Infection Control In Health Care Settings

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Infection Control in Health Care Settings Sagar Desai, Esther Chung, Harshal Lal, Emerald Yuan,

Josh Finley, Roma Panjwani, Hari Purushothaman

ABSTRACT Healthcare-associated infections are a broad class of

preventable conditions that affect patients during clinical treatment. These conditions cause a significant number of illnesses and incur high costs to the healthcare system in the United States. Current diagnostics attempt to identify HAIs in order to improve patient treatment and prevent further spread of pathogens. This report examines these diagnostic methods, including cultures, immunoassays, and nucleic acid tests, to determine their effectiveness. By analyzing current literature and commercialized devices, this report explores improvements to the existing diagnostic framework. Preventative measures in the healthcare setting can significantly reduce both the economic and medical impact of HAIs.

CLINICAL UTILITY Healthcare-associated infections (HAIs) are defined by the

Centers for Disease and Control (CDC) and the National Healthcare Safety Network (NHSN) as adverse conditions caused by infectious agent(s) that were not present or incubating at the time of admission (1). HAIs represent a major preventable threat to patients and include infections acquired from healthcare settings outside of the hospital, such as long term care facilities and rehabilitation centers (2).

HAIs are caused by patient exposure to a variety of pathogens while undergoing care in the healthcare setting. Most of these pathogens are introduced through invasive procedures and devices, such as surgery, urinary catheters, central lines, or mechanical ventilators. HAIs are responsible for 1.7 million infections and 99,000 deaths per year (3). HAIs also represent a significant financial burden on patients, care providers, and payors for treatment.

Average per patient treatment costs are $11,285 to treat Clostridium difficile related infection, $45,814 for central line associated blood stream infections (CLABSI), $40,144 for ventilator acquired pneumonia (VAP), and $20,785 for surgical site infections (SSI) (2). These costs only account for the in-hospital treatment, the loss of productivity and patient wages are not accounted for.

Many HAIs can be prevented, prompting the Centers for Medicare and Medicaid Services (CMS) to change its reimbursement rules for HAIs. The CMS will no longer provide reimbursement for treatment of HAIs, such as SSIs, CLABSIs, and catheter associated urinary tract infections (CAUTIs) (3).

An additional threat to patients is the emergence of HAIs cause by antibiotic resistant pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococci. As antibiotics continue to be readily prescribed for patient well-being, even for viral pathogens, many antibiotics have begun to lose their effectiveness as

pathogens become resistant, making it increasingly difficult for caregivers to treat HAIs (4).

Figure 1. Calculation of estimates of health care-associated infections in U.S. hospitals among adults and children outside of intensive care units, 2002. BSI: bloodstream infections, UTI: urinary tract infections, PNEU: pneumonia, SSI: surgical site infections. There were 1,195,142 estimated cases of HAIs in the United States in 2002. Urinary tract infections are the most common HAI (5).

The majority of common HAIs are preventable by implementing regulations requiring healthcare workers to wash their hands prior to patient contact, re-assessing the length of times patients should be placed on ventilators, or how long a central line or catheter must be in place. Other requirements dictated by the CMS include that every US hospital must designate at least one infection preventionist (IP) who is responsible for implementing recommended policies and practices aimed at the prevention and control of infectious communicable diseases (6). To help in distinguishing HAIs from other causal diseases, reporting of present on admission (POA) conditions is required for hospitals. The CDC has identified methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) as the top priorities for screening of incoming patients (3). Ventilator Associated Pneumonia Ventilator associated pneumonia (VAP) is caused by pathogens that pass to the lungs of patients on mechanical ventilator support. The endotracheal tube is inserted into the airway of the patient facilitates pathogen entry into the lower respiratory tract, either through colonization of the inside, or leakage around the outside of the endotracheal tube. Many different pathogens can give rise to VAP, with Pseudomonas aeruginosa and S. aureus accounting for approximately 44% of the observed cases (7). The main symptoms of VAP are fever, purulent (pus containing) sputum, and hypoxemia (low oxygen in blood) (7, 8). However, these symptoms are often difficult to observe in patients on ventilators because they are frequently sedated (9). VAP diagnosis based on clinical symptoms and chest radiography has low sensitivity and specificity. Culture based methods are the gold standard but entail long turn-around times. The primary treatment for VAP is broad spectrum antibiotics. Treatment is adjusted to more effective targeted antibiotics once the causative pathogen is identified.

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Central Line Associated Bloodstream Infections Central line associated bloodstream infections (CLABSI)

have the highest morbidity and mortality of HAIs, while also occurring in 3-5 per 100 cases of CL use. The primary symptoms of CLABSIs are bacteremia (bacteria in the blood), or fungemia (fungus in the blood) without a documented source, a fever greater than 38 degrees Celsius, hypotension, and oliguria (low urine output) (10). The pathophysiology of CLABSIs includes 4 main pathways of infection. The major pathways are internal and external colonization of the catheter surface by a pathogen. A patient’s glycoproteins (fibrinogen, fibronectin, collagen, and laminin) absorbed on the surface of the CL may form a layer that enhances the adherence of certain bacteria such as S. aureus. Additionally, bacteria that colonize the interior of the CL as biofilm are inherently more resistant to antimicrobial agents. Frequent opening of the CL may also be conducive to bacterial colonization. Through external colonization, bacteria may proliferate on the skin surrounding the CL insertion site and move into the patient via capillary action. Additionally, contamination of the fluids and drugs administered by CL may allow pathogens to enter the body (11).Once bacterial sepsis occurs in systemic circulation, a CLABSI is increasingly difficult to treat and chances of morbidity and mortality increase. Catheter Associated Urinary Tract Infections Catheter associated urinary tract infections (CAUTI) account for nearly 40% of HAIs in the US (12). CAUTIs occur primarily due to unnecessary or prolonged use of a catheter (13). The catheter disrupts the patient’s innate defense mechanisms and provides pathogens with a pathway into the bladder and upper urinary tract, allowing for the spread of a pathogen via the catheter-mucosa interface. Also, two-thirds of uropathogens are acquired extraluminally of the catheter (14). Additionally, CAUTIs are seen more in women than in men. The primary symptoms of a CAUTI are burning sensation during micturition, a frequent need to urinate, pain while urinating, and fever, nausea, and vomiting may occur in upper UTIs (15, 16). Clostridium difficile

One of the most common HAIs is a gastrointestinal infection due to the Gram positive bacterium Clostridium difficile, which is an opportunistic pathogen that invades patients on broad-spectrum antibiotics to treat diarrhea or different infections. Broad-spectrum antibiotics eliminate colonic bacteria, enabling C. difficile to overgrow in the intestine. The C. difficile spores most likely enter the patient via the hands of healthcare workers. Ingested C. difficile spores germinate and can freely colonize the vacant colon (17, 18). The main symptoms of a C. difficile infection are flu-like symptoms such as a high fever, chills, fatigue, and body aches, as well as bloating, diarrhea and abdominal pain (19). A C. difficile infection is diagnosed by detection of the binary toxin genes, tcdB and tcdC, which are produced by the spores (20). Left untreated, an infection can develop into pseudomembranous colitis, which has a higher morbidity and mortality than the original infection, and is also much more difficult to treat.

Methicillin-resistant Staphylococcus aureus (MRSA) MRSA is the most common infectious agent in HAIs and

is given priority by the CDC because of its prevalence in healthcare settings. It is highly virulent due to its broad disease spectrum and multi-drug resistance (21). Colonized patients do not present signs or symptoms of infection with MRSA. Therefore, asymptomatic patients need to be identified through pre-admission screening.

Methicillin resistance is acquired through horizontal gene transfer. The most common transferred element is the SCCmec cassette, containing the mecA gene, which codes a specific methicillin-resistant transpeptidase, known as penicillin-binding protein 2a (PBP2a) (22). This transpeptidase causes low-affinity binding of β-lactam antibiotics resulting in resistance to methicillin (23). Nowadays MRSA is more commonly acquired through community infections. Nasal carriage is the main cause of clinically significant infections, therefore patients are identified by nasal or oropharyngeal swabs. Once samples are obtained, they are either tested by culture or by polymerase chain reaction (PCR) based methods (3). Vancomycin-resistant Enterococci (VRE)

Enterococci are another key infectious agent causing HAIs. Most carriers are healthy members within a community. Most human enterococcal infections are cause by E. faecium (95%) and E. faecalis (5%) (3). The antibiotic vancomycin is used frequently to treat Enterococci with inherent high resistance to β-lactams. Vancomycin acts by binding to peptidoglycan chain precursors, preventing them from growing and cross-linking. Vancomycin-resistant enterococcal species can be classified into 5 van genotypes, with vanA and vanB being the most pathogenic and responsible for HAIs (24). The vanA gene (along with genes vanR, vanS, vanH, vanX, and vanZ) is acquired through horizontal gene transfer via the transposon Tn1546 (24). These genes collectively result in the synthesis of abnormal peptidoglycan precursors which vancomycin cannot bind (24). Enterococcal vanA- strains have high-level resistance to vancomycin and teicoplanin, while vanB strains have more modest levels of resistance to vancomycin, but are susceptible to teicoplanin. For VRE, the ratio of infected symptomatic patients to colonized asymptomatic patients is 1:10 in most hospital settings, making it critical that all patients are screened before admission (3). One risk factor for VRE colonization is the use of oral vancomycin, since it inhibits the growth of the normal gram-positive bowel flora (24). To test for the presence of VRE, a stool sample from a rectal swab is cultured. Most tests look for the presence of the vanA gene, but to definitively test for VRE an assay should also be able to detect for the presence of enterococci and vanB (3). Pre-admission Screening

Both MRSA and VRE do not present symptoms upon colonization, making pre-admission screening critical to prevent incidence of HAIs. Pre-admission screening is done by microbiology labs, which serve as the gatekeepers against further spread of the pathogen (25). Active surveillance cultures are commonly used and are designed to identify all

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patients colonized with a given multi-drug resistant organism. This method is mainly used to detect MRSA and VRE. Ideally these active surveillance culture programs should have a fast turnaround time of less than 24 hours. Standard cultures require 48-72 hours, which requires isolating patients for up to 3 days due to contact precautions regardless of the test results. Contact isolation and contact precautions require physicians to wear gowns and gloves before examining patients (26), and it has been shown that physicians are less likely to examine these patients. Reducing HAI outbreaks improves overall patient care and is economically beneficial for hospitals, but unnecessary isolation places a burden on patients, families, and care providers, and leads to logistical challenges for the hospital. Furthermore, certain patients such as those undergoing psychiatric treatment, are not placed under contact isolation measures, which can exacerbate symptoms and interfere with treatment of the patient (25). Hence there is a need for faster methods that prevent the violation of the ethical principle of nonmaleficence (25). Preventative Measures

In a recent study, universal decolonization of all patients in the intensive care unit (ICU) has been found to be more effective at preventing hospital-acquired bloodstream infections from any type of pathogen, compared to MRSA screening and isolation, or to targeted decolonization of only patients identified to be MRSA carriers (21). Decolonization involves removing transmittable bacteria from a patient, which is accomplished via bathing in chlorhexidine and intranasal administration of mupirocin over the course of several days. Chlorhexidine is an antiseptic agent that has activity against a broad array of pathogens. In comparison to other antiseptics, it has residual antibacterial activity, which prevents secondary infections from the environment and reduces microbial burden on the patient’s system (27). However, because chlorhexidine is an antiseptic, there needs to be careful monitoring of potentially resistant pathogens in the future (27). Mupirocin

Figure 2. Strength of evidence for general infection prevention practices targeted against MRSA: A. Alcohol-based hand rub. B. Antimicrobial stewardship program. C. Chlorhexidine gluconate cleansing cloth. D. Active surveillance cultures for MRSA. E. Nose and skin MRSA decolonization prior to surgery. The varying amounts of evidence correspond to the number of hospitals that saw an effect through implementing those programs (6).

specifically kills staphylococci and is available only for topical application. It is used in treating nasal carriage of MRSA. Combined use with chlorhexidine bathing is more

effective than using mupirocin alone. However, resistance to this antimicrobial agent has been reported (22). Universal decolonization therefore may not be a sustainable strategy.

Other effective methods used to prevent HAIs are summarized in Figure 2 (6). The use of alcohol-based hand rubs and implementation of antimicrobial stewardship programs have the highest level of evidence, while active surveillance cultures for MRSA had a lower level of evidence for effectiveness. Another preventative measure that can be implemented relatively easily, decontamination of all surfaces and medical equipment, is often overlooked or inadequately performed (28).

TECHNICAL PRODUCT ANALYSIS

Culture-based Testing for Healthcare Associated Infections (HAIs)

Despite long turnaround times, culture-based pathogen identification and antimicrobial susceptibility testing is still considered to be the gold standard of HAI testing (29).

Upon suspicion of HAIs, cultures of the infected area, blood, sputum, urine, or other bodily fluids/tissue are performed on general or specific media to identify the causative organism (30). Vancomycin resistant Enterococci (VRE)

Enterococci grow under specific conditions, and can be isolated, detected and enumerated on selective media. Rectal swabs or stool samples are typically plated on mEnterococcus agar or KF Streptococcus agar. Enterococci will grow on most blood containing agar, forming colonies that are alpha-hemolytic (green margins) or non-hemolytic (no margins). Bile-esculin-azide agar is used as a confirmation test, since Enterococci can hydrolyze esculin in the presence of bile (31). mEnterococcus agar contains Triphenyl tetrazolium chloride (TTC) dye, which is reduced to formazan in the bacterial cell to give red/maroon colonies.

Enterococci are gram positive, meaning the bacteria appear purple under the a microscope after gram staining due to uptake and retention of crystal violet into the peptidoglycan layer in the cell wall. Isolates are further characterized using biochemical tests such as the catalase test. Enterococci are catalase negative, meaning no bubbles are formed when a dilute peroxide solution is added to a bacterial isolate, since Enterococci do not produce the enzyme catalase which converts hydrogen peroxide to water and oxygen gas. However, Enterococci are PYR positive, meaning they produce the enzyme pyrrolidonase, which converts a substrate in the PYR reagent into a compound that undergoes a secondary reaction, generating a red product. To determine if an enterococcal isolate is vancomycin resistant, agar dilution antimicrobial susceptibility testing (AST) is performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The bacterium is plated on agar plates containing different concentrations of vancomycin, enabling the minimum inhibitory concentration to be determined (32). Methicillin-resistant Staphylococcus aureus (MRSA)

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S. aureus can be identified using standard microbiological methods such as growth characteristics, colony morphology, Gram’s staining, catalase and coagulase tests. Similar to Enterococci, Staphylococci are gram positive ball shaped bacteria that give rise to partial hemolysis when grown on blood agar. However, Staphylococci are catalase positive, as opposed to Enterococci. S. aureus is also coagulase positive, meaning it produces a coagulase enzyme that clots blood plasma, acting as one of the virulence factors of S. aureus. The formation of a clot around an infection caused by these bacteria likely protects it from phagocytosis by the host cells. The coagulase test makes it possible to differentiate S. aureus from other less pathogenic coagulase negative Staphylococci which are part of the normal human skin flora.

Methicillin resistance can be tested using a standard disk diffusion assay (Figure 3A), such as the Kirby-Bauer disk diffusion method, which is performed by plating bacterial inoculum on the surface of an agar plate. A paper disk containing a known concentration of the antibiotic oxacillin is placed on the inoculated agar surface and the plates are incubated for 16 to 24 hours at 35°C. The antibiotic leaches into the surrounding agar by diffusion. If the inoculated bacteria are susceptible to the antibiotic from the disk, a zone of growth inhibition (halo) is formed around the disk where the bacteria do not grow. The diameter of this zone enables classification into susceptible, intermediate, or resistant, using criteria published by the CLSI (33). MRSA is resistant to oxacillin, hence no halo is observed in Figure 3A. Alternatively, the bacteria can be grown directly on antibiotic containing agar (Figure 3B), wherein growth indicates that the bacteria are resistant (33).

Figure 3. (A) Oxacillin disk diffusion plate showing methicillin-resistant Staphylococcus aureus(34). (B) Mannitol salt agar plate with oxacillin showing methicillin-resistant S. aureus (34)

These manual methods are relatively time consuming and can be prone to error. Newer automated testing technologies such as the bioMérieux’s VITEK® 2 system, are aimed at same day bacterial identification and AST using standardized protocols. Clostridium difficile

To detect C. difficile via culture, stool samples are treated with heat shock or ethanol to kill off all bacteria except spores. The spores are then inoculated and incubated on selective media under anaerobic conditions (35). After incubation the colonies exhibit characteristics unique to C. diffcile such as fluorescence under Wood’s Lamp (which uses black light to detect bacterial infections), and the production of a horse manure-like odor.

The major virulence factors of C. difficile are toxins A and B. Toxin B is ~1000 times more toxic than A because it has 100-fold higher enzymatic activity than toxin A (36).

Culture methods cannot distinguish between toxin-producing and non-toxin producing isolates. Hence, positive culture tests require additional toxin screening (37), for example, via cell culture Cytotoxicity Neutralization Assays (CCCNAs). C. difficile toxins can be identified using tissue cultures. A monolayer of human or mammalian cells in culture is inoculated with a filtrate of the stool sample and incubated. The cytopathic effect (CPE) causes the cells to round up and slough off the monolayer. The sample is positive for C. difficile toxin B if the CPE is neutralized by an antiserum from either C. sordellii or C. difficile. (38).

This test is very sensitive and specific and is considered the gold standard in C. difficile testing (37).

Immunoassays Enzyme linked immunosorbent assays (ELISAs) are use to

detect antigens produced by HAI pathogens, or antibodies produced by the host in response to a pathogen, with enzymatic signal amplification.

Figure 3. Interpretation of C. diff Quik Chek Complete results. (A) positive result for non-toxigenic C.diff; (B) positive result for toxigenic C.diff; (C) Negative result; (D)-(G) Invalid results; (H) Indeterminate. Due to low bacterial load, the specimens may test negative for antigen but positive for toxin. Adapted from (39).

One example, the C. diff Quik Chek Complete by Alere tests for the presence of C. diff glutamate dehydrogenase

(E)

Tox Ag

C

C

Tox

Tox Tox

Tox

Tox Tox

Tox

Ag

Ag

Ag

Ag

Ag

Ag

Ag

C

C

C

C

C

C

(A)

(F)

(C)

(D)

(G)

(H)

(B)

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(GDH) and for toxin A and B specific to C. diff (39). The assay uses mouse monoclonal antibody specific for GDH and goat polyclonal antibodies for toxins A and B. The detection antibodies are coupled to HPR (39). The capture antibodies are immobilized on a membrane with three lines (

Figure 3): the antigen line (Ag) contains the antibodies against C. diff glutamate dehydrogenase, the dotted control line (C) contains anti-horseradish peroxidase antibodies, and the A and B test lines (Tox) contain the antibodies against C. diff toxins A and B (39). The stool sample is diluted and added to a solution containing the respective detection antibodies (39). This solution is then added to the sample well on top of the membrane and incubated for 15 minutes at room temperature. This incubation process allows for formation of the sandwich complex between the respective capture and detection antibodies with their antigens GDH and toxins A and B (39). Regardless of the presence of C.diff antigen, the anti-HRP antibody on the control line should always bind to HRP antibody conjugated detection antibodies in solution. The reaction window is then washed with the wash buffer, to remove unbound detection antibodies, and then a solution containing the HRP substrate tetramethylbenzidine is added. After another 10 minute incubation period, the test results are interpreted as illustrated in Figure 4. The C. diff Quik Chek Complete assay has sensitivity to GDH and toxins A and B of 92.4% and 93.9% and specificity of 83.5% and 99.3% (40). This assay allows for quick results with diagnosis in less than 1 hour. It is less expensive and easier to use than PCR (40, 41). This immunoassay has lower biohazard risks than culture method and no isolation of organisms is needed because the test can be performed directly from the sample.

Immunoassays are also used for the identification of MRSA. One study developed a novel screening test for MRSA that detects penicillin-binding protein 2a (PBP2) with 94.4% sensitivity and 100% specificity (42). The method uses anti-PBP2 monoclonal IgM and anti-mouse IgG antibodies in conjunction with standard ELISA or other immunoassay methods to detect MRSA. Matsui et al. developed an immunochromatographic test (ICT) for the detection of PBP2-producing cells for use in clinical laboratories. Two monoclonal antibodies, 10G2 and 1G12 were used to form an antigen sandwich. The 10G2 antibody was combined with a colloidal gold particle that served as a detector of PBP2. The 1G12 antibody was immobilized on a nitrocellulose membrane that captured the 10G2-gold colloid-PBP2 complex. To measure sensitivity, recombinant PBP2 (rPBP) was used. Results showed that the test strip was able to detect purified rPBP at concentrations as low as 1.0ng/50ul/test but was unable to detect protein levels below 0.5ng/test. Such rapid immunoassay tests may allow for better screening in hospital settings. The Luminex xTAG multiplex system offers a way to detect for at a sensitivity and specificity of 97.7% and 94.9% respectively (43). Test results are delivered in under five hours.

Nucleic Acid Testing Nucleic Acid Testing (NAT) is one of the most rapidly

developing IVD market segments, and is used to diagnose

infectious diseases such as HAIs. While immunoassays can typically be performed directly on clinical samples, nucleic acid testing requires the nucleic acids to be separated and purified due to the risk of nucleic acid degradation or polymerase inhibition (44). NAT can reduce the time to diagnose a patient to as little as 1-2 hours, compared to 24-48 hours required for current gold standard culture-based methods. NAT therefore offers rapid and accurate detection which is crucial in diagnosing HAIs to prevent transmission.

Cepheid GeneXpert The GeneXpert system by Cepheid is a fully automated

platform that conducts sample preparation followed by target amplification via the Polymerase Chain Reaction (PCR), with real time fluorescence detection. The GeneXpert uses disposable, single-use cartridges. Different cartridges are available to test for MRSA, C. difficile, and VRE.

The GeneXpert MRSA test targets the staphylococcal cassette chromosome mec (SCCmec), which contains the mecA gene that is responsible for resistance to β-lactams such as meticillin in S. aureus (45, 46). The cartridge contains multiple chambers, which enable reagent storage, nucleic acid extraction, PCR amplification, and real time detection. The cartridge contains a piston and rotating valve to enable fluid transfer between the 11 chambers. Within the chambers are freeze-dried enzymes, DNA building, and other reagents required for the reaction mixture. The system enables ultrasonic lysis of the pathogen, and nucleic acid extraction.

Figure 4. (A) Cepheid GeneXpert Cartridge Parts (B) Cutaway of Cartridge. The cartridge contains 11 chambers that hold sample, diluents, and reagents. Reagents and samples are transferred between the chambers using a plunger in the middle of the cartridge, in conjunction with a rotating valve. Most of the cartridge is dedicated to reagent storage and sample preparation, with PCR amplification and detection performed in the small square chamber at the back of the cartridge. Adapted from (49).

Once the sample is processed, it is mixed with the PCR reagents and moved into the reaction tube. The reaction tube allows for thermal cycling, optical excitation, and detection of the generated amplicons (47). The assay is started after the cartridge containing the patient stool sample and required reagents are inserted into the GeneXpert system. Compared to culture methods, the GeneXpert MRSA provides sensitivity

(A) (B) Cover

Liquid reservoirs

PCR reaction vessel

Syringe Rotary valve

Ultrasonic interface

Base

Plunger Bead retaining material Internal control bead

Bead retaining material

Primers & Probes

Enzyme reagents

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and specificity values of 95%-100% and 89.7%-97%, respectively (45, 46, 48).

Along with MRSA, GeneXpert has a kit that tests for Vancomycin-resistant enterococci (VRE). VRE grow resistant due to possession of vanA and vanB genes from other organisms (50, 51). GeneXpert vanA/vanB detects for these genes in the sample through RT-PCR just like the assays for MRSA and C. diff. Sensitivity and specificity for vanA and vanB were reported to be 95.8%-100% and 83.2%-99.5%, respectively compared to culture methods (50, 51).

GeneXpert also tests for C. difficile, which is the cause for the majority of healthcare acquired infections leading to antibiotic-associated diarrhea and pseudomembranous colitis (52). C. difficile carry toxin A (tcdA) or B (tcdB) genes, which are assay targets for diagnosis. There are two assays offered by GeneXpert to test for C. difficile. GeneXpert C. difficile PCR assay detects tcdB using RT-PCR. GeneXpert C. difficile /Epi PCR assay is a multiplex RT-PCR that detects binary toxin gene (cdt), tcdC gene, along with the tcdB leading to the identification of the epidemic 027/NAP1/B1 strain (20). Illumigene C. difficile

The Illumigene C. difficile assay by Meridian Bioscience uses loop-mediated isothermal amplification (LAMP) to detect a conserved 204-bp sequence within tcdA (20). Manual sample extraction consists of stool collection using the sample brush provided in the kit, placing the brush in diluent, and vortexing for 10 seconds. Lyophilized S.aureus is added to the sample, serving as the extraction and external amplification control. The solution is squeezed into the extraction tube and heated at 95° C for 10-minutes, then vortexed for an additional 10 seconds. The extracted solution is added to the reaction buffer tube and vortexed for another 10 seconds. This solution is then transferred into the test and control tubes, which contain primers for C. difficile and S.aureus, respectively. The reaction tubes are placed into the Illumipro-10 device where the sample undergoes automated isothermal amplification and detection (50). Along with the sample, the control consisting of S. aureus DNA target is amplified and detected to determine results read by the Incubator/Reader (51). Compared to culture methods, Ilumigene C. diff showed a sensitivity and specificity of 94%-95.2% and 95.3%, respectively (20, 51). Unlike the Cepheid GeneXpert, the Illumigene C. diff assay is not able to detect the hypervirulent 027/NAP1/BI strain (20). Portrait Toxigenic C. difficile Assay

The Portrait Toxigenic C. diff. Assay by Great Basin diagnoses the presence of C. diff. in stool samples. Similar to the Cepheid GeneXpert system, extraction, amplification, and detection occur in a single use test cartridge. The test is initiated once the cartridge is inserted into the analyzer. The assay uses isothermal helicase-dependent amplification (HDA) to unwind the double-stranded DNA (52, 53) . In this process, DNA helicase enzymes are used to separate the DNA strands instead of heat. Single strand binding proteins present in the mastermix keep the now single stranded DNA separated. In the Portrait C.diff assay, the 78-nucleotide 3’ region of the

tcdB gene is amplified, and then detected using immobilized capture probes on a slide array (54, 55). The addition of tetramethylbenzidine (TMB) added to the bound conjugate forming a colored precipate at the probe/target sequence complex location. Results are based these colored spots which form on the chip surface, which are then read by the optical reader (52). The sensitivity and specificity of the assay has been reported to be 97.6% and 96.4%, respectively (56).

Table 1. Nucleic Acid Tests to diagnose Healthcare-Associated Infections

Company - Platform Pathogen Target Molecular

method Extraction Time

MRSA SCCmec PCR Automated <2h

Cepheid - GeneXpert C. diff tcdA PCR Automated 45min

C. diff/EPI tcdB/tcdC PCR Automated 45min

VRE vanA/vanB PCR Automated ~50min

Illumigene C. diff tcdA LAMP Manual 1h

Portrait C. diff tcdB HDA Automated 1.5h

MARKET / COMPETITOR ANALYSIS Most hospitals have a policy of self-oversight regarding

decisions about HAIs. A group of professionals, called an Infection Control Board or a similar name, is tasked with oversight and decision-making regarding HAIs at the particular hospital. The structure and organization of these Infection Control Boards varies from hospital to hospital (57). However, the professionals in these groups are expected to be trained and certified in the safe handling of infectious materials. The Certification Board of Infection Control and Epidemiology, Inc., or CBIC, is one organization that offers this certification (58). These certified professionals are then tasked with making decisions regarding infection control protocols, treatment, and diagnostics.

Poor infection control policies can impact a hospital’s finances in a number of ways. Reimbursement schedules from the CMS are tied to a hospital’s performance in controlling HAIs. A policy, enacted in 2008, specifically denies payment for readmissions attributable to certain preventable HAIs—specifically, CAUTIs, SSIs and CLABSIs (59). Interestingly, though, some evidence indicates that this has not actually affected infection rates (60).

Another factor that plays into the financial motivation is consumer (i.e. patient) satisfaction. Like any company, a hospital must provide high-quality service to attract and retain customers. In fact, evidence shows that increased patient satisfaction, as measured by patient surveys, is tied to a hospital’s financial performance (61, 62).

Significant direct costs are incurred as a result of HAIs in the United States. Estimates place the nationwide costs of HAIs between $28.4 and $33.8 billion for 2007, when adjusted for inflation (63). Per patient, this equals between $16,359 and $19,430 on average. These costs can be broken down by infection site (Figure 5). The potential cost savings

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through HAI prevention have been estimated between $5.7-6.8 billion (low estimate) and $25.0-31.5 billion (high estimate) (63). The current analyses assume that 65%-70% of CLABSIs and CAUTIs and 55% of VAP and SSIs are preventable. CLABSI prevention has been singled out as having a higher cost impact than the other infection types due to the high cost combined with the high fatality rate and preventability (64). As shown in Figures 6 and 7, CLABSI and VAP both have high fatalities and average costs per patient, but low prevalence, complicating the cost impact estimates.

Figure 5. Mean Cost of HAIs per Patient by Infection Site. SSI = Surgical Site Infection; CLABSI = Central Line-Associated Blood Stream Infection; VAP = Ventilator-Associated Pneumonia; CAUTI = Catheter-Associated Urinary Tract Infection; CDI = Clostridium difficile-associated Infection (63).

The patient as a consumer is beginning to play a more important role in the HAI landscape, primarily through patient satisfaction surveys. The only government-utilized satisfaction survey is the HCAHPS, or Hospital Consumer Assessment of Healthcare Providers and Systems (65).

Figure 6. Fatality Rate of HAIs by Infection Site. CDI (Clostridium difficile-associated Infection) is omitted due to lack of reliable data.

Hospital adaption of this survey has been increasing steadily, and has grown from 1.1 million completed surveys from 2,421 hospitals to 3.1 million surveys from 3,928 hospitals (66).

Acute-care hospitals that participate in the IPPS (Inpatient Prospective Payment System), a reimbursement system

created by CMS for Medicare-qualified acute hospital stays, receive payments from CMS based on patient discharges (67). As of 2006, CMS has levied percentage penalties to a hospital’s payments if their HCAHPS performance is too low (59).

Figure 7. Infection Prevalence of HAIs by Infection Site. Prevalence measured as total number of infections per year (68).

Other surveys, such as the Leapfrog survey, exist but do not affect a hospital’s reimbursement and are voluntary (69). Furthermore, there is increasing confusion as a result of too many disparate surveys being developed—a single hospital can have a different rating for each survey (70). Further complicating matters, at least one study has indicated that patient satisfaction scores, on average, are not affected by the patient getting a hospital-acquired condition (71).

Still, by completing these surveys, patients have a direct role in pressuring hospitals to perform better HAI control. A hospital’s HCAHPS score affects its reimbursements, and evidence suggests the LeapFrog initiatives have had an overall positive effect on the performance of participating institutions, despite hurdles such as limited participation nationwide (72).

Another source of pressure from patients is the use of social media, which is becoming increasingly important to hospitals. Patients have indicated a desire for their health care providers to engage in social media platforms (73). As a marketing and surveying strategy, social media is more cost-effective than current solutions (74). Finally, the widespread visibility of social media-based information puts pressure on health care providers in the face of a potentially large audience, as opposed to patient survey data (75). Regulations to control HAIs

To monitor and control HAIs, in 2005, the National Healthcare Safety Network (NHSN) was established under the Healthcare Quality Promotion Division at the CDC. The CDC incorporated three former systems, the National Nosocomial Infections Surveillance system, the Dialysis Surveillance Network, and the National Surveillance System for Healthcare Workers, into a common national database (77). The goals of this consolidation were to estimate the magnitude of HAIs, and to develop and evaluate strategies to prevent HAIs. The NHSN also aids in improving quality by providing risk- adjusted data for both inter-facility and intra-facility

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Table 2. Prevalence of Healthcare-Associated Infections, based on pathogen type, accounted to the NHSN, by Type of HAI, 2009–2010

CLABSI - Central Line-Associated Blood Stream Infection; CAUTI - Catheter-Associated Urinary Tract Infection; VAP - Ventilator-Associated Pneumonia; SSI - Surgical Site Infection (76).

comparisons. Healthcare facilities receive assistance in developing surveillance and analysis methods to acknowledge patient safety problems as well as prevent HAIs (77), (78). According to the CDC, the NHSN is the gold standard for HAI surveillance (79). NHSN functions and workflow

In 2008, NHSN began enrolling all healthcare facilities in the United States (80). Data is collected and reported by the NHSN-trained healthcare facility personnel, on a monthly

Figure 8. Deaths due to Clostridium difficile infection between 1999 and 2010 (82).

basis using standardized methods. Information is either manually entered into the NHSN database, or uploaded as xml

files. NHSN categorized these data into two modules. The device-associated module includes CLABSIs, CAUTIs and VAP. The procedure-associated module includes SSIs and post procedure pneumonia (76). In addition to reporting based on HAI types, HAIs are also reported based on the pathogen causing infection (Table 2) (81). With C. difficile infections causing increased mortality each year (Figure 9), the federal government is mandating the reporting of C. diff infections (82), to facilitate implementation of appropriate epidemiological control strategies.

The reported infections are calculated based on the number of patient-days and infection rate. These infections are also categorized based on the nature of the healthcare facility and based on the age of patients (76). To hold active status, healthcare facilities must submit at least data for 6 months each year per module (83). Measures to overcome drawbacks

To incentivize the reduction of HAIs, the CMS terminated reimbursing healthcare providers for treating SSIs, CAUTIs and CLABSIs (84). Even with such policies, payment reductions were negligible and healthcare providers do not show much variation in the services they provide. The Federal government is attempting to implement more stringent mandates by means of the Affordable Care Act (ACA) to overcome this problem (84). A claim is denied reimbursement if the patient does not present with symptoms of the HAI at the point of admission (POA) as well as 30 days from discharge (85). This reimbursement change aims to alter staff behavior with the top management insisting on preventive measures. By modifying organizational structures, hospitals

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can bring about changes in patient care methods and clinician behavior, such as hand hygiene. By accepting the best HAI measures, consumers, clinicians, administrators and policy makers, can eliminate the preventable HAIs and identify various other benefits such as better quality of life (86).

The ACA tries to prevent HAIs by introducing incentives to improve measurement and reporting. It also ensures that

transparent information concerning HAI prevalence is available online so that the public can better evaluate their healthcare providers. Hospitals, with higher rates of HAI will be imposed a penalty and with such mandates it is expected that the cost of Medicare will reduce by $3.2 billion over the next decade (87).

Table 3. Overview of Different Diagnostic Tests Used to Screen for HAIs

Assay Mol. Method Pathogen Target Time Sensitivity Specificity Ease of Use*

Bile-Esculin-Azide Agar Culture VRE Pathogen 24-48 hours 86% 92% 2

CCCNAs Culture C. diff Toxin B 24- 48 hours 98% 100% 2

Blood Agar Culture MRSA Pathogen 18 to 24 hours 99% 99% 2

Kirby-Bauer Disk Diffusion

Method Culture MRSA

Susceptibility of organism to Antibiotics

Depends on growth of the organism (18-

36 hours)

99% 100% 2

Vitek 2 Culture S.aureus Pathogen and AST 24 hours 97.5% 100% 1

aBCYE Culture Legionellosis NA ~ 5 days 80% 100% 2

C. diff QuikChek Immunoassays C. diff A and B Toxins <30m 94.40% 100% 2

Luminex xTAG Immunoassays C. diff PBP2 <5h 97.70% 94.90% 2

NAT MRSA SCCmec <2h 95% - 100% 89.7% - 97% 2 Cepheid

GeneXpert NAT VRE VanA / VanB 45 min 95.8% - 100% 83.2% - 99.5% 2

NAT C. diff tcdA 45 min 93.50% 94.00% 2

NAT C. diff / EPI tcdB ~50 min 93.39% 94.02% 2

Illumigene NAT C. diff tcdA 1h 94% - 95.2% 95.30% 2

Portrait NAT C. diff tcdB 1.5h 97.60% 96.40% 2

*Ease of Use scoring: 1—requires extensive training and technical knowledge, 2—some training and technical knowledge is necessary, 3—anyone can run the assay after reading instructions.

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80. [Webpage] National healthcare safety network (NHSN)Healthy peopleMarch 28, 2013. Retrieved on: September 28, 2013. Available from: http://www.healthypeople.gov/2020/data/datasource.aspx?id=206 81. [Article] Sievert, D. M., Ricks, P., Edwards, J. R., Schneider, A., Patel, J., Srinivasan, A., Kallen, A., Limbago, B., Fridkin, S., Natl Healthcare Safety Network, and Participating NHSN Facilities. (2013) Antimicrobial-Resistant Pathogens Associated with Healthcare-Associated Infections: Summary of Data Reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection Control and Hospital Epidemiology. 34, 1-14, http://dx.doi.org/10.1086/668770 82. CDC Vital Signs. (Retrieved on: December 9, 2013) Centers for Disease Control and Prevention, 83. Operational Guidance for Acute Care Hospitals to Report Central LineAssociated BloodstreamInfection (CLABSI) Data to CDC’s NHSN for the Purpose of Fulfilling CMS’s Hospital Inpatient Quality Reporting (IQR) Requirements. (September 26, 2013) Centers for Disease Control and Prevention, 84. [Article] McNair PD, FAU - Luft, H. S., Luft HS, FAU - Bindman, A. B., and Bindman AB. Medicare's Policy Not to Pay for Treating Hospital-Acquired Conditions: The Impact. - Health Aff (Millwood).2009 Sep-Oct;28(5):1485-93.doi: 10.1377/hlthaff.28.5.1485. 85. [Webpage] Readmissions reduction programThe Centers for Medicare & Medicaid Services. Last Updated: August 2, 2013, . Retrieved on: December 9, 2013. Available from: http://www.cms.gov/Medicare/Medicare-Fee-for-Service-Payment/AcuteInpatientPPS/Readmissions-Reduction-Program.html 86. [Article] Stone, P. W., Glied, S. A., McNair, P. D., Matthes, N., Cohen, B., Landers, T. F., and Larson, E. L. (2010) CMS Changes in Reimbursement for HAIs Setting A Research Agenda. Med. Care. 48, 433-439, http://dx.doi.org/10.1097/MLR.0b013e3181d5fb3f 87. Affordable Care Act Update: Implementing Medicare Cost Savings. (Retrieved on: December 9, 2013) Centers for Medicare & Medicaid Services, 88. [Article] Jenkins, S. G., Raskoshina, L., and Schuetz, A. N. (2011) Comparison of Performance of the Novel Chromogenic Spectra VRE Agar to that of Bile Esculin Azide and Campylobacter Agars for Detection of Vancomycin-Resistant Enterococci in Fecal Samples. J. Clin. Microbiol. 49, 3947-3949, http://dx.doi.org/10.1128/JCM.00180-11 89. [Webpage] Legionella - diagnostic testingCentre for Disease Control and Prevention. Last Updated: February 5, 2013, 2013. Retrieved on: 12/10/2013. Available from: http://www.cdc.gov/legionella/diagnostic-testing.html