Towards Eliminating Bias in Cluster Analysis of TB Genotyped Data

The relative contributions of transmission and reactivation of latent infection to TB cases observed clinically has been reported in many situations, but always with some uncertainty. Genotyped data from TB organisms obtained from patients have been used as the basis for heuristic distinctions between circulating (clustered strains) and reactivated infections (unclustered strains). Naïve methods previously applied to the analysis of such data are known to provide biased estimates of the proportion of unclustered cases. The hypergeometric distribution, which generates probabilities of observing clusters of a given size as realized clusters of all possible sizes, is analyzed in this paper to yield a formal estimator for genotype cluster sizes. Subtle aspects of numerical stability, bias, and variance are explored. This formal estimator is seen to be stable with respect to the epidemiologically interesting properties of the cluster size distribution (the number of clusters and the number of singletons) though it does not yield satisfactory estimates of the number of clusters of larger sizes. The problem that even complete coverage of genotyping, in a practical sampling frame, will only provide a partial view of the actual transmission network remains to be explored

Associations between human leukocyte antigen class I variants and the Mycobacterium tuberculosis subtypes causing disease

BACKGROUND. The development of active tuberculosis disease has been shown to be multifactorial. Interactions between host and bacterial genotype may influence disease outcome, with some studies indicating the adaptation of M. tuberculosis strains to specific human populations. Here we investigate the role of the human leukocyte antigen (HLA) class I genes in this biological process. METHODS. Three hundred patients with tuberculosis from South Africa were typed for their HLA class I alleles by direct sequencing. Mycobacterium tuberculosis genotype classification was done by IS6110 restriction fragment length polymorphism genotyping and spoligotyping. RESULTS. We showed that Beijing strain occurred more frequently in individuals with multiple disease episodes (P < .001) with the HLA-B27 allele lowering the odds of having an additional episode (odds ratio, 0.21; P = .006). Associations were also identified for specific HLA types and disease caused by the Beijing, LAM, LCC, and Quebec strains. HLA types were also associated with disease caused by strains from the Euro-American or East Asian lineages, and the frequencies of these alleles in their sympatric human populations identified potential coevolutionary events between host and pathogen. CONCLUSIONS. This is the first report of the association of human HLA types and M. tuberculosis strain genotype, highlighting that both host and pathogen genetics need to be taken into consideration when studying tuberculosis disease development.Web of Scienc

Validation and Optimization of Host Immunological Bio-Signatures for a Point-of-Care Test for TB Disease.

The development of a non-sputum-based, point-of-care diagnostic test for tuberculosis (TB) is a priority in the global effort to combat this disease, particularly in resource-constrained settings. Previous studies have identified host biomarker signatures which showed potential, but there is a need to validate and refine these for development as a test. We recruited 1,403 adults presenting with symptoms suggestive of pulmonary TB at primary healthcare clinics in six countries from West, East and Southern Africa. Of the study cohort, 326 were diagnosed with TB and 787 with other respiratory diseases, from whom we randomly selected 1005 participants. Using Luminex® technology, we measured the levels of 20 host biomarkers in serum samples which we used to evaluate the diagnostic accuracy of previously identified and novel bio-signatures. Our previously identified seven-marker bio-signature did not perform well (sensitivity: 89%, specificity: 60%). We also identified an optimal, two-marker bio-signature with a sensitivity of 94% and specificity of 69% in patients with no history of previous TB. This signature performed slightly better than C-reactive protein (CRP) alone. The cut-off value for a positive diagnosis differed for human immuno-deficiency virus (HIV)-positive and -negative individuals. Notably, we also found that no signature was able to diagnose TB adequately in patients with a prior history of the disease. We have identified a two-marker, pan-African bio-signature which is more robust than CRP alone and meets the World Health Organization (WHO) target product profile requirements for a triage test in both HIV-negative and HIV-positive individuals. This signature could be incorporated into a point-of-care device, greatly reducing the necessity for expensive confirmatory diagnostics and potentially reducing the number of cases currently lost to follow-up. It might also potentially be useful with individuals unable to provide sputum or with paucibacillary disease. We suggest that the performance of TB diagnostic signatures can be improved by incorporating the HIV-status of the patient. We further suggest that only patients who have never had TB be subjected to a triage test and that those with a history of previous TB be evaluated using more direct diagnostic techniques

Diagnostic accuracy of the Cepheid 3-gene host response fingerstick blood test in a prospective, multi-site study: interim results.

BACKGROUND: The development of a fast and accurate, non-sputum-based point-of-care triage test for tuberculosis (TB) would have a major impact on combating the TB burden worldwide. A new fingerstick blood test has been developed by Cepheid (the Xpert-MTB-Host Response (HR)-Prototype), which generates a 'TB score' based on mRNA expression of 3 genes. Here we describe the first prospective findings of the MTB-HR prototype. METHODS: Fingerstick blood from adults presenting with symptoms compatible with TB in South Africa, The Gambia, Uganda and Vietnam was analysed using the Cepheid GeneXpert MTB-HR prototype. Accuracy of the Xpert MTB-HR cartridge was determined in relation to GeneXpert Ultra results and a composite microbiological score (GeneXpert Ultra and liquid culture) with patients classified as having TB or other respiratory diseases (ORD). RESULTS: When data from all sites (n=75 TB, 120 ORD) were analysed, the TB score discriminated between TB and ORD with an AUC of 0·94 (CI, 0·91-0·97), sensitivity of 87% (CI, 77-93%) and specificity of 94% (88-97%). When sensitivity was set at 90% for a triage test, specificity was 86% (CI, 75-97%). These results were not influenced by HIV status or geographical location. When evaluated against a composite microbiological score (n=80 TB, 111 ORD), the TB score was able to discriminate between TB and ORD with an AUC of 0·88 (CI, 0·83-0·94), 80% sensitivity (CI, 76-85%) and 94% specificity (CI, 91-96%). CONCLUSIONS: Our interim data indicate the Cepheid MTB-HR cartridge reaches the minimal target product profile for a point of care triage test for TB using fingerstick blood, regardless of geographic area or HIV infection status

Four-Gene Pan-African Blood Signature Predicts Progression to Tuberculosis.

Rationale: Contacts of patients with tuberculosis (TB) constitute an important target population for preventive measures because they are at high risk of infection with Mycobacterium tuberculosis and progression to disease.Objectives: We investigated biosignatures with predictive ability for incident TB.Methods: In a case-control study nested within the Grand Challenges 6-74 longitudinal HIV-negative African cohort of exposed household contacts, we employed RNA sequencing, PCR, and the pair ratio algorithm in a training/test set approach. Overall, 79 progressors who developed TB between 3 and 24 months after diagnosis of index case and 328 matched nonprogressors who remained healthy during 24 months of follow-up were investigated.Measurements and Main Results: A four-transcript signature derived from samples in a South African and Gambian training set predicted progression up to two years before onset of disease in blinded test set samples from South Africa, the Gambia, and Ethiopia with little population-associated variability, and it was also validated in an external cohort of South African adolescents with latent M. tuberculosis infection. By contrast, published diagnostic or prognostic TB signatures were predicted in samples from some but not all three countries, indicating site-specific variability. Post hoc meta-analysis identified a single gene pair, C1QC/TRAV27 (complement C1q C-chain / T-cell receptor-α variable gene 27) that would consistently predict TB progression in household contacts from multiple African sites but not in infected adolescents without known recent exposure events.Conclusions: Collectively, we developed a simple whole blood-based PCR test to predict TB in recently exposed household contacts from diverse African populations. This test has potential for implementation in national TB contact investigation programs

Distinct serum biosignatures are associated with different tuberculosis treatment outcomes.

Biomarkers for TB treatment response and outcome are needed. This study characterize changes in immune profiles during TB treatment, define biosignatures associated with treatment outcomes, and explore the feasibility of predictive models for relapse. Seventy-two markers were measured by multiplex cytokine array in serum samples from 78 cured, 12 relapsed and 15 failed treatment patients from South Africa before and during therapy for pulmonary TB. Promising biosignatures were evaluated in a second cohort from Uganda/Brazil consisting of 17 relapse and 23 cured patients. Thirty markers changed significantly with different response patterns during TB treatment in cured patients. The serum biosignature distinguished cured from relapse patients and a combination of two clinical (time to positivity in liquid culture and BMI) and four immunological parameters (TNF-?, sIL-6R, IL-12p40 and IP-10) at diagnosis predicted relapse with a 75% sensitivity (95%CI 0.38-1) and 85% specificity (95%CI 0.75-0.93). This biosignature was validated in an independent Uganda/Brazil cohort correctly classifying relapse patients with 83% (95%CI 0.58-1) sensitivity and 61% (95%CI 0.39-0.83) specificity. A characteristic biosignature with value as predictor of TB relapse was identified. The repeatability and robustness of these biomarkers require further validation in well-characterized cohorts

Potential of Host Markers Produced by Infection Phase-Dependent Antigen-Stimulated Cells for the Diagnosis of Tuberculosis in a Highly Endemic Area

CITATION: Chegou, N. N. et al. 2012. Potential of host markers produced by infection phase-dependent antigen-stimulated cells for the diagnosis of tuberculosis in a highly endemic area. PLoS ONE, 7(6): e38501, doi:10.1371/journal.pone.0038501.The original publication is available at http://journals.plos.org/plosoneBackground: Recent interferon gamma (IFN-γ)-based studies have identified novel Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-γ, were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the diagnosis of TB disease. Methodology and Principal Findings: Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-α were the most promising diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under the receiver operating characteristics plots, (1.0 to 1.0). Conclusions: Multiple cytokines other than IFN-γ in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified in well-designed diagnostic studies employing short-term culture assays. © 2012 Chegou et al.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038501Publisher's versio

Analysis and application of evolutionary markers in the epidemiology of Mycobacterium tuberculosis

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008.This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment

A study of a distal lesion of substantia nigra neurons in the rat brain : a model for Parkinsonism

Thesis (M.Sc.(Biochemistry)) -- University of Stellenbosch, 1991.Full text to be digitised and attached to bibliographic record

Transmission of tuberculosis in a high incidence urban community in South Africa.

BACKGROUND: The objective of this study was to identify risk factors for ongoing community transmission of tuberculosis (TB) in two densely populated urban communities with a high incidence rate of TB in Cape Town, South Africa. METHODS: Between 1993 and 1998 DNA fingerprints of mycobacterial isolates from TB patients were determined by restriction fragment length polymorphism (RFLP). Cases whose isolates shared identical fingerprint patterns were considered to belong to the same cluster and to be attributable to ongoing community transmission. RESULTS: The average annual notification rate of new smear positive TB was 238/100000. In all, 1023/1526 reported patients were culture positive, and RFLP was available for 768 (75%) of the isolates from these patients. Since some patients experienced more than one infection during the study period, 797 cases were included in the analysis. Of the cases, 575/797 (72%) were clustered. Smear-positive cases and those who were retreated after default were more likely to be clustered than smear-negative and new cases, respectively. Patients from Uitsig were more often part of large clusters than were patients from Ravensmead. Age, sex, year of diagnosis, and outcome of disease were not risk factors for clustering, nor for being the first case in a cluster, although various analytical approaches were used. CONCLUSIONS: The incidence and proportion of cases that are clustered in this area are higher than reported elsewhere. An overwhelming majority of TB cases in this area is attributed to ongoing community transmission, and only very few to reactivation. This may explain the lack of demographic risk factors for clustering