Comparison of a clinical prediction rule and a LAM antigen-detection assay for the rapid diagnosis of TBM in a high HIV prevalence setting

Background/Objective

The diagnosis of tuberculous meningitis (TBM) in resource poor TB endemic environments is challenging. The accuracy of current tools for the rapid diagnosis of TBM is suboptimal. We sought to develop a clinical-prediction rule for the diagnosis of TBM in a high HIV prevalence setting, and to compare performance outcomes to conventional diagnostic modalities and a novel lipoarabinomannan (LAM) antigen detection test (Clearview-TB®) using cerebrospinal fluid (CSF).

Methods

Patients with suspected TBM were classified as definite-TBM (CSF culture or PCR positive), probable-TBM and non-TBM.

Results

Of the 150 patients, 84% were HIV-infected (median [IQR] CD4 count = 132 [54; 241] cells/µl). There were 39, 55 and 54 patients in the definite, probable and non-TBM groups, respectively. The LAM sensitivity and specificity (95%CI) was 31% (17;48) and 94% (85;99), respectively (cut-point ≥0.18). By contrast, smear-microscopy was 100% specific but detected none of the definite-TBM cases. LAM positivity was associated with HIV co-infection and low CD4 T cell count (CD4<200 vs. >200 cells/µl; p = 0.03). The sensitivity and specificity in those with a CD4<100 cells/µl was 50% (27;73) and 95% (74;99), respectively. A clinical-prediction rule ≥6 derived from multivariate analysis had a sensitivity and specificity (95%CI) of 47% (31;64) and 98% (90;100), respectively. When LAM was combined with the clinical-prediction-rule, the sensitivity increased significantly (p<0.001) to 63% (47;68) and specificity remained high at 93% (82;98).

Conclusions

Despite its modest sensitivity the LAM ELISA is an accurate rapid rule-in test for TBM that has incremental value over smear-microscopy. The rule-in value of LAM can be further increased by combination with a clinical-prediction rule, thus enhancing the rapid diagnosis of TBM in HIV-infected persons with advanced immunosuppression.     

Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.There is broad heterogeneity in the ability of HIV-infected individuals to control virus replication, ranging from elite controllers, who maintain undetectable viral loads without treatment, to rapid progressors, who progress to AIDS within 2 years of infection (9, 22, 32). Many interrelated factors, including host and viral genetic factors involved in antiviral immunity and the viral life cycle, may partially account for the differences in the course of disease progression (10, 11, 30, 41). The complex interplay between host genetic factors and viral factors is exemplified by human leukocyte antigen (HLA) class I-restricted cytotoxic T-lymphocyte (CTL) responses, which exert considerable immune pressure on the virus, resulting in escape mutations that affect the interaction of viral and host proteins, thereby influencing infection outcome.The exact mechanisms by which some HLA class I alleles, such as HLA-B*57 and HLA-B*27, are associated with slower progression to AIDS, while others, such as B*5802 and B*18, are associated with accelerated disease progression (6, 20, 42), are unclear. The magnitude and/or breadth of HLA-restricted CTL responses to the conserved Gag protein has been correlated inversely with disease progression or markers of disease progression in several studies (12, 21, 28, 31, 35, 43, 46), although there are some exceptions (4, 16, 37), while preferential targeting of the highly variable envelope protein (as occurs in HLA-B*5802-positive individuals) correlates with higher viral loads (21, 29). Protective HLA alleles restrict CTL responses that impose a strong selection pressure on a few specific Gag p24 epitopes, resulting in escape mutations (14) for which fitness costs have been demonstrated either through site-directed mutations introduced into a reference strain background (2, 8, 25, 38) or through in vivo reversion of these mutations after transmission to an HLA-mismatched individual (8, 24). Recent evidence suggests that Gag escape mutations with a fitness cost, particularly those in p24, are a significant determinant of disease progression: the transmitted number of HLA-B-associated polymorphisms in Gag was found to significantly impact the viral set point in recipients (although an associated fitness cost was not shown) (7, 15), and in a small number of infants, decreased fitness of the transmitted virus with HLA-B*5703/5801-selected mutations in Gag p24 epitopes resulted in slower disease progression (33, 39). Also, the number of reverting Gag mutations (thought to revert as a consequence of fitness costs) associated with individual HLA-B alleles was strongly correlated with the HLA-linked viral set point in chronically infected patients (26). A recent in vitro study showed that HLA-associated variation in Gag-protease, with resulting reduced replication capacity, may contribute to viral control in HIV-1 subtype B-infected elite controllers (27). Taken together, these studies suggest that CTL responses restricted by favorable HLA alleles select for escape mutations in conserved epitopes, particularly those in Gag, resulting in a fitness cost to HIV and therefore at least partly explaining the slower disease progression in individuals carrying these alleles.To date, many of the studies investigating the fitness cost of Gag escape mutations and their clinical relevance have concentrated on escape mutations associated with protective HLA alleles, have not assessed fitness consequences in the natural sequence background (in the presence of other escape and compensatory mutations), and/or have focused on a limited number of patients. Most importantly, the majority of studies have focused on HIV-1 subtype B. The present study is the first to use a large population-based approach and clinically derived Gag-protease sequences to investigate comprehensively the relationships between immune-driven sequence variation in Gag, viral replication capacity, and markers of disease progression in chronic infection with HIV-1 subtype C, the most predominant subtype in the epidemic. We assayed the replication capacity of recombinant viruses encoding patient Gag-protease in an HIV-1-inducible green fluorescent protein (GFP) reporter cell line and found associations between lower replication capacities, protective HLA alleles, protective HLA-associated mutations, lower baseline viral loads, and higher baseline CD4 counts. However, Gag-protease replication capacity did not correlate with the subsequent rate of CD4 decline.     

HLA-Cw*03-Restricted CD8+ T-Cell Responses Targeting the HIV-1 Gag Major Homology Region Drive Virus Immune Escape and Fitness Constraints Compensated for by Intracodon Variation

The potential importance of HLA-C-restricted CD8⁺ cytotoxic T lymphocytes (CTL) in HIV infection remains undetermined. We studied the dominant HLA-Cw*03-restricted CTL response to YVDRFFKTL_296-304 (YL9), within the conserved major homology region (MHR) of the Gag protein, in 80 HLA-Cw*03-positive individuals with chronic HIV infection to better define the efficacy of the YL9 HLA-C-restricted response. The HLA-Cw*03 allele is strongly associated with HIV sequence changes from Thr-303 to Val, Ile, or Ala at position 8 within the YL9 epitope (P = 1.62 × 10⁻¹⁰). In vitro studies revealed that introduction of the changes T303I and T303A into the YL9 epitope both significantly reduced CTL recognition and substantially reduced the viral replicative capacity. However, subsequent selection of the Val-303 variant, via intracodon variation from Ile-303 (I303V) or Ala-303 (A303V), restored both viral fitness and CTL recognition, as supported by our in vivo data. These results illustrate that HLA-C-restricted CTL responses are capable of driving viral immune escape within Gag, but in contrast to what was previously described for HLA-B-restricted Gag escape mutants, the common Cw*03-Gag-303V variant selected resulted in no detectable benefit to the host.Human leukocyte antigen (HLA) class I is the most polymorphic region of the human genome. HLA class I genes are found at the A, B, and C loci of chromosome 6 and have been shown to play an important role in control of infections by intracellular pathogens (3). Of these three loci, HLA-B has had many more unique molecules identified than has HLA-A or -C (http://www.anthonynolan.org.uk/HIG/index.html). This locus variability is likely to reflect functional differences among HLA-B alleles and disease progression (20). In the context of HIV infection, several studies have demonstrated that HLA-B alleles have the greatest impact on HIV-1 replication control (7, 22). The reason why particular HLA-B, and not HLA-A or -C, alleles have been associated with improved outcome in HIV is unknown but may be explained by the numbers and regions of viral proteomes presented in the context of HLA-B molecules (19, 23, 46).Regardless of the importance of HLA-B-restricted responses in HIV control, the roles of the HLA-A and -C class I alleles have not been fully investigated. A genomewide analysis investigating single-nucleotide polymorphisms (SNP) has associated both the presence of certain HLA-B alleles and a dimorphism upstream of the HLA-C gene (−35C/T) as two of the strongest predictors of the plasma viral load in HIV infection (14). In addition, the −35 SNP has recently been associated with levels of HLA-C expression and may indicate a new role for HLA-C alleles in HIV-1 control (40).One reason that has been postulated to explain the lack of HLA-C association with immune pressure is the lower expression of HLA-C on the cell surface (18, 36, 37). However, HLA-C, unlike HLA-A and HLA-B, is not downregulated by the Nef protein, and this factor, therefore, may explain the lower expression level (9).Analyses of large numbers of HIV sequences have identified associations between particular HLA-C polymorphisms in the HIV pol gene and the set point viral load (27). These polymorphisms were within or in close proximity to defined HLA-C-restricted epitopes, suggesting that HLA-C-restricted responses could be driving HIV evolution (22, 27). Confirmation that all HLA-C-restricted epitopes are not fundamentally ineffective was recently provided by a study of an HLA-Cw*01-restricted epitope targeted to p15 Gag, which was found to drive selection of a 3-amino-acid insertion in a HIV-infected patient (6). Additionally, in vitro studies have demonstrated antiviral activities of HLA-C-restricted cytotoxic T lymphocytes (CTL) clones against HIV comparable to other HLA-B-restricted responses (2). In spite of all these data, the contribution of HLA-C responses to HIV control is still unknown.Our study was undertaken to address the role of an HLA-Cw*0303/0304-restricted response to YVDRFFKTL_296-304 (YL9) in HIV infection. This response was of particular interest due to its dominance and because it targets a highly conserved region of the Gag protein among different retroviruses, namely, the major homology region (MHR). In the study cohort of 778 individuals with chronic HIV infection, 80 were found to carry Cw*0303/0304. Gag viral sequences were obtained from HLA-Cw*0303/0304 subjects and used to identify and define HIV immune escape driven by the YL9-especific responses. We then studied the impacts of these mutations on viral recognition by CD8⁺ T cells and on virus replicative capacity (RC).     

Additive Contribution of HLA Class I Alleles in the Immune Control of HIV-1 Infection

Previous studies have identified a central role for HLA-B alleles in influencing control of HIV infection. An alternative possibility is that a small number of HLA-B alleles may have a very strong impact on HIV disease outcome, dominating the contribution of other HLA alleles. Here, we find that even following the exclusion of subjects expressing any of the HLA-B class I alleles (B*57, B*58, and B*18) identified to have the strongest influence on control, the dominant impact of HLA-B alleles on virus set point and absolute CD4 count variation remains significant. However, we also find that the influence of HLA on HIV control in this C-clade-infected cohort from South Africa extends beyond HLA-B as HLA-Cw type remains a significant predictor of virus and CD4 count following exclusion of the strongest HLA-B associations. Furthermore, there is evidence of interdependent protective effects of the HLA-Cw*0401-B*8101, HLA-Cw*1203-B*3910, and HLA-A*7401-B*5703 haplotypes that cannot be explained solely by linkage to a protective HLA-B allele. Analysis of individuals expressing both protective and detrimental alleles shows that even the strongest HLA alleles appear to have an additive rather than dominant effect on HIV control at the individual level. Finally, weak but significant frequency-dependent effects in this cohort can be detected only by looking at an individual''s combined HLA allele frequencies. Taken together, these data suggest that although individual HLA alleles, particularly HLA-B, can have a strong impact, HIV control overall is likely to be influenced by the additive effect of some or all of the other HLA alleles present.HIV-specific CD8⁺ T cells play a central role in resolution of primary viremia and the long-term suppression of viral replication (13). Supporting this notion is the observed correlation between possession of particular human leukocyte antigen (HLA) class I alleles and control of HIV, measured both directly by time-to-AIDS (5, 6) and indirectly via clinical markers of disease progression (viral load [VL] and CD4 count) (15, 26, 28). Specific HLA class I alleles have been associated with relatively successful control of viral replication and slow disease progression, most notably, alleles HLA-B*57 and HLA-B*27 (1, 7, 12, 15, 21, 23), and also with relatively ineffective control of viral replication and rapid disease progression [B*35(Px), B*5802, and B*18] (5, 15, 17, 23). In addition, general trends suggesting an HLA class I heterozygote advantage (5) and rare allele advantage (28) and, most recently, a correlation between levels of surface expression linked to certain HLA-Cw alleles (11, 27) and HIV control has also been described.Among the different HLA class I loci, the HIV-specific CD8⁺ T-cell responses restricted by HLA-B alleles are thought to play the central role in determining disease outcome: the majority of detectable HIV-specific CD8⁺ T-cell responses are restricted by HLA-B alleles (3, 15, 16), HLA-B-restricted responses typically express a more effective “polyfunctional” phenotype (14), the strongest HLA-associations with either slow or rapid progression are with HLA-B alleles (5, 10, 11, 15), and HLA-B-restricted CD8⁺ T cells exert the strongest selection pressure on the virus (15, 19, 24). However, whether this apparent association between HIV immune control and HLA-B is a general and causal trend or, rather, is biased by the coincidence that the strongest HLA associations with either extreme of disease control happen, by chance, to involve HLA-B alleles remains uncertain.In order to further investigate the correlation between HLA type and HIV infection control, we here examine a cohort now comprising >1,200 chronically HIV C-clade-infected, treatment-naïve subjects from Durban, South Africa, in an extended analysis following from our previous studies of a smaller cohort (15). We first address the question of whether the dominant role of HLA-B in this population compared to the roles of HLA-A or HLA-C results from the influence of HLA-B alleles in general or is dependent on a few known strong associations, such as that between HLA-B*57 alleles and low viremia. Second, in light of recent data (11, 27), we assess the impact of HLA-C alleles on HIV disease outcome and examine the effect of HLA haplotypes on observed HLA associations with disease control. Third, we investigate the question of whether the impact of certain HLA-B alleles on HIV outcome dominates that of other HLA-B alleles to negate the contribution of the latter or whether the impact of individual HLA alleles can be additive. Finally, we compare the impact of individual HLA alleles on HIV on immune control to the impact of heterozygote and rare allele advantage in this cohort.     

<Emphasis Type="Italic">HLArestrictor</Emphasis>—a tool for patient-specific predictions of HLA restriction elements and optimal epitopes within peptides

Traditionally, T cell epitope discovery requires considerable amounts of tedious, slow, and costly experimental work. During the last decade, prediction tools have emerged as essential tools allowing researchers to select a manageable list of epitope candidates to test from a larger peptide, protein, or even proteome. However, no current tools address the complexity caused by the highly polymorphic nature of the restricting HLA molecules, which effectively individualizes T cell responses. To fill this gap, we here present an easy-to-use prediction tool named HLArestrictor (), which is based on the highly versatile and accurate NetMHCpan predictor, which here has been optimized for the identification of both the MHC restriction element and the corresponding minimal epitope of a T cell response in a given individual. As input, it requires high-resolution (i.e., 4-digit) HLA typing of the individual. HLArestrictor then predicts all 8–11mer peptide binders within one or more larger peptides and provides an overview of the predicted HLA restrictions and minimal epitopes. The method was tested on a large dataset of HIV IFNγ ELIspot peptide responses and was shown to identify HLA restrictions and minimal epitopes for about 90% of the positive peptide/patient pairs while rejecting more than 95% of the negative peptide-HLA pairs. Furthermore, for 18 peptide/HLA tetramer validated responses, HLArestrictor in all cases predicted both the HLA restriction element and minimal epitope. Thus, HLArestrictor should be a valuable tool in any T cell epitope discovery process aimed at identifying new epitopes from infectious diseases and other disease models.     

Serological evidence of single and mixed infections of Rift Valley fever virus,Brucella spp. and Coxiella burnetii in dromedary camels in Kenya

Camels are increasingly becoming the livestock of choice for pastoralists reeling from effects of climate change in semi-arid and arid parts of Kenya. As the population of camels rises, better understanding of their role in the epidemiology of zoonotic diseases in Kenya is a public health priority. Rift Valley fever (RVF), brucellosis and Q fever are three of the top priority diseases in the country but the involvement of camels in the transmission dynamics of these diseases is poorly understood. We analyzed 120 camel serum samples from northern Kenya to establish seropositivity rates of the three pathogens and to characterize the infecting Brucella species using molecular assays. We found seropositivity of 24.2% (95% confidence interval [CI]: 16.5–31.8%) for Brucella, 20.8% (95% CI: 13.6–28.1%) and 14.2% (95% CI: 7.9–20.4%) for Coxiella burnetii and Rift valley fever virus respectively. We found 27.5% (95% CI: 19.5–35.5%) of the animals were seropositive for at least one pathogen and 13.3% (95% CI: 7.2–19.4%) were seropositive for at least two pathogens. B. melitensis was the only Brucella spp. detected. The high sero-positivity rates are indicative of the endemicity of these pathogens among camel populations and the possible role the species has in the epidemiology of zoonotic diseases. Considering the strong association between human infection and contact with livestock for most zoonotic infections in Kenya, there is immediate need to conduct further research to determine the role of camels in transmission of these zoonoses to other livestock species and humans. This information will be useful for designing more effective surveillance systems and intervention measures.     

Knowledge of HIV Serodiscordance,Transmission, and Prevention among Couples in Durban,South Africa

Objective

Couples’ voluntary HIV counseling and testing (CVCT) significantly decreases HIV transmission within couples, the largest risk group in sub-Saharan Africa, but it is not currently offered in most HIV testing facilities. To roll out such an intervention, understanding locale-specific knowledge barriers is critical. In this study, we measured knowledge of HIV serodiscordance, transmission, and prevention before and after receipt of CVCT services in Durban.

Design

Pre- and post-CVCT knowledge surveys were administered to a selection of individuals seeking CVCT services.

Methods

Changes in knowledge scores were assessed with McNemar Chi-square tests for balanced data and generalized estimating equation methods for unbalanced data.

Results

The survey included 317 heterosexual black couples (634 individuals) who were primarily Zulu (87%), unemployed (47%), and had at least a secondary level education (78%). 28% of couples proved to be discordant. Only 30% of individuals thought serodiscordance between couples was possible pre‐CVCT compared to 95% post-CVCT. One-third thought there was at least one benefit of CVCT pre‐CVCT, increasing to 96% post‐CVCT. Overall, there were positive changes in knowledge about HIV transmission and prevention. However, many respondents thought all HIV positive mothers give birth to babies with AIDS (64% pre-CVCT, 59% post-CVCT) and that male circumcision does not protect negative men against HIV (70% pre-CVCT, 67% post-CVCT).

Conclusions

CVCT was well received and was followed by improvements in understanding of discordance, the benefits of joint testing, and HIV transmission. Country-level health messaging would benefit from targeting gaps in knowledge about serodiscordance, vertical transmission, and male circumcision.     

Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression.     

Linking Human Health and Livestock Health: A “One-Health” Platform for Integrated Analysis of Human Health,Livestock Health,and Economic Welfare in Livestock Dependent Communities

Background

For most rural households in sub-Saharan Africa, healthy livestock play a key role in averting the burden associated with zoonotic diseases, and in meeting household nutritional and socio-economic needs. However, there is limited understanding of the complex nutritional, socio-economic, and zoonotic pathways that link livestock health to human health and welfare. Here we describe a platform for integrated human health, animal health and economic welfare analysis designed to address this challenge. We provide baseline epidemiological data on disease syndromes in humans and the animals they keep, and provide examples of relationships between human health, animal health and household socio-economic status.

Method

We designed a study to obtain syndromic disease data in animals along with economic and behavioral information for 1500 rural households in Western Kenya already participating in a human syndromic disease surveillance study. Data collection started in February 2013, and each household is visited bi-weekly and data on four human syndromes (fever, jaundice, diarrhea and respiratory illness) and nine animal syndromes (death, respiratory, reproductive, musculoskeletal, nervous, urogenital, digestive, udder disorders, and skin disorders in cattle, sheep, goats and chickens) are collected. Additionally, data from a comprehensive socio-economic survey is collected every 3 months in each of the study households.

Findings

Data from the first year of study showed 93% of the households owned at least one form of livestock (55%, 19%, 41% and 88% own cattle, sheep, goats and chickens respectively). Digestive disorders, mainly diarrhea episodes, were the most common syndromes observed in cattle, goats and sheep, accounting for 56% of all livestock syndromes, followed by respiratory illnesses (18%). In humans, respiratory illnesses accounted for 54% of all illnesses reported, followed by acute febrile illnesses (40%) and diarrhea illnesses (5%). While controlling for household size, the incidence of human illness increased 1.31-fold for every 10 cases of animal illness or death observed (95% CI 1.16–1.49). Access and utilization of animal source foods such as milk and eggs were positively associated with the number of cattle and chickens owned by the household. Additionally, health care seeking was correlated with household incomes and wealth, which were in turn correlated with livestock herd size.

Conclusion

This study platform provides a unique longitudinal dataset that allows for the determination and quantification of linkages between human and animal health, including the impact of healthy animals on human disease averted, malnutrition, household educational attainment, and income levels.