HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection.

BACKGROUND: The potential role of antibodies in protection against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody (NAb) responses in three individuals, who were superinfected within one year of primary infection, to ten matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length envs from a previous study showed limited diversification in the individuals who became superinfected with the same HIV-1 subtype within year one post-seroconversion. We hypothesized that this reflected a blunted NAb response, which may have made these individuals more susceptible to superinfection. RESULTS: Neutralization assays showed that autologous plasma NAb responses to the earliest, and in some cases transmitted/founder, virus were delayed and had low to undetectable titers in all three superinfected individuals prior to superinfection. In contrast, NAbs with a median IC50 titer of 1896 were detected as early as three months post-seroconversion in non-superinfected controls. Early plasma NAbs in all subjects showed limited but variable levels of heterologous neutralization breadth. Superinfected individuals also exhibited a trend toward lower levels of gp120- and V1V2-specific IgG binding antibodies but higher gp120-specific plasma IgA binding antibodies. CONCLUSIONS: These data suggest that the lack of development of IgG antibodies, as reflected in autologous NAbs as well as gp120 and V1V2 binding antibodies to the primary infection virus, combined with potentially competing, non-protective IgA antibodies, may increase susceptibility to superinfection in the context of settings where a single HIV-1 subtype predominates.

Timing and source of subtype-C HIV-1 superinfection in the newly infected partner of Zambian couples with disparate viruses.

BACKGROUND: HIV-1 superinfection occurs at varying frequencies in different at risk populations. Though seroincidence is decreased, in the negative partner of HIV-discordant couples after joint testing and counseling in the Zambia Emory HIV Research Project (ZEHRP) cohort, the annual infection rate remains relatively high at 7-8%. Based on sequencing within the gp41 region of each partner's virus, 24% of new infections between 2004 and 2008 were the result of transmission from a non-spousal partner. Since these seroconvertors and their spouses have disparate epidemiologically-unlinked viruses, there is a risk of superinfection within the marriage. We have, therefore, investigated the incidence and viral origin of superinfection in these couples. RESULTS: Superinfection was detected by heteroduplex mobility assay (HMA), degenerate base counting of the gp41 sequence, or by phylogenetic analysis of the longitudinal sequences. It was confirmed by full-length env single genome amplification and phylogenetic analysis. In 22 couples (44 individuals), followed for up to five years, three of the newly infected (initially HIV uninfected) partners became superinfected. In each case superinfection occurred during the first 12 months following initial infection of the negative partner, and in each case the superinfecting virus was derived from a non-spousal partner. In addition, one probable case of intra-couple HIV-1 superinfection was observed in a chronically infected partner at the time of his seroconverting spouse's initial viremia. Extensive recombination within the env gene was observed following superinfection. CONCLUSIONS: In this subtype-C discordant couple cohort, superinfection, during the first year after HIV-1 infection of the previously negative partner, occurred at a rate similar to primary infection (13.6% [95% CI 5.2-34.8] vs 7.8% [7.1-8.6]). While limited intra-couple superinfection may in part reflect continued condom usage within couples, this and our lack of detecting newly superinfected individuals after one year of primary infection raise the possibility that immunological resistance to intra-subtype superinfection may develop over time in subtype C infected individuals.

Reduced frequency of HIV superinfection in a high-risk cohort in Zambia.

Rates of HIV-1 superinfection, re-infection with a genetically distinct virus despite HIV-1 specific immune responses, vary in different risk populations. We previously found the rates of superinfection were similar to primary HIV infection (PHI) in a Zambian heterosexual transmission cohort. Here, we conduct a similar analysis of 47 HIV-positive Zambians from an acute infection cohort with more frequent follow-up, all infected by non-spousal partners. We identified only one case of superinfection in the first two years, significantly fewer than in our previous study, which was likely due to increased counseling during acute infection and an overall population-wide decline in factors associated with HIV transmission. The predominant virus detected after superinfection was a recombinant of the transmitted founder (TF) and the superinfecting strain. The superinfected individual mounted a neutralizing antibody response to the primary TF virus, which remained TF-specific over time and even after superinfection, did not neutralize the superinfecting variant.

HIV transmission in discordant couples in Africa in the context of antiretroviral therapy availability.

OBJECTIVE: The study aims to understand the basis of continued HIV-1 transmission in Zambian and Rwandan HIV-1-discordant couples in the context of antiretroviral therapy (ART). DESIGN: We identified nine Zambian and seven Rwandan acutely infected, epidemiologically-linked couples from government couples' voluntary counseling and testing (CVCT) clinics where transmitting partners reported being on ART near the time of transmission. METHODS: We quantified viral load and plasma antiretroviral drug concentrations near the time of transmission and used these as surrogate measures for adherence. We also sequenced the polymerase gene from both donor and recipient partners to determine the presence of drug resistance mutations (DRMs). RESULTS: In Zambia, all transmitting partners had detectable viral loads, and 8/9 were not on therapeutic antiretroviral regimens. In the remaining couple, despite being on a therapeutic regimen, DRMs were present and transmitted. In Rwanda, although six of seven transmitting partners had detectable viral loads, therapeutic levels of antiretroviral drugs were detected in four of seven, but were accompanied by DRMs. In the remaining three couples, either no antiretrovirals or subtherapeutic regimens were detected. CONCLUSIONS: A reduction of ART effectiveness in nontrial settings was associated with lack of antiretrovirals in plasma and detectable viral load, and also drug resistance. In Zambia, where CVCT is not widely implemented, inconsistent adherence was high in couples unaware of their HIV discordance. In Rwanda, where CVCT is deployed country-wide, virologic failure was associated with drug resistance and subsequent transmission. Together, these findings suggest that increasing ART availability in resource-limited settings without risk reduction strategies that promote adherence may not be sufficient to control the HIV epidemic in the post-ART era.

XtracTB Assay, a Mycobacterium tuberculosis molecular screening test with sensitivity approaching culture.

Nucleic acid amplification tests are increasingly used to diagnose tuberculosis (TB) due to their speed and sensitivity compared to sputum smear microscopy. However, these tests fail to equal culture's sensitivity with sputum smear microscopy negative specimens and therefore cannot be used to rule out TB disease. For molecular tests to match culture's sensitivity, they must detect ≤10 genomic copies of Mycobacterium tuberculosis (MTB) DNA, the limit of detection of culture, process ≥1 ml of sputum ensuring sufficient number of MTB are in the reaction, and efficiently remove sputum associated inhibitors from this large sample. Here we report the preliminary characterization of XtracTB Assay, a MTB testing protocol designed for inclusion in either an integrated point-of-care platform or a high throughput automated central laboratory system. The test combines DNA sequence specific sample prep to reduce the co-extraction of qPCR inhibitors with the amplification of two MTB specific loci (IS6110 and senX3-regX3) to increase test sensitivity and minimize the likelihood of false negatives. The analytical sensitivity of the XtracTB Assay was 5 genomic copies/ml of sputum rivaling that of culture. Furthermore, 142 valid test results yield clinical sensitivity of 94.9% (95% CI: 90.1-99.9) and specificity of 100% (95% CI: 90.0-100.0).

Improved method for collection of sputum for tuberculosis testing to ensure adequate sample volumes for molecular diagnostic testing.

The quality and quantity of sputum collected has an important impact on the laboratory diagnosis of pulmonary TB. We conducted a pilot study to assess a new collection cups for the collection of sputum for the diagnosis of pulmonary tuberculosis. The pilot study utilized the standard collection cup in South Africa demonstrating a mean collection volume of 2.86±2.36SDml for 198 samples; 19% of the specimens contained <1ml and 12% contained >5ml. We designed and tested two novel sputum cups with a narrow bottom section and clear minimum and maximum markings to allow patients and clinicians to know whether sufficient sputum volume has been produced. The cups differed in their shape and manufacturing approach. The two options also support different mixing approaches being considered for a highly sensitive companion TB-screening assay being developed at Northwestern University (XtracTB assay). Sputum was collected from 102 patients at Nolungile Youth Centre, Khayelitsha, Cape Town, South Africa for a total of 204 samples. The mean volumes collected from the two cups were 2.70±0.88SDml and 2.88±0.89SDml. While the mean volumes of current and novel cups are similar, the volume ranges collected with the novel cups were narrower, and 98% of the specimen volumes were within the target range. Only 4 samples contained >5ml, but none were >6ml, and none of the specimens contained <1ml. The number of coughs that produced the samples, patient HIV and TB status plus qualitative descriptions of the sputum specimens were also evaluated.

Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens.

Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear-negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tuberculosis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to specifically address this diagnostic gap is reported. First, human genomic DNA is identified as a significant qPCR inhibitor. To circumvent this problem, a novel, streamlined sample preparation method utilizing detergent and proteolysis to thin the sputum and DNA sequence specific MTB DNA isolation was developed. Additionally, a multiplexed qPCR assay targeting two MTB complex-specific loci: the potentially multi-copy IS6110 and the single-copy senX3-regX3, combined with the cotJC gene from Bacillus atrophaeus spores amplified as a process control was developed. The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay.

Low antibody-dependent cellular cytotoxicity responses in Zambians prior to HIV-1 intrasubtype C superinfection.

We have previously shown that HIV-1 superinfected Zambian seroconverters mount low binding and neutralizing antibody responses to their primary HIV-1 infecting virus, which could increase susceptibility to re-infection. Here, we investigated if antibody-dependent cellular cytotoxicity (ADCC), a process by which virus-infected cells are killed, was also reduced. Superinfected individuals exhibited low ADCC activity compared to non-superinfected individuals, but similar levels of CMV-reactive binding antibodies, suggesting superinfected individuals are capable of generating and maintaining virus-specific antibodies.