Field evaluation of a point-of-care triage test for active tuberculosis (TriageTB).

BACKGROUND: To improve tuberculosis (TB) diagnosis, the World Health Organisation (WHO) has called for a non-sputum based triage test to focus TB testing on people with a high likelihood of having active pulmonary tuberculosis (TB). Various host or pathogen biomarker-based testing devices are in design stage and require validity assessment. Host biomarkers have shown promise to accurately rule out active TB, but further research is required to determine generalisability. The TriageTB diagnostic test study aims to assess the accuracy of diagnostic test candidates, as well as field-test, finalise the design and biomarker signature, and validate a point-of-care multi-biomarker test (MBT). METHODS: This observational diagnostic study will evaluate sensitivity and specificity of biomarker-based diagnostic candidates including the MBT and Xpert® TB Fingerstick cartridge compared with a gold-standard composite TB outcome classification defined by symptoms, sputum GeneXpert® Ultra, smear and culture, radiological features, response to TB therapy and presence of an alternative diagnosis. The study will be conducted in research sites in South Africa, Uganda, The Gambia and Vietnam which all have high TB prevalence. The two-phase design allows for finalisation of the MBT in Phase 1 in which candidate host proteins will be evaluated on stored serum from Asia, South Africa and South America and on fingerstick blood from 50 newly recruited participants per site. The MBT test will then be locked down and validated in Phase 2 on 250 participants per site. DISCUSSION: By targeting confirmatory TB testing to those with a positive triage test, 75% of negative GXPU may be avoided, thereby reducing diagnostic costs and patient losses during the care cascade. This study builds on previous biomarker research and aims to identify a point-of-care test meeting or exceeding the minimum World Health Organisation target product profile of a 90% sensitivity and 70% specificity. Streamlining TB testing by identifying individuals with a high likelihood of TB should improve TB resources use and, in so doing, improve TB care. TRIAL REGISTRATION: NCT04232618 (clinicaltrials.gov) Date of registration: 16 January 2020.

The recombination landscape of the Khoe-San likely represents the upper limits of recombination divergence in humans.

BACKGROUND: Recombination maps are important resources for epidemiological and evolutionary analyses; however, there are currently no recombination maps representing any African population outside of those with West African ancestry. We infer the demographic history for the Nama, an indigenous Khoe-San population of southern Africa, and derive a novel, population-specific recombination map from the whole genome sequencing of 54 Nama individuals. We hypothesise that there are no publicly available recombination maps representative of the Nama, considering the deep population divergence and subsequent isolation of the Khoe-San from other African groups. RESULTS: We show that the recombination landscape of the Nama does not cluster with any continental groups with publicly available representative recombination maps. Finally, we use selection scans as an example of how fine-scale differences between the Nama recombination map and the combined Phase II HapMap recombination map can impact the outcome of selection scans. CONCLUSIONS: Fine-scale differences in recombination can meaningfully alter the results of a selection scan. The recombination map we infer likely represents an upper bound on the extent of divergence we expect to see for a recombination map in humans and would be of interest to any researcher that wants to test the sensitivity of population genetic or GWAS analysis to recombination map input.

Cascade Immune Mechanisms of Protection against Mycobacterium tuberculosis (IMPAc-TB): study protocol for the Household Contact Study in the Western Cape, South Africa.

BACKGROUND: Natural immunity against Mycobacterium tuberculosis exists, and > 90% of those infected remain disease-free. Innate and adaptive immune responses required to mediate such protection against tuberculosis (TB) are, however, poorly understood. METHODS: This is an analytical study exploring protective and non-protective pathways of immunity against Mycobacterium tuberculosis. Adults without HIV infection are recruited at community healthcare clinics in high TB incidence areas of the Western Cape Province, South Africa. Data regarding participants' medical, social and medication usage will be collected, and clinical examinations and point-of-care tests documented. Reference tests for TB (chest radiographs and sputum tests for GeneXpert MTB/RIF Ultra®, Auramine smear and liquid cultures) and investigations to classify infection states [interferon-gamma release assay (IGRA) and SARS-CoV-2 polymerase chain reaction (PCR) nasopharyngeal swab and IgG], are done on all participants who meet the inclusion criteria. 18F-Fluorodeoxyglucose positron emission tomography combined with computerized tomography will be done on all close contacts (contacts) and healthy control (controls) participants. Participants are divided into 12 study groups representing a spectrum of TB clinical phenotypes and prior SARS-CoV-2 infection based on their TB status, exposure history, results of IGRA test at baseline and 3 months, SARS-CoV-2 serology, and PCR results, and for contacts and controls, PET-CT imaging findings indicative of sub-clinical TB lesions. Samples for experimental assays include whole blood for isolation of peripheral blood mononuclear cells and blood in PAXgene® tubes for RNA isolation. All SARS-CoV-2 PCR negative study participants undergo bronchoscopy for collecting bronchoalveolar lavage samples. DISCUSSION: The paired blood and BAL samples will be used for comprehensive analyses of the tissue-specific and systemic immunity that will include e.g., cytometry by time-of-flight analyses, RNA-sequencing, multiplex immunoassays, epigenetic analysis, and mechanistic studies of control of infection by Mycobacterium tuberculosis. Results will be integrated with those from mice and non-human primate studies to provide a comprehensive analysis of protective pathways in natural and vaccine-induced immunity against Mycobacterium tuberculosis.

A multi-phenotype genome-wide association study of clades causing tuberculosis in a Ghanaian- and South African cohort.

Despite decades of research and advancements in diagnostics and treatment, tuberculosis remains a major public health concern. New computational methods are needed to interrogate the intersection of host- and bacterial genomes. Paired host genotype datum and infecting bacterial isolate information were analysed for associations using a multinomial logistic regression framework implemented in SNPTest. A cohort of 853 admixed South African participants and a Ghanaian cohort of 1359 participants were included. Two directly genotyped variants, namely rs529920 and rs41472447, were identified in the Ghanaian cohort as being statistically significantly associated with risk for infection with strains of different members of the MTBC. Thus, a multinomial logistic regression using paired host-pathogen data may prove valuable for investigating the complex relationships driving infectious disease.

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.

Host urine immunological biomarkers as potential candidates for the diagnosis of tuberculosis.

OBJECTIVE: To investigate the potential of host urinary biomarkers as diagnostic candidates for tuberculosis (TB). METHODS: Adults self-presenting with symptoms requiring further investigation for TB were enrolled in Cape Town, South Africa. Participants were later classified as having TB or other respiratory diseases (ORD) using results from TB confirmatory tests. The concentrations of 29 analytes were evaluated in urine samples from participants using the Luminex platform, and their diagnostic potential was assessed using standard statistical approaches. RESULTS: Of the 151 study participants, 34 (22.5%) were diagnosed with TB and 26 (17.2%) were HIV-positive. Seven biomarkers showed potential as TB diagnostic candidates, with accuracy improving (in HIV-positives) when stratified according to HIV status (area under the receiver operating characteristics curve; AUC ≥0.80). In HIV-positive participants, a four-marker biosignature (sIL6R, MMP-9, IL-2Ra, IFN-γ) diagnosed TB with AUC of 0.96, sensitivity of 85.7% (95% confidence interval (CI) 42.1-99.6%), and specificity of 94.7% (95% CI 74.0-99.9%). In HIV-negatives, the most promising was a two-marker biosignature (sIL6R and sIL-2Ra), which diagnosed TB with AUC of 0.76, sensitivity of 53.9% (95% CI 33.4-73.4%), and specificity of 79.6% (95% CI 70.3-87.1%). CONCLUSIONS: Urinary host inflammatory biomarkers possess TB diagnostic potential but may be influenced by HIV infection. The results of this study require validation in larger studies.

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-ß, 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.

Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis.

We investigated host-derived biomarkers that were previously identified in QuantiFERON supernatants, in a large pan-African study. We recruited individuals presenting with symptoms of pulmonary TB at seven peripheral healthcare facilities in six African countries, prior to assessment for TB disease. We then evaluated the concentrations of 12 biomarkers in stored QuantiFERON supernatants using the Luminex platform. Based on laboratory, clinical and radiological findings and a pre-established algorithm, participants were classified as TB disease or other respiratory diseases(ORD). Of the 514 individuals included in the study, 179(34.8%) had TB disease, 274(51.5%) had ORD and 61(11.5%) had an uncertain diagnosis. A biosignature comprising unstimulated IFN-γ, MIP-1ß, TGF-α and antigen-specific levels of TGF-α and VEGF, identified on a training sample set (n = 311), validated by diagnosing TB disease in the test set (n = 134) with an AUC of 0.81(95% CI, 0.76-0.86), corresponding to a sensitivity of 64.2%(95% CI, 49.7-76.5%) and specificity of 82.7%(95% CI, 72.4-89.9%). Host biomarkers detected in QuantiFERON supernatants can contribute to the diagnosis of active TB disease amongst people presenting with symptoms requiring investigation for TB disease, regardless of HIV status or ethnicity in Africa.

Changes in Host Immune-Endocrine Relationships during Tuberculosis Treatment in Patients with Cured and Failed Treatment Outcomes.

A bidirectional communication between the immune and endocrine systems exists and facilitates optimum responses in the host during infections. This is in part achieved through changes in secretion patterns of hypothalamic hormones induced by inflammatory cytokines. The aim of this study was to elucidate the immune-endocrine alterations during tuberculosis (TB) treatment in patients with cured and failed TB treatment outcomes. Blood samples were collected from 27 cured and 10 failed patients and hormone as well as cytokine concentrations quantified at baseline, week 4, and month 6 of TB treatment. Hormone profiles of the two treatment outcome groups were different from each other prior to as well as during TB treatment. Treatment response effects were observed for cortisol, estradiol, T3, T4 ghrelin, leptin, amylin, adiponectin, and dehydroepiandrosterone (DHEA). Trends suggest that T4, amylin, and DHEA concentrations were different between treatment outcomes, although these did not reach statistical significance. Relationships between endocrine and inflammatory markers and the biological pathways involved differed between cured and failed treatment patients. These results highlight the complex interaction between the endocrine and immune system during active TB disease and throughout treatment and suggest that endocrine markers in conjunction with inflammatory markers may be useful in predicting unfavorable treatment outcomes.

Association of toll-like receptors with susceptibility to tuberculosis suggests sex-specific effects of TLR8 polymorphisms.

BACKGROUND: Toll-like receptors (TLRs) are involved in the recognition of conserved microbial structures, leading to activation of an inflammatory response and formation of an adaptive immune response. METHODS: Twenty-three polymorphisms in five TLR genes were genotyped in 729 tuberculosis cases and 487 healthy controls in a population-based case-control association study in a South African population. RESULTS: We detected sex-specific associations for TLR8 polymorphisms, with rs3761624 (OR=1.54, p<0.001), rs3764879 (OR=1.41, p=0.011) and rs3764880 (OR=1.42, p=0.011) associated in females and rs3764879 (OR=0.72, p=0.013) and rs3764880 (OR=0.75, p=0.036) associated in males. Epistatic interactions between the TLR genes were investigated and the TLR1_rs4833095 polymorphism was shown to interact with TLR2_rs3804100 and (GT)n microsatellite (p=0.002) and alter susceptibility to TB. We also studied the role of TLRs in disease caused by different Mycobacterium tuberculosis genotypes in 257 tuberculosis cases, and identified associations between specific TLR polymorphisms and disease caused by specific strains. CONCLUSION: This study provides further evidence that the TLRs play an important role in the outcome of tuberculosis disease, and suggests a partial explanation for the male bias in tuberculosis ratios.

The temporal dynamics of relapse and reinfection tuberculosis after successful treatment: a retrospective cohort study.

BACKGROUND: There is increasing evidence from tuberculosis high-burden settings that exogenous reinfection contributes considerably to recurrent disease. However, large longitudinal studies of endogenous reactivation (relapse) and reinfection tuberculosis are lacking. We hypothesize a relationship between relapse vs reinfection and the time between treatment completion and recurrent disease. METHODS: Population-based retrospective cohort study on all smear-positive tuberculosis cases successfully treated between 1996 and 2008 in a suburban setting in Cape Town, South Africa. Inverse gaussian distributions were fitted to observed annual rates of relapse and reinfection, distinguished by DNA fingerprinting of Mycobacterium tuberculosis strains recultured from diagnostic samples. RESULTS: Paired DNA fingerprint data were available for 130 (64%) of 203 recurrent smear-positive tuberculosis cases in the 13-year study period. Reinfection accounted for 66 (51%) of 130 recurrent cases overall, 9 (20%) of 44 recurrent cases within the first year, and 57 (66%) of 86 thereafter (P < .001). The relapse rate peaked at 3.93% (95% confidence interval [CI], 2.35%-5.96%) per annum 0.35 (95% CI, .15-.45) years after treatment completion. The reinfection tuberculosis rate peaked at 1.58% (95% CI, .94%-2.46%) per annum 1.20 (95% CI, .55-1.70) years after completion. CONCLUSIONS: To our knowledge, this is the first study of sufficient size and duration using DNA fingerprinting to investigate tuberculosis relapse and reinfection over a lengthy period. Relapse occurred early after treatment completion, whereas reinfection dominated after 1 year and accounted for at least half of recurrent disease. This temporal relationship may explain the high variability in reinfection observed across smaller studies. We speculate that follow-up time in antituberculosis drug trials should take reinfection into account.

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.

Differential expression of host biomarkers in saliva and serum samples from individuals with suspected pulmonary tuberculosis.

The diagnosis of tuberculosis remains challenging in individuals with difficulty in providing good quality sputum samples such as children. Host biosignatures of inflammatory markers may be valuable in such cases, especially if they are based on more easily obtainable samples such as saliva. To explore the potential of saliva as an alternative sample in tuberculosis diagnostic/biomarker investigations, we evaluated the levels of 33 host markers in saliva samples from individuals presenting with pulmonary tuberculosis symptoms and compared them to those obtained in serum. Of the 38 individuals included in the study, tuberculosis disease was confirmed in 11 (28.9%) by sputum culture. In both the tuberculosis cases and noncases, the levels of most markers were above the minimum detectable limit in both sample types, but there was no consistent pattern regarding the ratio of markers in serum/saliva. Fractalkine, IL-17, IL-6, IL-9, MIP-1 ß , CRP, VEGF, and IL-5 levels in saliva and IL-6, IL-2, SAP, and SAA levels in serum were significantly higher in tuberculosis patients (P < 0.05). These preliminary data indicate that there are significant differences in the levels of host markers expressed in saliva in comparison to those expressed in serum and that inflammatory markers in both sample types are potential diagnostic candidates for tuberculosis disease.

Potential of host markers produced by infection phase-dependent antigen-stimulated cells for the diagnosis of tuberculosis in a highly endemic area.

BACKGROUND: 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.

Population structure of multi- and extensively drug-resistant Mycobacterium tuberculosis strains in South Africa.

Genotyping of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in four South African provinces (Western Cape, Eastern Cape, KwaZulu-Natal, and Gauteng) revealed a distinct population structure of the MDR strains in all four regions, despite the evidence of substantial human migration between these settings. In all analyzed provinces, a negative correlation between strain diversity and an increasing level of drug resistance (from MDR-TB to extensively drug-resistant TB [XDR-TB]) was observed. Strains predominating in XDR-TB in the Western and Eastern Cape and KwaZulu-Natal Provinces were strongly associated with harboring an inhA promoter mutation, potentially suggesting a role of these mutations in XDR-TB development in South Africa. Approximately 50% of XDR-TB cases detected in the Western Cape were due to strains probably originating from the Eastern Cape. This situation may illustrate how failure of efficient health care delivery in one setting can burden health clinics in other areas.

Improving nutritional status of children with cystic fibrosis at Red Cross War Memorial Children's Hospital.

AIM: To determine the nutritional status of children attending a cystic fibrosis clinic in a tertiary hospital in South Africa and compare it to previously reported 10-year rates. METHODS: Weights and heights were measured of 69 (37 male and 32 female) children aged between 1 year and 18 years. Expected weight-for-age, expected height-for-age, expected weight-for-height and body mass index (BMI) were compared with international standards for underweight, stunting, wasting and BMI goal. RESULTS: The nutritional status of the patients has improved over the last 10 years, most significantly for wasting, which decreased from 58.3% in 1996 to 15.9% in 2006 (95% confidence interval (CI), 1.315-14.09, P < 0.05). Fifty-two percent of the children were underweight in 2006, compared with 66.7% in 1996 (95% CI, 0.044-13.96, P < 0.05). Stunting was found in 31.9% of the current sample. Females over 15 years had expected weight-for-age 25.9% lower than those between 10 years and 15 years, while no difference was found between the male age groups. Female height-for-age was 7.06 percentage points greater than males between 10 years and 15 years (95% CI, 2.16-11.96, P < 0.01). Males between 10 years and 15 years had significantly lower BMIs than the corresponding female group. Coloured patients had significantly lower BMIs than white patients in all age groups. CONCLUSIONS: These children demonstrated continuing improvement in nutritional status, although deficits remain. The normalisation of mean weight-for-age and weight-for-height with far fewer wasted patients is encouraging. Interventions are needed in some areas to ensure that all children show progress.

Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns.

RATIONALE: Multiple infections with different strains of Mycobacterium tuberculosis may occur in settings where the infection pressure is high. The relevance of mixed infections for the patient, clinician, and control program remains unclear. OBJECTIVES: This study aimed to describe reinfection and mixed infection as underlying mechanisms of changing drug-susceptibility patterns in serial sputum cultures. METHODS: Serial M. tuberculosis sputum cultures from patients diagnosed with multi-drug-resistant (MDR) tuberculosis were evaluated by phenotypic drug-susceptibility testing and mutation detection methods. Genotypic analysis was done by IS6110 DNA fingerprinting and a novel strain-specific polymerase chain reaction amplification method. MEASUREMENTS AND MAIN RESULTS: DNA fingerprinting analysis of serial sputum cultures from 48 patients with MDR tuberculosis attributed 10 cases to reinfection and 1 case to mixed infection. In contrast, strain-specific polymerase chain reaction amplification analysis in 9 of the 11 cases demonstrated mixed infection in 5 cases, reinfection in 3 cases, and laboratory contamination in 1 case. Analysis of clinical data suggests that first-line therapy can select for a resistant subpopulation, whereas poor adherence or second-line therapy resulted in the reemergence of the drug-susceptible subpopulations. CONCLUSIONS: We have shown that, in some patients with MDR tuberculosis, mixed infection may be responsible for observations attributed to reinfection by DNA fingerprinting. We conclude that treatment and adherence determines which strain is dominant. We hypothesize that treatment with second-line drugs may lead to reemergence of the drug-susceptible strain in patients with mixed infection.

Rate of reinfection tuberculosis after successful treatment is higher than rate of new tuberculosis.

RATIONALE: In a high-tuberculosis (TB) incidence area of Cape Town, South Africa, there is a very high rate of unexplained recurrent TB. The incidence of new bacteriologically confirmed disease in the area is 313 per 100,000 individuals. OBJECTIVE: To estimate the rate of recurrent TB attributable to reinfection after successful treatment. METHODS: All patients with reported TB in the area between 1993 and 1998 were followed up to 2001 for disease needing retreatment (recurrences). Patients who were multi-drug-resistant or who had treatment failure, were transferred, or died during treatment were excluded. Analysis was restricted to patients for whom DNA fingerprinting of their Mycobacterium tuberculosis isolates was obtained. Reinfection TB was defined as a recurrent TB episode in which the strains of the separate episodes differed by more than four bands. MEASUREMENTS AND MAIN RESULTS: 612 of 897 (68%) patients had a DNA fingerprint available at enrollment. Median duration of follow-up was 5.2 years. Recurrent TB occurred in 108 of 612 (18%) patients, of whom 61 of 447 (14%) experienced recurrence after successful treatment, and 47 of 165 (28%) experience recurrence after default. Of the 108 patients with recurrent TB, 68 (63%) had a DNA fingerprint in the second episode. Among these patients, 24 of 31 (77%) recurrences after successful treatment and 4 of 37 (11%) recurrences after default were attributable to reinfection. The reinfection disease rate after successful treatment was estimated at 2.2 per 100 person-years. CONCLUSIONS: The age-adjusted incidence rate of TB attributable to reinfection after successful treatment was four times that of new TB. People who had TB once are at a strongly increased risk of developing TB when reinfected.

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.

Stability of polymorphic GC-rich repeat sequence-containing regions of Mycobacterium tuberculosis.

Mycobacterium tuberculosis cultures were subjected to DNA fingerprinting with IS6110- and polymorphic GC-rich sequence (PGRS)-containing probes. The PGRS banding patterns remained highly stable during multiple cultures of specimens from one disease episode (0.5% changed) and during transmission in patients with close contact (1.9% changed). Characteristic PGRS-restriction fragment length polymorphism motifs for different strain groupings may indicate distant evolutionary events leading to the differentiation of M. tuberculosis strain lineages.