Friday, October 28, 2011

Applications of Molecular Diagnostic Identification of Pathogens

Molecular identification should be considered in three scenarios, namely (a) for the identification of an organism already isolated in pure culture, (b) for the rapid identification of an organism in a diagnostic setting from clinical specimens or (c) for the identification of an organism from non-culturable specimens, e.g. culture negative endocarditis (Millar et al., 2003).

Molecular Diagnosis of Viral Infections
The diagnosis of viral infections has been hampered for many years due to the cost, laboratory time and skilled personnel required for the cell culture systems used, together with the generally low sensitivity and slow growth of many viruse in artificial media (Takeuchi, Y et al., 2001)
Serology is often unhelpful in the early stages of infection, specific antisera for the serology tests can be difficult to obtain, and the clinical detection of antibodies is relatively insensitive for a number of viruses.
Recent advances in molecular biology have made possible the detection and characterization of viral nucleic acids. Methods such as PCR enable the amplification of specific regions of interest
The implementation of molecular methods has resulted in progress regarding the diagnosis of many viruses and the monitoring of antiviral therapy, especially HIV-1 (Human Immunodeficiency Virus type 1), HBV (Hepatitis B Virus) and HCV (Human Cytomegalovirus). It has also led to the development of amplification assays on nearly all human viruses, including those that are more easily be cultivated, such as HSV-1 and HSV-2 (Herpes Simple Virus type 1 and 2) (Espy, M.J et al., 2000)
1- Herpes simplex virus (HSV)
Encephalitis is a serious infection but diagnosis previously required brain biopsy in certain cases due to the low sensitivity of cerebrospinal fluid (CSF) culture and serology (Gilbert. GL, et al., 1999) PCR now allows the detection of HSV DNA from CSF with 95% sensitivity (Lakeman. FD et al., 1995) thus avoiding invasive brain biopsy.
Viral meningitis, commonly caused by either enteroviruses or HSV, is more reliably detected by PCR when compared to culture (van Vliet.KE et al., 1998) and in a shorter time (one versus up to five days). HSV PCR can be multiplexed with other pathogens responsible for meningitis (Read.SJ et al., 1997)
Genital ulceration due to HSV, usually due to HSV type 2 infection, is now routinely detected by PCR in many clinical microbiology laboratories due to its increased sensitivity over viral culture.
2-Blood borne virus
The detection of blood borne virus infection is also improved by both PCR and non-PCR molecular methods.
1- HCV
Active hepatitis C virus (HCV) infections are diagnosed by the presence of HCV RNA since the detection of antibody to HCV cannot distinguish between past and present infection.
In terms of infectiousness only those with detectable HCV RNA have a significant risk of transmitting HCV by transfusion, organ transplantation, needle-stick injury or vertically to the child (Dore.GJ et al., 1997)
2- HIV
Although infection with the human immunodeficiency virus (HIV) is routinely diagnosed by serology, early HIV infection can be detected by HIV pro-viral DNA detection before HIV antibodies are confirmed by Western Blot serology (Dax EM.2004) Vertical transmission of HIV infection is also detected in the infant using HIV pro-viral DNA detection (Luzuriaga. K, Sullivan.JL, 1994)
3-Intrauterine viral infection of the foetus
Intrauterine infection of the foetus with viruses e.g:
- cytomegalovirus (CMV) (Palasanthiran P, Jones C, Garland S, 2002)
- rubella (Nourse C. Rubella, 2002) and
- varicella zoster virus (Heuchan A, Isaacs D, 2002) can be detected by PCR testing of amniocentesis fluid.
4- Respiratory viral infection
Molecular detection of respiratory viral pathogens from both upper respiratory specimens such as nasopharyngeal aspirates or throat swabs and lower respiratory specimens such as sputum or bronchoalveolar lavage fluid is cost-effective due to the prevention of hospitalisation, decreasing unnecessary testing and procedures, directing specific therapy, and reducing unnecessary antibiotic use (Henrickson KJ, 2005).
Large multiplex or tandem PCR assays testing for all the common respiratory viruses along with fastidious bacterial causes of pneumonia are now feasible providing a thorough yet cost-effective alternative to conventional detection methods.
Uncommon yet significant respiratory viruses such as severe acute respiratory syndrome (SARS) -coronavirus (SARS-CoV) and -influenza A/H5N1 (avian influenza) virus can also be incorporated into these assays thus acting as an in-built early detection system.
During the SARS epidemic due to the SARS-CoV, PCR testing of respiratory specimens for other respiratory viruses was crucial to exclude a number of suspected cases which fulfilled the case definition for SARS. PCR detection was most helpful due to the ability to rapidly screen for many respiratory viruses.
Subsequently a specific SARS CoV PCR has been developed for the early detection of SARS-CoV infection with a sensitivity of 50-87% early in the disease (Ng EK et al., 2003) Serology for SARS-CoV is up to 100% sensitive but of limited diagnostic value early in the disease when the risk of transmission is greatest (Ho PLet al., 2005)
The recent avian influenza (H5N1) outbreaks in South East Asia and beyond have also illustrated the need for rapid viral diagnosis. Molecular detection methods were developed following the 1997 Hong Kong outbreak (Yuen KY et al., 1998) and have the advantage of being rapid and able to be performed in many clinical microbiology laboratories. Specific serology needs live virus for the microneutralisation assay which is currently classed as a Biosafety Level 4 organism in Australia. Likewise direct immunofluorescence detection requires influenza type A/H5-specific monoclonal antibodies (. World Health Organisation 2005)
With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant.
Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009 .(Lee HK et al.,2010)
A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements.( Wu LT.,2010)
5- Viral diarrhoeal disease
Viruses cause more infectious diarrhoea worldwide than bacteria and other pathogens. The diagnosis of viral diarrhoeal disease has improved with the development of PCR detection. The method of choice for microbiological diagnosis of rotavirus from stool samples is PCR. Norovirus, a calicivirus formerly known as Norwalk virus and responsible for large outbreaks both in the community and health care facilities, can be diagnosed by electron microscopy, enzyme immunoassay and PCR but PCR is the most sensitive and rapid method. PCR is also the most sensitive method for the diagnosis of astroviruses and enteric adenoviruses (serotypes 40 and 41) (Clark B, McKendrick M.2004)

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