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Overview

HIV/AIDS laboratory in the Department of Clinical Research has been in the forefront providing high quality diagnostic and research support for various HIV and HIV/TB clinical trials and research projects conducted by the National Institute for Research in Tuberculosis (NIRT). The laboratory has been accredited by International Agencies like the NIH and WHO, and has been participating successfully for many years in External Quality Assurance programs such as NIH-VQA and CDC-GAP for HIV-1 DNA PCR, NIH-VQA and RCPA-QAP for HIV-1 viral load assay, NIH-VQA and WHO for HIV-1 drug resistance genotyping, QASI (NARI) for CD4, and HIV Serology EQAS with NARI for diagnostic ELISA. The focus of research in the laboratory include immunopathogenesis of HIV/TB co-infection, molecular characterization of subtype C strains circulating in the local population, and identification of novel compounds with anti-HIV activity.The workload of the department is managed by a professional team of 2 scientists, 5 technical officers, 5 technical assistants, 2 technicians and 2 support staff who have permanent tenure and 1 technical officer, 1 technical assistant, and a senior research fellow who work for the institute in fixed tenure. At present, four research scholars are pursuing research for Ph.D. degree in the HIV/AIDS Laboratory. The laboratory has also been imparting project training and internships to selected eligible undergraduate and postgraduate students from different universities.

The laboratory has various dedicated sections that function to carry out Routine (Diagnostic) and Research activities that include

•  Serology

•  Flow Cytometry facility

•  Viral Load Laboratory

•  Cellular immunology Lab

•  Virology Section

•  Molecular Biology Laboratory

•  Drug resistance testing Laboratory

•  Central Instrumentation and Cold Room facility

 

Serology Lab

This is the central specimen processing unit of the lab where samples are received and processed in a methodical way. The lab undertakes diagnostic testing for HIV-1 and 2 infections, and Hepatitis B and C infections by means of serological tests that utilize rapid immunoassay methods. The lab is equipped with a Beckman Coulter Act5 Diff CP analyzer that fully automates the Complete Blood Count (CBC) testing. A Standardized Manual Differential Staining and Counting methodology is also in place for verifying doubtful results and samples yielding abnormal DC values. The section also undertakes sample preparation for CD4/CD8 cell count estimation by flow cytometry and sample storage for further analyses and future studies.

 

Flow Cytometry Facility

The laboratory is equipped with two state-of-the-art flow cytometers: Beckman Coulter's Epics XL and Beckton Dickinson's FACS Count System, which are used extensively to estimate absolute counts and percentages of CD4 T lymphocytes to guide treatment decisions for HIV-infected patients, to stage progression of HIV/AIDS, and to assess patient prognosis. The facility is also utilized for various research applications and offers an unlimited breadth of application possibilities.

 

 

Viral Load Lab

Since inception, this section of the laboratory has been providing services of molecular diagnosis of HIV-1 infection and quantification of circulating virus for monitoring response to antiretroviral treatment in HIV-1-infected individuals enrolled in various clinical trials and studies carried out at NIRT. Besides, it has been serving as a centralized testing laboratory for molecular diagnosis of HIV infection in infants known to be exposed to HIV infection, and viral load estimation in HIV-infected individuals on ART to determine eligibility for second line ART, since 2009. The lab is equipped with a COBAS (Comprehensive BioAnalytical System) Amplicor HIV-1 Monitor assay system that requires manual RNA extraction and uses the COBAS Amplicor analyzer for RT-PCR amplification, and detection via magnetic particles coated with oligonucleotides specific for the target. The lab has recently implemented the advanced method of “real time PCR” in full time, with a fully automated process of RNA extraction, PCR amplification and detection. Two new high-end FDA approved technologies “COBAS AMPLIPREP/COBAS TAQMAN HIV test” developed by Roche diagnostics and “M2000sp/ M2000rt” developed by Abbott Molecular Technologies have been acquired to increase the sensitivity, throughput of the laboratory and improvement in turn-around-time (TAT).

 

Cellular Immunology Lab

The Cellular immunology laboratory is equipped with a Class II Biosafety cabinet, water bath, two CO2 incubators, a refrigerated centrifuge, and a fluorescent and a light microscope. A dedicated thermal cycler Gene Amp 9700 (Applied Biosystems) adds significance to this facility. The facility has been extensively used to carry out cellular assays, multiplex ELISAs, sample preparation for advanced flow cytometric analyses, ELISPOT assay and other research applications. Besides, this facility is also used for maintenance of cell lines.

 

Virology Section: BSL2+ CLASS 10000

The BSL2+ Class Virology Section is a negative pressure room with an ante-room at differential pressure. The lab is equipped with a Class II Biosafety cabinet fitted with a virus burn out, CO2 incubator, fluorescent microscope, refrigerated multipurpose centrifuge, water bath and other amenities required to conduct almost all types of cell culture work with international standards. The facility houses a dedicated freezer and refrigerator to stock media, media supplements, serum, antibiotics and other items required for the work. A shared facility for cryopreservation is also available for storing cells and cell lines. Currently the facility is utilized for research applications such as viral culture, isolation, evaluation of anti-viral activity of new compounds, cloning and characterization of HIV-1 subtype C isolates circulating in the local population, and identification of broadly neutralizing antibodies with newer specificities in HIV-1C infected individuals.

Molecular Biology Lab: BSL2 CLASS 10000

The molecular biology facility is well designed to ensure unidirectional workflow in order to prevent cross-contamination. There are four dedicated rooms – Pre-PCR room, Extraction room, Amplification Room and Post-PCR room connected by an ante-room. Differential air pressure is maintained in each room for contamination containment. The facility is equipped with a dead air box in the reagent preparation area, three dedicated Class II Biosafety cabinets in the extraction area, a dedicated laminar flow hood and a Bio Rad XR Gel documentation System in the Post PCR area. Other amenities like separate freezers and refrigerators for storing processed and unprocessed samples and amplified PCR products, dry incubators, dry baths, refrigerated centrifuges tailor-made for various purposes, electrophoresis units, two thermal cyclers (a Gene Amp 9700 and a PTC-100 from Applied Biosystems and MJ Research Inc.) and one Rotor Gene 6000 Real Time PCR system from Corbet Research are available for continuous throughput. The infrastructure along with the trained staff is capable of providing technical capabilities to carry out routine and specialized molecular biology techniques.

 

HIV-1 Drug Resistance Testing Facility

This facility undertakes HIV drug resistance testing by genotyping. Currently, the lab is equipped with a 4 capillary ABI Avant 3100 DNA Sequencer which will be replaced soon with a new 8 capillary sequencer. The lab was set up to support the various clinical trials untaken by NIRT. The lab was accredited by WHO in 2007 and identified as a National Reference Laboratories for HIV-1 drug resistance genotyping to support National and Global programs for monitoring HIV drug resistance in the South Indian population for NACO and WHO DR surveillance programs. The facility is also used extensively for genetic studies aimed at identifying genetic polymorphisms by direct sequencing.

 

Central Instrumentation Facility

The central instrumentation facility is equipped to provide solution to various types of workflow with an expansive portfolio of instrumentation. The facility enables to advance the research in a multitude of application areas, and houses a variety of equipments such as plate reader, automated ELISpot imaging system, shaker incubator and controlled rate freezing.

The HIV Lab also has a 100 sq ft walk-in cold room facility that is backed up by continuous power supply. There are six -20 0 C freezers, six -80 0 C freezers, one -150°C freezer and a Liquid Nitrogen Sample Storage System.

 

Ongoing Research Activities

•  Predictors and immunologic characterization of tuberculosis-associated Immune Reconstitution Inflammatory Syndrome in HIV-TB patients started on antiretroviral therapy

•  Molecular characterization of full-length envelope gene of HIV-1 subtype C clinical isolates

•  Identification of broadly neutralizing antibodies with novel specificities in HIV- 1 subtype C infected individuals

•  High density lipoprotein cholesterol and gene polymorphisms among HIV infected South Indians on first line antiretroviral therapy

•  Identification of viral and host determinants responsible for the clinical differences seen in HIV-1 and HIV-2 infections

•  Molecular cloning and characterization of full-length HIV-1 subtype C and HIV-2 isolates

 

Service to National Programs

The lab has been functioning as a Regional Reference Laboratory for National AIDS Control Organization (NACO) for two National Programs viz. ‘Early Infant Diagnosis Program' and ‘Second Line ART Program'. The HIV/AIDS laboratory is also a National Reference Laboratory for WHO for HIV-1 Drug Resistance Genotyping.

 

   

 

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