Detection of Acute and Early HIV-1 Infections in an HIV Hyper-Endemic Area with Limited Resources

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CHAPTER 2 Detection of Acute and Early HIV-1 Infections in an HIV Hyper- Endemic Area with Limited Resources

The editorial style of the PLOS ONE journal was followed in this chapter – (minor post- publication amendments have been included).

Introduction

Halting and reversing the spread of HIV was part of Millennium Development Goal (MDG) 6A [1]. Although good progress has been made in achieving MDG 6A, there were still too many new HIV infections by the end of 2014 and 2015, and two thirds of these infections were found in sub-Saharan Africa [2,3] Acute HIV infection (AHI) refers to the time of virus acquisition until the appearance of HIV antibodies. Early or primary HIV infection, which includes AHI, is regarded as the interval between virus acquisition and the establishment of HIV viral load (VL) set-point [4]. Chronic HIV stage follows after the set-point is established [5]. People with early HIV infection contribute significantly to the transmission of HIV, as they have very high VLs in blood and genital secretions. It is estimated that early HIV infection stage is 26 times more infectious compared to the chronic stage [6]. This early stage of HIV infection is also known to predominantly produce C-C chemokine receptor type 5 (CCR-5) HIV strains, which are efficiently transmitted across the genital mucosa [7].
Rapid HIV tests play a crucial role in detecting HIV infections, and thereby initiating a cascade of linking infected patients to care. These rapid tests are commonly used for diagnosis of HIV infection in low resource settings such as in sub-Saharan Africa, but have poor sensitivity for detection of early HIV infection [8,9], which results in giving false negative results to highly infectious individuals. The addition of p24 antigen to some rapid HIV tests has led to a slight improvement in sensitivity for detection of early HIV infections, as the p24 antigen component on these tests performs poorly [8,9]. Tests that have shortened the HIV window period such as enzyme-linked immunosorbent assays (ELISAs) and NAATs are costly and not readily available for point-of-care testing [10].
Management of early HIV infection has benefits for the infected individual, and prevents secondary spread of HIV in the population [4,5]. This study aimed to detect acute and early HIV infections in an HIV hyper-endemic setting with limited resources.

Materials and Methods

Recruitment and enrollment

This was a cross-sectional diagnostic study, conducted between March 2012 to June 2015, which enrolled individuals who had negative rapid HIV results and were 14 years or older. Participants were recruited and enrolled from 5 HIV counseling and testing (HCT) clinics in the Tshwane district of South Africa (SA). Four of these HCT clinics were antenatal clinics and one was a general HCT clinic. Rapid HIV testing was done according to the SA HIV testing guidelines, which recommend a serial HIV testing strategy at the points-of-care [11]. Testing at the HCT clinics was done by HIV counselors, who had received training in HIV testing and counseling. The rapid HIV test that was commonly used for screening during the course of this study was Advanced Quality (Intec Products Inc). Abon (Abon Biopharm) was used for screening in 2014; however, this was replaced with Advanced Quality at the end of 2014. At enrolment, study samples and participants’ cell phone numbers were collected. Plasma was separated from whole blood through centrifugation at 1700 relative centrifugal force (RCF) for 20 minutes, and stored at -70ºC within 24 hours after collection until the time of testing.

Ethics statement

Written consent was obtained from all participants before enrollment. The study was approved by the University of Pretoria’s Faculty of Health Sciences Ethics Committee (Protocol number 295/2015) and by Tshwane Research Ethics Committee (TMREC 2010/26). The legal ages for consenting to HIV testing and medical treatment in South Africa are 12 and 14 years, respectively [12]. During the course of the study we noticed that some people who came for HIV testing were younger than 18 years of age, and came alone without parents or guardians. Hence, we amended our study protocol to include this group, in order to extend the benefit of earlier diagnosis of HIV to them. We applied for inclusion of this group with our Ethics Committee, and were granted So all study participants, including 14-17 years, signed the same written consent forms that were used for participants older than 18 years.

Sample testing

Roche CAP/CTM HIV VL version 2 assay (Roche Diagnostics, Mannheim, Germany) with a lower detection limit of 20 copies/ml was used for NAAT, in a mini-pool of 5 samples, using 200 μl from each sample to constitute a 1 ml sample volume required for testing. An additional volume of about 20 μl from one of the pool samples was used for top-up in order to avoid sample rejection due to insufficient volume. Pools that had undetectable VL were considered negative for HIV. Individual sample testing was done in pools that had detectable VL. A VL threshold of ≥5000 copies/ml in an individual sample was considered as diagnostic for HIV infection. If HIV VL was <5000 copies/ml, a repeat test was done in a follow-up sample in order to exclude a possible contamination in the initial test [5]. All participants were encouraged to voluntarily collect NAAT results; however, those who tested positive on NAAT were contacted on their cell phones to come back to the clinic for further management. During this follow-up visit, HIV counseling and repeat rapid testing were done, follow-up samples collected, and participants referred for appropriate management.
The following serology tests were done in NAAT-positive samples: 3rd generation Genscreen HIV-1/2 version 2 ELISA (BioRad, Marnes-la-Coquette, France) and HIV Western Blot (Bio- Rad Laboratories, Redmond WA, USA) for antibody detection; p24 antigen (Roche Diagnostics, Mannheim, Germany); and limiting antigen (LAg) HIV avidity assay {Maxim Biomedical Inc., Rockville, USA} for confirmation of early HIV infection in samples with detectable antibodies. LAg avidity assay was repeated on follow-up samples. Acute HIV infection was defined as the presence of HIV RNA with or without p24 antigen in the absence of HIV antibodies. Early HIV infection was defined as the presence of HIV RNA with or without p24 antigen, and presence of HIV antibodies with low avidity as reflected by values <1.5 normalised optical density (OD-n) on LAg avidity assay. Samples found to have HIV RNA with or without p24 antigen, and HIV antibodies with high avidity of >1.5 OD-n were classified as having chronic infection.
Rapid HIV testing was later repeated from stored plasma samples in the laboratory using the same tests that were used at the points-of-care. All the tests were performed and analyzed according to manufacturer’s instructions. CD4 count results were later enumerated from the laboratory information system and patient records for NAAT-positive participants.

Statistical analysis

A descriptive analysis was used to present summary statistics (median, proportions and 95% confidence intervals) for the parameters. Proportions of acute, early and chronic HIV infections that were missed at the points-of-care were computed. Comparison of the proportions of newly diagnosed HIV infections between males and females was done using Fisher’s exact test. In addition, median values of HIV VL and CD4 count were compared between the groups of participants with early and chronic HIV infections using the Wilcoxon rank-sum test. All the statistics were performed on the STATA version 14.1 software (StataCorp LP, College Station, TX, USA). A p-value of ≤0.05 was considered statistically significant.

Results

Demographics and newly diagnosed HIV infections

From March 2012 to June 2015, the study enrolled and tested 6910 participants who had negative rapid HIV test results (Fig 2.1). Their median age was 27 years (IQR: 23 – 31). Females formed a large proportion (87%, n = 6011) of the study group, and 88% (n = 5271) of the female participants were pregnant. NAAT detected HIV RNA in 55 samples, resulting in 0.8% of newly diagnosed HIV-infected individuals (95% CI: 0.6-1.0). This showed a negative predictive value (NPV) of 99.2% (95% CI: 99.0-99.4) for rapid HIV testing at the points-of- care. The newly diagnosed HIV infections were detected in all five study clinics, and were slightly higher in pregnant females (0.9%) compared to non-pregnant females and males, 0.7% (p = 0.83) and 0.6% (p = 0.54), respectively.

Serological characterization of NAAT-positive samples

Of the 55 NAAT-positive participants, 52 (95%) tested positive for HIV antibodies on Genscreen HIV ELISA. Western Blot (W. Blot) was positive in 48 (92%) of these antibody- positive participants. P24 antigen testing was performed on 53 participants, and was positive in 12 (23%) participants. The two samples that were insufficient for p24 antigen test were positive on HIV ELISA. Limiting antigen avidity assay identified 16 antibody-positive participants as having early HIV infection, and classified the remainder (n = 36) as having chronic infection. Limiting antigen avidity was repeated on follow-up samples of participants who came for a follow-up visit, and the results remained the same as in initial testing. The combination of HIV RNA, p24 antigen, HIV antibody, LAg avidity and W. Blot test results enabled staging of HIV infections. Amongst those with early HIV infection, few participants were detected very early before the appearance of HIV antibodies, some were detected at the peak of HIV vireamia, and many others were near the time of HIV VL set-point (Fig 2.2). A significant proportion of HIV-infected individuals were classified as having chronic HIV infection (Fig 2.2).

HIV viral loads and CD4 counts of NAAT-positive participants

Median HIV VL was slightly higher in participants with early HIV infection (4.5 log, IQR: 3.7-5.7) compared to those with chronic infection (4.2 log, IQR: 3.8-4.9) (p = 0.33). Most participants (89%) had HIV VLs >1500 copies/ml. CD4 count results were available for 44 participants. Median CD4 count was significantly higher in participants with early HIV infection (434 cells/μl, IQR: 287-706) compared to those with chronic infection (351 cells/μl, IQR: 228-469) (p = 0.03) (Tables 2.1 and 2.2).

Declaration
Acknowledgements
Summary
Presentations and publications
Table of contents
Abbreviations and symbols
List of figures
List of tables
CHAPTER 1: Introduction and Study Aims
1.1 HIV prevalence.
1.2 Transmission modes of HIV.
1.3 HIV structure and taxonomy
1.4 HIV genome and diversity
1.5 HIV replication
1.6 Natural course of HIV infection
1.7 Diagnosis of HIV infection
1.8 Treatment of HIV infection
1.9 Prevention of HIV
1.10 The importance of diagnosing early HIV infection
1.11 Study hypothesis
1.12 Study aims and objectives
1.13 Sample size
1.14 References
CHAPTER 2: Detection of Acute and Early HIV-1 Infections in an HIV Hyper-Endemic Area with Limited Resources
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Recruitment and enrolment.
2.3.2 Ethics statement
2.3.3 Sample testing
2.3.4 Statistical analysis
2.4 Results
2.4.1 Demographics and newly diagnosed HIV infections
2.4.2 Serological characterization of NAAT-positive samples
2.4.3 HIV viral loads and CD4 counts of NAAT-positive participants.
2.4.4 Repeat rapid testing from stored plasma samples
2.4.5 Follow-up of NAAT-positive participants
2.5 Discussion
2.5.1 Frequency of new HIV infections
2.5.2 Earlier detection of HIV-infected individuals misdiagnosed at the points-of-care
2.5.3 Alternative strategies for detecting HIV-infected individuals misdiagnosed at the points-of-care
2.5.4 Implications of misdiagnosing HIV infections at the points-of-care
2.5.5 Possible explanations for misdiagnosis of HIV-infected people at the points-of-care
2.6 Conclusions
2.7 References
CHAPTER 3: High Risk Exposure to HIV among Sexually Active Individuals who Tested Negative on Rapid HIV Tests in the Tshwane District of South Africa – the importance of behavioural prevention measures
3.1 Abstract
3.2 Introduction
3.3 Method
3.4 Results
3.5 Discussion
3.6 Conclusions
3.7 References
CHAPTER 4: Transmitted Minority and Majority Antiretroviral Drug-Resistance Variants of HIV in the Tshwane District of South Africa
4.1 Abstract
4.2 Background
4.3 Objective
4.4 Study design
4.5 Results
4.6 Discussion
4.7 Conclusions
4.8 References
CHAPTER 5: Vertical Transmission of HIV among Pregnant Women who were Initially Misdiagnosed by the Rapid HIV Tests in Four South African Antenatal Clinics
5.1 Abstract
5.2 Introduction
5.3 Methods
5.4 Results
5.5 Discussion
5.6 Conclusions
5.7 References
CHAPTER 6: Field Performance of the INSTI Rapid HIV-1/2 Antibody Test in Two South African Antenatal Clinics
6.1 Abstract
6.2 Introduction, Materials and Methods, Results, and Discussion
6.3 References
CHAPTER 7: Perspectives
7.1 References
Appendix 
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