Impact of HLA selection pressure on HIV fitness at a population level in Mexico and Barbados.

UNLABELLED: Previous studies have demonstrated that effective cytotoxic T lymphocyte (CTL) responses drive the selection of escape mutations that reduce viral replication capacity (VRC). Escape mutations, including those with reduced VRC, can be transmitted and accumulate in a population. Here we compared two antiretroviral therapy (ART)-naive HIV clade B-infected cohorts, in Mexico and Barbados, in which the most protective HLA alleles (HLA-B*27/57/58:01/81:01) are differentially expressed, at 8% and 34%, respectively. Viral loads were significantly higher in Mexico than in Barbados (median, 40,774 versus 14,200; P < 0.0001), and absolute CD4(+) T-cell counts were somewhat lower (median, 380/mm(3) versus 403/mm(3); P = 0.007). We tested the hypothesis that the disparate frequencies of these protective HLA alleles would be associated with a higher VRC at the population level in Mexico. Analysis of VRC in subjects in each cohort, matched for CD4(+) T-cell count, revealed that the VRC was indeed higher in the Mexican cohort (mean, 1.13 versus 1.03; P = 0.0025). Although CD4 counts were matched, viral loads remained significantly higher in the Mexican subjects (P = 0.04). This VRC difference was reflected by a significantly higher frequency in the Barbados cohort of HLA-B*27/57/58:01/81:01-associated Gag escape mutations previously shown to incur a fitness cost on the virus (P = 0.004), a difference between the two cohorts that remained statistically significant even in subjects not expressing these protective alleles (P = 0.01). These data suggest that viral set points and disease progression rates at the population level may be significantly influenced by the prevalence of protective HLA alleles such as HLA-B*27/57/58:01/81:01 and that CD4 count-based guidelines to initiate antiretroviral therapy may need to be modified accordingly, to optimize the effectiveness of treatment-for-prevention strategies and reduce HIV transmission rates to the absolute minimum. IMPORTANCE: Immune control of HIV at an individual level is strongly influenced by the HLA class I genotype. HLA class I molecules mediating effective immune control, such as HLA-B*27 and HLA-B*57, are associated with the selection of escape mutants that reduce viral replicative capacity. The escape mutants selected in infected patients can be transmitted and affect the viral load and CD4 count in the recipient. These findings prompt the hypothesis that the frequency of protective alleles in a population may affect viral set points and rates of disease progression in that population. These studies in Mexico and Barbados, where the prevalence rates of protective HLA alleles are 8% and 34%, respectively, support this hypothesis. These data suggest that antiretroviral therapy (ART) treatment-for-prevention strategies will be less successful in populations such as those in Mexico, where viral loads are higher for a given CD4 count. Consideration may therefore usefully be given to ART initiation at higher absolute CD4 counts in such populations to optimize the impact of ART for prevention.

Adaptation of HIV-1 to human leukocyte antigen class I.

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Improving Laboratory Efficiency in the Caribbean to Attain the World Health Organization HIV Treat All Recommendations.

Scientific evidence showing the benefits of early initiation of antiretroviral therapy (ART) prompted World Health organization (WHO) to recommend that all persons diagnosed as HIV positive should commence ART irrespective of CD4 count and disease progression. Based on this recommendation, countries should adopt and implement the HIV "Treat All" policy to achieve the UNAIDS 90-90-90 targets and ultimately reach epidemic control. Attaining this goal along the HIV treatment cascade depends on the laboratory to monitor progress and measure impact. The laboratory plays an important role in HIV diagnosis to attain the first 90 and in viral load (VL) and HIV drug resistance testing to reinforce adherence, improve viral suppression, and measure the third 90. Countries in the Caribbean region have endorsed the WHO HIV "Treat all" recommendation; however, they are faced with diminishing financial resources to support laboratory testing, seen as a rate-limiting factor to achieving this goal. To improve laboratory coverage with fewer resources in the Caribbean there is the need to optimize laboratory operations to ensure the implementation of high quality, less expensive evidence-based approaches that will result in more efficient and effective service delivery. Suggested practical and innovative approaches to achieve this include: (1) targeted testing within HIV hotspots; (2) strengthening sample referral systems for VL; (3) better laboratory data collection systems; and (4) use of treatment cascade data for programmatic decision-making. Furthermore, strengthening quality improvement and procurement systems will minimize diagnostic errors and guarantee a continuum of uninterrupted testing which is critical for routine monitoring of patients to meet the stated goal.

Evaluation of two enzyme-linked immunosorbent assay methods for detection of immunoglobulin M antibodies in acute leptospirosis.

Leptospirosis is a common zoonosis of worldwide distribution. Diagnosis of leptospirosis is usually accomplished by serology, but the microscopic agglutination test (MAT) generally requires paired sera for detection of seroconversion and is considered too complex for routine use. A number of rapid assays have been developed in recent years. In the present study, 2 immunoglobulin (Ig) M enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the early diagnosis of acute leptospirosis in Barbados. A total of 103 patients admitted to the Queen Elizabeth Hospital for diagnosis of suspected leptospirosis were investigated. A case of leptospirosis was confirmed by a 4-fold rise in titer between 2 sera tested by MAT, an initial titer of > or = 800 in the MAT, or by isolation of leptospires from blood or urine. A total of 48 cases of leptospirosis were confirmed. In 33 cases, both commercial assays were positive in the first sample, taken at admission, a mean of 6.7 days after onset of symptoms, whereas seroconversion was detected in a further 9 cases. Both assays were negative in 5 cases, and the remaining case gave discordant results in the 2 assays. False-positive IgM results were detected in 4 patients without leptospirosis. The sensitivity of the 2 assays was 89.6 and 97.5%, respectively, and specificities were 92.7 and 96.4%, respectively. The positive predictive values were 87.8 and 95.5%, and the negative predictive values were 90.7 and 89.5%, respectively. Either of these assays can be used for early diagnosis of leptospirosis, particularly in laboratories that cannot perform more specialized leptospiral serology.

Strengthening national laboratory health systems in the Caribbean Region.

The President's Emergency Plan for AIDS Relief (PEPFAR) programme for the Caribbean Region was established in 2008 to address health system challenges, including fragile laboratory services and systems. The laboratory component of this programme consisted of several phases: assessment of laboratory needs of all 12 countries engaged in the programme; addressing gaps identified during the assessment; and monitoring and evaluation of the progress achieved. After one year of PEPFAR collaboration with national governments and other partners, laboratory services and systems greatly improved. Some of the milestones include: (1) the accreditation of a public laboratory; (2) improved access to HIV diagnosis with faster turnaround time; (3) establishment of capacity for platforms for DNA PCR, viral load and HIV drug resistance; (4) development of the laboratory workforce; and (5) establishment of a framework for implementation of sustainable quality management systems for laboratory accreditation. The progress recorded in strengthening laboratory health systems after one year of initiating this collaboration shows that with a rigorous initial assessment, programme design and intervention and strategic partnership, national laboratory health systems can be greatly enhanced to support programme implementation. Continued collaboration and country leadership is critical to create an integrated and sustainable laboratory network in the Caribbean.

Performance of the Panleucogating protocol for CD4+ T cell enumeration in an HIV dedicated laboratory facility in Barbados.

OBJECTIVE: To compare the Panleucogating (PLG) protocol with the routinely used four-color protocol for CD4+ T cell count enumeration. DESIGN AND METHODS: One hundred fifty-three blood samples were randomly selected from samples received at the National HIV Laboratory for routine immunological monitoring. Samples were prepared using Coulter CYTO-STAT tetraCHROME monoclonal antibodies and FlowCARE PLG CD4 reagent for four-color and PLG, respectively, and analyzed on the Beckman Coulter EPICS XL flow cytometer. The PLG protocol used a sequential gating strategy where CD4+ T cells were identified using side scatter properties of cells and CD45 staining. The four-color protocol used CD45 and CD3 to identify CD4+ T cells. RESULTS: Absolute CD4+ T cell counts and percentages ranged from 4 to 1,285 cells/microL and 0.9 to 46.7%, respectively. Linear regression analyses revealed good correlation of PLG with the four-color protocol (absolute counts, R2 = 0.95; percentages, R2 = 0.98) over the entire range including the clinically relevant range. Bland Altman statistics revealed no bias for CD4 counts <500 cells/microL and a slight underestimation by PLG for counts >500 cells/microL (Bias = -32.7 cells/microL; 95% agreement limits = -151.3- +86.0). CD4+ T cell percentages were the similar over the entire range (Bias = 0.6%; 95% agreement limits = -1.97 +/- 3.18). CONCLUSIONS: PLG is an accurate method for enumerating CD4+ T cells and has resulted in major cost savings to the Government of Barbados. This has implications for the sustainability of the National HIV containment program in Barbados and the other resource limited Caribbean countries. The PLG technique is now being routinely used in Barbados.