The human immunodeficiency virus type-2 (HIV-2) prevalence in South Africa (SA) is unknown, however, sporadic cases have been reported. Human immunodeficiency virus -1 and 2 differentiation is not part of most South African public laboratories’ testing algorithm. Human immunodeficiency virus -2 diagnosis using serology assays may be complicated by HIV-1 and HIV-2 antibody cross-reactivity.
To determine the proportion of HIV-2 infections in specimens that tested HIV-1/2 positive at a public laboratory in Tshwane.
A total of 480 specimens that were previously tested with fourth generation ELISA platforms (Modular E170 [Roche, Switzerland] and Architect i2000 [Abbott, Germany]) were randomly selected. Human immunodeficiency virus -1 and 2 antibody differentiation testing was carried out using the Multispot HIV-1/2 rapid assay (Bio-Rad Laboratories, USA). An in-house nested HIV-2 PCR assay targeting the 5′-long terminal repeats (5′-LTR) region was evaluated and used as a confirmatory test.
The study tested 480 HIV-1/2 seropositive patients and their mean age was 36.7 years (range 3–82 years). Of the 480 patients, 292 (60.8%) were female, 182 (37.9%) were male and 6 (1.3%) were not specified. Human immunodeficiency virus differentiation results were as follows: 466 (97.1%) were positive for only HIV-1 antibodies, 11 (2.3%) [95%CI: (0.98%; 3.74%)] were positive for both HIV-1 and HIV-2 antibodies, 3 (0.6%) were negative for both antibodies and none were positive for only HIV-2 antibodies. Of the 11 specimens with both HIV-1 and HIV-2 antibodies, seven had sufficient volume for confirmatory testing and were all negative on the in-house HIV-2 PCR assay.
The multispot HIV-1/2 rapid assay demonstrated cross-reactivity between HIV-1 and HIV-2 antibodies. Human immunodeficiency virus -2 infections were not detected.
Human immunodeficiency virus type 2 (HIV-2) belongs to the
South African data on HIV-2 infections is limited. Sporadic cases have been reported in the past, which is an indication that HIV-2 may be circulating in South Africa (SA).
Routine diagnostic serology assays that are widely used in SA national public sector health laboratories do not include HIV-1 and HIV-2 differentiation.
Dolutegravir is effective against HIV-2, but strains with raltegravir mutations may have low levels of resistance.
The virology diagnostic laboratory is currently using a fourth generation enzyme-linked immunosorbent assay (4th gen ELISA) for HIV-1/2 testing. The fourth generation ELISA can detect both HIV-1 and HIV-2 antibodies and p24 antigen, but it does not differentiate between the two types. The World Health Organization (WHO) HIV testing guidelines recommend HIV-2 differentiation with serological and/or molecular assays in settings where HIV-2 infections have been detected.
This study aimed to determine the proportion of HIV-2 infections in specimens that tested HIV-1/2 positive in a public laboratory in Tshwane.
Human immunodeficiency virus -1 and -2 differentiation using the Multispot HIV-1/2 rapid test.
Evaluation of the in-house HIV-2 nested polymerase chain reaction (PCR) assay.
Confirmatory testing using the in-house HIV-2 PCR assay.
This was a retrospective, cross-sectional study conducted between February 2013 and July 2016.
This study was conducted in the National Health Laboratory Services (NHLS), Tshwane Academic Division (TAD) Virology Laboratory at the University of Pretoria, Faculty of Health Sciences. The specimens used came from the public sector hospitals and clinics around Tshwane Metropolitan Municipality, submitted for routine HIV screening.
A total of 480 plasma and serum specimens that had previously tested positive for HIV-1/2 antibody, were randomly selected. The inclusion criteria were age of more than 24 months and HIV-1/2 antibody positive results on two different fourth generation ELISA platforms, the Modular E170 (Roche, Switzerland) and the Architect i2000 (Abbott, Germany).
The HIV-2 prevalence is unknown in SA. Based on the HIV-2 prevalence data from other parts of the world outside the West African region,
The Multispot HIV-1/2 rapid assay (Bio-Rad Laboratories, USA) was used for HIV-1 and HIV-2 differentiation.
The procedure was performed according to the manufacturer’s instructions. Four known HIV-2 antibody positive specimens, from a laboratory in Portugal, that were previously tested using only the Western Blot assay, and 52 HIV-1 and HIV-2 antibody negative specimens that were previously tested using HIV-1/2 fourth generation ELISA, Modular E170 (Roche, Switzerland) were included to evaluate performance of the Multispot HIV-1/2 rapid assay.
Two sets of primers, targeting the conserved 5′ long terminal repeat (5′-LTR) region of HIV-2, were designed and numbered according to the HIV-2 ROD reference strain, accession number M15390.1 (
Designed primers used for the in-house nested human immunodeficiency virus-2 polymerase chain reaction and sequencing.
Primers | Positions | Sequences | Length (number of bases) | Tm |
---|---|---|---|---|
MMFw-1 (outer) | 11–27 | 5՛-CGGAGAGGCTGGCAGAT-3՛ | 17 | 62 |
MMRv-1 (outer) | 566–583 | 5՛-AGTTTCTCGCGCCCATCT-3՛ | 18 | 59.9 |
MMFw-2 (inner) | 72–94 | 5՛-GTGTTCCCTGCTAGACTCTCACC-3՛ | 23 | 66.3 |
MMRv-2 (inner) | 392–409 | 5՛-GGAGCACTCCGTCGTGGT-3՛ | 18 | 64.5 |
Tm, melting temperature;
°C, degree celsius.
Except for the initial centrifugation step to get a purified pellet, the extraction of total nucleic acids was performed manually using the QIAamp UltraSens Virus kit (Qiagen, Germany) according to the manufacturer’s instructions.
Centrifugation was optimised at 800 relative centrifugal force (RCF) for 3 min instead of the recommended 1200 RCF. Optimisation to a lower RCF produced an easily dissolvable pellet. Sample input volume for extraction was 500 µL, and lower input volume of 250 µL was used for samples with insufficient volume. The final eluate volume was 60 µL.
A nested PCR was carried out using the Superscript III One-Step RT-PCR kit with platinum Taq deoxyribonucleic acid polymerase (5 U/µL) (Life technologies, USA) according to the manufacturer’s instructions. The total reaction volumes for both first and second round PCR were 50 µL. First round PCR contained 25 µL of 2x reaction mix (a buffer containing 0.4 mM of each dNTP, 2.4 mM MgSO4); 1 µL of each primer (outer forward and reverse) (10 µM); 1 µL of Taq enzyme (5 U/µL), 5 µL of template and 17 µL of nuclease-free H2O. The second round PCR reaction contained 25 µL of 2x reaction mix (a buffer containing 0.4 mM of each dNTP, 2.4 mM MgSO4); 1 µL of each primer (inner forward and reverse)(10 µM); 0.2 µL of Taq enzyme (5 U/µL), 0.5 µL of template and 22.3 µL of nuclease-free H2O.
The first round PCR conditions were as follows: cDNA synthesis for 1 cycle at 50 °C for 30 min; denaturation for 1 cycle at 94 °C for 2 min; amplification 40 cycles (denature at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 68 °C for 1 min); final extension for 1 cycle at 68 °C for 7 min and hold at 4 °C. The second round PCR conditions were as follows: denaturation for 1 cycle at 94 °C for 2 min; amplification 40 cycles (denature at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 68 °C for 30 s); final extension for 1 cycle at 68 °C for 7 min and hold at 4 °C. Polymerase chain reaction amplicons were visualised on a 1.5% agarose gel by ultraviolet (UV) illumination.
Polymerase chain reaction amplicons were sequenced to evaluate the in-house HIV-2 nested PCR, at Inqaba Biotechnical Industries (Pty) Ltd using the second round PCR primers (MMFw-2 and MMRv-2) (
This study was approved by the research ethics committee, Faculty Health Sciences, University of Pretoria (ethics reference number: 11/2013). Permission to use the blood specimens and to retrieve demographics information from the laboratory information system (LIS) was obtained from Tshwane Academic Division of the NHLS. The information of each specimen was managed with strict confidentiality and each specimen was given a unique study number to ensure patient anonymity.
The study tested 480 HIV-1 and HIV-2 seropositive patients, and their mean age was 36.7 years (range: 3–82 years). Of the 480, 292 (60.8%) were female, 182 (37.9%) were male and 6 (1.3%) were not specified. All four known HIV-2 antibody positive specimens used to evaluate the Multispot HIV-1/2 rapid assay were positive for HIV-2 antibodies only. All 52 known HIV-1 and HIV-2 ELISA negative specimens were negative. Differentiation results of the 480 specimens were as follows: 466 (97.1%) were positive for only HIV-1 antibodies, 11 (2.3%) 95%CI: 0.98%; 3.74% were positive for both HIV-1 and HIV-2 antibodies, 3 (0.6%) were negative for both antibodies and none were positive for only HIV-2 antibodies (
Algorithm of the specimens tested with Multispot HIV-1/2 rapid assay.
Evaluation results of the in-house nested HIV-2 PCR, on four known HIV-2 antibody positive specimens, were comparable to a commercial real-time PCR, which was carried out in a private laboratory (
HIV-2 polymerase chain reaction results of the four known HIV-2 antibody positive specimens.
Known HIV-2 specimens | In-house nested HIV-2 PCR | Genesig standard kit real time HIV-2 PCR (Private laboratory) | Internal Control for Real-Time PCR |
---|---|---|---|
83541 | Positive | Positive | Positive |
82176 | Negative | Negative | Positive |
77515 | Negative | Negative | Positive |
77538 | Negative | Negative | Positive |
HIV, human immunodeficiency virus; PCR, polymerase chain reaction.
Gel Electrophoresis with a 100 bp DNA ladder. Specimens 83541, 82176, 77515 and 77538 are known HIV-2 antibody positive. Specimen 83541 tested positive for HIV-2 PCR (fragment size = 338 bp). HIV-1 specimens (9820 and 3447) with high viral load were undetectable.
The PCR products of the confirmed HIV-2 subtype A/B specimen (83541) were sent for sequencing. Phylogenetic analysis revealed that this sequence clustered with subtype A of HIV-2 (
Phylogenetic tree showing the sequence of the known human immunodeficiency virus-2 sample that was positive on in-house polymerase chain reaction (labelled with a black dot). This sequence clustered with subtype A human immunodeficiency virus-2 isolates.
This study aimed to determine the proportion of HIV-2 infections in specimens that tested HIV-1/2 antibody positive at a virology diagnostic laboratory using the Multispot HIV-1/2 rapid assay for differentiation. The Multispot rapid test has been reported as satisfactory with regard to the detection of HIV-1 and HIV-2.
Differentiation results showed HIV-1 and HIV-2 dual seropositivity in 11 (2.3%) of the specimens. Of the 11, seven specimens with sufficient volume for the in-house HIV-2 PCR tested negative. This may indicate HIV-1 and HIV-2 antibody cross-reactivity. These results were not surprising, as HIV-1 and HIV-2 antibody cross-reactivity have been observed in other studies.
The first two reported HIV-2 cases in SA were documented in 1988
The in-house nested HIV-2 PCR had comparable results, on the four known HIV-2 antibody positive specimens, with the commercial assay (Genesig Standard kit) processed in a private laboratory (
The negative HIV-2 PCR on specimens with dual HIV seropositivity may not completely rule out dual HIV infections, as there are many factors that may contribute to false-negative HIV-2 PCR. Such factors include naturally low viral load in HIV-2 infections because of the low replication rate.
The limitations of this study include small sample size, quality and type of the specimens used as described here.
Low specimen volumes (250 µL – 500 µL) were used for extraction because of specimen insufficiency. Nucleic acid quantification and quality assessment were also not done. This may have led to false negative results. Polymerase chain reaction failure of the in-house PCR could not be excluded because of the lack of internal controls; however, the commercial HIV-2 PCR with comparable results and with internal controls, amplified. The developed in-house nested HIV-2 PCR needs further evaluation with more HIV-2 positive specimens, including other subtypes and addition of more HIV-1 only specimens. The commercial assay can only detect HIV-2 subtypes A and B.
The Multispot HIV-1/2 rapid assay demonstrated some cross-reactivity between HIV-1 and HIV-2 antibodies. Human immunodeficiency virus-2 infections were not detected. Dual infections could not be excluded in this study. The in-house HIV-2 PCR assay needs further evaluation to determine its sensitivity and specificity. These results cannot be generalised because the sample size was too small. Further studies are needed to explore HIV-2 infections and treatment response in SA.
The Multispot HIV-1 and HIV-2 rapid assay was later discontinued in 2016 and was replaced by the Geenius HIV-1 and HIV-2 Supplemental Assay.
(1) Tshwane Academic division of the NHLS and the University of Pretoria Virology Department for support and supply of all patients’ specimens, (2) Dr Osman for arranging the known HIV-2 specimens from a Portugal laboratory and (3) Dr Allison Glass (private laboratory) for testing HIV-2 PCR on the four known HIV-2 specimens.
This research was conducted for the fulfilment of the MMed in Pathology (Clinical Virology) and supervised by Simnikiwe Horatious Mayaphi and Lynne Margaret Webber.
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
S.H.M. and L.M.W. developed the study concept, design, manuscript editing and they were also supervisors. P.B. contributed in data analysis, statistics and manuscript editing. R.T.M. conducted laboratory tests, data collection, data analysis and writing manuscript.
This work was supported by the NHLS Research Trust (GRANT004 94395), Discovery Foundation (reference number 034211) and the University of Pretoria Research grant.
The data that support the findings of this study are available from the corresponding author, R.T.M., upon reasonable request.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.