Discriminatory ability of hypervariable variable number tandem repeat loci in population-based analysis of Mycobacterium tuberculosis strains, London, UK

To address conflicting results about the stability of variable number tandem repeat (VNTR) loci and their value in prospective molecular epidemiology of Mycobacterium tuberculosis, we conducted a large prospective population-based analysis of all M. tuberculosis strains in a metropolitan setting. Optimal and reproducible conditions for reliable PCR and fragment analysis, comprising enzymes, denaturing conditions, and capillary temperature, were identified for a panel of hypervariable loci, including 3232, 2163a, 1982, and 4052. A total of 2,261 individual M. tuberculosis isolates and 265 sets of serial isolates were analyzed by using a standardized 15-loci VNTR panel, then an optimized hypervariable loci panel. The discriminative ability of loci varied substantially; locus VNTR 3232 varied the most, with 19 allelic variants and Hunter-Gaston index value of 0.909 . Hypervariable loci should be included in standardized panels because they can provide consistent comparable results at multiple settings, provided the proposed conditions are adhered to

The importance of recent infection with Mycobacterium tuberculosis in an area with high HIV prevalence: a long-term molecular epidemiological study in Northern Malawi.

BACKGROUND: The proportion of cases of tuberculosis due to recent infection can be estimated in long-term population-based studies using molecular techniques. Here, we present what is, to our knowledge, the first such study in an area with high human immunodeficiency virus (HIV) prevalence. METHODS: All patients with tuberculosis in Karonga District, Malawi, were interviewed. Isolates were genotyped using restriction-fragment-length polymorphism (RFLP) patterns. Strains were considered to be "clustered" if at least 1 other patient had an isolate with an identical pattern. RESULTS: RFLP results were available from 83% of culture-positive patients from late 1995 to early 2003. When strains with <5 bands were excluded, 72% (682/948) were clustered. Maximum clustering was reached using a 4-year window, with an estimated two-thirds of cases due to recent transmission. The proportion clustered decreased with age and varied by area of residence. In older adults, clustering was less common in men and more common in patients who were HIV positive (adjusted odds ratio, 5.1 [95% confidence interval, 2.1-12.6]). CONCLUSIONS: The proportion clustered found in the present study was among the highest in the world, suggesting high rates of recent transmission. The association with HIV infection in older adults may suggest that HIV has a greater impact on disease caused by recent transmission than on that caused by reactivation

MIRU-VNTR genotyping of Mycobacterium tuberculosis strains using QIAxcel technology: a multicentre evaluation study

Molecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB. The standard 24-loci MIRU-VNTR typing includes PCR amplification followed by the detection and sizing of PCR fragments using capillary electrophoresis on automated sequencers or using agarose gels. The QIAxcel Advanced system might offer a cost-effective medium-throughput alternative.Performance characteristics of the QIAxcel Advanced platform for the standard 24 VNTR loci panel was evaluated at two centres on a total of 140 DNA specimens using automated capillary electrophoresis as a reference method. Additionally 4 hypervariable MIRU-VNTR loci were evaluated on 53 crude DNA extracts. The sizing accuracy, interlaboratory reproducibility and overall instrument's performance were assessed during the study.An overall concordance with the reference method was high reaching 98.5% and 97.6% for diluted genomic and crude DNA extracts respectively. 91.4% of all discrepancies were observed in fragments longer than 700bp. The concordance for hypervariable loci was lower except for locus 4120 (96.2%). The interlaboratory reproducibility agreement rates were 98.9% and 91.3% for standard and hypervariable loci, respectively. Overall performance of the QIAxcel platform for M.tuberculosis genotyping using a panel of standard loci is comparable to that of established methods for PCR fragments up to 700bp. Inaccuracies in sizing of longer fragments could be resolved through using in-house size markers or introduction of offset values. To conclude, the QiaXcel system could be considered an effective alternative to existing methods in smaller reference and regional laboratories offering good performance and shorter turnaround times

Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study

BACKGROUND: A large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster. METHODS AND FINDINGS: We sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998-22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation. CONCLUSIONS: Our study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases

Time-and-motion tool for the assessment of working time in tuberculosis laboratories: a multicentre study

SETTING: Implementation of novel diagnostic assays in tuberculosis (TB) laboratory diagnosis requires effective management of time and resources. OBJECTIVE: To further develop and assess at multiple centres a time-and-motion (T&M) tool as an objective means for recording the actual time spent on running laboratory assays. DESIGN: Multicentre prospective study conducted in six European Union (EU) reference TB laboratories. RESULTS: A total of 1060 specimens were tested using four laboratory assays. The number of specimens per batch varied from one to 60; a total of 64 recordings were performed. Theoretical hands-on times per specimen (TTPS) in h:min:s for Xpert® MTB/RIF, mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping, Ziehl-Neelsen staining and manual fluorescence microscopy were respectively 00:33:02 ± 00:12:32, 00:13:34 ± 00:03:11, 00:09:54 ± 00:00:53 and 00:06:23 ± 00:01:36. Variations between laboratories were predominantly linked to the time spent on reporting and administrative procedures. Processing specimens in batches could help save time in highly automated assays (e.g., line-probe) (TTPS 00:14:00 vs. 00:09:45 for batches comprising 7 and 31 specimens, respectively). CONCLUSIONS: The T&M tool can be considered a universal and objective methodology contributing to workload assessment in TB diagnostic laboratories. Comparison of workload between laboratories could help laboratory managers justify their resource and personnel needs for the implementation of novel, time-saving, cost-effective technologies, as well as identify areas for improvement

The oral mucosal and salivary microbial community of Behçet's syndrome and recurrent aphthous stomatitis.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Behçet's syndrome (BS) is a multisystem immune-related disease of unknown etiology. Recurrent aphthous stomatitis (RAS) is characterized by the presence of idiopathic oral ulceration without extraoral manifestation. The interplay between the oral microbial communities and the immune response could play an important role in the etiology and pathogenesis of both BS and RAS

Clinical and ultrasonographic features of abdominal tuberculosis in HIV positive adults in Zambia

Background: The diagnosis of abdominal tuberculosis (TB) is difficult, especially so in health care facilities in developing countries where laparoscopy and colonoscopy are rarely available. There is little information on abdominal TB in HIV infection. We estimated the prevalence and clinical features of abdominal (excluding genitourinary) TB in HIV infected adults attending the University Teaching Hospital, Zambia. Methods: We screened 5,609 medical inpatients, and those with fever, weight loss, and clinical features suggestive of abdominal pathology were evaluated further. A clinical algorithm was used to specify definitive investigations including laparoscopy or colonoscopy, with culture of biopsies and other samples. Results: Of 140 HIV seropositive patients with these features, 31 patients underwent full evaluation and 22 (71%) had definite or probable abdominal TB. The commonest presenting abdominal features were ascites and persistent tenderness. The commonest ultrasound findings were ascites, para-aortic lymphadenopathy (over 1 cm in size), and hepatomegaly. Abdominal TB was associated with CD4 cell counts over a wide range though 76% had CD4 counts <100 cells/μL. Conclusion: The clinical manifestations of abdominal TB in our HIV-infected patients resembled the well-established pattern in HIV-uninfected adults. Patients with fever, weight loss, abdominal tenderness, abdominal lymphadenopathy, ascites and/or hepatomegaly in Zambia have a high probability of abdominal TB, irrespective of CD4 cell count. © 2009 Sinkala et al; licensee BioMed Central Ltd

Variability in interpretation of chest radiographs among Russian clinicians and implications for screening programmes: observational study.

OBJECTIVE: To determine variability in interpretation of chest radiographs among tuberculosis specialists, radiologists, and respiratory specialists. DESIGN: Observational study. SETTING: Tuberculosis and respiratory disease services, Samara region, Russian Federation. PARTICIPANTS: 101 clinicians involved in the diagnosis and management of pulmonary tuberculosis and respiratory diseases. MAIN OUTCOME MEASURES: Interobserver and intraobserver agreement on the interpretation of 50 digital chest radiographs, using a scale of poor to very good agreement (kappa coefficient: < or = 0.20 poor, 0.21-0.40 fair, 0.41-0.60 moderate, 0.61-0.80 good, and 0.81-1.00 very good). RESULTS: Agreement on the presence or absence of an abnormality was fair only (kappa = 0.380, 95% confidence interval 0.376 to 0.384), moderate for localisation of the abnormality (0.448, 0.444 to 0.452), and fair for a diagnosis of tuberculosis (0.387, 0.382 to 0.391). The highest levels of agreement were among radiologists. Level of experience (years of work in the specialty) influenced agreement on presence of abnormalities and cavities. Levels of intraobserver agreement were fair. CONCLUSIONS: Population screening for tuberculosis in Russia may be less than optimal owing to limited agreement on interpretation of chest radiographs, and may have implications for radiological screening programmes in other countries

Survival of drug resistant tuberculosis patients in Lithuania: retrospective national cohort study

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement FP7-223681.

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

Mycolic acids (MAs), which play a crucial role in the architecture of mycobacterial cell walls, were analyzed using electrospray ionization tandem mass spectrometry. A targeted analysis based on the 10 most abundant and characteristic multiple reaction monitoring pairs was used to profile the crude fatty acid mixtures from Mtb and several nontuberculous mycobacterial strains. Comparative analysis yielded unique profiles for MAs, enabling the reliable identification of mycobacterial species. In a case-control study of tuberculosis (TB) and non-TB Polish patients, we demonstrated the potential diagnostic utility of our approach for the rapid diagnosis of active TB with sensitivity and specificity surpassing those of existing methods. This robust method allows the identification of TB-positive patients after 2 h of sample preparation in the case of direct sputum analysis or 10 days of culturing, both of which are followed by 1 min of liquid chromatography– tandem mass spectrometry analysis