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

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

Simultaneous genotyping and species identification using hybridization pattern recognition analysis of generic Mycobacterium DNA arrays

High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 165 genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure

The Directly Observed Therapy Short-Course (DOTS) strategy in Samara Oblast, Russian Federation

BACKGROUND: The World Health Organisation (WHO) defines Russia as one of the 22 highest-burden countries for tuberculosis (TB). The WHO Directly Observed Treatment Short Course (DOTS) strategy employing a standardised treatment for 6 months produces the highest cure rates for drug sensitive TB. The Russian TB service traditionally employed individualised treatment. The purpose of this study was to implement a DOTS programme in the civilian and prison sectors of Samara Region of Russia, describe the clinical features and outcomes of recruited patients, determine the proportion of individuals in the cohorts who were infected with drug resistant TB, the degree to which resistance was attributed to the Beijing TB strain family and establish risk factors for drug resistance. METHODS: prospective study RESULTS: 2,099 patients were recruited overall. Treatment outcomes were analysed for patients recruited up to the third quarter of 2003 (n = 920). 75.3% of patients were successfully treated. Unsuccessful outcomes occurred in 7.3% of cases; 3.6% of patients died during treatment, with a significantly higher proportion of smear-positive cases dying compared to smear-negative cases. 14.0% were lost and transferred out. A high proportion of new cases (948 sequential culture-proven TB cases) had tuberculosis that was resistant to first-line drugs; (24.9% isoniazid resistant; 20.3% rifampicin resistant; 17.3% multidrug resistant tuberculosis). Molecular epidemiological analysis demonstrated that half of all isolated strains (50.7%; 375/740) belonged to the Beijing family. Drug resistance including MDR TB was strongly associated with infection with the Beijing strain (for MDR TB, 35.2% in Beijing strains versus 9.5% in non-Beijing strains, OR-5.2. Risk factors for multidrug resistant tuberculosis were: being a prisoner (OR 4.4), having a relapse of tuberculosis (OR 3.5), being infected with a Beijing family TB strain (OR 6.5) and having an unsuccessful outcome from treatment (OR 5.0). CONCLUSION: The implementation of DOTS in Samara, Russia, was feasible and successful. Drug resistant tuberculosis rates in new cases were high and challenge successful outcomes from a conventional DOTS programme alone

Microevolution of extensively drug-resistant tuberculosis in Russia.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72%belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted. © 2012 by Cold Spring Harbor Laboratory Press

Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10−11 for rs4733781; P = 1.0 × 10−10 for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis–infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

BACKGROUND: Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. METHODS AND FINDINGS: We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42-4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43-5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. CONCLUSIONS: We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian reg

Ertapenem and Faropenem against Mycobacterium tuberculosis: in vitro testing and comparison by macro and microdilution

BACKGROUND: Interest in carbapenems has been rising in the last few years due to the emergence of drug resistant tuberculosis. Ertapenem (ETP), given once a day parenteral, and faropenem (FAR), oral, have a better administration profile than meropenem (MEM), imipenem (IPM) and doripenem (DOR). The addition of amoxicillin-clavulanate (AMC) inhibits the hydrolysis by the carbapenemase present in Mycobacterium tuberculosis (MTB). The aim of this study was to determine the in vitro activity of ETP and FAR against susceptible and resistant clinical MTB strains by two widely use methodologies, the BACTEC960 MGIT and microdilution. RESULTS: 19 clinical isolates with different susceptibility profiles and H37Rv were included. Minimal inhibitory concentration (MIC) testing was performed using two methods of different concentrations of ETP and FAR with and without AMC. MIC50 was 2 and 8 for FAR with and without AMC by both methods. MIC90 was > 16 and > 8 by microdilution and MGIT respectively and did not change after AMC addition. 18/20 samples were resistant to the highest concentration of ETP, with and without AMC. Half of the samples had some susceptibility to FAR; addition of AMC further reduced the MIC level in seven isolates. 10/20 isolates showed susceptibility to FAR and the addition of AMC further reduced the MIC in 7 isolates. However, most of the MICs were near the limit of effectiveness (8 μg/mL). Resistance to FAR was associated with resistance to MEM (p = 0.04) but not to resistance profiles of other drugs, including M/XDR status. CONCLUSIONS: The lack of ETP activity may be associated with its degradation, independent of carbapenemase, during incubation. No susceptibility pattern to traditional drugs can predict susceptibility to FAR and susceptibility testing is not routinely available. PK/PD studies are needed as reaching the concentrations tested in these experiments may be challenging. This work highlighted some of the limitations of carbapenem use. More evidence is needed to clarify their true impact in TB treatment and outcome, considering the financial burden, complications and microbiota changes associated with their use