira-tory specimens that can be used for testing include nasopharyngeal swabs and washes, sputum, bronchial specimens and bron-choalveolar lavages. Oropharyngeal speci-mens and rayon swabs appear to yield fewer viruses than nasopharyngeal specimens and nylon flocked swabs, respectively, in the older adult patient [16]. Nasal washes may be dif-ficult to obtain in older patients, who may be frail or cognitively impaired. When collect-ing nasopharyngeal swabs, it should be noted that older adult patients may have very dry mucosa, especially in cold winter conditions or if they are on oxygen supplementation. In these cases, there can be difficulty in collect-ing a high quality, cellular nasopharyngeal

specimen for testing. Adequate cellularity is required for DFA, as the respiratory tract epithelial cells need to be directly visualised. Other test modalities do not require speci-men cellularity, so a poor quality specimen will not be noticed, per se, with culture, rapid antigen testing or RT-PCR. Molecular meth-ods are highly sensitive and may still detect a respiratory virus in spite of poor specimen quality [12]. Therefore, in older adults, when a diagnosis of viral RTI is to be carried out, molecular methods such as RT-PCR are generally the most reliable [Figure 2].

Other issues in rTIlaboratory testing in older adultsConcern arises as to whether test results from nucleic acid amplification methods are representative of active viral infection. In immunocompromised patients, for exam-ple, prolonged shedding of viral nucleic acid can occur for months after infection. Detec-tion of viral nucleic acid in a given patient may represent asymptomatic viral shedding (subclinical infection) or the tail-end of a respiratory viral infection unrelated to the current respiratory symptoms, among other possibilities. Detection of respiratory virus in asymptomatic older adults occurs less fre-quently than in young children, in the order of 1-5% [17]. Viral shedding usually ceases after 1-2 weeks of infection [1, 17]. As with all patients undergoing evaluation for a res-piratory infection, in older adult patients the laboratory test results must be interpreted together with the clinical and radiological findings, and current epidemiological trends.

Interestingly, with the increased sensitivity of RT-PCR, it is not uncommon to detect more than one viral pathogen in a respiratory sample. In paediatric patients this occurs in 10 – 20% of cases [18]. The incidence of mixed viral RTI in older adults has only infrequently been studied using molecular

methods [12], and has been reported with very low frequency using other test meth-ods such as serology and culture [3, 4]. It is also of note that there were fewer reported cases of the 2009 pandemic H1N1 influenza in older adults. This is thought to be due to pre-existing immunity from cross-reactive, protective antibodies against the novel strain [19]. When considering the diagnosis of viral RTI in older adult patients, it is criti-cal to consider the interplay between their immune system, their comorbid conditions and the viral pathogen.

ConclusionsFor older adults, a highly sensitive assay is necessary to minimise false negative test results. Rapid antigen testing for RSV is not recommended in this patient group. Negative rapid antigen, DFA and culture results should be interpreted with caution as these methods are insensitive, particularly in the older adult population. Nucleic acid amplification-based testing, with its superior sensitivity, is the most robust method of detecting respiratory viruses in the older adult population, and further study is warranted to determine the clinical, epidemiological and financial bene-fits compared with more traditional methods of diagnosis.

references1. Falsey AR et al. J Clin Virol 2006; 35(1):46-50.2. Fulton RB, Varga SM. Aging health 2009; 5(6):775.3. Flamaing J et al. Eur J Clin Microbiol Infect Dis 2003;

22(12):720-5.4. Falsey AR et al. J Am Geriatr Soc 1995; 43(1):30-6.5. Rosewell A et al. Epidemiol Infect 2010; 138(8):1126-34.6. Walsh EE et al. J Infect Dis 2007; 195(7):1046-51.7. Greenberg SB. Semin Respir Crit Care Med 2007;

28(2):182-92.8. Hicks LA et al. J Am Geriatr Soc 2006; 54(2):284-9.9. Barenfanger J et al. J Clin Microbiol 2000; 38(8):2824-8.10. Woo PC et al. J Clin Microbiol 1997; 35(6):1579-81.11. Falsey AR et al. J Am Geriatr Soc 1996; 44(1):71-3.12. She RC et al. Diagn Microbiol Infect Dis 2010; 67(3):246-50.13. Rouleau I et al. J Clin Microbiol 2009; 47(9):2699-703.14. Talbot HK et al. Infect Control Hosp Epidemiol 2010;

31(7):683-8.15. Rothberg MB et al. Ann Intern Med 2003; 139(5 Pt

1):321-9.16. Hernes SS et al. Eur J Clin Microbiol Infect Dis 2011;

30(2):159-65.17. Jartti T et al. Pediatr Infect Dis J 2008 Dec; 27(12):1103-7.18. Gruteke P et al. J Clin Microbiol 2004; 42(12):5596-603.19. Hancock K et al. N Engl J Med 2009; 361(20):1945-52. The authorRosemary C. She, MDDepartment of PathologyUniversity of Utah School of MedicineSalt Lake City, UT, USAe.mail: [email protected]

– April/May 2011 Virology30

Figure 2. More sensitive assays, such as nucleic acid amplification techniques, detect more cases of older adults with viral respiratory tract infections. In this study, 67 older adult patients with respiratory tract infection were evaluated by different test methods. The multiplex RT-PCR (RVP) detected a respiratory virus infection in 16 cases, while culture and direct fluorescent antibody testing detected many fewer cases [12].

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what are immunodiagnostic assays?All immunodiagnostic assays rely on spe-cific antigen-antibody binding for accurate results. ELISA (enzyme-linked immuno-sorbant assays), immunofluorescent anti-bodies (FA) and lateral flow chromato-graphic immunoassay are often utilised in diagnosing infectious diseases [Figure 1].

In ELISA, an antibody with a specific affinity to a viral antigen is attached to a solid sur-face (usually polystyrene). A secondary anti-body is covalently linked to an enzyme which produces a colorimetric result indicating the presence of target antigen. These assays often use a 96-well plate format, allowing a large number of samples to be tested in a short time-frame (2-4 hours). Confirmation of positive ELISA results may be carried out using a blocking antibody assay.

In FA testing, cells bound on glass micro-scope slides are stained with specific anti-bodies directed against a target viral antigen. A fluorochrome (usually FITC) linked to the

antibody emits an apple-green fluorescence when examined using appropriate light wavelengths, and this can be used to identify viral antigen directly from patient specimens [Figure 2], or in shell-vial cultures. FA assays typically demonstrate greater sensitivity and specificity (fewer false negative and false pos-itive results, respectively) than other immu-nodiagnostic assays, but equipment and tech-nical training is required to perform them.

Lateral Flow immunoassays (aka chromato-graphic assays) dominate the diagnostic test-ing market. Although varying in format, these assays use a hydrophobic reaction membrane strip to which anti-target antibodies are bound. Specimens are treated with reagents to produce target antigen-conjugates con-taining coloured particles (i.e. colloidal gold) and are allowed to migrate across the mem-brane. If captured by membrane-anti-target antibodies, a coloured line indicative of a positive test is produced.

An advantage of lateral flow immunoas-says is that they permit point-of-care testing

(POCT) in emergency rooms and physician’s office settings on a 24 hour, 7-days-a-week basis. These assays require no equipment, little technical training, are inexpensive and produce rapid results (about 15 minutes).

what are influenza viruses?Influenza viruses have a genome consist-ing of eight single stranded RNA segments (influenza type C has seven RNA seg-ments) enclosed within a protein capsid and enveloped by a lipid membrane. Viral membrane proteins, haemagglutinin and neuraminidase enable virus-to-cell attachment, fusion and infection.

Influenza respiratory disease occurs in annual epidemics during the winter in both the northern and southern hemispheres. The virus is transmitted via aerosols and contact with infected secretions. There are three types of influenza: A, B and C. Type C virus only causes mild paediatric res-piratory illness and is not tested in the lab. Influenza type B only triggers human infec-tion, producing moderate symptoms. Influ-enza type A can cause severe, fatal disease in avian and mammalian species.

The symptoms of influenza infection may include fever, cough, headache, body aches and sinusitis. Lower respiratory tract symp-toms, including bronchitis, pleurisy and viral or secondary bacterial pneumonia are more severe and potentially lethal.

what makes detecting influenza viruses so important? Annual influenza mortality is estimated between 250,000 and 500,000 deaths glob-ally, with increased risk in patients who are very young or elderly, immunosuppressed, or who suffer from chronic cardiac, neu-rodevelopmental or pulmonary conditions.

Pharmacological agents are available for the treatment of influenza. Amantadine and its closely-related derivative, riman-tadine, specifically act on influenza type A virus. Other drugs, namely oseltami-vir and zanamivir, can exert effects on both influenza A and B. The effectiveness of drugs is dependent on specific virus identification and rapid drug administra-tion, usually within 48 hours following the onset of symptoms. Influenza viruses can

Immunodiagnostic assays, influenza and the H1N1 pandemicImmunodiagnostic assays are important for identifying infectious diseases. For influenza viruses, accurate diagnosis permits appropriate anti-viral drug administration and prevents nosocomial transmission in hospitals and healthcare facilities. This article highlights the role of immunodiagnostic assays, their importance for influenza testing and their performance during the 2009 influenza A / H1N1 pandemic.

by Dr G. P. Leonardi

– April/May 2011 Immunodiagnostics32

Figure 1. Immunoassays used in clinical virology. ELISA formats can include tube (a) and 96-well plate (b) formats. In both, a colour change indicates a positive result. The 96-well format allows testing of large numbers of patient samples but requires instrumentation to perform. Lateral flow chromatographic assays (c) produce coloured lines on hydrophobic membranes to demonstrate positive results (influenza B positive).

readily become resistant to one or all anti-viral drugs, complicating treatment and necessitating ongoing pharmacological viral surveillance.

Lateral flow immunoassays are a corner-stone of influenza testing due to their use-fulness in hospital and in a doctor’s office setting. Patients can be tested and if neces-sary treated with the appropriate antiviral medication at the point of care. Rapid test-ing prevents nosocomial viral transmis-sion in hospital and long-term care facili-ties, where patients are at greatest risk for influenza complications. In one study of influenza A outbreaks in care homes, cases of preventable infection ranged from 9% to 38% when combining immunodiagnostic and culture testing, compared with solely relying on culture to determine who was given amantadine prophylaxis [1].

The unpredictability of the influenza genome also permits the evolution of new, potentially lethal viruses that can cause worldwide epi-demics, or pandemics. Genetic variation in all influenza strains occurs by genetic drift, leading to gradual changes in viral antigens. For influenza A viruses, a second mecha-nism, namely genetic shift, also occurs. Here a reassortment of RNA segments from bird,

mammal and human viruses may occur, pro-ducing a new strain to which humans have no prior exposure. After recombination, successful animal-to-human viral transmis-sion, followed by efficient human-to-human transmission can lead to an explosive epi-demic or pandemic outbreak. New pan-demic strains are unpredictable with respect to symptoms, risk groups, drug sensitivity, transmission efficiency and morbidity and mortality. In March 2009 a new recombinant influenza subtype emerged in Mexico and rapidly spread worldwide. Pandemic 2009 H1N1 virus had arrived.

The 2009 H1N1 experienceImportant distinctions between the 2009/H1N1 and co-circulating seasonal H3 and H1 influenza A strains became apparent. Independent risk factors associated with the 2009 H1N1 infection were morbid obesity, pregnancy and young age, whereas a low incidence of illness was observed in those aged 65 years or older [2-3]. An increased frequency of nausea/vomiting and diarrhoea was reported in the 2009 H1N1 infection. Drug sensitivity between viruses also differed. Both seasonal H3N2 and the 2009 H1N1 was sensitive to osleta-mivir and zanamivir, but resistant to aman-tadine and rimantadine. The reverse was

true for the seasonal H1N1 virus.There was a dramatic surge of patient visits to hospital emergency departments follow-ing the pandemic outbreak. Isolation facili-ties for 2009 H1N1-infected patients were in short supply. Definitive diagnosis of 2009 H1N1 became necessary both for appropri-ate therapy and selective patient isolation.

Diagnostic testing for the pandemic 2009 H1N1 virusThe sensitivity of lateral flow immunoassays for identifying the new influenza subtype was unacceptable. A comparison of three lateral flow immunoassays to identify the 2009 H1N1 virus found sensitivities rang-ing between 40 and 70% [4]. Even if sen-sitivity were 100 %, these immunoassays could not specifically differentiate influenza A subtypes. Molecular reverse-transcriptase polymerase chain reaction assays (rt-PCR) can provide a highly sensitive diagnostic alternative to lateral flow immunoassays, but cannot be used at point-of-care.

A 2009 H1N1 rt-PCR assay was Emergency Use Authorized (EUA) and distributed to various regional laboratories across the United States. These facilities soon received so many requests for testing that reporting of results was seriously delayed. A mean


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time interval of 5.6 + 0.37 days from speci-men collection to the availability of rt-PCR results was reported in one study [3]. This reporting time-frame did not result in improved patient care, nor did it reduce patient numbers in hospital isolation areas.

Manufacturer-developed rt-PCR assays soon received EUA clearance and became available for laboratory use. However, most hospital laboratories lacked the ability to perform these molecular assays. Although molecular assays are much more sensitive than other virology assays, high equip-ment, maintenance and reagent costs, together with the need for specific techni-cal training, often precluded their adoption by laboratories.

Fluorescent antibody techniques offered an alternative to both lateral flow and molecular assays. Many hospital labo-ratories (i.e. microbiology, serology and immunology) already performed fluo-rescence microscopy. These techniques could be used to identify viral antigens directly in patient nasopharyngeal speci-mens, re-test lateral immunoassay-nega-tive samples, or identify viruses in rapid shell vial cultures.

The issue of specific 2009 H1N1 identifi-cation remained unresolved until a fluo-rescent antibody reagent for the new pan-demic virus (D3Ultra H1N1 Influenza A virus ID Kit; DHI, Athens OH, USA) was EUA cleared. This reagent provided an accurate, rapid and inexpensive assay to specifically identify the 2009 H1N1 virus in hospital laboratories lacking molecular diagnostic capabilities [5]. However, fol-lowing the first 2009 H1N1 influenza sea-son, the pandemic emergency was lifted, as was the EUA status for assays used to indentify A/2009 H1N1 virus.

Highlights of the recent 2010-2011 flu seasonPandemic 2009 H1N1 displaced the sea-sonal A/H1N1 viral subtype and currently co-circulates with seasonal H3N2 virus. Both viruses have identical drug sensitiv-ity to oseltamivir and zanamivir, so rt-PCR subtyping is far less necessary.

Studies of 2009 H1N1 oseltamivir resistance in Japan and the United States have demon-strated only minimal levels (0.5 to 1.2%) of resistance, predominantly among severely immunocompromised patients [6-7]. These findings suggest that zanimivir should be

the drug of choice for these patients.Lateral flow immunoassays still remain a mainstay for influenza testing despite reduced sensitivity with 2009 H1N1 virus. At point-of-care, positive lateral flow results are reliable and useful for initiating antivi-ral drug therapy. Negative results should be confirmed by re-testing. An algorithm has been developed for this purpose using FA or shell-vial culture techniques [Figure 3]. Positive influenza A virus results can be subtyped using FA or rt-PCR methods.

Finally, the unpredictability of influenza A viruses remain a constant challenge to public health. A recent study characterised genetic reassortment between seasonal H3N2 and 2009 H1N1 viruses as a potential cause of increased pathogenicity [8]. It would greatly benefit public health if diagnostic manufac-turers could develop new and more sensitive point-of-care immunoassays and provide testing alternatives for laboratories lacking molecular capabilities. We know that the next great influenza pandemic is not a matter of “if” but rather “when”.

references1. Leonardi GP. et al. Comparison of rapid detection meth-

ods for influenza A virus and their impact on the health-care management of institutionalized geriatric patients. J. Clin Microbiol 1994; 32: 70-74.

2. Dawood FS, Jain S, Finelli L and members of the novel swine-origin influenza A (H1N1) virus investigation team. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009; 360(25): 2605-2615.

3. Leonardi GP, Mitrache I, Pigal A, Freedman L. Public hos-pital-based experience during an outbreak of pandemic influenza A (H1N1) virus infections. J Clin Microbiol 2010; 48(4): 1189-1194.

4. Centers for Disease Control and Prevention. 2009. Evalu-ation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) virus- United States, 2009. MMWR 2009; 58: 826-829

5. Leonardi, G.P. Rapid identification of 2009 H1N1 Influ-enza A virus using fluorescent antibody methods. Am J Clin Pathol 2009; 134: 910-915.

6. Graitcer SB et al. Characteristics of patients with oseltami-vir-resistant pandemic (H1N1) 2009, United States. Emerg Infect Dis 2011; 17(2): 255-257.

7. Ujike M et al. Monitoring and characterization of oseltami-vir-resistant pandemic (H1N1) 2009 virus, Japan, 2009-2010. Emerg Infect Dis 2011; 17(3): 470-479.

8. Schrauwen EJA et al. Possible increased pathogenicity of pandemic (H1N1) 2009 influenza virus upon reassort-ment. Emerg Infect Dis 2011; 17(2): 200-208.

The authorDr G.P. LeonardiDepartment of Pathology and LaboratoriesNassau University Medical CenterEast Meadow, NY, USAe-mail: [email protected]

– April/May 2011 Immunodiagnostics34

Figure 2. Fluorescent immunoassay testing of a nasopharyngeal specimen using the D3 Ultra 2009 H1N1 reagent (DHI Inc, Athens, OH, USA). Positive cells emit apple-green fluorescence. This product allowed the definitive identification of the 2009 pandemic H1N1 virus without the need for molecular diagnostics.

Figure 3. Respiratory virus testing algorithm permitting the specific identification of pandemic 2009 H1N1. Patient nasopharyngeal swabs are collected, placed in viral transport medium [VTM] and tested using a lateral flow immunoassay at the point of care. Laboratory retesting of negative results can be done using fluorescent antibodies [FA] specifically directed against influenza A & B antigen. Positive influenza A specimens are sub-typed using the D3 Ultra 2009 H1N1 FA kit or by rt-PCR. Negative direct specimen FA results can again be retested using shell vial cultures [R-mix too;

NEGATIVE POSITIVE INFLUENZA A&B

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Traditionally, the gold standard for diagno-sis in the clinical microbiology laboratory has been culture – the organism is isolated and identified definitively, and a patient report is generated. Where culture is not feasible, for example in the diagnosis of Hepatitis B infection, the accepted stand-ard has been the detection of markers, either antigen or antibody, in the patient’s serum. For diagnosis of fungal infection, it is accepted in the case of the difficult-to-culture Malassezia furfur for example, that microscopy is sufficient, given that its appearance is sufficiently distinctive to allow its identification solely by direct microscopy. The gold standards for diag-nosis therefore have evolved on the basis of the most sensitive and specific techniques available at the time.

It is widely accepted that the earlier an accurate microbiology report can be gen-erated, the more significant the impact it is likely to have on patient care. However, sample culture takes a minimum of 18 hours, followed by further subculture for identification and susceptibility testing. At the other end of the scale, a sample sus-pected of containing Mycobacterium tuber-culosis may be cultured for 42 days before a negative report can be generated.

The first molecular assays to be used have been those where culture has not been possible in the routine laboratoryCurrently, in clinical diagnostic laboratories, molecular detection of pathogens is begin-ning to replace traditional culture-based techniques. One example of where molec-ular-based diagnosis has already become well-established is in the detection of Chla-mydia trachomatis infection. The 1980s saw direct fluorescent antibody (DFA) tests and enzyme immunoassay replace tissue culture – the gold standard of the time - particularly

in the absence of specialised and time-con-suming tissue culture facilities. In the 1990s the commercial development of Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) methods for chlamydia detection, although more expensive than DFA/EIA, were nonetheless shown to be comparable to tissue culture in terms of sensitivity and specificity [1]. More recently, Real Time PCR (RT PCR) methods for chla-mydia detection have been developed, and large throughput of samples and commer-cial competition have helped to drive down costs incurred by the testing laboratory, so that most diagnostic laboratories can now expect to pay less than €8 per test. The wide-spread use of relatively user-friendly molec-ular methods for chlamydia testing may have helped to pave the way, albeit rather slowly, for the uptake of further molecular assays as they became available.

Challenges associated with developing in-house assaysThe development of in-house molecular assays has been undertaken by some diag-nostic laboratories. The advantages of devel-oping an in-house assay over the use of a similar commercial assay include lower cost and a degree of flexibility in the choice of open-platform technology. Furthermore, particularly in the case of reference labora-tories, the development of an in-house assay may allow a rapid response to a pandemic for example, whereby a national diagnostic service for a novel infectious agent can be provided quickly, without waiting for a com-mercial company to offer a suitable product. The ready availability of sequences in inter-national genetic databases, together with the rapid dissemination of such information by agencies such as the US Centers for Disease Control and Prevention, has made assay development possible on a global basis. The extensive evaluations and validations required by the diagnostic laboratory before

adopting a new in-house assay, when com-pared to a commercial CE-marked assay for in vitro diagnostic (IVD) use, might how-ever be seen as a disadvantage.

why are commercial molecular diagnostic assays so expensive?The inherent cost to the end user of using CE-IVD molecular assays can be attrib-uted to a number of factors that are borne by the manufacturer. Companies seeking to bring diagnostic kits to the market for the purpose of IVD testing must achieve clear-ance according to the criteria set down by IVD Directive 2007/47/EC. Inherent in this process is the requirement to work to Good Manufacturing Practice (GMP) standards and to maintain a Quality Management Sys-tem (QMS) as described in ISO13485:2003 which is prescriptive in terms of medical device QMS requirements for regulatory purposes. The development of a CE-IVD assay incorporates a number of extensive phases including research, analytical and clinical validation, certification and ongoing post-market approval requirements. Fur-thermore, significant costs are incurred from developing novel technologies for an assay or through license fees for the use of patented technologies. As an example, a multiplex real-time PCR assay may incur license fees for gene targets, fluorophores, quenchers, modi-fied polymerases, amplification technology and preparation processes such as lyophilisa-tion. The costs of GMP grade raw materials, manufacturing, direct/indirect distribution and product support also bring with them important financial considerations.

That certain routine diagnostics tests remain traditionally culture-based is dictated largely by the cost of introducing new techniques (as discussed above), staff training and increased space requirements. Such factors are all too often the primary causes for stag-nation when implementing novel molecular diagnostics in the clinical laboratory.

Diagnostic microbiology, as we have long known, is complicated by bio-diversityDespite the shortcomings outlined above, diagnostic molecular biology is argu-ably the fastest growing area in current laboratory-based medicine, and has the potential to change the course of clinical

Molecular diagnostics: the changing culture of medical microbiologyTraditional culture-based diagnosis of infection is gradually being replaced by molecular detection methods. This article examines the challenges, considerations and some new developments in the field of molecular clinical microbiology that may both affect and be effected by the medical scientist as part of the collective drive to improve healthcare.

by Dr S. Bullman, Dr R. Sleator and Dr B. Lucey

– April/May 2011 Microbiology36

medicine dramatically over the next dec-ade. An emerging field within the world of molecular microbiology is metagenomics which encompasses a relatively new sphere of genetic research, enabling the study of organisms that are not easily cultured in the laboratory [2]. Metagenomics is adopted on the basis that a vast majority of microbial bio-diversity is overlooked by traditional culture-based techniques. This in turn has led to the development of pathogenomics – the application of genomic and metagen-omics data to understand microbe diversity and interaction as well as host-microbe interactions in disease states.

who has responsibility for effecting new strategies for laboratory-based diagnosis?Overall, the recent rapid advances in molec-ular-based detection methods, described earlier, underline the need for tripartite col-laborations between clinical, research and commercial laboratories. Collectively, we can hope to continue to design and develop ever-more sophisticated, rapid and auto-mated assays targeting clinically significant organisms whilst still ideally conforming to the working day of the medical scien-tist. There is a fundamental need to iden-tify those pathogens, both established and emerging, which impose the highest clini-cal and economic cost. Thus, the source of infection can be identified and effective infection control measures implemented quickly enough to be effective.

A serendipitous consequence of molecular-based detection methods is that, in addition to their speed, high specificity and sensi-tivity, they may also facilitate the detection

of previously unknown and undetected pathogens. In support of this has been the identification of Campylobacter ureolyticus as a novel gastrointestinal putative patho-gen using a commercial multiplex-based PCR detection system in the Republic of Ireland [3, 4]. Given that campylobacter is currently the most commonly-isolated bac-terial pathogen worldwide, the detection of a new species in more than 20% of the total campylobacter detections among patients with acute gastroenteritis may be deemed a significant finding.

Indeed, this study revealed that C. ureo-lyticus now surpasses C. coli as the second most common campylobacter species in the faeces of patients presenting with gas-troenteritis (at least in Southern Ireland). This organism, which is incapable of growth on routine campylobacter culture, has until now, never been reported in the faeces of patients with diarrhoeal illness. This is an example of the benefit that molecular diag-nostics can bring to bear on the detection of fastidious organisms, which by routine culture would have been otherwise reported as false negatives. Naturally however, the detection of C. ureolyticus has necessitated further research to establish the source and true virulence potential of this novel enteric agent (work which is currently ongoing in our laboratory). Interestingly, the majority of faecal samples routinely tested for enteric pathogens return negative results, begging the question - what else is being missed?

ConclusionWith a time-to-result of hours rather than days, molecular detection promises, in addition, higher specificity and sensitivity.

However, given that molecular biology tar-gets DNA rather than live cells, the approach can sometimes be too sensitive – making no designation between DNA from live or dead organisms. Furthermore, the application of molecular diagnostic approaches requires specialised expertise for quality assessment and control, and for auditing and accredita-tion purposes. Ultimately, the effective use of molecular diagnostic methods will rely on the same criteria as for traditional culture methods - maintaining an affordable level of sensitivity and specificity, seeking not to exclude the less common pathogens, while selecting for those deemed to be the most significant in human infectious disease.

references1. Schachter J. DFA, EIA, PCR, LCR and other technologies: what

tests should be used for diagnosis of chlamydia infections? Immunol Invest 1997; 26:157-161.

2. Sleator RD, Shortall C, Hill C. Metagenomics. Lett Appl Micro-biol 2008; 47: 361-6.

3. O’Leary J, Corcoran D, Lucey B. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bac-terial enteric pathogens. J Clin Microbiol 2009; 47: 3449-3453

4. Bullman S, Corcoran D, O’Leary J, Lucey B, Byrne D, Sleator RD. Campylobacter ureolyticus: an emerging gastrointestinal patho-gen? FEMS Immunol Med Microbiol. 2011; 61:228-30.

The authorsDr Susan Bullman, Dr Roy Sleator and Dr Brigid Lucey*Department of Biological Sciences Cork Institute of Technology Bishopstown, Cork, Ireland*Corresponding author: Dr Brigid Lucey, FAMLSLecturerTel: +353 21 4326306e-mail: [email protected]


0094_Mycoplamsa_RevolutioN_AD_2_EXE.indd 1 15.04.2011 09:59:38 www.cli-online.com & search 25519

The clinical diagnosis of an infectious disease is routinely confirmed in the laboratory by two standard approaches. One approach is to detect serum antibody that is specific for the agent of interest. Typically IgM is measured by ELISA, since the presence of pathogen-specific IgM is usually only observed follow-ing a recent infection or vaccination. A sig-nificant rise (> four-fold) in antigen-specific IgG titre can be used as an alternative to IgM testing in some instances. The second main approach is to detect the pathogen itself. This can be done in a number of ways including culture, detection of pathogen-derived anti-gens (the basis for many rapid tests), or detec-tion of pathogen-specific nucleic acid (PCR). All of these methods have their unique limi-tations. However, these limitations can be

compounded when the methods are used to test specimens from individuals who have been previously exposed to the infec-tious agent through vaccination or naturally acquired infection.

The problem of standard diagnostics with mumpsAs one illustration, mumps paramyxovirus causes the classic childhood illness that is predominantly characterised by swelling of the parotid (salivary) glands and fever. The most frequent complications include orchitis (in post-pubertal males), meningitis, hear-ing loss and pancreatitis. Fortunately, the incidence of mumps disease has been dra-matically reduced in countries that provide good vaccination coverage [1]. However, it

has been known for some time that one can acquire wild-type mumps virus infection more than once [2]. Moreover, the efficacy of two doses of MMR vaccine is estimated to be ~90% (79-95%) for protection against mumps [3]. It is, therefore, not surprising that certain vaccinated individuals remain susceptible. This has been demonstrated in recent years by a few limited outbreaks among two-dose vaccine recipients [1]. The reasons for these breakthrough infections are not well understood since the correlate of protection for mumps has not been defined and since there is only one known serotype of mumps virus. However, the reasons may include a failure to respond to immunisation, priming of inefficient immune responses, loss of immunity over time or other intrinsic differences among virus strains.

When individuals who have been previ-ously vaccinated or naturally infected are (re)infected with mumps, they may respond somewhat differently from someone who has never been exposed to the virus. In contrast to someone who experiences a pri-mary mumps infection, previously exposed individuals may or may not have a detect-able IgM response. If they do have an IgM response, it may be blunted in amount and duration. Furthermore, there is considerable variation in the sensitivity and specificity of individual IgM testing methods and kits (typically based on ELISA or immunofluo-rescence staining). As a result of all these fac-tors, a negative test for mumps-specific IgM response does not rule out the possibility that the individual is actually infected. In addition, previously exposed individuals often have a high mumps-specific IgG titre at the time of symptom onset and it usually does not rise enough to confirm the infection. Waiting several days or weeks to obtain a convales-cent serum specimen for comparison with the acute serum IgG level also defeats the immediate purpose of a preferred diagnostic.

Similarly, a negative result by RT-PCR also does not rule out the possibility of mumps infection. Previous studies indicate that sam-ples collected within the first three days of symptom onset are the most likely to be posi-tive [4, 5 and our unpublished observations].

Detection of antigen-specific B-cells by ELISPOT as an alternative to standard diagnosticsFor reasons that are not always clear, some infectious diseases can cause symptomatic re-infections in individuals who have been previously vaccinated or naturally infected. When this occurs, standard diagnostic methods based on serology or detection of the infectious agent can be unreliable or uninformative. In these circumstances, detection of activated antigen-specific B-cells by ELISPOT (Enzyme Linked Immunosorbent Spot) is a sensitive and reliable alternative that can overcome many limitations of standard diagnostics, but certain obstacles currently hinder its routine clinical application.

by Dr D. Latner

– April/May 2011 Molecular diagnostics38

Figure 1. Scheme showing how antibody-secreting B-cells are detected by ELISPOT.

This suggests that the amount and duration of virus shedding may be limited in vacci-nated individuals. Moreover, it underscores the fact that virus detection is critically dependent on a number of other factors that include the timing of specimen col-lection, the method of specimen collection (including by massage of the parotid prior to obtaining oral fluid) and the diligent pro-cessing, storage and transportation of sam-ples. In addition, unexpected sequence poly-morphisms have the potential to negatively affect the efficiency of PCR primer binding and amplification.

These diagnostic challenges are not unique to mumps but are shared with other diseases that are known to cause breakthrough infections, such as pertussis (whooping cough) and varicella (chicken-pox). For the physician, this can be par-ticularly frustrating because breakthrough disease among vaccinated individuals can have a mild presentation, thus making diagnosis even less certain. Furthermore, a low frequency of laboratory confirmation among suspect cases can lead to an under-estimation of disease incidence and frus-trate the implementation of epidemiologic control measures.

ELISPOT: an alternative approachThe detection of activated antigen-specific B-cells by ELISPOT is a robust alternative approach that can overcome the limita-tions of serology and pathogen detection in breakthrough cases. The rationale for using this method is based on the different stages of the B-cell response to infection [6]. Naïve B-cells do not secrete antibody unless they become activated. Once activated, they begin to proliferate and secrete antibody. These activated cells are called “Antibody-Secreting B-Cells (ASC)” or “plasmablasts.” The ASC continue to proliferate until the pathogen is cleared from the body, and then most of the cells simply die. Some of the ASC migrate to germinal centers in the spleen where they interact with T-cells, receive cytokine signals, and undergo the process of affin-ity maturation. From the germinal centres some of the cells go on to become memory B-cells and others become long-lived plasma cells. Memory B-cells remain in the circula-tion but do not secrete antibody unless they become re-stimulated by antigen. In contrast, long-lived plasma cells migrate to the bone marrow where they are sequestered from circulation and they continue to secrete anti-body that is the source of antibody found in the serum. Thus, B-cells that are found in the

blood do not secrete antibody unless they have been recently stimulated by antigen (i.e., infection), [Figure 1].

The method of detecting activated B-cells by ELISPOT was initially developed in 1983 by two separate groups [7, 8] and it is performed as briefly follows. Peripheral blood mononuclear cells (PBMCs) are iso-lated from whole blood (5-10mL), then these cells are cultured for 6-24 hours in a 96-well ELISPOT plate that has been coated with the antigen of interest. Gravity causes the cells to settle in the bottom of the wells. Antibody that is secreted from activated B-cells and that is specific for the antigen on the plate will bind to the bottom of the well in the immediate vicinity of the cell. After the incubation period, the cells and non-specific antibodies are washed away. Anti-body that is bound to the plate is detected using an enzyme-conjugated secondary antibody that causes the precipitation of a colorimetric reagent. The spots that develop can be counted and directly represent the presence of individual activated cells. This process is extraordinarily sensitive and spe-cific. One activated antigen-specific B-cell can be detected among several million PBMCs [Figure 2].



The application of B-cell ELISPOT as a diagnostic tool is also not a new idea. In fact, a review by H. Arvilommi in 1996 describes its use for detection of the B-cell response to a wide variety of bacterial, viral and para-sitic diseases [9]. A similar concept has been employed to diagnose latent TB infection. The T-SPOT.TB test (T-Spot) produced by Oxford Immunotec Limited (Abingdon, UK) is similar in that it detects the presence of antigen-specific cells in the blood [10]. However, this method is fundamentally dif-ferent from B-cell ELISPOT in that it meas-ures interferon gamma production from memory T-cells following a brief stimula-tion with peptide antigens.

B-cell ELISPOT and mumpsIn a recent proof-of-concept publication [6], it was shown that mumps-specific B-cell responses could be detected in the majority of individuals for 1-2 weeks following adminis-tration of MMR vaccine even though most of them had previously received two MMR vac-cinations. In addition, it was used to detect wild-type infection in seven out of seven individuals who had been previously vacci-nated. Only one of these individuals was IgM positive and two were RT-PCR positive. The time of testing post-symptom onset ranged from 1-2 weeks for most individuals. These results illustrate the main advantages of the technique which are: a) the B-cell response can be detected independently of serum antibody levels, b) convalescent samples are not necessary and c) activated B-cells can be detected for a much longer period of time than virus is likely to be shed.

If B-cell ELISPOT has such clear advantages over standard methods, why then has the method not become commonplace in clini-cal laboratories? There are a few obstacles that

must be overcome. Perhaps the single biggest issue is that the activated ASC do not sur-vive for an extended period of time follow-ing blood collection. Unlike memory T-cells, whivh are detected with the T-SPOT.TB test, freezing severely affects the viability of B-cell plasmablasts. This could be particularly det-rimental if the frequency of the cells is low to begin with and it necessitates the immediate testing of fresh samples. Since most physicians’ offices are not equipped to perform ELISPOT testing on-site, there is a need to improve the viability of the cells after transportation or to modify the technique so that it can be easily performed in the field without specialised laboratory equipment. It might be possible to include special additives in the blood collec-tion tubes or in the freezing media to increase the viability of the ASC. Alternatively, on-site testing could be facilitated by cell culture media that does not require CO2 to maintain pH balance or by utilisation of a micro-fluidic (lab-on-a-chip) device.

Another obstacle that could affect routine use of this method is the blood volume required for testing. Although the technique could be performed with as little as 1-2 mL of blood, 5-10 mL is more desirable to allow for adequate testing of positive and negative controls and to increase the odds of detecting cells with very low frequencies. Some indi-viduals, particularly young children, may not be amenable to having this amount of blood collected. However, as described above, the most likely application of B-cell ELISPOT would be in older individuals who experi-ence breakthrough disease and who may be more willing to provide the sample. It should also be pointed out that ASC may cross-react with antigens derived from unrelated viruses and thus lead to an inaccurate result. This could be addressed by using recombinant or

single protein antigens, but it would require careful quality assurance steps and the inclu-sion of a differential panel of antigens to rule out other possible causes.

Although not necessarily intended as a replacement for standard diagnostic meth-ods, B-cell ELISPOT has the potential to overcome many limitations of standard methods for certain applications. Theo-retically, the method could also be used to detect any adaptive B-cell response – not just those specific for infectious diseases.

DisclaimerThe views and conclusions in this article are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention.

references1. Barskey AE, Glasser JW, LeBaron CW. Mumps resur-

gences in the United States: A historical perspective on unexpected elements. Vaccine 2009; 27: 6186-95.

2. Gut JP et al. Symptomatic mumps virus reinfections. J Med Virol 1995; 45:17-23.

3. Plotkin SA, Orenstein WA, Offit PA. Vaccines 2008; 5th ed. Saunders, Philadelphia, Pa.

4. Bitsko RH et al. Detection of RNA of mumps virus during an outbreak in a population with a high level of measles, mumps, and rubella vaccine coverage. J Clin Microbiol 2008; 46:1101-3.

5. Rota JS et al. Investigation of a mumps outbreak among university students with two measles-mumps-rubella (MMR) vaccinations, Virginia, September-December 2006. J Med Virol 2009; 81:1819-25.

6. Latner DR et al. ELISpot Detection of Mumps-Specific Antibody Secreting B-cells as an Alternative Method of Laboratory Diagnosis. Clin Vaccine Immunol 2010.

7. Czerkinsky CC et al. A solid-phase enzyme-linked immu-nospot (ELISPOT) assay for enumeration of specific anti-body-secreting cells. J Immunol Methods 1983; 65:109-21.

8. Sedgwick JD, Holt PG. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods 1983; 57:301-9.

9. Arvilommi H. ELISPOT for detecting antibody-secreting cells in response to infections and vaccination. APMIS 1996; 104:401-10.

10. Mazurek M et al. Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuber-culosis infection - United States, 2010. MMWR Recomm Rep 2010; 59:1-25.

The authorDon Latner, Ph.D.MicrobiologistCenters for Disease Control and PreventionNational Center for Immunization and Res-piratory DiseasesMMRHLB1600 Clifton Road, NE MS-G18Atlanta, GA 30333, USATel: +1 404-639-2771 e-mail: [email protected]

– April/May 2011 Molecular diagnostics40

Figure 2. An outline of the B- cell ELISPOT method.

The scientific proof of a technology breakthrough is not established by a single study. The technology must be evaluated and the study results duplicated in numerous settings to be considered scientifically valid. In the last two years, 50 published studies by leading diabetes hospitals throughout the world validate that Nova’s StatStrip glucose sensor technology dramatically improves accuracy by reducing hematocrit and other interferences. These studies have been conducted at some of the most prestigious diabetes centers in the world including the Mayo Clinic, John Hopkins University School of Medicine, University of California Davis Center for Point of Care Technology, University of Toronto Sunnybrook Health Sciences Center, Addenbrooke’s Hospital Cambridge University Hospitals, UK, WEQAS and University Hospital, Cardiff, Wales, Isala Klinieken, Netherlands. Some conclusions:

www.statstrip.com

“Here we further demonstrate for the first time that anemia is the primary cause of glucometer error in hemodynamically stable adult ICU patients and that eliminating hematocrit error decreases the frequency of hypoglycemia.”Pidcoke M et al. Crit Care Med 2010

“The new generation StatStrip glucose meter, which has been designed to compensate for hematocrit and chemical interferences, reduces the likelihood of erroneous results arising from interference factors that influence current conventional glucose meters.”Bewley B et al. Point of Care 2009

“The StatStrip system was not susceptible to hematocrit, ascorbate or maltose interferences, either alone or in combination with one another. The other strip meter systems tested were significantly influenced by these interferences.”Lyon ME. AACC, Annual Meeting 2008

“With the exception of the Nova StatStrip, all meters were affected by variable hematocrit.”Mohn B. NZJ Med Lab Sci 2010

Copies of this booklet are available by contacting Nova Biomedical Tel: 781-894-0800www.novabiomedical.com

Nova Multi-Well™

Glucose StatStrip® Technology

Publications and Presentations

GLU

Europe Cont’d

The Nova StatStrip® Blood Glucose Meter

Evaluation: Hematocrit Dependency,

Method Comparison, Interfering

Substances and Neonatal Samples

Clinical Evaluation of a Point of Care Device

for Creatinine measurements in the Follow-

up of Kidney Transplant Patients

Validation of two Point-of-Care Testing

devices for fetal lactate assessment

during labour

Evaluation of the Nova Biomedical

StatStrip® Glucose Meter

Assessment of the Performance of Hand-

held POC Sensors for Measuring

3-Hydroxybutyrate

Four Step Validation Procedure for

Evaluating POCT Meters

Comparison of Four Hospital Based

Glucose Meter Technologies for Accuracy,

Precision and Interferences Encountered

in Hospitalized Patients

An Evaluation of the Analytical

Specificity and Clinical Application of a

New Generation Hospital Based Glucose

Meter in a Dialysis Setting

Improved POC Meter Accuracy for

Monitoring and Managing Glucose Levels

in Dialysis Patients

Nova StatStrip®: Could This Device be used

to Effectively Implement Tight Glycemic

Control and Triage Blood Glucose and

Insulin Management in Critical Illness

An Evaluation of the Analytical

Performance of a New Generation

Hospital Based Glucose Meter in a

Neonatal Care Unit

Reliability of Glucose Meters in Hospitals

in Austria

Performance of the Nova StatStrip® Point of

Care Glucose Meter in a Neonatal Intensive

Care UnitMulti-National Sites

Multi-site Evaluation of Point of Care

Glucose Meters in a Neonatal Intensive

Care Unit

A Multi-Site Analytical Assessment of a

New Hospital POC Glucose Meter for

Accuracy, Precision, Correlation, and

Interferences Encountered in

Hospitalized Patients

Canada (Cont’d)

Evaluation of a Point-Of-Care (POC) Glucose

Meter Suitable for Use in Complex Tertiary

Care Facilities Asia

Performance of a StatStrip® Meter

Europe

Comparison of Accuracy of a Glucometer

and a Blood Gas Analyzer in an Adult ICU

Comparison of Accuracy of Three

POC Glucometers in an Adult ICU

Performance Of The Statstrip Glucose Meter

In Inpatient Management Of Diabetes Mellitus

Testování Glukometrů a Jejich Porvnání

Evaluation d’un Nouveau Lecteur de

Glycémie Intégrant une Correction

Automatique de l’Hématocrite

Galatose Interference on POCT Glucose

Analysis

Interference of Hematocrit and Maltose

Plasma Concentrations on the Accuracy of

Different Blood Glucose Measuring Systems

Das Blutzuckermessystem StatStrip® ist nicht

empfindlich für Interferenzen durch

Hämatokrit oder andere bekannte

Störsubstanzen

Genauigkeit des Blutzuckermess-systems

StatStrip® im Vergleich zu anderen

Messsystems und zu einer Standard- Labor-

methode

Analytical Performance of an Interference-

Resistant Glucose Meter

Suitability Assessment of a New Bedside

Interference Free Glucose System for Use

in Critical Care

United States

Comparison Evaluation of Three

Point-of-Care Glucose Meters with

Neonatal Patient Samples Exhibiting

Varied Hematocrit and Triglyceride

Concentrations

Comparative Testing for Better

Glycemic Control

Accuracy and Reliability of the Nova

StatStrip® Glucose Meter for Real-Time

Blood Glucose Determinations

during Glucose Clamp Studies

Evaluation of the Impact of

Hematocrit and Other

Interference on the Accuracy of

Hospital-Based Glucose Meters

Use of Samples from Indwelling Venous

Catheters for Glucose Meter Testing

Comparison of Four Hospital Based

Glucose Meter Technogies Accuracy,

Precision, and Interference Encountered

in Critically Ill Patient

Evaluation of a New POCT Bedside

Glucose Meter and Strip with Hematocrit

and Interference Corrections

Evaluations of Nova StatStrip® Blood

Glucose Monitoring System in Neonates

Evaluation and Implementation of the

Nova StatStrip® Bedside Glucose

Monitor for Patients Undergoing Cardiop-

monary Bu-pass Graft Surgery (CABG)

Development and Use of a Methodology

for the Evaluation and Implementation of

POCT Devices

RGH’s Method for Evaluation and Imple-

mentation of Data-managed Bedside

Glucose (POCTG) Monitoring

Evaluation of Point of Care Bedside

Glucose Monitors for Use in a Specialty

and Transplant Hospital

Hematocrit Effect Outweighs Other

Sources of Glucometer Error in

Critical Care

Assessing the Performance of Handheld

Glucose Testing for Critical Care

Canada

Evaluation of a Glucose Meter with Negli-

gible Hematocrit or Chemical Interference

Predicted Discrepancies Between Direct

Reading Whole Blood Biosensors and

Central Lab Plasma Methods: Predicting

and Avoiding Medical Error

Estimates of Total Analytical Error in

Consumer and Hospital Glucose Meters

Contibuted by Hematocrit, Maltose and

Ascorbate

Interference Studies with Two

Hospital-Grade and Two Home-Grade

Glucose Meters

No. 169 Rev. 1/20/11

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Waltham, MA 02454-9141 U.S.A.

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Scientific Papers Validate Nova’s Technology Breakthrough In Glucose Sensor Accuracy


The search for biomarkers of immune restoration disease asso-ciated with Mycobacterium tuber-culosis in HIV patients beginning antiretroviral therapy.Immune restoration disease associated with Mycobacterium tuberculosis is a com-plication of antiretroviral therapy seen in a subset of HIV-1 patients soon after they commence antiretroviral therapy. It is characterised by a ‘paradoxical’ worsening of treated tuberculosis or an ‘unmasking’ of subclinical tuberculosis. As antiretroviral therapy becomes increasingly available in countries with a high prevalence of tuber-culosis, this form of immune restoration disease will become more common. This article summarises the biomarkers that may illuminate immunopathogenesis and assist in diagnosis.Oliver BG, Price P. Biomark Med. 2011 Apr;5(2):149-54.

Surrogate markers of infection: interrogation of the immune system.Infectious diseases remain the great-est causes of morbidity and mortality in global terms. As much of the burden occurs in the developing world, limited access to diagnostic testing in these parts of the worldhas hampered the diagnosis and treatment of these conditions, while, in the developed world, the cost of man-aging infectious diseases remains con-siderable. Despite the size of the problem there remains an ongoing need for tests

that improve diagnostic sensitivity and specificity, provide more rapid diagnoses, are available for point-of-care testing in remote regions, and can help inform ther-apeutic decision-making by identifying resistance patterns or patient outcomes. This article discusses the background to biomarker development for infectious diseases, some current assays that are pro-viding useful information regarding the host’s response to infection (using exam-ples such as Cytomegalovirus and Myco-bacterium tuberculosis), as well as likely future technologies and their limitations.Chakera A, Lucas A, Lucas M. Biomark Med. 2011 Apr;5(2):131-48.

Is it appropriate to routinely use a nucleic acid amplification test for the diagnosis of tuberculosis?The study described in this paper com-pared the usefulness of the nucleic acid amplification (NAA) test with conven-tional tests under normal laboratory operational conditions. The NAA test was performed on the first sputum specimen from all patients. Liquid media culture, solid media culture and Ziehl-Neelsen stain for an acid-fast bacilli (AFB) smear were performed on three sputum speci-mens. The results were calculated using the gold standard of either the culture results or the clinical diagnosis. Of the 593 patients tested, 151 (25.5%) were diagnosed with pulmonary tuberculosis. The sensitivity of the first specimen only was 64% for the NAA test, 54% for the AFB smear, 77% for BACTEC MGIT 960 culture, 40% for Lowenstein-Jensen (LJ) culture and 25% for 7H11 culture. The sensitivity when using all three speci-mens increased to 63% for the AFB smear, 87% for the BACTEC MGIT 960 culture, 51% for the LJ culture and 40% for 7H11 culture. The specificity was 100% for all culture tests, 99% for the AFB smear, and 99.5% for NAA test. The mean turna-round time was 1.34 days for NAA, 0.59 days for AFB smear, 11 days for BACTEC MGIT 960 culture, 23 days for LJ culture, and 20 days for 7H11 culture. The sensi-tivity of NAA is still far from ideal, and the test is not cost-effective. Lin CB et al. Kaohsiung J Med Sci. 2011 Apr;27(4):138-43.

Clinical application and limitations of interferon gamma release assays for the diagnosis of latent tuberculosis InfectionInterferon gamma-release assays (IGRAs) represent advances in tuber-culosis immunology and evolutionary

biology and were designed to replace the tuberculin skin test (TST) for the diag-nosis of latent tuberculosis infection because of their logistical advantages and enhanced specificity over TST. Although IGRAs and TST have been useful in epi-demiologic studies, they lack the sen-sitivity and reproducibility normally expected from diagnostic tests in clini-cal practice. In this review, an overview of the current recommendations and knowledge in the field are presented, and the practical approaches are discussed in areas of uncertainty associated with dis-cordant IGRA results. Herrera V, Perry S, Parsonnet J, Banaei N. Clin Infect Dis. 2011 Apr;52(8):1031-7.

A multicentre evaluation of the accuracy and performance of IP-10 for the diagnosis of infection with M. tuberculosis.Interferon gamma inducible protein 10 (IP-10) has potential as a diagnostic marker for infection with Mycobacterium tuberculosis, with comparable accuracy to QuantiFERON-TB Gold In-Tube test (QFT-IT). This study assessed the sensi-tivity and specificity of IP-10, and evalu-ated the impact of co-morbidity on IP-10 and QFT-IT. One hundred and sixty-eight cases with active TB, 101 healthy controls and 175 non-TB patients were included. IP-10 and IFN-γ were meas-ured in plasma of QFT-IT stimulated whole blood and analysed using previ-ously determined algorithms. A sub-group of 48 patients and 70 healthy con-trols was tested in parallel with T-SPOT.TB. It was found that IP-10 and QFT-IT had comparable accuracy. Sensitivity was 81% and 84% with a specificity of 97% and 100%, respectively. Combining IP-10 and QFT-IT improved sensitivity to 87% (p < 0.0005), with a specificity of 97%. T-SPOT.TB was more sensitive than QFT-IT, but not IP-10. Among non-TB patients IP-10 had a higher rate of posi-tive responders (35% vs 27%, p < 0.02) and for both tests a positive response was associated with relevant risk fac-tors. IFN-γ but not IP-10 responses to mitogen stimulation were reduced in patients with TB and non-TB infection. This study confirms and validates previ-ous findings and adds substance to IP-10 as a novel diagnostic marker for infec-tion with M. tuberculosis. IP-10 appeared less influenced by infections other than TB; further studies are needed to test the clinical impact of these findings.ruhwald M et al on behalf of TBNET. Tuberculosis (Edinb). 2011 Apr 1.

SCIENTIfIC LITErATUrE rEVIEw – April/May 2011 42

There are many peer-reviewed papers covering TB diagnosis and treatment monitoring, and it is frequently difficult for healthcare professionals to keep up with the literature. As a special service to our readers, CLI presents a few key literature abstracts from the clinical and scientific literature chosen by our editorial board as being particularly worthy of attention.

MPT 64 antigen detection for rapid confirmation ofM. tuberculosis isolates.A new rapid immunochromatographic test kit for the detection of MPT 64 antigen in M. tuberculosis isolates using mouse monoclonal MPT 64 Antibody was evaluated for rapid identification of M.tuberculosis isolates. The sensitivity, specificity and predictive values of the kit were assessed. Fifty-four culture iso-lates of M.tuberculosis in broth and on LJ medium, 12 non-mycobacterial isolates, 10 non-tubercular (NTM) rapidly grow-ing Mycobacteria isolated from pus and five smear positive sputum samples were tested for detection of MPT64 antigen using the SD Bioline immunochromatog-raphy (ICT) test kit. The H37 RV strain was employed as the positive reference control. this strain showed the presence of an MPT64 antigen band. A similar band was formed in all the 54 MTB isolates tested. MPT64 band formation was not detected in any of the other test isolates.Rapid identification of MTB culture iso-lates is very important for drug susceptibil-ity testing. This test kit was found to have excellent sensitivity, specificity, negative predictive value and positive predictive value. This rapid test is a reliable and cost-effective method for confirming MTB cul-ture isolates in resource-poor laboratories.Kumar VG, Urs TA, ranganath rr. BMC res Notes. 2011 Mar 24;4(1):79.

New and improved diagnostics for detection of drug-resistant pulmonary tuberculosis.Drug-resistant TB is now well established throughout the world and most TB patients are not screened for drug resistance due to lack of laboratory resources and rapid accurate point-of-care tests. Accurate and rapid diagnosis of TB and drug-resistant TB is of paramount importance in estab-lishing appropriate clinical management and infection control measures. During the past decade, there have been significant advances in diagnostic technologies for TB and drug-resistant TB. This article is to review the current data, recommendations and evidence base for these tests.Second-line drug susceptibility testing (DST) is complex and expensive. Auto-mated liquid culture systems and molecu-lar line probe assays are recommended by the WHO as the current ‘gold standard’ for first-line DST. Liquid culture DST for ami-noglycosides, polypeptides and fluoroqui-nolones has been shown to have relatively good reliability and reproducibility for diagnosis of extensively drug-resistant TB.

However, DST for other second-line drugs (ethionamide, prothionamide, cycloserine, terizidone, para-aminosalicylic acid, clofa-zimine, amoxicillin-clavulanate, clarithro-mycin, linezolid) is not recommended. Automated liquid culture systems are currently recommended by the WHO as the ‘gold standard’ for second-line DST. In this review, the phenotypic and genotypic methods currently available for the diag-nosis of TB and drug-resistant forms of Mycobacterium tuberculosis are described, and future prospects for TB diagnostics are discussed. Current technologies for the detection of drug resistant M. tubercu-losis vary greatly in terms of turnaround time, cost and complexity. Ultimately, the ‘holy grail’ diagnostic test for TB must ful-fil all technical specifications for a good point-of-care test, screen for drug resist-ance concurrently and be adaptable to the various health system levels and to coun-tries with diverse economic resources and TB burden.O’Grady J et al. Curr Opin Pulm Med. 2011 May;17(3):134-141.

Prevalence of multidrug-resistant tuberculosis among newly diag-nosed cases of sputum-positive pulmonary tuberculosis.The prevalence of multidrug-resistant tuberculosis (MDR-TB) is increasing throughout the world. Although previous treatment for TB is the most important risk factor for development of MDR-TB, treatment-naive patients are also at risk due to either spontaneous mutations or transmission of drug-resistant strains. This study ascertained the prevalence of MDR-TB among new cases of sputum-positive pulmonary TB. This prospective, observational study involving newly diag-nosed cases of sputum-positive pulmonary tuberculosis diagnosed between 2008 and 2009 was carried out in New Delhi, India. All sputum-positive TB cases were sub-jected to mycobacterial culture and first-line drug-susceptibility testing (DST). MDR-TB was defined as TB caused by bacilli showing resistance to at least isonia-zid and rifampicin. DST was carried out in 177 cases, and only two cases of MDR-TB were detected. MDR-TB prevalence is low among new cases of sputum-positive pul-monary TB treated at primary care level in Delhi. Nation-wide and State-wide repre-sentative data on the prevalence of MDR-TB are lacking. Efforts should be directed towards continued surveillance for MDR-TB among newly diagnosed TB cases.Sharma SK et al. Indian J Med res. 2011 Mar;133(3):308-11.

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wireless connectivity for glucose and creatinine meters

A wireless con-nectivity solution for StatStrip Glu-cose and StatSen-sor Creatinine meters is now available. The Nova StatStrip Wireless Tote provides bi-direc-tional, wireless

connectivity of the test results and patient data to/from a Lab or Hospital Information System (LIS). With a convenient, lightweight carrying case for meters and testing supplies, the Wireless Tote, provides rapid charting of test results and patient data to enable the fre-quent therapy decisions needed for patients on glycaemic control protocols. In addition, it provides admission/transfer/discharge feeds of patient name, ID, date of birth, gender, and bed location to the meter for bedside con-firmation of multiple patient identifiers and positive patient ID. With other handheld glu-cose meters, patient test data are frequently not uploaded until the point of care device is placed in a dock — sometimes occurring only at the end of a shift. As glucose tests and therapy decisions are being made frequently on hospitalised patients, the need for contin-uous, secure, wireless connectivity is becom-ing essential. As hospitals move to upgrade their IT infrastructure to support intercon-nected devices, many are looking to wireless technology to improve charting of point of care test results, reduce the costs of hard-wiring a facility, and to support additional capabilities such as asset and patient tracking. This wireless solution addresses these needs through transmission over 802.11 standard

protocol with data encryption and additional security measures.

NovA BioMedicAlWaltham, MA, USA www.cli-online.com & search 25557

fully auto-mated HEr2 fISH testDesigned for easy and accurate assess-ment of HER2 gene status in breast can-cer tissue, the the

fully automated Leica HER2 FISH system combines the use of the gold standard Path-Vysion HER2 FISH probes (Abbott Molec-ular Inc) with Leica’s BOND automated advanced staining system, delivering accu-rate results for diagnostic confidence.Automation of labour-intensive FISH techniques reduces process variation while offering walk-away convenience. Samples can be processed continuously, eliminat-ing the need to batch cases, offering fast turnaround times, saving valuable hands-on time and allowing rapid reporting of patient results. This fully automated sys-tem uses an optimised ready-to-use Leica HER2 FISH reagent kit with the robust BOND protocol to produce consistent, high quality stained slides. The system enhances laboratory workflow, increas-ing efficiency and enabling the laboratory to provide a responsive service to their clinicians and clients.

leicA MicroSySteMS GMBHWetzlar, Germany www.cli-online.com & search 25565

Histology simulation software programme

The need for histopathology laboratories to work faster and more efficiently is becoming more and more necessary. SMART Automa-tion, the Tissue-Tek premium product line from Sakura Finetek, meets and exceeds this increasing demand. Together with Incon-trol Simulation Solutions, the company has developed the SMART analysis tool to visualise and quantify the effects of automa-tion and change to a continuous workflow. The software programme guides the user through all relevant steps in the laboratory and parameters. The model shows the labo-ratory in its current situation as well as with the new automated set-up. It also provides a direct comparison between the two options. A range of parameters and graphs shows key performance indicators such as turnaround times, productivity, waiting time, utilisation of systems and working of staff. The simu-lation tool provides histology professionals with a valuable insight into the influence of SMART Automation in their specific laboratory setting.

SAkUrA FiNetek eUrope B.v.Alphen Aan den rijn, The Netherlands www.cli-online.com & search 25499

PrODUCT NEwS – April/May 2011 44

Joint IfCC worldLab and EuroMedLab 2011 congress and exhibitionThis major joint event of laboratory medicine will take place at the International Conference Centre (ICC) in Berlin, Germany from 15th to 19th May 2011. An attractive scientific programme will be offered: in addition to the classical types of scientific sessions including plenary sessions, symposia, workshops and poster presentations, new types of scientific sessions such as “the year in review”, pro-con sessions, and clinical case conferences will also be offered. All of this is designed to develop a highly interactive forum to discuss cutting-edge science in the field. A major focus of the scientific programme will centre around the

post-genomic area and its impact on the patient so that a much deeper understanding of genomic, transcriptomic and metabolomic regulation with regard to the pathophysiology of diseases can be gained. Major challenges in laboratory diagnostic medicine remain the risk assessment of the patient, the prediction of disease development and the evaluation of preventive strategies. Another major focus of the conference will be aimed at young scientists and clinicians from around the world with a forum for scientific and social exchange being organised of particular interest to the next generation of specialists in the field of laboratory medicine and clinical chemistry. A comprehensive exhibition of products from the diagnostic industry will also be organised with companies from all over the world exhibiting their latest equipment and instruments and underlining the global partnership between industry and specialists working in laboratories. For more information visit http://www.berlin2011.org/

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reagents for automated systems

Coinciding with the company’s celebration of 30 years of designing, developing, manu-facturing and marketing reagents, Biosys-tems S.A is launching a new line of reagents: automated systems reagents (ASR). These clinical chemistry and turbidimetry reagents are ready-to-use liquids specifically designed for direct use, without any manipulation or transfer, on different instruments on the market, so helping to improve operational efficiency and workflow in clinical laborato-ries. The new line of reagents is compatible with Modular, Hitachi 917, Olympus AU Series, Advia Series and Dirui Series analys-ers. The barcodes corresponding to direct use in Olympus AU series are available. Substitu-tion by ASR of the reagents currently used ion these analysers is highly cost-effective.

BioSySteMSBarcelona, Spain www.cli-online.com & search 25558

POC HbA1c analyser with added connectivity and security functions

A new enhanced version of Sie-mens’ DCA Vantage Ana-lyzer, a point-of-care (POC) immunoassay analyser for dia-betes manage-ment, is avail-

able. The system now provides enhanced operator management and connectivity capabilities to meet the growing demands in POC testing for improved compliance management and data capture into patient records. The DCA Vantage Analyzer with Version 3.0 Software has the capacity to manage a larger number of operators while providing the required security access modes to prevent unauthorised use. In addition, the analyser includes the POCT1-A2 communication protocol, which allows supervisors to remotely manage operator lists when interfaced with the POC data management software. Use of this stand-ard interface simplifies the connection to

POC data management systems, enabling results to be automatically transmitted to the patient electronic medical record. The operator management capability has been expanded to support up to 1000 opera-tors, each with their unique access level, as defined by their supervisor. Another enhancement to the analyzer is the ability to display, print and transmit the HbA1c refer-ence ranges with the patient results, aiding in improved results interpretation. With its added security access modes and customis-able operator access, the enhanced analyser minimises risk to the institution while pro-tecting patient information.

SieMeNS MedicAl SolUtioNS diGNoSticStarrytown, Ny, USA www.cli-online.com & search 25553

HIV / CD4 dual test

A novel point of care rapid test is avail-able that can simultaneously determine a patient’s HIV infection status as well as their current immune status. The Retro-STATUS HIV/CD4 350 rapid test detects HIV 1/2 antibodies as the indicator of HIV infection and, with the same device, indi-cates whether the patient’s CD4 count is above or below 350 CD4 cells per microli-tre, the cutoff recommended by the WHO as the CD4 count indicative of the need to start anti-retroviral drug therapy. As this test can be run in almost any setting, it brings affordable HIV and immune status testing capabilities to those resource-poor countries where there is a lack of infra-structure, lab facilities and expensive and hard-to-maintain equipment. Together with a shortage of skilled lab personnel, this makes screening of those patients who are most in need of it, difficult if not impossible. The test requires only a few drops of sample, with results available in 20 minutes. Testing can be carried out on site while the patient is present, which eliminates problematic call backs and the long delays typical when sending samples to remote centralised laboratories.

MilleNNiUM BiotecHNoloGy, iNc. plaistow, NH, USA www.cli-online.com & search 25551

Biomarker test for early onset pre-eclampsia

Pre-eclampsia is a leading cause of mater-nal and peri-natal mortality and morbidity. Currently, the condition is diagnosed by an increase in

blood pressure and the presence of pro-tein in the urine. However, these signs are non-specific for pre-eclampsia and are not always present in women who develop severe complications (e.g. the HELLP syn-drome). A diagnostic tool to facilitate the diagnosis of early onset pre-eclampsia is now available. The Triage PLGF test meas-ures Placental Growth Factor (PlGF), a bio-marker involved in placental development, which provides a highly sensitive and spe-cific indication of the placental dysfunc-tion that leads to early onset pre-eclampsia. The new test, for use with the Triage Meter-Pro, provides rapid and accurate quantifi-cation of PlGF in plasma. The test can be performed in a laboratory or clinic setting and provides results within 15 minutes.

Alere iNterNAtioNAl Morges, Switzerland www.cli-online.com & search 25494

freezing containers for larger tube sizes

Designed for use with the complete range of Thermo Sci-entific Nunc and Nalgene cryogenic stor-age tubes, the Mr. Frosty line of freezing con-

tainers now accommodates 3.6, 4.5 and 5.0 mL cryotubes, thus providing a flexible solution for any tube size. The freezing con-tainers are safe, easy-to-use and enable sim-ple cooling of samples at an optimal rate of -1 °C per minute, so effectively maintaining cell viability. Requiring only 100 percent isopropyl alcohol and a -70 to -80 °C freezer, the containers are ideal for any laboratory requiring cost-effective and repeatable cryopreservation of cells.

tHerMo FiSHer ScieNtiFicWinchester, vA, USA www.cli-online.com & search 25497


PCr kit for detection of dermatophytes and Trichophyton rubrum

Nail infections are caused by der-matophytes, most commonly by Trichophyton rubrum, followed by Trichophyton mentagrophytes. As onychomycosis requires long-term systemic antifungal treat-ment, the correct identification of causal fungi is mandatory. The time required for traditional species identification by culture ranges from two to four weeks. This CE-marked, PCR-based in vitro diagnostic method can detect dermatophytes in general, and T. rubrum specifically, within five hours. A study also showed that this multiplex PCR method increases the detection of pan-dermatophytes by 4.3% and T. rubrum by 18.6% compared to conventional methods. This PCR-based method will thus be more specific, simpler and rapid, and will potentially reduce costs.

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Immunoassay test kits

Providing outstanding sensitivity, specificity, accuracy and preci-sion, ReQuest immunoassay kits meet the needs of a wide-range of laboratories. The kits pro-vide reliable, high performance, user-friendly and cost effective diagnostic test methods for con-ditions including TORCH, auto-immune diseases and infectious diseases. The kits can be used with Awareness Technology’s

ChemWell automated analyser, as well as with the company’s Stat Fax and ChroMate microplate readers for Cost Effective by Design diagnostic test systems, all available through a global net-work of distributors.

AWAreNeSS tecHNoloGy iNcpalm city, Fl, USA www.cli-online.com & search 25564

Multi-gas incubator

Offering exceptional control and an industry-first decon-tamination system, the MCO-19M multi-gas incubator provides extremely accurate control of O2 and CO2 levels combined with a precisely reg-ulated temperature for a wide range of cell culture applica-tions. By achieving a stable, uni-form culturing environment the new incubator ensures ideal growth conditions and excel-lent reproducibility of experi-mental results. The incubator also introduces the option of a unique H2O2 decontamina-tion system, which enables full decontamination and incuba-tor cleaning to be completed within five hours.

SANyo eUrope Bvetten-leur, The Netherlands www.cli-online.com & search 25569

Procalcitonin ELISAProcalcitonin (PCT) is the pre-cursor of the hormone calci-tonin. PCT can be produced by numerous cell types and organs, mainly by the thyroid parafolli-cular cells (C cells) and the neu-roendocrine cells of the lungs

and intestine, after proinflamma-tory stimulation, especially when caused by a bacterial challenge. The level of procalcitonin in the blood of healthy individuals is low. When the value of procal-citonin exceeds 0.25 ng/mL, a bacterial infection is indicated. Measurement of procalcitonin can be used to facilitate diagnosis of severe sepsis and the level is closely correlated with the degree of sepsis. One major advantage of PCT compared with other analytes is its early and highly specific increase in response to severe systemic bacterial infec-tions and sepsis. Increased PCT levels can be observed 3–6 hours after an infectious challenge. The

Human Procalcitonin ELISA kit extends BioVendor’s portfolio of diagnostic kits for sepsis. Suitable for serum and urine samples, the 96 well-microtitre plate format can be used with standard ELISA equipment or robotic systems.

BioveNdorModrice, The czech republic www.cli-online.com & search 25571

– April/May 201147PrODUCT NEwS

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fast-clotting serum blood collection tube

The BD Vacutainer Rapid Serum Tube is a blood collection device designed to help acute healthcare facilities rapidly analyse blood serum for patient diagnosis, and fea-tures the BD Hemogard safety-engineered closure, which enhances healthcare worker safety as well as compatibility with clini-cal analysers utilising front-end automa-tion. With a five-minute clotting time and as little as three minutes of centrifugation (at 4000 g), the blood collection tube can offer savings of up to 32 minutes of labora-tory time. Standard serum separator tubes require a 30-minute clot time followed by 10 minutes of centrifugation before clini-cal laboratory professionals can begin ana-lysing the sample and getting test results. The 5 mL-draw, 13x100mL plastic sterile blood-collection tube combines a throm-bin-based additive with gel technology, which creates a high quality serum sample by minimising haemolysis and fibrin for-mation. Haemolysis and clotted specimens are two common examples of sub-optimal quality, both of which can lead to labora-tory errors. Studies conducted by BD show a 59 percent reduction of haemolysis and fibrin strand formation with this tube com-pared with standard tubes. This observed sample quality improvement may help lower the number of repeat collections by phlebotomy, which in turn can increase the productivity of the laboratory.

Bd diAGNoSticSerembodegem, Belgium www.cli-online.com & search 25546

Complete range of Quality Control strains for antimicrobial susceptibility testingAll of the quality control strains recom-mended in the new EUCAST (European Committee on Antimicrobial Suscepti-bility Testing) harmonised disc diffusion method for antimicrobial susceptibility testing are available as Oxoid Culti-Loops. This range of quality control organisms, which includes all EUCAST-recommended strains, are disposable bacteriological loops

containing stabilised, viable microorgan-isms, supplied in packs of five. Each loop is individually foil-wrapped and ready-to-use, and is designed for quick, easy and safe preparation of standardised cultures. tHerMo FiSHer ScieNtiFicBasingstoke, Hants, Uk www.cli-online.com & search 25547

All-in-one water purification system for high-volume clinical analysers

Providing up to 100 litres per hour and 2000 litres per day of clinical labora-tory reagent water (CLRW), the Elix Gulfstream Clini-cal all-in-one water purifica-tion system is designed specifi-cally for clinical analysers. The system includes Merck Millipore’s well-known Elix

electrodeionisation technology, as well as improved reverse osmosis cartridges. These complementary technologies help extend the lifetime of the purification car-tridges, thus reducing running costs. Biomedical labs need to avoid costly downtime. When problems occur due to inconsistency in the quality of pure water, however, this can disrupt sample process-ing, so halting or reducing a lab’s output and resulting in unforeseen expenses. This system provides biomedical labs with a reliable, constant source of pure water for their analysers. Water produced by the sys-tem meets the stringent standards of the Clinical and Laboratory Standards Insti-tute (CLSI) for CLRW.

Merck MilliporeBillerica, MA, USA www.cli-online.com & search 25559

Giardia monoclonal antibodiesGiardia is the leading cause of parasitic gastro-intestinal disease in the US and much of Europe. It occurs most com-monly following

the ingestion of water contaminated with the Giardia lamblia organism, and is passed from human to human via the faecal-oral route although domestic and wild animals can con-taminate water sources as well. ViroStat has introduced several new monoclonal antibod-ies specific to Giardia. These are reactive with the cyst form of the organism and target the GSA65 antigen. ELISA pairing recommen-dations are given on the data sheet which can be found on the company’s website.

viroStAtportland, Me, USA www.cli-online.com & search 25548

research pipettes in single or duo pack

Developed as an extension to the well estab-lished Acura line of manual pipettes, the Acura manual XS 826 integrates all the best features of this family of pipettes, together with unique characteristics such as weight reduction and ultra low activation forces. The short pipette shaft with narrow, conical end allows for superior instrument drivabil-ity and easy access to microtubes. Aiming at excellence in pipetting, the instrument is a most reliable tool particularly for research laboratories. The new pipette is supplied in a single box or in an economical TwiXS pack containing two units and one free shelf pipette holder. There are four different packs from which to choose, each covering spe-cific volumes ranging from 0.1 µL to 1000 µL, allowing any research lab to find the most suitable combination of volumes.

Socorexecublens, Switzerland www.cli-online.com & search 25570


– April/May 201149BOOK rEVIEwS


Cytopathology

Ed by Behdad Shambayati, Pub. by Oxford University Press Feb. 2011, 448 pp., £31.99

The book ‘Cytopa-thology’ is one of the Fundamentals of Biomedical Science series published by Oxford University Press and written to reflect the chal-lenges of practicing biomedical science

today. Books in this series draw together essential basic science with insights into laboratory practice to show how an under-standing of the biology of disease is cou-pled to the analytical approaches that lead to diagnosis. The book provides a broad-ranging over-view of the microscopic study of normal and abnormal cells, which embraces the latest imaging and visualisation methods to study the structure of cells. The full colour presen-tation features over 400 figures. Written with the needs of the biomedical scientist centre-stage, the book provides a firm grounding in normal cell structure, as well as the abnormal features that are indicative of different clinical

conditions. It also explains how screening programmes can be used to detect changes early, so giving an invaluable opportunity for treatment regimes to be implemented in a timely way. Crucially, the book demon-strates throughout how an understanding of cellular physiology underpins the key inves-tigations carried out by a biomedical scien-tist to forge a clear link between science and practice. Intended for undergraduates at all levels studying a practice-oriented biomedi-cal science degree programme, the book is also suitable for biomedical postgraduates and anyone studying cytopathology as part of their course.

oxFord UNiverSity preSSoxford, Uk www.cli-online.com & search 25561

ABC of COPD, 2nd Edition

Ed by Graeme P. Currie, Pub. by Wiley-Blackwell Dec. 2010, 88 pp., € 27.60 Chronic Obstructive Pulmonary Disease (COPD) is a progressive, largely irreversi-ble lung condition characterised by airflow obstruction. Although cigarette smoking is the single most important risk factor

in its development, other associations and risk factors are thought to have increasing rel-evance throughout the world. COPD is usually managed in primary care, although it is com-

monly under-diagnosed, and is one of the most common medical conditions neces-sitating admission to hospital. The second edition of the ABC of COPD pro-vides the entire multidisciplinary team with a reliable, up-to-date and accessible account of COPD. Extensively updated by experi-enced clinicians — including new chap-ters on spirometry, inhalers, oxygen, death, dying and end of life issues — this ABC is an authoritative and practical guide for gen-eral practitioners, practice nurses, specialist nurses, medical students, paramedical staff, junior doctors, non-specialist doctors and all other health professionals working in both primary and secondary care.

Wiley-BlAckWellchichester, West Sussex, Uk www.cli-online.com & search 25562

Binding Site relocates to Birmingham city centre

In March 2011 The Binding Site Group, a manufacturer of immunodiagnostic prod-ucts (with a global turnover of around £43 million), moved its headquarters to larger, more eco- friendly premises in the cen-tre of Birmingham, UK. Having achieved double digit growth for many years, well above the industry average, this Special-ist Protein Company has re-housed its 380 UK-based staff into a new six-storey building that will fulfil the company’s needs for growth over the foreseeable future. The new site is a reflection of the company’s success and shows its commit-ment to Birmingham (having its roots at the University in the 1960s), to its staff and customers, and to its future growth.

The new building just off Five Ways in Birmingham has an impressive range of sustainable features and a BREEAM (Building Research Establishment’s Environmental Assessment Method, the world’s most widely used means of reviewing the environmental perfor-mance of buildings) rating of ‘Excellent’. It was built with a focus on key issues such as energy and water consumption and has provisions for rainwater harvest-ing. The building meets strict carbon emission restrictions and has solar con-trol glass to minimise heat gains, while ensuring natural levels of daylight. The provision of bicycle racks and excel-lent local public transport links ena-bles employees to limit their impact on the environment when travelling to and from work.An opening ceremony was held in March together with a celebration of Binding Site’s 3rd Queen’s Award for Enterprise.

New genetic test for hereditary breast cancerUK geneticists have developed new DNA sequencing methods and data analysis techniques for the faster identification of hereditary breast cancer.NewGene‚ a specialist molecular diag-nostic company jointly owned by New-castle Hospitals NHS Foundation Trust and Newcastle University‚ UK has

developed an advanced gene sequenc-ing process to successfully identify all mutations in the coding regions of two genes associated with inherited breast cancer – BRCA1 and BRCA2. In the first application of its type‚ NewGene is suc-cessfully using the Roche 454 GS-FLX platform for complete sequencing of all BRCA genes. The breakthrough follows two years of assay development work with specially developed data analysis software to enable high volume testing of gene sequences to be undertaken at a level not previously possible. This tech-nology platform represents a much faster and higher capacity DNA sequencing process than is associated with the tra-ditional Sanger technique used for this type of testing.The availability of the advanced test to UK and European healthcare provid-ers will mean the earlier identification of family members at risk of develop-ing breast cancer. In addition‚ by reduc-ing the cost of testing‚ health trusts will be able to extend hereditary breast cancer screening to those who may not currently qualify for gene sequencing.The new breast cancer test has already been successfully used in gene testing work car-ried out for the Northern Genetics Service‚ an NHS regional clinical genetics ser-vice serving NHS trusts across the North of England.

INDUSTry NEwS – April/May 2011 50

CALENDAr Of EVENTS

May 15-19, 2011IFCC-Worldlab-Euromedlab Berlin 2011 CongressBerlin, GermanyTel. +39 02 66802323fax +39 02 6686699e-mail: [email protected]

May 23-26, 2011FOCUS 2011Harrogate, UKTel. +44 141 434 1500fax +44 141 434 1519 e-mail: [email protected]

May 24-27, 2011Hospitalar 2011São Paulo, Brazilwww.hospitalar.com/ingles/

July 24-28, 20112011 AACC Annual MeetingAtlanta, GA, USAwww.aacc.org/events/2011am/

September 21-24, 201114th Annual Meeting of the European Society for Clinical Virology (ESCV)funchal, Madeira, Portugalwww.escv.org/meetings/meetings.asp

September 25-28, 201150th Annual ESPE Meet-ing – European Society of Paediatric EndocrinologyGlasgow, Scotland, UKTel. +46 8 459 66 00fax +46 8 661 91 25e-mail: [email protected]

October 2-6, 201112th International Congress of Therapeutic Drug Monitor-ing and Clinical ToxicologyStuttgart, GermanyTel. +49 711 8931 636fax +49 711 8931 370e-mail: [email protected]

October 15 – 16, 2011The Lancet / ESCMID Conference on Healthcare-Associated Infections and Antimicrobial ResistanceBeijing, ChinaTel. +86 21 61333077fax +86 21 52980210 e-mail: [email protected]

October 24–26, 2011ESCMID Conference on Diag-nosing Infectious Diseases: Future and InnovationVenice, ItalyTel. +39 041 52 62 530fax +39 041 52 71 129 e-mail: [email protected]

November 8-10, 2011JIB 2011 – Journées Interna-tionales de BiologieParis, franceTel. +33 1 4756 5079 fax +33 1 4756 5258 e-mail: [email protected] www.jib-sdbio.fr

November 16-19, 2011MEDICADüsseldorf, Germanye-mail: [email protected]

November 17–18, 2011ESCMID Conference on Infec-tions in Immunocompromised HostsIstanbul, TurkeyTel. +90 312 440 5011fax +90 312 441 4562 www.escmid.org

December 4-8, 201122nd World Allergy CongressCancún, MexicoTel. +1 414 276 1791fax +1 414 276 3349e-mail: [email protected]/wac2011/

Dates and descriptions of future events have been obtained from official industrial sources. CLi cannot be held responsible for errors, changes or cancellations.

For more events see:www.cli-online.com/events/

A91DX-9105-A1-4A00 © 2011 Siemens Healthcare Diagnostics. All rights reserved.

Answers for life.

Only Siemens has the innovative solutions your lab needs to reach the top and the vision to keep you there.

Planning for your future starts with choosing the right diagnostics partner today. Siemens provides comprehensive andcustomizable solutions so laboratorians and clinicians can improve productivity every day. And, with a 130-year tradition of innovation, you can trust Siemens to stay on the leading edge of emerging trends and technologies, so together wecan set a new standard in patient care for years to come. www.siemens.com/diagnostics

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| April/May 2011 Volume 35 Biomarkers for Chronic Obstructive … · 2016-04-25 · The development of drug-resistant bacteria is the inevitable result of natural selection, but imprudent

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