Molecular Microbiology Tests Based On PCR Offered At ABSN Kolkota Molecular Microbiology Laboratory

It is necessary to institute a specific therapy at the earliest in infectious diseases. An early specific method of diagnosis is essential for this purpose.PCR is a more sensitive and rapid method and is also specific for the infectious agent being tested for. The results help physicians and surgeons to institute the specific therapy required for the treatment of the disease at the earliest possible time.

A negative result is likely to help the physicians to exclude a specific infectious agent as the cases of the disease process.

PCRs performed at Molecular Microbiology laboratory for detection of genomic DNA of different infectious agents in clinical specimens by qualitative and quantitative (real time PCR)

Mycobacterium tuberculosis (M. tuberculosis)

Mycobacterium tuberculosis is an obligate, acid – fast, Gram – positive bacilli infecting human beings. Its growth is slow and needs 3-4 weeks for a visible growth. The organism causes “human type” of tuberculosis in man. Primary infection is usually accompanied by mild or no symptoms, but this infection might get complicated due to the spread of organism to kidney, spleen, bone marrow, central nervous system and other organs including bone. The standard conventional methods of diagnosis are time consuming, (4-6 weeks) and are not always successful, especially in cases of extra pulmonary tuberculosis. By application of the nested PCR, M. tuberculosis genome can be detected within a day which aids in initiating the appropriate therapy immediately.

PCR for the detection of Eubacterial genome and Panfungal genome can be performed with clinical specimens particularly aspirates collected from sterile sites.This would enable one to broadly differentiate the infection as bacterial or fungal. The method is rapid, highly sensitive compared to conventional microbiological methods.

Propionibacterium acnes is among the most common causes of postoperative endophthalmitis. Cultivation of this bacterium is difficult as it is an anaerobic bacterium several days for its growth. PCR on vitreous / aqueous aspirates is a rapid, highly sensitive and specific method of detection of this bacterium.

Real time PCR:

Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility, and the reduced risk of carry-over contamination as it does not require any post PCR handling. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude) and is extremely accurate and less labor-intensive than current quantitative PCR methods.

The assay significantly has higher reliability of the results compared with conventional PCR, because with real-time PCR, the whole amplification profile is known. Individual reactions deviating in their amplification efficiency (e.g. owing to the presence of polymerase inhibitors) can be identified easily. Quantitative real time PCR is more precise than end-point determinations. The assay is commonly recommended wherever the exact number of the target is required and also to monitor prognosis in patients on therapy.

Real-time PCR has gained wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility, and the reduced risk of carry-over contamination as it does not require any post PCR handling. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude) and is extremely accurate and less labor-intensive than current quantitative PCR methods.

The assay significantly has higher reliability of the results compared with conventional PCR, because with real-time PCR, the whole amplification profile is known. Individual reactions deviating in their amplification efficiency (e.g. owing to the presence of polymerase inhibitors) can be identified easily. Quantitative real time PCR is more precise than end-point determinations. The assay is commonly recommended wherever the exact number of the target is required and also to monitor prognosis in patients on therapy.

Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that can lead to acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system begins to fail, leading to life-threatening opportunistic infections. Infection with HIV occurs by the transfer of blood, semen, vaginal fluid or breast milk. Within these body fluids, HIV is present as both free virus particles and virus within infected immune cells. The four major routes of transmission are unprotected sexual intercourse, contaminated needles, breast milk, and transmission from an infected mother to her baby at birth (Vertical transmission). The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection

Clinical Specimen: Body fluids, Peripheral Blood

Chikungunya is a RNA virus belongs to the family Togaviridae, genus Alphavirus. The disease Chikungunya also known as Chikungunya virus disease or Chikungunya fever is characterized by severe, sometimes persistent, joint pain (arthritis), as well as fever and rash. The disease is spread by the bite of infected mosquitoes and the clinical symptom resembles that of dengue fever. It is rarely life-threatening. In India the disease is more prevalent in Andhra Pradesh, Karnataka, Maharasthra, Tamil Nadu, Madhya Pradesh, Gujarat, Kerala, A & N Island, GNCT of Delhi, Rajasthan, Pondicherry, Goa. The virus isolation technique is cumbersome, and serological diagnosis is so often not reliable. The best method of detection of the virus is real time PCR.

Clinical Specimen: Peripheral Blood

Cytomegalovirus is a DNA beta-herpes virus and is ubiquitous in nature. Infection caused by the virus is usually silent in immunocompetent individuals, although acute CMV infection may also cause a brief mononucleosis-like malaise in immunocompetent adults. It is proven to establish latency in macrophages. Following infection, the virus resides in endothelial cells, macrophages, or granulocyte stem cells and may cause re infection if the host is rendered immunosuppressed, as by HIV or by immunosuppressive agents used during transplantation and chemotherapy. From infected pregnant women the virus gets transmitted to fetuses causing congenital defects, as they have poorly developed immune systems. As normal population is known to harbor baseline copy number of virus, estimation of quantity of virus is very important to distinguish normal subjects from diseased especially in blood samples. Real time PCR is the best method to quantify the virus copy numbers.

Clinical Specimens: Peripheral Blood, Body fluids

Hepatitis B virus is an hepadnavirus — hepa from hepatotrophic and dna because it is a DNA virus. The virus has a circular genome composed of partially double-stranded DNA. The viruses replicate through an RNA intermediate form by reverse transcription.Hepatitis B causes hepatitis an infection and inflammation of the liver. The disease may lead to carcinoma of liver if not intervened.Although serological markers are available for the detection of the viral antigens, DNA amplification techniques are preferred due to the marked sensitivity of the assay. Quantification by real time PCR is advised for monitoring viral load for the prognosis.

Clinical Specimens: Peripheral Blood, Body fluids

The Hepatitis C virus (HCV) is a small (50 nm in size), enveloped, single-stranded, positive sense RNA virus. HCV causes Hepatitis C, an infectious disease of the liver, The infection is often asymptomatic, but once established, chronic infection can progress to fibrosis, and cirrhosis . HCV is spread by blood-to-blood contact. Most people have few symptoms after the initial infection, yet the virus persists in the liver in about 80% of those infected. Persistent infection can be treated with medication, such as interferon and ribavirin, but only a minority is cured. The disease can be diagnosed by serological markers. The HCV viral load is an important factor in determining the probability of response to interferon-based therapy which is done by real time PCR.

Clinical Specimens: Peripheral Blood, Body fluids

Mycobacterium tuberculosis is an obligate, acid fast, Gram positive bacilli infecting human beings. Its growth is slow and needs 3-4 weeks for a visible growth. Primary infection is usually accompanied by mild or no symptoms, but this infection might get complicated due to the spread of organism to kidney, spleen, bone marrow, central nervous system and other organs including bone. The standard conventional methods of diagnosis are time consuming, (4-6weeks) and are not always successful, especially in cases of extra pulmonary tuberculosis. By application of the real time PCR, M. tuberculosis genome can be detected within a day with quantitation which aids in initiating the appropriate therapy immediately.

Clinical Specimens: Sputum, Bronchoalveolar Lavage, Biopsy, Sterile Body fluids, etc