Comprehensive investigation of sources of misclassification errors in routine HIV testing in Zimbabwe.

INTRODUCTION: Misclassification errors have been reported in rapid diagnostic HIV tests (RDTs) in sub-Saharan African countries. These errors can lead to missed opportunities for prevention-of-mother-to-child-transmission (PMTCT), early infant diagnosis and adult HIV-prevention, unnecessary lifelong antiretroviral treatment (ART) and wasted resources. Few national estimates or systematic quantifications of sources of errors have been produced. We conducted a comprehensive assessment of possible sources of misclassification errors in routine HIV testing in Zimbabwe. METHODS: RDT-based HIV test results were extracted from routine PMTCT programme records at 62 sites during national antenatal HIV surveillance in 2017. Positive- (PPA) and negative-percent agreement (NPA) for HIV RDT results and the false-HIV-positivity rate for people with previous HIV-positive results ("known-positives") were calculated using results from external quality assurance testing done for HIV surveillance purposes. Data on indicators of quality management systems, RDT kit performance under local climatic conditions and user/clerical errors were collected using HIV surveillance forms, data-loggers and a Smartphone camera application (7 sites). Proportions of cases with errors were compared for tests done in the presence/absence of potential sources of errors. RESULTS: NPA was 99.9% for both pregnant women (N = 17224) and male partners (N = 2173). PPA was 90.0% (N = 1187) and 93.4% (N = 136) for women and men respectively. 3.5% (N = 1921) of known-positive individuals on ART were HIV negative. Humidity and temperature exceeding manufacturers' recommendations, particularly in storerooms (88.6% and 97.3% respectively), and premature readings of RDT output (56.0%) were common. False-HIV-negative cases, including interpretation errors, occurred despite staff training and good algorithm compliance, and were not reduced by existing external or internal quality assurance procedures. PPA was lower when testing room humidity exceeded 60% (88.0% vs. 93.3%; p = 0.007). CONCLUSIONS: False-HIV-negative results were still common in Zimbabwe in 2017 and could be reduced with HIV testing algorithms that use RDTs with higher sensitivity under real-world conditions and greater practicality under busy clinic conditions, and by strengthening proficiency testing procedures in external quality assurance systems. New false-HIV-positive RDT results were infrequent but earlier errors in testing may have resulted in large numbers of uninfected individuals being on ART.

Characterization of gp120 hydrolysis by IgA antibodies from humans without HIV infection.

Antibody hydrolysis of the superantigenic gp120 site and HIV-1 neutralization was studied as a potential anti-HIV mechanism in uninfected humans. gp120 hydrolysis by purified serum and salivary antibodies was determined by electrophoresis and peptide sequencing, the proteolytic mechanism was analyzed using electrophilic peptide analogs, and viral neutralization was studied using peripheral blood mononuclear cells as hosts. Polyclonal and monoclonal IgA but not IgG preparations selectively catalyzed the cleavage of HIV gp120 at rates sufficient to predict biologically relevant protection against the virus. The IgA hydrolytic reaction proceeded by noncovalent recognition of gp120 residues 421-433, a component of the superantigenic site of gp120, coordinated with peptide bond cleavage via a serine protease-like mechanism. The Lys-432-Ala-433 bond was one of the cleavage sites. Infection of peripheral blood mononuclear cells by a primary isolate of HIV was neutralized by the IgA but not IgG fractions. The neutralizing activity was specifically inhibited by an electrophilic inhibitor of the catalytic activity. The existence of catalytic IgAs to gp120 in uninfected humans suggests their role in resistance to HIV.

HIV type 1 subtype C gag and nef diversity in Southern Africa.

Several HIV-1 subtype C-specific gag- and/or nef-based vaccines are currently intended for clinical trial in southern Africa. Here we provide sequences of 64 gag and 45 nef genes sampled in Malawi, Zambia, Zimbabwe, and South Africa and assess the degree of southern African HIV-1 diversity that will confront these vaccines. Whereas reasonable phylogenetic evidence exists for geographical clustering of subtype C gag and nef sequences from various other parts of the world, there is little evidence of similar population founder effects in the southern African epidemic. The entire breadth of subtype C diversity is represented in the southern African genes suggesting there may be no advantage in producing region- or country-specific subtype C vaccines. We do not, however, find much evidence of intersubtype recombination in the Southern African genes, implying that the likelihood of vaccine failure due to the emergence of intersubtype recombinants is probably low.

Viral dynamics and CD4+ T cell counts in subtype C human immunodeficiency virus type 1-infected individuals from southern Africa.

Defining viral dynamics in natural infection is prognostic of disease progression and could prove to be important for vaccine trial design as viremia may be a likely secondary end point in phase III HIV efficacy trials. There are limited data available on the early course of plasma viral load in subtype C HIV-1 infection in Africa. Plasma viral load and CD4+ T cell counts were monitored in 51 recently infected subjects for 9 months. Individuals were recruited from four southern African countries: Zambia, Malawi, Zimbabwe, and South Africa and the median estimated time from seroconversion was 8.9 months (interquartile range, 5.7-14 months). All were infected with subtype C HIV-1 and median viral loads, measured using branched DNA, ranged from 3.82-4.02 log10 RNA copies/ml from 2-24 months after seroconversion. Viral loads significantly correlated with CD4+ cell counts (r=-0.5, p<0.0001; range, 376-364 cells/mm3) and mathematical modeling defined a median set point of 4.08 log10 (12 143 RNA copies/ml), which was attained approximately 17 months after seroconversion. Comparative measurements using three different viral load platforms (bDNA, Amplicor, and NucliSens) confirmed that viremia in subtype C HIV-1-infected individuals within the first 2 years of infection did not significantly differ from that found in early subtype B infection. In conclusion, the course of plasma viremia, as described in this study, will allow a useful baseline comparator for understanding disease progression in an African setting and may be useful in the design of HIV-1 vaccine trials in southern Africa.

Inactivated- or killed-virus HIV/AIDS vaccines.

Inactivated or "killed" virus (KV) is a "classical" approach that has produced safe and effective human and veterinary vaccines but has received relatively little attention in the effort to develop an HIV/AIDS vaccine. Initially, KV and rgp120 subunit vaccines were the two most obvious approaches but, unfortunately, rgp120 has not been efficacious and the KV approach has been limited by a variety of scientific, technical, and sociological factors. For example, when responses to cellular antigens, present on SIV grown in human cells, proved to be largely responsible for efficacy, the KV approach was widely discounted. Similarly, when lab-adapted HIV-1 appeared to lose envelope glycoprotein during preparation (not the case for primary isolates), this was viewed as a fundamental barrier to the KV concept. Also, a preference for "safer", genetically-engineered vaccines, and emphasis on cellular immunity, have left KV low on the priority list for funding agencies and investigators. The recent suggestion that "native" trimeric gp120 displays conserved conformational neutralization epitopes, along with the failure of rgp120, and difficulties in raising strong cellular responses with DNA or vectored vaccines, has restored some interest in the KV concept. In the past 15 years, several groups have initiated pre-clinical development of KV candidates for SIV or HIV and promising, albeit limited, information has been produced. In this chapter we discuss the rationale (including pros and cons) for producing and testing killed-HIV vaccines, the prospects for success, the nature and scope of research needed to test the KV concept, what has been learned to date, and what remains undone.

Effects of CCR5-Delta32 and CCR2-64I alleles on HIV-1 disease progression: the protection varies with duration of infection.

OBJECTIVE: To examine temporal variation in the effects of CCR5-Delta32 and CCR2-64I chemokine receptor gene polymorphisms on HIV-1 disease progression. DESIGN: Pooled analysis of individual patient data from 10 cohorts of HIV-1 seroconverters from the United States, Europe, and Australia. METHODS: We studied HIV-1 seroconverters of European (n = 1635) or African (n = 215) ancestry who had been genotyped for CCR5-Delta32 and CCR2-64I. We used Cox proportional hazards models with time-varying coefficients to determine whether the genetic protection against AIDS (1987 case definition) and death varied with time since seroconversion. RESULTS: Protection against AIDS conferred by CCR5-Delta32 held constant at a 31% (RH 0.69, 95% CI 0.54, 0.88) reduction in risk over the course of HIV-1 infection, whereas protection against death held constant at a 39% reduction in risk (RH 0.61, 95% CI 0.45, 0.88). When the period from AIDS to death was isolated, the survival benefit of CCR5-Delta32 diminished 2 years after AIDS. Protection against AIDS conferred by CCR2-64I was greatest early in the disease course. Compared with individuals without CCR5-Delta32 or CCR2-64I, individuals with one or two copies of CCR2-64I had a 58% lower risk of AIDS during the first 4 years after seroconversion (RH 0.42, 95% CI 0.23, 0.76), a 19% lower risk during the subsequent 4 years (RH 0.81, 95% CI 0.59, 1.12), and no significant protection thereafter. CONCLUSION: The protection against AIDS provided by CCR5-Delta32 is continuous during the course of infection. In contrast, the protection provided by CCR2-64I is greatest early in the course of infection.

High incidence and prevalence of HIV-1 infection in high risk population in Calcutta, India.

HIV-1 infection in India has been increasing steadily over the last decade. In the absence of potent antiviral therapy, estimates of HIV infection are needed to monitor the epidemic, institute prevention strategies in target populations and determine the suitable populations for vaccine studies. In this report we present the HIV-1 seroprevalence and annual estimates of seroincidence in a high risk population from Calcutta, the most populous city in the eastern part of India. In 1206 high risk subjects tested over two years between February of 1999 and December 2000, we have determined an overall seroprevalence of 40.1% using enzyme-linked immunosorbent assay followed by a confirmatory Western blot testing. Furthermore, using a newly described Standardized Testing Algorithm for Recent HIV-1 Seroconversion (STARHS), we have estimated an annual seroincidence rate of about 7% in this population during this two-year study. Such a high annual seroincidence rate makes this population well suited for studies of HIV-1 prevention, including vaccine trials.

Absolute and percent CD4+ T-cell enumeration by flow cytometry using capillary blood.

INTRODUCTION: CD4+ T-cell counting is usually performed on whole blood obtained from standard venipuncture. Venipuncture requires expertise, results in discomfort and generates biological waste. Capillary blood could be used to measure the levels of CD4+ T-cell in children, elderly and very ill patients. We studied the agreement between CD4+ T-cell counts and percent generated using venous blood with those obtained with capillary blood in HIV-infected adults and children in a resource-limited tropical setting. METHODS: This cross-sectional study consecutively enrolled a total of 152 adult and pediatric HIV-positive patients attending two outpatient clinics in Maputo City, Mozambique. We recruited individuals presenting for their routine clinical follow-up that included the determination of CD4+ T-cell counts in peripheral blood. For each subject, peripheral blood specimens were obtained by both venipuncture and finger prick. Specimens were tested using two flow cytometers, the FACSCount and the FACSCalibur. RESULTS: Absolute CD4+ T-cell counts obtained using capillary blood were in close agreement with those from venous blood both on the FACSCalibur (absolute bias=+12.3 cells/mm³, limits of agreement: -259.2 to +283.9, R²=0.96) and the FACSCount (absolute bias=+16.1 cells/mm³, limits of agreement: -209.2 to +241.5, R²=0.97). Percent CD4+ T-cell counts were measured only on the FACSCalibur also showed a good agreement with a bias of +0.6% and limits of agreement of -3.1 to +4.3. CONCLUSIONS: Absolute CD4+ T-cell counts and percent generated using capillary blood are in close agreement with those from venous blood. Point-Of-Care assays and standard flow cytometers can be deployed in a tiered laboratory network where both venous and capillary blood collection can be used for CD4+ T-cell enumeration.

Absence of reproducibly detectable low-level HIV viremia in highly exposed seronegative men and women.

OBJECTIVE: Transient HIV infections have been invoked to account for the cellular immune responses detected in highly virus-exposed individuals who have remained HIV-seronegative. We tested for very low levels of HIV RNA in 524 seronegative plasma samples from 311 highly exposed women and men from three longitudinal HIV cohorts. DESIGN: Two thousand and seventy-three transcription-mediated amplification (TMA) HIV RNA tests were performed for an average of 3.95 TMA assays per plasma sample. Quadruplicate TMA assays, analyzing a total of 2 ml of plasma, provided an estimated sensitivity of 3.5 HIV RNA copies/ml. RESULTS: Four samples from individuals who did not seroconvert within the following 6 months were positive for HIV RNA. For one sample, human polymorphism DNA analysis indicated a sample mix-up. Borderline HIV RNA detection signals were detected for the other three positive samples but further replicate TMA testing yielded no positive results. Nested PCR assays (n = 254) for HIV proviral DNA in peripheral blood mononuclear cells (PBMCs) from these three individuals were negative. CONCLUSION: Transient viremia was not reproducibly detected in highly HIV-exposed seronegative men and women. If transient infections do occur, plasma HIV RNA levels may remain below the detection limits of the sensitive assay used here, be of very short duration, or viral replication may be restricted to mucosal surfaces or their draining lymphoid tissues.

Flash-heat inactivation of HIV-1 in human milk: a potential method to reduce postnatal transmission in developing countries.

BACKGROUND: Up to 40% of all mother-to-child transmission of HIV occurs by means of breast-feeding; yet, in developing countries, infant formula may not be a safe option. The World Health Organization recommends heat-treated breast milk as an infant-feeding alternative. We investigated the ability of a simple method, flash-heat, to inactivate HIV in breast milk from HIV-positive mothers. METHODS: Ninety-eight breast milk samples, collected from 84 HIV-positive mothers in a periurban settlement in South Africa, were aliquoted to unheated control and flash-heating. Reverse transcriptase (RT) assays (lower detection limit of 400 HIV copies/mL) were performed to differentiate active versus inactivated cell-free HIV in unheated and flash-heated samples. RESULTS: We found detectable HIV in breast milk samples from 31% (26 of 84) of mothers. After adjusting for covariates, multivariate logistic regression showed a statistically significant negative association between detectable virus in breast milk and maternal CD4+ T-lymphocyte count (P=0.045) and volume of breast milk expressed (P=0.01) and a positive association with use of multivitamins (P=0.03). All flash-heated samples showed undetectable levels of cell-free HIV-1 as detected by the RT assay (P<0.00001). CONCLUSIONS: Flash-heat can inactivate HIV in naturally infected breast milk from HIV-positive women. Field studies are urgently needed to determine the feasibility of in-home flash-heating breast milk to improve infant health while reducing postnatal transmission of HIV in developing countries.

Genetic characteristics of HIV-1 subtype C envelopes inducing cross-neutralizing antibodies.

This study aimed to characterize genetic features of HIV-1 subtype C envelope glycoproteins capable of eliciting cross-reactive neutralizing antibodies during natural infections. The gp160 sequences were determined for 36 HIV-1 subtype C isolates (donor viruses) from infected individuals residing in Malawi, Zimbabwe, Zambia and South Africa, whose sera displayed a range of cross-neutralizing activities against a panel of 5 subtype C and 5 subtype B viruses (panel viruses). Hierarchical clustering analysis of neutralization data of the panel viruses predicted phylogenetic relationships between subtype B and C panel viruses, suggesting some subtype-specific neutralization determinants. A similar comparison of subtype C donor viruses showed no significant correlation; however of three donor sequence pairs resolvable by phylogenetic analysis, two were also associated within the neutralization clustering dendrogram, suggesting that closely related viruses may elicit antibodies targeting common neutralization determinants. Significantly, viruses that had shorter V1-V4 loops induced antibodies that showed more neutralization breadth against the subtype C panel viruses (p=0.0135). This study indicates that that some structural features of envelope, such as shorter variable loops, may facilitate the elicitation of cross-reactive neutralizing antibodies in natural infections. Collectively these data provide some insights into design features of an envelope immunogen aimed at inducing neutralizing antibodies.

Sequential turnover of human immunodeficiency virus type 1 env throughout the course of infection.

We examined the rates of variant population turnover of the V1-V2 and V4-V5 hypervariable domains of the human immunodeficiency virus type 1 (HIV-1) gp120 molecule in longitudinal plasma samples from 14 men with chronic HIV-1 infection using heteroduplex tracking assays (HTA). Six men had high rates of CD4+ T-cell loss, and eight men had low rates of CD4+ T-cell loss over 2.5 to 8 years of infection. We found that V1-V2 and V4-V5 env populations changed dramatically over time in all 14 subjects; the changes in these regions were significantly correlated with each another over time. The subjects with rapid CD4 loss had significantly less change in their env populations than the subjects with slow CD4 loss. The two subjects with rapid CD4 loss and sustained low CD4 counts (<150/microl for at least 2 years) showed stabilization of their V1-V2 and V4-V5 populations as reflected by low levels of total change in HTA pattern and low HTA indices (a novel measure of the emergence of new bands and band distribution); this stabilization was not observed in other subjects. The stabilization of env variant populations at low CD4 counts following periods of rapid viral evolution suggests that selective pressure on env, likely from new immune responses, is minimal when CD4 counts drop dramatically and remain low for extended periods of time.

HIV seroincidence estimates among at-risk populations in Buenos Aires and Montevideo: use of the serologic testing algorithm for recent HIV seroconversion.

Using the serological testing algorithm for recent HIV seroconversion, we estimated annualized incidences (per 100 person-years) of HIV-1 infection in different at-risk groups in Buenos Aires and Montevideo, during a 5-year period between 1998 and 2003. HIV-positive serum samples from 9 serosurveys conducted among men who have sex with men, patients attending clinics for a sexually transmitted infections consult (STIs), female commercial sex workers, injecting drug users (IDUs), noninjecting cocaine users (NICUs), asymptomatic women screened for HIV infection, and patients with tuberculosis were used. HIV incidences were as follows: 6.7 for men who have sex with men, 2.0 for STIs, 1.3 for female commercial sex workers, 0.0 for Argentinean IDUs, 10.3 for Uruguayan IDUs, 3.1 for Argentinean NICUs, 4.4 for Uruguayan NICUs, and 2.4 for patients with tuberculosis. Among asymptomatic women screened for HIV infection, incidence rose from 0.4 in 1998 to 4.6 in 1999 and to a high of 10.2 in the year 2000. Unexpectedly, high HIV incidences were detected among at-risk groups in Buenos Aires and Montevideo. This pattern shows an emerging HIV epidemic among heterosexuals stemming from core HIV-infected at-risk groups. There is an urgent need for development and implementation of specific prevention strategies to address this burgeoning epidemic.

Rate and incidence estimates of recent human immunodeficiency virus type 1 infections among pregnant women in Sao Paulo, Brazil, from 1991 to 2002.

The serological testing algorithm for recent human immunodeficiency virus (HIV) seroconversion (STARHS) was employed to estimate HIV incidence among pregnant women from Sao Paulo, Brazil. A cross-sectional study (1999 to 2002) showed an incidence of infection of 0.2 per 100 pregnant women per year (95% confidence interval, 0.041 to 0.608). Western blot profiles suggested an association between results of the STARHS analysis and gp41/gp31 bands.

Viral, nutritional, and bacterial safety of flash-heated and pretoria-pasteurized breast milk to prevent mother-to-child transmission of HIV in resource-poor countries: a pilot study.

BACKGROUND: Heat-treated breast milk of HIV-positive mothers has potential to reduce vertical transmission. This study compared the impact of flash-heating (FH) and Pretoria pasteurization (PP) on HIV, nutrients, and antimicrobial properties in human milk. METHODS: Milk samples were spiked with 1 x 10 (8) copies/mL of clade C HIV-1 and treated with FH and PP. We measured HIV reverse transcriptase (RT) activity before and after heating (n = 5). Heat impact on vitamins A, B6, B12, and C; folate, riboflavin, thiamin, and antimicrobial proteins (lactoferrin and lysozyme) was assessed. Storage safety was evaluated by spiking with Escherichia coli or Staphylococcus aureus. RESULTS: Both methods inactivated > or = 3 logs of HIV-1. FH resulted in undetectable RT activity. Neither method caused significant decrease in any vitamin, although reductions in vitamins C and E were noted. Heat decreased immunoreactive lactoferrin (P < 0.05) but not the proportions of lactoferrin and lysozyme surviving digestion. FH seems to retain more antibacterial activity. Both treatments eliminated spiked bacteria. CONCLUSIONS: FH may be superior to PP in eliminating all viral activity; both methods retained nutrients and destroyed bacterial contamination. Heat-treated breast milk merits further study as a safe and practical infant feeding option for HIV-positive mothers in developing countries.

High incidence of HIV-1 in South Africa using a standardized algorithm for recent HIV seroconversion.

Southern Africa has among the highest rates of HIV-1 infection in the world as judged by cross-sectional HIV-1 prevalence surveys carried out among women attending antenatal clinics. Incidence rates, which provide information on the number of new cases of infection, are more informative of the current state of the epidemic than estimates of prevalence, which provide information on the rates averaged over some previous time. Cohort studies to measure incidence rates are expensive and difficult to carry out, however, and few have been done in Africa. A recently developed standardized algorithm for recent HIV-1 seroconversion (STARHS) based on a sensitive/less-sensitive enzyme-linked immunosorbent assay was used to determine the incidence of HIV-1 subtype C infection among women attending public sector antenatal clinics in Hlabisa, a rural district in KwaZulu-Natal, South Africa. The STAHRS results were confirmed by using a mathematic model to obtain an independent estimate of the age-specific incidence rates from the age-specific prevalence data. The data reveal extraordinarily high HIV-1 incidence rates in South Africa. In 1999, the annual incidence of HIV-1 among susceptible women aged 15 to 49 years standardized to the age distribution of adult women in Hlabisa was 17%. Incidence peaked among 22-year-old women at 24% per year. The HIV-1 incidence rates provide valuable additional information indicating that new infections are continuing unabated and that the HIV-1 epidemic is still growing in rural South Africa.

Development of a new less-sensitive enzyme immunoassay for detection of early HIV-1 infection.

The sensitive/less-sensitive (S/LS) enzyme immunoassay (EIA) testing strategy for discriminating "early" from "longstanding" HIV infection has been widely applied for detecting recent seroconverters and estimating HIV incidence rates. The originally developed assay (3A11-LS EIA; Abbott Laboratories, Abbott Park, IL) involved performance of LS EIAs using a bead-based assay that required specialized equipment and reagents of limited availability. In contrast, 96-microwell-based EIAs are more universally applied for HIV serodiagnosis throughout the world. The authors report development and preliminary validation of an LS protocol using an EIA in a 96-well format: the Vironostika HIV-1 MicroElisa System (Vironostika-LS EIA; Bio Merieux, Raleigh, NC). The results with samples from recent HIV-1 seroconverters, persons with longstanding HIV-1 asymptomatic infection, patients on highly active antiretroviral therapy, and AIDS patients show a high degree of correlation between the Vironostika-LS EIA and 3A11-LS EIA. The authors also demonstrate that the Abbott 3A11-LS EIA and Vironostika-LS EIA performed comparably on HIV-1-positive samples from persons infected with non-B HIV-1 subtypes. These results support the potential use of the Vironostika-LS EIA for detection of recent HIV-1 infections for incidence projections and for other epidemiologic, clinical, and molecular surveillance applications.