Serology

HIV Antibody Rapid Test:

HIV1/2 antibody test is done using NACO, New Delhi testing algorithms and the test result is given to the client within 3 hours along with pre-test and post-test counseling by the trained counselors. The HIV antibodies are generally detectable within 21 days after HIV infection in humans with the minimum window period of 3 months. The efficient HIV testing algorithm for the diagnosis of HIV infection is determined based on the sensitivity and specificity of the screening assays and the established algorithm is commonly used. Although HIV Western blot is one of the confirmatory tests, but this is not commonly practiced in developing countries.

HIV-1 Western Blot:

An antibody test with very high specificity is required as a supplemental/confirmatory assay. This assay shows the interpreter precisely, which HIV antigens have elicited an antibody response in the patient.

Diagnosis of Other Infections:

• Diagnosis of Sexually Transmitted Infections (STIs) such as Herpes Simplex Virus-2 viral antibody by ELISA, Syphilis by RPR and TPPA/TPHA, Chlamydial and gonococcal infections by Multiplex PCR.
• Diagnosis of Hepatitis Viruses by ELISA (Anti-HCV and HBsAg).
• Diagnosis of Opportunistic Infections (OIs) such as antibodies to Toxoplasma gondii (differentiation of IgG and IgM is also done); detection of Cryptococcus neoformans antigen in serum or CSF is done by rapid card test.
• Typhoid diagnosis done by WIDAL testing – for Salmonella typhi and Salmonella paratyphi infections.
• For neuro-syphilis RPR is done in CSF specimen.

Detuned Assay for Recent HIV Infection:

Detuned ELISA (less sensitive assay) for HIV-1 incidence estimation is widely applied as one of the epidemiological research tools. This assay has been developed by researcher, Dr.Bharat Parekh, PhD in Centre for Disease Control and Prevention (CDC), Atlanta US.

Non-NAT Plasma Viral Load Assay:

We have also successfully standardized a low-cost HIV viral load assay, which quantifies reverse trancriptase activity of HIV using an ExaVir load assay kit. Currently, this assay is being evaluated for the use in resource-limited countries in collaboration with Prof. Suzanne Crowe, Burnet Institute, Melbourne, Australia.

The laboratory is equipped with 2 ELISA Readers, 2 ELISA Washers, Rotator, Rocker, Centrifuges, Biological Incubator, and Electronic Pipettes.

Microbiology

Division of Microbiology:

Early accurate diagnosis of opportunistic infections (OIs), the common presenting symptom of the patients, is the key for success of effective management and preventive measures. The Microbiology Division specializes in diagnosing opportunistic infections in HIV infected patients caused by bacteria, fungi and parasites. It is well equipped with instruments such as BD-MIGIT for rapid culture and identification of Mycobacterium tuberculosis and anti-TB drug sensitivity testing, The facility has Light/Fluorescent Microscope (Leica) attached to a digital camera, Class II BioSafety Cabinet, BOD Incubator, Biosafety centrifuges and GeneXpert PCR system for the diagnosis of TB infection.

Diagnostic Services offered:

• TB Diagnosis by AFB staining method, AFB Culture & Anti-TB drug susceptibility test by rapid, BD-MIGIT.
• Identification of bacterial infections by conventional culture based methods and antibiotic susceptibility testing for the recovered bacteria.
• Fungal identification by special staining methods and culture.
• Parasite identification by special staining and microscopic examination.

In order to give the utmost confidence in our capabilities and results, we participate in an national and international EQAS programs such as College of American Pathologists (CAP),USA; UK-NEQAS, UK, One World Accuracy (OWA) and, USA; Duke University, USA, and have an excellent track record of top scores in these programmes.

Virology & Molecular Biology

Virology & Molecular Biology Laboratory:

The lab is equipped with automated high-end instruments for molecular diagnosis of STIs and HIV. Quality of the testing is assured throughout the testing process. The current test menu includes HIV-1 RNA and DNA PCR and CT/NG DNA Multiplex PCR.

HIV-1 RNA PCR – Quantitative (Viral Load Testing):

Accurate monitoring of viral load levels, in conjunction with clinical presentation and other laboratory markers may be used to assess prognosis, monitor anti-retroviral therapy and clinical management of HIV-1 infected patients.
This assay quantitatively measures the amount of HIV-1 RNA in the peripheral blood (viral load). The ABBOTT m2000rt RealTime PCR is a FDA (USA) approved methodology and it is an in vitro nucleic acid amplification test for the quantification of HIV-1 RNA in human plasma

The test facilitates the quantification of HIV-1 RNA over the range of 40/150 -10 million copies/mL.
Required specimen is anticoagulated plasma in EDTA tube. Heparin separated plasma sample should not be used. Turn around time (TAT) for this assay is 5-7 days.

ABBOTT Multiplex PCR for Gonococcus and Chlamydia:

The ABBOTT Chlamydia trachomatis / Neisseria gonorrhoae (CT/NG) test is a multiplex qualitative in vitro test for the detection of its DNA in the clinical specimens (urine / vaginal swab). The test is based on Real Time PCR using m2000rt system.

HIV-1 Drug Resistance Assay:

Drug resistance has been identified as a major factor contributing to therapy failure. The genetic basis of drug resistance is the high mutation rate and very high replication rate of HIV. Persistent viral replication in the presence of sub-inhibitory drug levels leads to the evolution of drug resistant variants and consequently to therapy failure. In order to find a new potent drug combination after therapy failure, current treatment guidelines recommend resistance testing. Different methods for resistance testing exist, which are either based on scanning the viral genome for resistance-associated mutations (genotypic assays) or on measuring viral activity in cell culture assays in the presence or absence of drug (phenotypic assays).
YRG CARE Laboratory performs Genotyping test which is used more commonly in clinical settings because of their wider availability, lower-cost, and short turnaround time. Required specimen is EDTA anticoagulated plasma. The test is done using validated and quality assured, in-house methodology with PCR amplification ( using ABI PCR 9700 system) and sequencing (using ABI 3500 Genetic Analyser) of the pol gene of the virus. Patient must have a viral load of more than 3000 copies/mL for genotyping to be performed. The report is provided within 10 days.

Hematology & Immunology

Hematology and Immunology:

A primary target of HIV is CD4 T-cells, which are preferentially depleted during the course of the disease. The utility of CD4 T-cell measurements involves clinical considerations for HIV disease classification and AIDS definition, assessment of prognosis, and the design of clinical trials. It is well recognised now that accurate and reliable enumeration of CD4 T-cell counts is very crucial for monitoring the rate of progression to AIDS, both for initiating prophylaxis for opportunistic infections as well as monitoring the impact of antiretroviral therapy (ART). The lab is equipped with different range of flow cytometries (FACSCount, Guava PCA, Beckman Coulter EPICS XL/MCL and Beckman Coulter FC400 for CD4 count/CD4%), Hematology analyzers (Sysmex KX-21 and Sysmex XT-1800i) and automated ESR Reader.

Other Diagnostic Services Offered:

1. CBC – Cell Counts (HGB, RBC, WBC, Platelet).
2. ESR – Erythrocyte Sedimentation Rate.
3. Urine Analysis, Stool Analysis and Body fluids Analysis.
4. Pregnancy Test.

Quality Control:

Daily internal QC is performed to monitor the instrument and reagent performance and daily precision is monitored for the instrument reproducibility.

Biochemistry

Division of Biochemistry

In the clinical management of HIV infection, highly active antiretroviral therapy (HAART) plays a major role. The success of the treatment achieved mainly through HAART is tempered significantly by drug toxicities. These toxicities often occur in patients who have been exposed to multiple drugs for prolonged periods of time, thus the monitoring of long-term toxicities is necessary for the better management of HIV infected individuals.

The main reason for monitoring toxicity is to ensure the safety for individuals who are on treatment and identify those who need to stop treatments that are harmful to their health and change to other treatments. Biochemistry is a clinical laboratory service that undertakes biochemical analysis to
provide data, which is used for the diagnosis and monitoring of HIV disease.
Laboratory analysis of blood (serum and plasma) and body fluids is performed using advanced scientific instrumentation, such as Beckman Coulter AU 400/480 Autoanalyzers and Roche AVL 9180 Electrolyte analyzer.

Quality Control:

In-house Internal Quality Controls are performed for every run and these results are plotted on Levy-Jennings charts and interpreted according to a Westgard Multi-rule algorithm. We are participating in EQAS with the College of American Pathologists (CAP), USA.

Liver function tests:

Liver toxicity is a growing problem among HIV patients, particularly those who are on HAART and among co-infected with hepatitis C or hepatitis B. Clinicians need to monitor potential symptoms of liver disease and/or drug-related effects in the patients.

Serum albumin tests look for a protein that is made by the liver and released into the blood and keep circulating in the blood rather than being drawn into the tissues. Reduced levels of albumin suggest that the liver is not functioning properly.

Low serum albumin is also a marker for poor nutritional status, and in some populations it may predict progression in HIV disease. This may be a reason for using the test to identify people at higher risk who should have priority access to treatment and other medical and social support.

Bilirubin is a protein released into the blood by the lyses of red blood cells. Bilirubin is the cause of jaundice, when a person’s eyeballs and skin go yellow and their urine is darkened. Jaundice happens when the liver is no longer doing its job of removing the bilirubin from the blood. Increased levels of bilirubin can be detected before jaundice is obvious, suggesting that liver damage is occurring.

Metabolic Complications:

Cholesterol levels in the blood are raised with long-term treatment with a number of protease inhibitors currently in uses and possibly with other drugs. Whether this will translate into a substantial risk of heart disease probably depends on the extent to which other risk factors (such as cigarette smoking, family history, etc) are present.

Lactic acidosis is a rare and very serious complication linked to nucleoside analogues in general although it may be more common with AZT (Zidovudine), d4T (Stavudine) and 3TC (Lamivudine). It is caused by damage to mitochondria – the systems inside cells that generate energy by combining sugars and oxygen. This makes the body switch to alternative energy systems that cause lactic acid to build up in the blood.

Renal function tests:

Two tests commonly performed to monitor the functioning of kidneys are Creatinine levels and BUN (Blood Urea Nitrogen) tests. If elevated, they mean that the kidneys may be damaged.

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