Abstract:
The Env gene of Human Immunodeficiency Virus (HIV) is vulnerable to mutations
that could contribute to variability of diagnostic sensitivity of antibody-based HIV
diagnostic tests. Previous studies established that some HIV Enzyme-linked
Immunosorbent Assay (ELISA) kits displayed different diagnostic sensitivities when
they were used to test blood samples from various geographical locations. Currently,
the most widely used testing platforms for HIV in Kenya are the ELISA and Lateral
Flow tests (LFTs) which are coated with peptides that have been developed using
global consensus sequence of HIV Env gp41 Immunodominant Region (IDR) (and
sometimes gp120) that may theoretically not be able to detect some local HIV strains.
This study was designed to develop and evaluate LFT and ELISA test kits for HIV 1/2
based on the prevailing consensus sequence of HIV Env gene in Kenya.
The study was a Laboratory-Based Experimental Design that involved collection of
200 HIV positive and a similar number of HIV negative blood samples from the four
Regional Blood Transfusion Centers (RBTCs) in Kenya over a period of eighteen
months. From the HIV positive samples, ribonucleic acid (RNA) was extracted, reverse
transcribed and then amplified. The resultant cDNA was sent to Macrogen Europe
(Amsterdam, Netherlands) for sequencing. The Consensus sequence of HIV Env gp41
IDR gene established in this study and the global consensus of HIV Env gp41 IDR
were sent to LifeTein LLC (Hillsborough, NJ) for production of corresponding bulk
peptides. These peptides were used with other commercially available reagents,
through a series of optimization experiments, to produce ELISA and LFTs which were
evaluated using the 400 characterized HIV panels prepared in this study. The study was
approved by KEMRI Ethical Review Committee. Blood samples collected from the
NBTCs concealed the identities of the donors. The data was collected and entered in
the Excel software. Sequences were analyzed using various bioinformatics' tools that
included Shannon-Two Entropy, Los Alamos PhyML 3.0 phylogenetic tree software,
“RIP” software for ddetermination of HIV sub-types and recombinants and Phyre2
software for peptide structure prediction, among others.
This study established the prevailing consensus sequence of Env gp41 IDR gene in
Kenya and also developed two HIV testing kits (LFT and ELISA) using Consensus
HIV Env gp41 IDR peptide from this gene. There was 100% sequence similarity in the
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Cysteine Loop and CTL epitope of HIV Env gp41 IDR peptides between Kenya and
global consensus peptide sequences. In general, 67.4% of the amino acid positions in
Consensus HIV Env gp41 IDR peptide (Kenya) were highly conserved with the
Entropy value of below 0.25 (baseline Entropy). Of the 91 samples that were
sequenced the sub-type distribution of HIV was A1 (76.9%), C (6.6%), D (14.3%) and
CRF A2.CY.94CY017_41 (2.2%). Samples that were tested in this study consisted of
31.9% “recent” HIV infections. In respect to Vironostika™ Uni-Form II Ag/Ab ELISA
the developed LFT had a diagnostic sensitivity (D-SN) of 95.2% (95% CI: 90.3-98.0%)
which was the same as that of Aware™ HIV-1/2 BSP LFT and close to the two kits
that are currently in the National HIV testing Algorithm in Kenya: First Response™1-
2.0 (95.9%, 95% CI: 91.2-97.4%) and KHB Colloidal Gold (95.9%, 95% CI: 91.2-
98.5%). The developed LFT had a higher D-SN than the “Tie Breaker” kit in the
Algorithm, Uni-Gold™ HIV Test (93.8%, 95% CI: 88.5-97.1). The ELISA test kit
developed in this study had a D-SN of 97.2% (95% CI: 93.1-99.2%). The study also
established that the overall D-SN of using LFTs in HIV testing in Kenya was 96.0%
(95% CI: 92.3-98.3%) in respect to Vironostika™ Uni-Form II Ag/Ab ELISA and that
among the LFTs, Determine™ HIV-1/2 showed the highest values of both D-SN and
analytical sensitivity, 96.6% (95% CI: 92.2-98.9%) and 63pg/ml respectively, making
it the LFT that would have been the most preferred choice of a Screening test in the
National HIV testing Algorithm in Kenya were not for its being 10-16% more
expensive than the current kits in this Algorithm. This study also established that
although there were some variations of the HIV Env gp41 IDR peptide in Kenya in
comparison with global consensus of the same, there were no differences in the
performance of the kits developed using Consensus HIV Env gp41 r-IDR (Kenya) and
Consensus HIV Env gp41 r-IDR (Global) peptides. The study also established that the
analytical sensitivity of Determine™ HIV-1/2 Ag/Ab Combo and Vironostika™ UniForm II Ag/Ab ELISA in detection of p24 antigen were the same at 63pg/ml.
This study recommends the following: return of Determine™ HIV-1/2 to the National
HIV Testing Algorithm due to its superior performance, regular monitor of the
effectiveness of the HIV testing kits in Kenya for the possible emergence of HIV
variants that could escape detection by these kits and seeking of more funds to support
the commercialization of the prototype HIV kits developed in this study.