1 CA García-Sepúlveda MD PhD Clinical Applications of Molecular Diagnosis Viral & Human Genomics Laboratory Facultad de Medicina, Universidad Autónoma.

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CA García-Sepúlveda MD PhD

Clinical Applications of Molecular Diagnosis

Viral & Human Genomics LaboratoryFacultad de Medicina, Universidad Autónoma de San Luis Potosí

Laboratorio de Genómica Viral & Humana - Facultad de Medicina - Universidad Autónoma de San Luis Potosí

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Introduction

Human Genome Project (2000) revolutionized the way in which scientists search for the mechanisms of diseases.

Tests that identify molecular and genetic markers for an individual patient.

These markers determine potential benefit from a specific therapy, or risk of developing a specific disease or other health condition.

From 100,000 to 23,000 genes.

Initial funds (DOE) of $3.5 billion USD


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Molecular Diagnostics Industry

$5.5 Billion industry

$8 Billion by 2010

40 million annual test volumes in the U.S.

Projected to be 1/3 of all diagnostic testing


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Industry Test Volumes & Applications

55% - Infectious disease

23% - Blood Screening

13% - Genetic Testing

7% - Cancer

.


Prediction of risk – Oncotype.

Early detection - Fragile X.

Classification of disease – Leukemias.

Therapeutic homming of presumptive

target.

Prediction of toxicity & response –

Herceptin.

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Breast Cancer and Targeted Therapy

211,000 women diagnosed with breast cancer and 40,000 deaths per year (US 2005 estimate).

Herceptin (trastuzumab) chemotherapy approved by the FDA in 1998.

Risk of congestive heart failure.

Herceptin could benefit women who over-expressed a protein – HER2/Neu.

Molecular diagnostic tests reveal who could and will not benefit from Herceptin.

Herceptin benefit test Cost $500 USD

Herceptin Tx costs $25,000 – $80,000

Getting the “right” women on Herceptin


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Coumarin Pharmacogenomics

Warfarin is an oral anticoagulant that inhibits vitamin K reductase.

Discovered 60 years ago and currently one of the most prescribed drugs in the world.

Used to prevent thromboembolisms due to atrial fibrilation, recurring miocardial strokes, Deep vein thrombosis, Pulmonary thromboembolism and that due to valve replacements.

Between 1 and 7% of treated patients will suffer lethal hemorrhagic complications (very tight therapeutical safety index).

CYP2C9 & VKORC1 polimorphisms define metabolic rates and might explain between 10 and 25% of interindividual therapeutic response variations.

“The FDA highlights the opportunity for healthcare providers to use genetic tests (CYP2C9 & VKORC1) to improve their initial estimate of what is a reasonable warfarin dose for individual patients”.


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Molecular Diagnostics in Infectology

Nucleic acid amplification methods have revolutionised conventional laboratory methods based on phenotypic expression of antigens or biochemical products.

Increasingly incorporated into the clinical microbiology laboratory, particularly for the detection and characterisation of virus infections and fastidious bacteria.

Rapid turn-around time, high sensitivity and high specificity are appealing.

Molecular detection is mostly known in the form of PCR technology.


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Applications

Virology: It has been applied to resistance testing, genotyping and viral load quantification in addition to routine viral detection.

Bacteriology:Applied to resistance testing, the detection of fastidious bacteria and faster detection of serious bacterial infections.

Parasitology and mycology:Applied to the rapid diagnosis of fungal infections in neutropenic patients and those in which diagnostic interventions are risky.

Other:Assessing and monitoring biohazards, molecular epidemiology and infection control.


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Virological detection

The diagnosis of viral infections has traditionally been:

Costly,

Laborious,

Highly skilled,

Slow.

Serology is often unhelpful in the early stages of infection.

Specific antisera for the serology tests can be difficult to obtain.

PCR technology has therefore improved the detection of a number of these viruses.


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Herpes Simplex Virus

HSV encephalitis required brain biopsy in some cases due to the low sensitivity of cerebrospinal fluid (CSF) culture and serology.

PCR now allows the detection of HSV DNA from CSF with 95% sensitivity thus avoiding invasive brain biopsy.

Viral meningitis, commonly caused by enteroviruses or HSV, is more reliably and more rapidly detected by PCR compared to culture (1 vs. 5 days).

PCR can be multiplexed for other pathogens that cause meningitis.


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Blood-borne pathogen detectionActive HCV infections are diagnosed by the presence of RNA as serology cannot distinguish between past and present infections.

Only individuals with detectable HCV RNA carry a risk of transmitting HCV by transfusion, organ transplantation, needle-stick injury or vertically.

Although HIV is routinely diagnosed by serology, early HIV infection can be detected through proviral DNA PCR before it can be confirmed via western blot.

Reduction of window period from 22 and 66 days to 9 and 7 days respectively.


Ctrl

CMVHBV

HIV

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Paediatric infectionsIntrauterine infection of the foetus with CMV, rubella and varicella zoster virus can be detected through PCR of amniocentesis fluid.

The method of choice for microbiological diagnosis of rotavirus from stool samples is PCR.

Norovirus PCR is the most sensitive and rapid detection method.

PCR is also the most sensitive method for the diagnosis of astroviruses and enteric adenoviruses (serotypes 40 and 41).


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Respiratory infections

Molecular detection of respiratory viral pathogens from NPA, TS, sputum or BAL fluid is cost-effective.

It decreases hospitalisation, unnecessary testing and procedures and guides specific therapy (reducing unnecessary antibiotic use).


Large multiplex or tandem PCR assays for common respiratory viral pathogens currently available.

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Viral load monitoringMonitoring viral DNA or RNA loads has become the standard of care for several chronic viral infections:

•HIV

•HBV

•HCV

•CMV

HIV viral load testing is an integral component of the management of HIV infection.

It is the major tool used to monitor the success of antiretroviral therapy and to detect the emergence of viral resistance

HIV viral loads also predict progression of disease, and gives prognostic information


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HBV/HCV viral load monitoring


HCV RNA viral loads are used to monitor response to combination interferon- & ribavirin therapy.

Patients who remain negative for HCV RNA 6 months after combination therapy usually achieve a sustained virological response.

If HCV RNA is undetectable after 12 weeks of therapy there is a 75% chance of sustained virological response.

In HBV carriers with active liver disease VL determine the need and effectiveness of either interferon-a or lamivudine antiviral therapy.

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CMV viral load monitoring

CMV infection is serious in bone marrow, solid organ transplant recipients and HIV-infected patients.

Poor sensitivity of traditional culture methods (& very slow).

VL testing is currently the accepted standard for monitoring the emergence of CMV infection during immunosuppression.

Allows pre-emptive therapy prior to the emergence of clinical disease with high sensitivity when compared to culture.


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Viral genotyping - HIV

HIV genotyping for the detection of drug resistance is the standard of care to guide antiretroviral therapy.

• Clinical failure (worsening of symptoms).

• Virological failure (increasing VL).

• Immunological failure (decreasing lymphocyte counts).

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Viral genotyping - HCV

Six HCV genotypes geographically distributed throughout the world.

HCV genotype is the single strongest determinant of success in combination therapy (all patient candidates undergo genotyping).


Those with HCV genotype 2 or 3 receive 6 months of therapy with a 76% chance of success.

Those with HCV genotype 1 only have a 56% chance of success after 12 months of therapy.

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Bacteriological applications

Speed and resolution of molecular methods have firmly established their utility in several applications:

• Detection of fastidious (hard to culture) bacteria.

• Rapid detection of severe bacterial diseases.

• Assessment of antibiotic resistance.

• Identification of bacterial etiology through broad-spectrum PCR


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Fastidious bacteria - Mycobacteria

M. tuberculosis is one of the few examples where conventional culture remains more sensitive than molecular testing.

Difficulties in DNA extraction from the bacterial cells.

Despite this limitation, it allows confirmation of acid-fast bacilli with up to 98% sensitivity in pulmonary tuberculosis within a day (versus two or more weeks by culture).

Traditional methods for detecting rifampicin & isoniazid resistance require culture, delaying the diagnosis & increasing the risk of transmission of resistant disease in the community.

A multiplex PCR for the sequencing of rpoB and hsp65 gene allows same day results of most multi-drug resistant strains.


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Rapid bacterial detectionSevere infectious diseases sometimes require a prompt & unequivocal diagnosis so as to guide therapeutical & preventitive measures (both individually and population-wide).

Meningococcal disease has devastating consequences and requires early diagnosis for correct antibiotic therapy as well as early chemoprophylaxis for close contacts.


Multiplex kits for the detection of common causes of meningitis.

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Mycology/Parasitology applications

Molecular testing not as frequently applied to eukaryotic infections.

Pneumocystis jiroveci causes severe pneumonia in immunosuppresed patients but detection is limited to microscopy of respiratory specimens.


Immunofluorescence is more sensitive but is more expensive and needs specialised facilities.

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Mycology/Parasitology applications

Another mycological example is the use of 18S rRNA gene PCR to detect Aspergillus spp. infection in neutropenic patients.

Disease is notoriously difficult to diagnose due to the poor sensitivity of culture in early disease (and the difficulty in obtaining histopathological specimens in patients with reduced platelet counts).

Early treatment is essential for the best outcomes resulting in empiric use of costly and toxic antifungal therapy.


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Emerging infectious disease

surveillanceRapid and reliable aetiological diagnosis underpins the effective management of contagious diseases.


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SARS-CoV & special pathogen

detectionSevere acute respiratory syndrome (SARS) coronavirus (SARS-CoV).

During the early outbreak, PCR testing of respiratory specimens for other respiratory viruses rapidly excluded likely causes.


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Pandemic influenza detection

Following the 1997 Hong Kong influenza outbreak rapid end-point PCR diagnostic tests proved their worth.

Specific serology for H5 Influenza requires live virus for microneutralisation assay (currently a Biosafety Level 4 organism).


Direct immunofluorescence assay (DFA) requires influenza-specific monoclonal antibodies.

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Biosecurity applications

Bioterrorism: Violent action using living matter to harm or kill people for political reasons.

Biothreats: Any bacteria, especially weaponisable (deliverable) ones.

Weaponisable agents such as Bacillus anthracis, variola major virus, Clostridium botulinum & Yersinia pestis.

The rapidity at which such an incident could escalate mandates rapid, reliable and sensitive detection methods.


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Concluding remarks

Largest single source of objective health and disease status

information

Less than 5% of global health cost

Drives up to 70% of clinical decisions

Diagnosis, therapy, prognosis

Admit, isolate, ICU, discharge

Acute, chronic, preventive.

Validates resource use, cost.

Enforces optimal resource use (particularly in developing

countries).


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Potential to Transform Health Care

More accurate disease diagnosis

Health care providers more efficient

Targeted medicine

Response, dosing, adverse events

New, better, safer medicines

Diagnostic, prognostic process

When, whether, how to treat


30Laboratorio de Genómica Viral & Humana - Facultad de Medicina - Universidad Autónoma de San Luis Potosí

Laboratorio de Genómica Viral y Humana

CA García-Sepúlveda MD PhDDM Hernández-Piña LTS

DL Alvarado-Hernández MSc QFBD Hernández-Ramírez MSc QFBSE Guerra-Palomares MSc QFBHI Contreras-Treviño BSc QFB

PG Hernández-Sánchez BSc IBQJL Ramírez García-Luna MD

https://www.genomica.uaslp.mx

Laboratorio de Virología, Depto. de Microbiología

DE Noyola-Cherpitel MD MScAE Hernández QFB

S Aranda-Romo Mest MScEE Godoy-Lozano MSc QFB

A Comas-García MD

1 CA García-Sepúlveda MD PhD Clinical Applications of Molecular Diagnosis Viral & Human Genomics Laboratory Facultad de Medicina, Universidad Autónoma.

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