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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mutavhatsindi, van der Spuy, Malherbe, Sutherland, Geluk, Mayanja-Kizza, Crampin, Kassa, Howe, Mihret, Sheehama, Nepolo, Günther, Dockrell, Corstjens, Stanley, Walzl, Chegou and the AE-TBC ScreenTB Consortia.)

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., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The South African Medical Research Council Centre for Tuberculosis Research has a rich history of high-impact research that has influenced our understating of this hyper-epidemic which is further exacerbated by the emergence and spread of drug-resistant forms of the disease. This review aims to summarise the past 30 years of research conducted in the Centre which has influenced the way that tuberculosis (TB) is diagnosed and treated. The review includes the development of new technologies for rapid screening of people with probable TB and the repurposing of human diagnostics for wildlife conservation.

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-β, 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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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-1β, 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.

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.

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&#95;rs4833095 polymorphism was shown to interact with TLR2&#95;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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

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 β , 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.

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.

Setting: Brewelskloof Hospital, Western Cape, South Africa., Objectives: To verify the perceived increase in rifampicin monoresistant tuberculosis (RMR-TB) in the Cape Winelands-Overberg region and to identify potential risk factors., Design: A retrospective descriptive study of trends in RMR-TB over a 5-year period (2004-2008), followed by a case-control study of RMR and isoniazid (INH) monoresistant TB cases, diagnosed from April 2007 to March 2009, to assess for risk factors., Results: The total number of RMR-TB cases more than tripled, from 31 in 2004 to 98 in 2008. The calculated doubling time was 1.63 years (95%CI 1.18-2.66). For the assessment of risk factors, 95 RMR-TB cases were objectively verified on genotypic and phenotypic analysis. Of 108 specimens genotypically identified as RMR cases, 13 (12%) were misidentified, multidrug-resistant TB. On multivariate analysis, previous use of antiretroviral therapy (OR 6.4, 95%CI 1.3-31.8), alcohol use (OR 4.8, 95%CI 2.0-11.3) and age ≥ 40 years (OR 5.8, 95%CI 2.4-13.6) were significantly associated with RMR-TB., Conclusion: RMR-TB is rapidly increasing in the study setting, particularly among patients with advanced human immunodeficiency virus (HIV) disease. Routine drug susceptibility testing should be considered in all TB-HIV co-infected patients, and absence of INH resistance should be confirmed phenotypically if genotypic RMR-TB is detected.

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.

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., (© 2011 The Authors. Journal of Paediatrics and Child Health © 2011 Paediatrics and Child Health Division (Royal Australasian College of Physicians).)

Temporal analysis of drug-resistant tuberculosis (TB) cases in the Western Cape, South Africa, showed a 1.5-fold increase over a 2-year period, suggesting a doubling time of 8.2 years. This increase was strongly associated with multidrug resistance and the Beijing genotype. Forty-two per cent of the overall increase was due to the Beijing genotype strain R220, suggesting that this strain had evolved unique properties that allowed for both acquisition and transmission of drug resistance. To curb the drug-resistant TB epidemic in this setting, it will be essential to implement rapid diagnostics and efficient infection control measures, improve contact screening and ensure treatment adherence.

Current anti-tuberculosis (anti-TB) drug sensitivity testing methods provide a dichotomous readout: isolates are reported as either drug susceptible or drug resistant. This report demonstrates that rapid molecular methods may provide information concerning both the level of resistance and cross-resistance to other anti-TB drugs that is important for optimal clinical management. Specific mutations detected by the Hain GenoType MTBDRplus test, recently approved by the World Health Organization (WHO) for rapid TB diagnosis and drug resistance testing, could inform the decision of whether to include high dose isoniazid (INH) when treating patients with INH mono-resistant TB, MDR-TB or XDR-TB. The presence of mutations in the inhA gene or promoter region generally confers low level INH resistance that can be overcome by high dose INH. The same mutations also confer resistance to ethionamide indicating little benefit from its inclusion in second line treatment regimens in such cases. This information has high clinical relevance since inhA mutations account for a large proportion of INH resistance, and optimized therapy regimens are crucial to improve patient outcomes and reduce the spread of drug resistant TB. This hypothesis needs to be tested in well controlled clinical and pharmacokinetic studies.

Many molecular epidemiological investigations of M. tuberculosis are reported using data collected over relatively short timeframes. We postulated that such studies would tend to under-estimate the amount of disease in a community attributable to ongoing transmission. To test this hypothesis we used 12-year datasets of both real and simulated epidemics with the latter being based on two possible models of transmission. We analysed the effect of viewing the datasets through time windows of varying sizes on the measured degree of strain clustering as an indicator of ongoing transmission. We found that shorter windows significantly under-estimated transmission and that this effect was inversely correlated with the size of a cluster. Accordingly, we recommend that molecular epidemiological studies of M. tuberculosis, for the purposes of estimating transmission, be conducted over a minimum of 3-4 years and that the distribution of cluster size be taken into account in the interpretation of such data.

Mycobacterium tuberculosis strains can be classified into a number of major clades according to defined evolutionary markers. It is hypothesised that strains comprising these clades have evolved different properties which may influence a local strain population structure. To investigate this, we analysed the incidence of tuberculosis caused by the predominant clades (Beijing, Haarlem, LAM, Quebec and the Low-Copy Clade) found in a community within the Cape Town metropole in South Africa over a 12-year period. We found that while the incidence of cases infected with strains of the Haarlem, LAM, Quebec and the Low-Copy Clades remained relatively stable, that of cases of the Beijing clade increased exponentially over time, with a doubling time of 4.86 years (P=0.018). This growth was exclusively attributable to drug-susceptible strains. Although drug-resistant Beijing cases remained constant in number, non-Beijing drug-resistant cases declined over time (P=0.007). Drug-susceptible Beijing-infected cases had a greater proportion of smear-positive sputa than their non-Beijing counterparts (P=0.013) and were less likely to be successfully treated (retreatment cases) (P=0.026). Recent evidence suggests that these differences likely reflect enhanced pathogenicity rather than transmissibility. The rapid emergence of Beijing strains demonstrates adaptation to conditions within the study community and poses a grave challenge to future TB control.

IS6110 restriction fragment length polymorphism (RFLP) genotyping is the most widely used genotyping method to study the epidemiology of Mycobacterium tuberculosis. However, due to the complexity of the IS6110 RFLP genotyping technique, and the interpretation of RFLP data, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping has been proposed as the new genotyping standard. This study aimed to determine the discriminatory power of different MIRU-VNTR locus combinations relative to IS6110 RFLP genotyping, using a collection of Beijing genotype M. tuberculosis strains with a well-established phylogenetic history. Clustering, diversity index, clustering concordance, concordance among unique genotypes, and divergent and convergent evolution were calculated for seven combinations of 27 different MIRU-VNTR loci and compared to IS6110 RFLP results. Our results confirmed previous findings that MIRU-VNTR genotyping can be used to estimate the extent of recent or ongoing transmission. However, molecular epidemiological linking of cases varied significantly depending on the genotyping method used. We conclude that IS6110 RFLP and MIRU-VNTR loci evolve independently and at different rates, which leads to discordance between transmission chains predicted by the respective genotyping methods. Concordance between the two genotyping methods could be improved by the inclusion of genetic distance (GD) into the clustering formulae for some of the MIRU-VNTR loci combinations. In summary, our findings differ from previous reports, which may be explained by the fact that in settings of low tuberculosis incidence, the genetic distance between epidemiologically unrelated isolates was sufficient to define a strain using either marker, whereas in settings of high incidence, continuous evolution and persistence of strains revealed the weaknesses inherent to these markers.

The emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have raised concern about diagnostic delay associated with culture-based drug susceptibility testing methods. The association between rifampin resistance and MDR-TB or XDR-TB makes it an important genetic marker for genotypic drug susceptibility testing. In this article, we describe the analysis of the physical properties of the rifampin resistance-determining region (RRDR) in the rpoB gene by high-resolution thermal melt analysis as a method for detecting rifampin resistance in Mycobacterium tuberculosis complex. The RRDR from the M. tuberculosis complex was amplified by PCR from DNA templates extracted from sputum cultures of M. tuberculosis or the laboratory strain (H37Rv) in the presence of a fluorescent DNA binding dye. Subsequent mixing of the amplification products from the respective sputum cultures and the laboratory strain and thermocycling allowed the formation of DNA duplexes. The thermal denaturation properties of these DNA duplexes were determined by measuring the derivative of the intensity of fluorescence at different temperatures. Analysis of DNA extracted from 153 sputum cultures showed a sensitivity of 98% and a specificity of 100% for the detection of rifampin resistance compared to the "gold standard" culture-based phenotyping method. No statistical difference was detected in the performance of the method when applied to crude DNA from 134 boiled cultures. This method, named "FAST-Rif" ("fluorometric assay for susceptibility testing of rifampin"), allowed the rapid, reliable, and easy detection of genotypic rifampin resistance as a marker for MDR-TB and XDR-TB.

This study describes a comparative analysis of the Beijing mycobacterial interspersed repetitive unit types of Mycobacterium tuberculosis isolates from Cape Town, South Africa, and East Asia. The results show a significant association between the frequency of occurrence of strains from defined Beijing sublineages and the human population from whom they were cultured (P < 0.0001).

This study aimed to reconstruct the evolutionary history of Beijing strains of Mycobacterium tuberculosis and to test the hypothesis that evolution has influenced the ability of the Beijing strains within the different Beijing sublineages to spread and cause disease. A PCR-based method was used to analyze the genome structure of 40 different loci in 325 Beijing isolates collected from new and retreatment tuberculosis patients from an urban setting and 270 Beijing isolates collected from high-risk tuberculosis patients from a rural setting in the Western Cape, South Africa. The resulting data were subjected to phylogenetic analysis using the neighbor joining algorithm. Phylogenetic reconstructions were highly congruent with the "gold standard" phylogenetic tree based on synonymous single-nucleotide polymorphisms, thereby allowing a prediction of the order in which the evolutionary events had occurred. A total of seven independently evolving Beijing sublineages were identified. Analysis of epidemiological data in relation to the Beijing sublineage suggested an association between recent evolutionary change and frequency of occurrence in an urban population (P<0.001) as well as in the rural population (P<0.001). This concept was further supported by an association between more recently evolved Beijing strains and an increased ability to transmit and to cause disease (odds ratio, 5.82; 95% confidence interval, 3.13 to 10.82 [P<0.001]). An association between Beijing sublineage and demographic and clinical parameters and drug resistance could not be demonstrated. From these data, we suggest that the pathogenic characteristics of Beijing strains are not conserved but rather that strains within individual lineages have evolved unique pathogenic characteristics.

Setting: Zimbabwe and Zambia., Objective: To determine the genetic diversity of Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in Zimbabwe and Zambia., Design: M. tuberculosis isolates cultured from TB patients presenting at referral hospitals in Zimbabwe and health care clinics in Zambia were characterised by IS6110 genotyping and/or spoligotyping using internationally standardised methods. Genotypic data were compared to those from Cape Town and the SpolDB3.0 database., Results: A predominant group of strains could be identified among 116/246 (47.2%) Zimbabwean isolates by their characteristic IS6110-banding pattern and unique spoligotype signature, where spacers 21-24, 27-30 and 33-36 were deleted. Comparison with strains from Cape Town showed that they were closely related to a family of strains present in 2.3% of Cape Town patients. Comparison of the spoligotypes with those obtained from 114 isolates from Zambia showed that 74 (65%) of these isolates had the same spoligotype signature. Spoligotypes in the SpolDB3.0 database showed that this group of strains was rarely isolated in other parts of the world, but was commonly isolated in Southern Africa., Conclusion: A predominant group of strains infecting approximately half of the patients in the study are major contributors to the TB epidemic in this region. We have designated this group of strains the Southern Africa 1 (SAF1) family.

Background: South Africa has a high burden of drug-resistant tuberculosis (TB)., Methods: Routine drug susceptibility testing was performed prospectively over a 2-year period on Mycobacterium tuberculosis isolates in two health districts of the Western Province, South Africa. A cluster of drug-resistant strains that shared a rare mutation in katG315 was found in 64 of the 450 cases identified as having been infected with drug-resistant TB. Isolates belonging to this cluster were phenotypically and genotypically characterised. Epidemiological and clinical characteristics were used to identify mechanisms leading to the acquisition and spread of this drug-resistant strain., Results: An outbreak of an emerging non-Beijing drug-resistant strain infecting 64 pulmonary tuberculosis (PTB) cases was identified. This previously undetected genotype (now designated DRF150) is characterised by five IS6110 insertions, specific spoligotypes and high levels of resistance to the first-line TB medications isoniazid, streptomycin and rifampicin. In 45% of the cases it is also resistant to ethambutol and pyrazinamide. Key factors leading to the development and spread of this drug-resistant genotype were inappropriate chemotherapy, poor adherence to treatment and prolonged periods of infectiousness due to delays in susceptibility testing., Conclusions: Molecular markers allowed early identification of an emerging non-Beijing drug-resistant strain.

Knowledge of the clonal expansion of Mycobacterium tuberculosis and accurate identification of predominant evolutionary lineages in this species remain limited, especially with regard to low-IS6110-copy-number strains. In this study, 170 M. tuberculosis isolates with

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.

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.

Genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from the Western Cape Province of South Africa showed that drug resistance is widespread and recently transmitted. Multidrug-resistant (MDR) isolates comprise 40% of this collection, and a large pool of isoniazid monoresistance may be a future source of MDR tuberculosis.

In order to fully understand the global tuberculosis (TB) epidemic it is important to investigate the population structure and dissemination of the causative agent that drives the epidemic. Mycobacterium tuberculosis strain family 11 (F11) genotype isolates (found in 21.4% of all infected patients) are at least as successful as the Beijing genotype family isolates (16.5%) in contributing to the TB problem in some Western Cape communities of South Africa. This study describes key molecular characteristics that define the F11 genotype. A data-mining approach coupled with additional molecular analysis showed that members of F11 can easily and uniquely be identified by PCR-based techniques such as spoligotyping and dot blot screening for a specific rrs491 polymorphism. Isolates of F11 not only are a major contributor to the TB epidemic in South Africa but also are present in four different continents and at least 25 other countries in the world. Careful study of dominant compared to rare strains should provide clues to their success and possibly provide new ideas for combating TB.

The stability of the genotypic marker IS6110, used to define the epidemiology of Mycobacterium tuberculosis, is one of the most important factors influencing the interpretation of DNA fingerprint data. We propose that evolved strains should be considered together with clustered strains to represent chains of ongoing transmission. For the present study we used a large set of fingerprint data for strains collected between 1992 and 1998 from residents of a community with a high incidence of tuberculosis in Cape Town, South Africa. Interstrain genetic distances were calculated by counting the banding pattern mismatches in the IS6110 DNA fingerprints of different isolates. These data demonstrate that the propensity to change by one or two bands is independent of the IS6110 copy number. Hence, the genetic distance between pairs of isolates can be simply expressed as the number of differences in the banding patterns. From this foundation, a data set which identifies newly evolved strains has been generated. Inclusion of these evolved strains into various molecular epidemiological calculations significantly increased the estimate of ongoing transmission in this study setting. The indication is that nearly all cases of tuberculosis in this community are due to ongoing transmission. This has important implications for tuberculosis control, as it indicates that the control measures used at present are unable to reduce the level of transmission. This technique may also be applicable to the study of low-incidence tuberculosis outbreaks as well as the analysis of epidemiological data from other disease epidemics.

This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.

Deciphering the structure of pathogen populations is instrumental for the understanding of the epidemiology and history of infectious diseases and for their control. Although Mycobacterium tuberculosis is the most widespread infectious agent in humans, its actual population structure has remained hypothetical until now because: (i) its structural genes are poorly polymorphic; (ii) adequate samples and appropriate statistics for population genetic analysis have not been considered. To investigate this structure, we analysed the statistical associations (linkage disequilibrium) between 12 independent M. tuberculosis minisatellite-like loci by high-throughput genotyping within a model population of 209 isolates representative of the genetic diversity in an area with a very high incidence of tuberculosis. These loci contain variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs). Highly significant linkage disequilibrium was detected among the MIRU-VNTR loci in this model. This linkage disequilibrium was also evident when the MIRU-VNTR types were compared with the IS6110 restriction fragment length polymorphism types. These results support a predominant clonal evolution of M. tuberculosis.

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS6110 RFLP profiles. In a single case, serial isolates with an unchanged IS6110 RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS6110 RFLP to define ongoing transmission clusters.

The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.

Setting: A retrospective study in an urban setting with a high tuberculosis incidence., Objective: To study the molecular epidemiology and disease dynamics of a prevalent Mycobacterium tuberculosis strain family, F29, a subset of the Beijing strains., Design: Multi-locus DNA fingerprinting and genomic analysis was used to characterise F29 isolates. Demographic and molecular epidemiologic data were correlated with those of other isolates from the setting. The geographic locations of five F29 strain clusters were plotted using a geographic information system (GIS) and an index for geographic distribution was calculated. Their cumulative temporal occurrence was also plotted., Results: The genomic similarity of F29 to the Beijing-strains was confirmed. A high degree of clustering predicted high disease transmission. Spatial distribution was mostly widespread except for one cluster. Smaller foci of transmission were observed. The temporal spread showed ongoing transmission., Conclusion: F29 belongs to the Beijing strain group. The prevalence and high degree of strain clustering, with limited geographic clustering, indicates that F29 strains are endemic in the study community. However, recent epidemiologic events also contributed to the disease spread. The combination of molecular epidemiologic, spatial and temporal data has enhanced our understanding of the disease dynamics of Beijing strains in our study community.

The spoligotyping method has become an important tool for the tracking of Mycobacterium tuberculosis strains in different epidemiological settings. In this study, we demonstrate the ability of the spoligotyping technique to accurately determine the pathogenetic mechanism of recurrent disease. This methodology has advantages over conventional restriction fragment length polymorphism methods which may be useful in large-scale intervention studies.

In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosis strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosis strains in the context of the molecular epidemiology of the study setting.

The interpretation of molecular epidemiologic data of Mycobacterium tuberculosis infection is dependent on the understanding of the stability and evolutionary characteristics of the DNA fingerprinting marker used to classify clinical isolates. This study investigated the stability of the IS6110 banding pattern in serial tuberculosis isolates collected from patients resident in an area with a high incidence of tuberculosis. Evolutionary changes were observed in 4% of the strains, and a half-life (t(1/2)) of 8.74 years was calculated, assuming a constant rate of change over time. This rate may be composed of a high rate of change seen during the early disease phase (t(1/2) = 0.57 years) and a low rate of change seen in the late disease phase (t(1/2) = 10.69 years). The early rate probably reflects change occurring during active growth prior to therapy, while the low late rate may reflect change occurring during or after treatment. We demonstrate that the calculation of these rates is strongly influenced by the time interval between onset of disease and sputum sampling. These calculations are further complicated by partial replacement of the original strain population, resulting in the sporadic appearance of clonal variants in sputum specimens. Therefore, the true extent of genetic diversity may be underestimated within each host, thereby influencing molecular epidemiological data used to establish transmission chains.

Setting: Patients with positive smears for acid-fast bacilli were enrolled at tuberculosis (TB) clinics in the Khartoum region of Sudan., Objective: To identify the presence of drug resistant genotypes in M. tuberculosis isolates which are difficult to treat., Methods: Genus specific PCR-SSCP was performed to confirm the presence of M. tuberculosis in clinical isolates. Genotypic drug resistance testing was performed by mutation analysis and spoligotyping was used to monitor transmission and to identify epidemic strains., Results: Fifty (48%) of the original 105 samples were classified as M. tuberculosis. Four (4%) of the samples were typed as mycobacteria other than TB, while the remaining (n =50) samples were refractory to further molecular analysis. The fifty amplifiable M. tuberculosis samples were used for subsequent mutation analysis and typing. Mutations were identified in the genes conferring resistance to INH (kat G, 12%), RIF (rpoB, 8%), SM (r psL and rrs, 30%) and EMB (embB, 4%). Two of the samples (4%) had mutations in genes associated to both INH and RIF and can be classified as MDR-TB. Thirty-three percent (13/39) of the persistant tuberculosis cases (5/18 treatment failure; 5/14 relapse; 3/7 defaulter) had mutations accounting for drug resistance. A total of 27 different spoligotypes were identified from 49/50 samples. Twenty-nine (59%) of the isolates were grouped into one of seven clusters, while 20 (41%) showed unique patterns. One patient was infected with M. bovis., Conclusion: This is the first molecular approach to characterize clinical isolates of M. tuberculosis from Sudan. The results show that drug resistance is indeed a serious problem and it may compliment the efforts of the National Tuberculosis Programme to improve strategies to control this disease., (Copyright 2002 The British Infection Society.)

Interpretation of the molecular epidemiological data of Mycobacterium tuberculosis is dependent on the validity of the assumptions that have been made. It is assumed that the IS6110 banding pattern is sufficiently stable to define epidemiological events representing ongoing transmission. However, molecular epidemiological data also support the observation that the IS6110 banding pattern may change over time. Factors affecting this rate may include the nature and duration of disease in a host and the opportunity to experience different host environments during the transmission cycle. To estimate the rate of IS6110 change occurring during the process of transmission, M. tuberculosis isolates from epidemiologically linked patients were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis. The identification of IS6110 banding pattern changes during ongoing transmission suggested that a rate could be estimated. IS6110 change was significantly associated with strains with >5 IS6110 elements (P = 0.013) and was not observed in low-copy-number isolates. The minimum rate of appearance of variant strains was calculated to be 0.14 variant cases per source-case per year. This data suggest that clustering of isolates based on identical RFLP patterns is expected to underestimate transmission in patients infected with high-copy-number isolates. A model based on the rate of appearance of both variant and invariant strains demonstrates that the genotypically defined population structure may change by 18.6% during the study period of approximately 6.5 years. The implications for the use of RFLP data for epidemiologic study are discussed.