Serology

First and second line ELISA assays for the detection of antibodies to HIV-1/-2 are conducted routinely on dried blood spots (DBS) or plasma for a variety of studies. HIV antibody detection in breast milk has  been optimized and performed. The Calypte® HIV-1 BED Incidence EIA has been successfully implemented in 2009 for use on plasma and DBS for determination of population estimates of HIV-1 incidence, and we have performed approximately 2100 tests during this period. In addition to routine GCLP compliant internal quality assurance practices, the laboratory subscribes to one external QC programme namely the National Health Laboratory Services Quality Assessment Programme (a panel of six samples, tested by both first and second line ELISA, three times per year).

Molecular - Viral Load Quantification

Historically, the Nuclisens Easy-Q (Biomérieux) HIV-1 viral load quantification (NASBA) platform has been used in our laboratory for quantification of HIV-1 RNA on plasma and DBS. The semi-automated silica-based nucleic acid extraction method used in this system (MiniMag), enables isolation of nucleic acid that may be used on alternative platforms, such as real-time PCR assays and sequencing reactions. We successfully established a realtime PCR platform (Bio-Rad MiniOpticon) and have performed HIV-1 RNA quantification on DBS, plasma, and breast milk using various extraction methods including MiniMag, QiaGen and Abbott ASPS assays. Thus we have at our disposal two unique technologies for nucleic acid quantification on a variety of specimen types. Qualitative and quantitative detection of viral DNA from DBS, whole blood and breast milk has been optimized and implemented in conjunction with a human reference gene normalization assay.  In-house methods for quantification of six other viruses associated with breast milk transmission have been made available and are under study through collaborators from University of Montpellier. Equipment to perform automated electrophoresis for determination of quantity, purity and integrity of extracted DNA, RNA and protein, is also available (Bio-Rad Experion) and has been made available for use by other laboratories at the DDMRI. The laboratory currently subscribes to two independent HIV-1 RNA viral load quantification external quality assurance panels, namely QCMD HIV RNA proficiency programme (a panel of ten samples, tested twice yearly), and the NEQAS panel (a panel of two samples, tested 3 times per year). For DNA quantification we subscribe to two panels per year of the QCMD HIV DNA proficiency programme.

Molecular – Genotypic Analysis

A 3130 XL Genetic Analyzer is available in the laboratory, one of two such instruments currently available at the UKZN Medical School laboratories. Determination of circulating subtypes as well as resistance genotyping has been performed on plasma using the FDA approved Viroseq method. In addition we are currently optimizing resistance genotyping using the  Viroseq and an in-house method on DBS. HIV-1 phylogeny was also performed on plasma using an established in-house method. The sequencing facility not only provides a service to the Africa Centre but also to other laboratories including Microbiology, HIV Pathogenesis Programme (HPP) and the Hasso Plattner Research Laboratory (HPRL) on a range of genomes such as HIV, Mycobacterium and the human host.  Output of sequences from these and Africa Centre studies using the ABI 3130xl averaged approximately  700-800 sequences per month in 2009. A fully functional cloning facility housed at the  HPRL will be used for studies investigating viral diversity, evolution and origin of the virus in MTCT. In addition, viral diversity has also been evaluated and confirmed using single genome amplification (SGA) in the laboratory. The laboratory now has two comprehensive bioinformatics workstations for advanced bioinformatics and sequence analysis. 

Bio-safety Level 2+ Unit

The Africa Centre Virology Laboratory has its own Bio-Safety Unit where culture of HIV in lymphocyte cell lines and related immunological assays have been initiated in a collaborative effort between three research groups, the others being HPP and HPRL. The unit functions at bio-safety level 2-plus, that requires scientific and other staff to adhere to stringent methodology to ensure their safety. At present it is the only such facility at the DDMRI and Nelson R Mandela School of Medicine.

Staff

Scientific staff members have completed a training course in Good Clinical Laboratory Practice.   Laboratory technologists are proficient in serology and real time PCR assays from a variety of sample materials including DBS, whole blood, plasma and breast milk. One staff member has received training in HIV and TB immunopathology/immunohistology through the Fogarthy International Training Scholarship programme and will transfer this technology to the Africa Centre laboratory.

Collaborations

The Africa Centre laboratory is currently involved in collaboratoive research with the following: Prof T.Ndung’u and Dr W.Carr (HPP) – innate immune mechanisms in limiting HIV-1 clade C trasmission among South African mother-infant pairs- the role of natural killer cells in HIV-1 pathogenesis (using stored DBS samples); Dr G.Kaplan (PHRI, NY, USA) – immune cells involved in the anti-TB response at the site of infection in spinal TB/HIV-1 co-infection; Prof S.Govender (Dept. Orthopaedic Surgery, UKZN) – CD4 and CD8 t-cell polyfunctionality in spinal TB and HIV co-infection.

Table of laboratory workload from 2007-2009

	2007	2008	2009	Total
DSS HIV ELISA	12,433	12,226	11,666	36,325
MDP HIV ELISA	320	236	54	610
OTHER HIV ELISA	141	37	445	623
ACASV HIV ELISA	-	-	2,576	2,576
ACASV HIV cBED	-	-	2,112	2,112
ACASV PLA HIV RNA	-	-	429	429
ACASV DBS HIV DNA	-	-	710	710
VTS BM HIV RNA	-	2,753	-	2,753
VTS BM HIV DNA	-	-	1,280	1,280
KB HIV RNA (DBS & PLA)	11	1,654	870	2,535
SEQUENCING REACTIONS	2,066	4,752	11,671	18,489

DSS – Demographic Surveillance System;
MDP – Microbicide Development Project;
ACASV – Africa Centre Antenatal Seroconverter Vertical Transmission Study;
VTS – Vertical Transmission Study;
BM – Breast milk;
DBS – Dried Blood Spot; 
cBED – HIV-1 BED Incidence Enzyme Immunoassay;
KB – Kesho Bora Study