Value of repeat testing using Cepheid GeneXpert CT/NG for indeterminate PCR results when diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae.

Nucleic acid amplification tests (NAATs) are the most sensitive method for diagnosing chlamydia and gonorrhoea. We use the COBAS 4800 CT/NG combined assay (Roche Molecular Diagnostics, CA, USA), and whilst the majority of samples yield definitive results, a small proportion are reported as indeterminate. In these instances, it is usual practice to request repeat samples which delays diagnosis. This audit was twofold: first to establish the proportion of indeterminate results with current NAAT testing requiring re-sampling. Second, to determine whether a second NAAT such as Cepheid GeneXpert CT/NG assay (Cepheid, CA, USA) could be used on initial indeterminate samples to resolve indeterminate results, therefore reducing need for repeat sampling. During 2012, 144/21,931 (0.66%) samples were indeterminate for Neisseria gonorrhoeae, Chlamydia trachomatis or both, and a repeat sample was received in only 51.77% of patients with final results being delayed for more than 24 h. Over the next six months, there were 77/9472 (0.81%) indeterminate results. After an evaluation and introduction of the Cepheid assay, the number of indeterminate results fell to 9 (0.10%). Thus, use of the Cepheid assay significantly reduced indeterminate results, reduced reliance on a repeat sampling and significantly improved turnaround time, laboratory workflow and patient experience.

Evolution of drug resistance during 48 weeks of zidovudine/lamivudine/tenofovir in the absence of real-time viral load monitoring.

OBJECTIVES: To describe the resistance mutations selected by a first-line regimen of zidovudine/lamivudine/tenofovir in the absence of real-time viral load monitoring. DESIGN: A substudy of 300 participants from the Development of Antiretroviral Therapy in Africa trial in Uganda and Zimbabwe, which compared managing antiretroviral therapy with and without laboratory monitoring. METHODS: Stored plasma samples from selected time points were assayed retrospectively for HIV-1 RNA. The pol gene in all baseline samples and those with HIV RNA >1000 copies per milliliter at weeks 24 and 48 were sequenced. RESULTS: The proportion with HIV RNA >1000 copies per milliliter increased from 15% at 24 weeks to 24% at 48 weeks. Eighteen of 31 (58%) genotyped samples at 24 weeks had ≥ 1 major nucleoside reverse transcriptase inhibitor-associated mutations compared with 41 of 47 (87%) at 48 weeks. Excluding 1 nonadherent patient, a mean of 2.0 (95% confidence interval: 1.3 to 2.8) thymidine analogue mutations (TAMs) developed between weeks 24 and 48 among 14 patients with HIV RNA >1000 copies per milliliter at both time points. K65R was detected in 8 of 63 (13%) patients and was negatively associated with number of TAMs (P = 0.01) but not viral subtype (P = 0.30). CONCLUSIONS: A high rate of acquisition of TAMs, but not of K65R, among patients with prolonged viraemia was observed. However, most patients were virologically suppressed at 48 weeks, and long-term clinical and immunological outcomes in the Development of Antiretroviral Therapy in Africa trial were favorable.

Novel subtype C human immunodeficiency virus type 1 envelopes cloned directly from plasma: coreceptor usage and neutralization phenotypes.

Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.

Viral rebound and emergence of drug resistance in the absence of viral load testing: a randomized comparison between zidovudine-lamivudine plus Nevirapine and zidovudine-lamivudine plus Abacavir.

BACKGROUND: We investigated virological response and the emergence of resistance in the Nevirapine or Abacavir (NORA) substudy of the Development of Antiretroviral Treatment in Africa (DART) trial. METHODS: Six hundred symptomatic antiretroviral-naive human immunodeficiency virus (HIV)-infected adults (CD4 cell count, <200 cells/mm(3)) from 2 Ugandan centers were randomized to receive zidovudine-lamivudine plus abacavir or nevirapine. Virology was performed retrospectively on stored plasma samples at selected time points. In patients with HIV RNA levels >1000 copies/mL, the residual activity of therapy was calculated as the reduction in HIV RNA level, compared with baseline. RESULTS: Overall, HIV RNA levels were lower in the nevirapine group than in the abacavir group at 24 and 48 weeks (P < .001), although no differences were observed at weeks 4 and 12. Virological responses were similar in the 2 treatment groups for baseline HIV RNA level <100,000 copies/mL. The mean residual activity at week 48 was higher for abacavir in the presence of the typically observed resistance pattern of thymidine analogue mutations (TAMs) and M184V (1.47 log(10) copies/mL) than for nevirapine with M184V and nonnucleoside reverse-transcriptase inhibitor mutations, whether accompanied by TAMs (0.96 log(10) copies/mL) or not (1.18 log(10) copies/mL). CONCLUSIONS: There was more extensive genotypic resistance in both treatment groups than is generally seen in resource-rich settings. However, significant residual activity was observed among patients with virological failure, particularly those receiving zidovudine-lamivudine plus abacavir.

Tat mutations in an African cohort that do not prevent transactivation but change its immunogenic properties.

Humoral responses against extra-cellular HIV-1 Tat may be beneficial as Tat has been implicated in the viral pathogenesis associated with HIV-1 disease progression. We determined the levels of anti-Tat IgG in sera of HIV-1 seropositive individuals from the Rural Clinical Cohort in Uganda using nine different Tat proteins representative of the major subtypes presently accounting for 97% of infections worldwide. We observed the presence of anti-Tat IgG able to react against the various subtypes tested, although none cross-reacted against all nine variants. We show that 46.25% of seropositive patients were able to recognise at least one Tat variant with 1:1000 sera dilution. We also show that the C terminus of Tat is the most variable region and an important epitope that might explain the limitation of cross-recognition of Tat antibodies regarding Tat variants. This study shows in seropositive patients that Tat can tolerate mutations without modification of its primary function but with changes in its immunogenic properties. These findings should be considered when designing Tat-based HIV-1 vaccines.

Relation between chemokine receptor use, disease stage, and HIV-1 subtypes A and D: results from a rural Ugandan cohort.

OBJECTIVES: To determine whether there are differences in coreceptor use in subjects infected with HIV-1 envelope subtypes A and D that could explain the differences in progression rates between these subtypes in a rural Ugandan cohort. METHODS: HIV-1 was subtyped in env by V3 sequencing or heteroduplex mobility assay. Coreceptor use was determined by the ability of the isolates to replicate in U87 CD4 cells expressing different coreceptors. The Fisher exact test was used to examine the relation between coreceptor use and subtype, clinical stage, and V3 charge. The Kruskall-Wallis nonparametric test was used to examine the association between median CD4 cell counts, coreceptor use, and subtype. Logistic regression was used to examine predicted coreceptor use at different CD4 groupings. RESULTS: Isolates from 66 participants were analyzed. Thirty-one were infected with subtype A, and 35 were infected with subtype D. Although this work was based on a small sample size, we found statistically significant differences. The probability of having an X4 virus was higher in subtype D infections than in subtype A infections among those with a non-AIDS clinical status (Fisher exact test, P = 0.040). Logistic regression analysis, in which we predicted X4 use by subtype and stratified by CD4 group, confirmed these findings among those with a CD4 count >200 cells/microL (likelihood ratio test, P = 0.003). R5 viruses were associated with higher median CD4 cell counts than X4 or X4/R5 (Kruskall-Wallis test, P = 0.0045). A V3 charge of +5 and greater was highly associated with X4 virus (Fisher exact test, P = 0.006). CONCLUSIONS: These subtype differences in coreceptor use may partially explain the faster progression rates we have previously reported in individuals infected with subtype D compared with subtype A. Our observations may have implications for the future use of coreceptor inhibitors in this population.

Genotypic variation in the pol gene of HIV type 1 in an antiretroviral treatment-naive population in rural southwestern Uganda.

The majority of studies of HIV-1 drug resistance have involved subtype B viruses. Here we have characterized subtype distribution and determined the levels of polymorphism at protease (PR) and reverse transcriptase (RT) drug resistance positions, in antiretroviral treatment-naive HIV-positive Ugandan patients. We have also investigated codon usage variability at these positions and assessed intersubtype recombination within the pol gene. The study population consisted of 187 patients, from a cohort established by the UK Medical Research Council Programme on AIDS in Uganda in 1990. Results indicate that 28.3% of patients were infected with subtype A (n = 53), 64.2% subtype D (n = 120), 6.4% A/D recombinant (n = 12), and 1.1% subtype C (n = 2). Variation in amino acid usage at drug resistance-associated positions was minimal between the two main subtypes (A and D) in RT, but there was appreciable variation in PR. Codon usage, however, was considerably more variable between subtypes A and D in both PR and RT. Thus, while no natural high-level resistance to antiretroviral therapy was detected in this cohort, subtypes A and D may possess different genetic barriers to be overcome in order to achieve resistance. With the increasing introduction of antiretroviral therapy into Africa, such information will be vital in our understanding and evaluation of the development of drug resistance as it occurs, and how to interpret resistance data the type of which has rarely previously been seen. This analysis also significantly increases the number of Ugandan PR and RT sequences characterized to date.

Full-length HIV-1 Tat protein necessary for a vaccine.

AIDS vaccines now use a truncated version of 86 residues of the Tat protein related to the HIV-1 HXB2 strain predominant in Europe and North America. We compared antibodies raised in rabbits using a B subtype short Tat HXB2(86) and a full-length Tat HXB2(100). Serum against HXB2(86) recognizes only B and D subtypes while serum against HXB2(100) recognizes B, D, and C subtype variants. Conformational epitopes appear to be involved in the capacity of anti-Tat HXB2 sera to recognized non-homologous Tat variants. A linear B-epitope identified in sequence 71-81 in HXB2(86) disappears in HXB2(100), which has a new linear B-epitope identified at the C-terminus. Anti-HXB2(100) serum has a higher titer in neutralizing antibody against homologous and non-homologous variants compared to anti-HXB2(86) serum. We suggest that a Tat vaccine should contain a Tat variant with regular size, up to 99-101 residues now found in the field.

The glutamine-rich region of the HIV-1 Tat protein is involved in T-cell apoptosis.

Human immunodeficiency virus (HIV) infection and the progression to AIDS are characterized by the depletion of CD4(+) T-cells. HIV-1 infection leads to apoptosis of uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated, in part, by the HIV-1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells. We chemically synthesized two 86-residue subtype D Tat proteins, Ug05RP and Ug11LTS, from two Ugandan patients who were clinically categorized as either rapid progressor or long-term survivor, with non-conservative mutations located essentially in the glutamine-rich region. Structural heterogeneities were revealed by CD, which translate into differing trans-activational and apoptotic effects. CD data analysis and molecular modeling indicated that the short alpha-helix observed in subtype D Tat proteins from rapid progressor patients such as Tat Mal and Tat Ug05RP is not present in Ug11LTS. We show that Tat Ug05RP is more efficient than Tat Ug11LTS in its trans-activational role and in inducing apoptosis in binding tubulin via the mitochondrial pathway. The glutamine-rich region of Tat appears to be involved in the Tat-mediated apoptosis of T-cells.

HIV-1 subtype dynamics over 10 years in a rural Ugandan cohort.

Our objective was to monitor changes in the subtypes of HIV-1 infecting a rural Ugandan cohort where the spread of HIV-1 is by unprotected heterosexual contact and subtypes A and D predominate. Should one subtype be better able to spread we would anticipate a rise in incidence of one subtype at the expense of the other over a decade of study. We employed a natural history cohort, which had been established by the Medical Research Council in 1990 and subtyped virus from 90% (139) incident cases by DNA sequencing in two separate genes. We found that viral subtype had no predilection for males, females or age at infection and that between 1990 and 2000 there was no significant change in the relative number of different subtypes. The only significant trend was a reduction in the proportion of viruses classified as recombinant. This may reflect the overall decline in prevalence of HIV-1 in Uganda over this period.

Tat HIV-1 primary and tertiary structures critical to immune response against non-homologous variants.

Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that mutations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzyme-linked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.

Inter- and intra-genic intersubtype HIV-1 recombination in rural and semi-urban Uganda.

OBJECTIVE: To investigate the number and variety of viruses with discrepant subtypes between env and gag and within gag in two cohorts in Uganda. METHODS: Sequences were generated from PCR products amplified directly (without cloning) from patient blood and compared in the v3/v4 region of env and the p17 and p24 regions of gag to reference subtype strains by phylogenetic analysis. Gag sequences with a discrepant subtype between p17 and p24 were analysed further to indicate approximate sites of recombination. RESULTS: Envelope subtypes D and A were predominant, but subtypes B, C and G were also found. From analysis of three short regions of the HIV genome we found 15 different combinations of subtype assortment, including 11 different recombinant permutations. Approximately 30% of viruses (29/104) in this part of Uganda appear to be recombinants between the env and gag genes and 10% (11/104) are recombinant within the gag gene. There was no clear pattern of crossover points within the gag gene. There seems to be no evidence of new circulating recombinant forms. CONCLUSION: Both inter-genic and intra-genic inter-subtype recombination appear to be a relatively common occurrence in this geographical region where two subtypes of virus co-circulate. These results have implications for cross-clade vaccine design.

Injecting drug users in Bangladesh: prevalence of syphilis, hepatitis, HIV and HIV subtypes.

Injecting drug users (IDU) were enrolled from two detoxification clinics and two needle/syringe exchange programmes (NEP) in central and northwest Bangladesh. Syphilis, hepatitis C and HIV rates were highest in IDU from the NEP of central Bangladesh (23, 66.5 and 1.4%, respectively), whereas current hepatitis B infection rates were highest in IDU from the NEP of northwest Bangladesh (12%). Five HIV strains were subtype C and one E/B. The 32 base pair (bp) deletion of the CCR5 gene was not detected.