A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing.

Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings. IMPORTANCE Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings. This article describes a new workflow that can diagnose drug-resistant TB in a shorter time, with less equipment, and for a lower price than current methods. The amount of TB DNA is first increased without the need for bulky and costly thermocycling equipment. The DNA is then read using a portable sequencer called a MinION, which indicates whether there are tell-tale changes in the DNA that indicate whether the TB strain is drug resistant. Our workflow could play an important role in the future in the fight against the public health challenge that is TB drug resistance.

Distribution of molecular strains of Mycobacterium tuberculosis in an intermediate burden Asia Pacific city.

Hong Kong is an intermediate tuberculosis (TB) burden city in Asia Pacific with slow decline of case notification in the last decade. By 24-loci mycobacterial interspersed repetitive units - variable number of tandem repeats genotyping, we examined 534 Mycobacterium tuberculosis isolates collected from culture-positive hospitalised TB patients in a 1.7 million population geographic region in the city. Overall, 286 (75%) were classified as Beijing genotype, of which 216 (76%) and 59 (21%) belonged to modern and ancient sub-lineage, respectively. Only two cases were genetically clustered while spatial clustering was absent. Male gender, permanent residency in Hong Kong and born in Hong Kong or Mainland China were associated with Beijing genotype. The high prevalence of Beijing modern lineage was similar to that in East Asia, which reflected the pattern resulting from population migration. The paucity of clustering suggested that reactivation accounted for most of the TB disease cases, which was and echoed by observation that half were 60 years old or above, and the presence of co-morbid medical conditions. The predominance of reactivation TB cases in intermediate burden localities implies that the detection and control of latent TB infection would be the major challenge in achieving TB elimination.

Estimation of the global burden of Mycobacterium tuberculosis lineage 1.

Tuberculosis is still problematic as it affects large numbers of people globally. Mycobacterium tuberculosis Lineage 1 (L1) or Indo Oceanic Lineage, one of widespread major lineages, has a specific geographic distribution and high mortality. It is highly diverse and endemic in several high burden countries. However, studies on the global burden of L1 and its sublineages remain limited. This may lead to the underestimation of the importance of its variance in developing and applying tuberculosis control measures. This study aimed to estimate the number of patients infected with M. tuberculosis L1 and its sublineages worldwide. The proportion of L1 among tuberculosis patients was searched in published reports from countries around the world and the number of patients was calculated based on a WHO report on country incidences and populations. The numbers of patients infected with the five major sublineages, namely L1.1.1, L1.1.2, L1.1.3, L1.2.1, and L1.2.2 were estimated where information was available. It was found that L1 accounted for 28% of global tuberculosis cases in 2012 and 2018. Over 80% of the L1 global burden was in India, the Philippines, Indonesia and Bangladesh, which are also among the countries with highest absolute numbers of tuberculosis patients in the world. Globally, the estimated number of patients infected with M. tuberculosis L1.2.1 and L1.1.2 was over 1.1 million and of patients infected with L1.1.1 was about 200,000. This study demonstrated that L1 contributes significantly to the global burden of tuberculosis. To achieve the End TB Strategy, more attention needs to be paid to the responses of M. tuberculosis L1 to various control measures.

Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost.

BACKGROUND: The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics. METHODS: A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports. RESULTS: Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities. CONCLUSION: Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

TB transmission is associated with prolonged stay in a low socio-economic, high burdened TB and HIV community in Cape Town, South Africa.

BACKGROUND: While several studies have assessed the associations between biological factors and tuberculosis (TB) transmission, our understanding of the associations between TB transmission and social and economic factors remains incomplete. We aimed to explore associations between community TB transmission and socio-economic factors within a high TB-HIV burdened setting. METHODS: We conducted a cross-sectional molecular epidemiology study among adult patients attending a routine TB clinic. Demographic and clinical data were extracted from TB registers and clinical folders; social and economic data were collected using interviewer-administered questionnaires; Mycobacterium tuberculosis isolates were genotyped and classified as clustered/non-clustered using IS6110-based Restriction Fragment Length Polymorphism. Composite "social" and "economic" scores were generated from social and economic data. Data were analyzed using StataCorp version 15.0 software. Stratified, bivariable analyses were performed using chi-squared. Wilcoxon signed rank tests; univariable and multivariable logistic regression models were developed to explore associations in the social, economic, traditional and composite TB risk factors with TB transmission. RESULTS: Of the 505 patient Mtb  strains, 348(69%) cases were classified as clustered and 157(31%) were non-clustered. Clustered cases were more likely to have lived longer in the study community, (odds ratio [OR] = 1.05, 95% Confidence interval [C.I]:1.02-1.09, p = 0.006); in the same house (OR = 1.04, C.I: 0.99-1.08, p = 0.06); and had increased household crowding conditions (i.e fewer rooms used for sleeping, OR = 0.45, C.I:0.21-0.95, p = 0.04). Although a higher proportion of clustered cases had a low economic score, no statistically significant association was found between clustering and either the economic score (p = 0.13) or social score (p = 0.26). CONCLUSIONS: We report a novel association between Mtb transmission and prolonged stay within a high burdened community. Transmission was also associated with fewer rooms for sleeping in a household. Increased social interaction and prolonged residence in a high burdened community are important factors linked to Mtb transmission, possibly due to increased probability of higher effective contact rates. The possible importance of degrees of poverty within low socio-economic setting warrants further study.

Elucidation of drug resistance mutations in Mycobacterium tuberculosis isolates from North India by whole-genome sequencing.

OBJECTIVES: Rapid diagnosis of drug-resistant tuberculosis (TB) is required for better patient management and treatment outcomes. Whole-genome sequencing (WGS) can be used to detect single nucleotide polymorphisms (SNPs) and deletions/insertions that are responsible for mostMycobacterium tuberculosis drug resistance. WGS is being performed at scale in high-income countries, but there are limited reports of its use in India. METHODS: In this study, 33 clinicalM. tuberculosis isolates from the Mycobacterial Repository in Chandigarh underwent WGS. Phenotypic drug susceptibility testing was performed according to World Health Organization (WHO) recommendations. Four isolates were excluded from the analysis due to culture contamination or mislabelling during the study. RESULTS: Among the remaining 29 isolates, 21 (72.4%) were multidrug-resistant TB (MDR-TB) and 1 (3.4%) was extensively-drug resistant TB (XDR-TB). The most common mutations observed for isoniazid, rifampicin, ofloxacin and kanamycin resistance werekatG(S315T), rpoB(S450L), gyrA(A90V) and rrs(A1401G), respectively. The isolates mainly belonged to lineages 2 and 3, with most MDR-TB among lineage 2 isolates. CONCLUSION: WGS ofM. tuberculosis isolates allows the detection of drug resistance to all drugs in a single test and also provides insight into the evolution and drug-resistant TB.

A Clinical TB Detection Method Based on Molecular Typing Technique with Quality Control.

The gold standard for diagnosing pulmonary Mycobacterium tuberculosis (TB) is the detection of tubercle bacillus in patient sputum samples. However, current methods either require long waiting times to culture the bacteria or have a risk of getting false-positive results due to cross-contamination. In this study, a method to detect tubercle bacillus based on the molecular typing technique is presented. This method can detect genetic units, variable number of tandem repeat (VNTR), which are the characteristic of tuberculosis (TB), and performs quality control using a mathematical model, ensuring the reliability of the results. Compared to other methods, the proposed method was able to process and diagnose a large volume of samples in a run time of six hours, with high sensitivity and specificity. Our method is also in the pipeline for implementation in clinical testing. Reliable and confirmed results are stored into a database, and these data are used to further refine the model. As the volume of data processed from reliable samples increases, the diagnostic power of the model improves. In addition to improving the quality control scheme, the collected data can be also used to support other TB research, such as that regarding the evolution of the tubercle bacillus.

Assessment of the use and need for an integrated molecular surveillance of tuberculosis: an online survey in Germany.

BACKGROUND: The implementation of an integrated molecular surveillance (IMS) of tuberculosis (TB) is of high priority for TB control. IMS is defined as the systematic inclusion of molecular typing results in the national TB surveillance system. Although not standardized, an IMS of TB is already implemented in several low TB incidence countries. Germany is in the process of implementing a nationwide IMS of TB. This requires close collaboration between national and local health authorities. We conducted an online survey to understand the current use of molecular typing results for TB surveillance among the local public health offices (PHO)s in Germany, and to collect their perception and expectations towards the implementation of a nationwide IMS of TB. METHODS: The online survey was developed using the software Voxco and included 31 questions. The survey was sent to all the 377 local PHOs in Germany in April 2017. Responses were collected until June 2017. RESULTS: A total of 174/377 (46.2%) local PHOs participated in our survey, and 88/377 (23.3%) used molecular typing results in their routine TB surveillance work. The PHOs used molecular typing results especially as support for epidemiological contact tracing (62/88, 70.4%). We found statistically significant differences between answers of PHOs that did not use molecular typing results (n = 86) vs. PHOs that did use molecular typing results (n = 88): the latter perceived the use of molecular typing results as more beneficial for their work compared to the former (65.9% vs. 34.9%, p < 0.05). Moreover, the PHOs using molecular typing results expect for the future more support and coordination from regional and national public health institutes, especially regarding the identification and analysis of molecular clusters. CONCLUSIONS: Our study is a step forward in the broader goal of implementing an IMS of TB in Germany. The local PHOs currently using the molecular typing results highlighted their positive attitude towards the implementation of an IMS, but also their needs of more support. Similar assessments might serve as an example for other countries which are on the way to implement a nationwide IMS of TB.

Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility.

BACKGROUND: Several technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5'3'FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5' and 3' IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5'3'FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility. OBJECTIVES: Here we introduced critical amendments to the original IS6110-5'3'FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses. METHODS: IS6110-5'3'FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in E. coli for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data. RESULTS: In doing so, the overall turnaround time of IS6110-5'3'FP was reduced to 4 hours. The new protocol allowed detecting almost all 5' and 3' IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5'3'FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4. CONCLUSIONS: The IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRU-VNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.

Multiplex PCR from Menstrual Blood: A Non-Invasive Cost-Effective Approach to Reduce Diagnostic Dilemma for Genital Tuberculosis.

AIM: Genital tuberculosis (GTB) is a potent contributor to irreversible damage to the reproductive system and infertility in females. As no gold standard diagnostic tool is yet available, clinical suspicion and relatively insensitive approaches such as histopathology, laparoscopy and hysterosalpingogram are currently critical determinants in the diagnosis of GTB. Although a polymerase chain reaction (PCR)-based assay using endometrial tissue seems promising, sampling does require an invasive procedure. OBJECTIVE: We hypothesized that menstrual blood may provide an alternate non-invasive source of samples for PCR-based GTB diagnosis. METHODS: We enrolled 195 women with primary infertility in whom GTB was suspected. We obtained ethics committee approval from our institution and written informed consent from subjects. Endometrial tissue and menstrual blood was collected from the subjects and culture, histopathology, and multiplex PCR with both sample type was performed for each subject. RESULTS: The sensitivity and specificity of multiplex PCR was, respectively, 90.2 and 86.1% for menstrual blood, 95.8 and 84.3% for endometrial tissue, and 64.8 and 93.2% for histopathology staining. CONCLUSIONS: A strong clinical suspicion aided with multiplex PCR using menstrual blood may significantly reduce the diagnostic dilemma for GTB diagnosis in a non-invasive, sensitive, rapid, and cost-effective manner.

Tuberculosis in the immigrant population in Italy: state-of-the-art review.

Although the incidence of tuberculosis (TB) has been decreasing in the European Union/European Economic Area (EU/EEA) in recent decades, specific subgroups of the population, such as immigrants, remain at high risk of the disease. Immigration from areas of high incidence is thought to have fuelled the resurgence of TB in areas of low incidence. Indeed, while immigrants have a high risk of acquiring TB prior to migration, after migration they are exposed to additional risk factors for acquiring or reactivating TB infection, such as poverty, stressful living conditions, social inequalities, overcrowded housing, malnutrition, substance abuse and limited access to health care. In Italy as well, TB has increasingly become a disease for specific population subgroups such as immigrants and in urban settings often driven by reactivation of imported latent TB infection (LTBI). In this paper we present an analysis of the national scientific literature from recent years in order to estimate the burden of TB in foreign-born populations, to establish the burden of TB in migrants by gender, age group and country of origin as well as other relevant subgroups, and evaluate the clinical manifestations of latent or active tuberculosis and treatment response.

Assessment of Bactericidal Drug Activity and Treatment Outcome in a Mouse Tuberculosis Model Using a Clinical Beijing Strain.

Mycobacterium tuberculosis Beijing strains are associated with lower treatment success rates in tuberculosis (TB) patients. In contrast, laboratory strains such as H37Rv are often used in preclinical tuberculosis models. Therefore, we explored the impact of using a clinical Beijing strain on treatment outcome in our mouse tuberculosis model. Additionally, the predictive value of bactericidal activity on treatment outcome was assessed. BALB/c mice were infected with a Beijing strain and treated with one of 10 different combinations of conventional anti-TB drugs. Bactericidal activity was assessed by determining reductions in mycobacterial load after 7, 14, and 28 days and after 2, 3, and 6 months of treatment. Treatment outcome was evaluated after a 6-month treatment course and was based on lung culture status 3 months posttreatment. None of the anti-TB drug regimens tested could achieve 100% treatment success. Treatment outcome depended critically on rifampin. Four non-rifampin-containing regimens showed 0% treatment success compared to success rates between 81 and 95% for six rifampin-containing regimens. Bactericidal activity was predictive only for treatment outcome after 3 months of treatment. Our data advocate the use of multiple mycobacterial strains, including a Beijing strain, to increase the translational value of mouse TB models evaluating treatment outcome. Additionally, our findings support the notion that bactericidal activity in the first 2 months of treatment, as measured in clinical phase IIa/b trials, has limited predictive value for tuberculosis treatment outcome, thus emphasizing the need for better parameters to guide future phase IIII trials.

Positive epistasis of major low-cost drug resistance mutations rpoB531-TTG and katG315-ACC depends on the phylogenetic background of Mycobacterium tuberculosis strains.

Mycobacterium tuberculosis Beijing genotype strains increasingly circulate in different world regions, either as historical endemic, e.g. in East Asia, or recently imported, e.g. in South America, and this family is regarded as the most successful lineage of the global tuberculosis (TB) epidemic. Here we analysed the transmission capacity of these strains in the context of their phylogenetic background and drug resistance mutations. The study collection included all multidrug resistant (MDR) strains of Beijing genotype isolated in Beijing Chest Hospital, the largest tertiary TB facility in North China, in 2011-2013 (n = 278). Strains were subjected to NTF/IS6110 and 24-loci MIRU-VNTR analysis. Drug resistance mutations were detected in rpoB, katG, inhA and oxyR-ahpC. A total of 58 and 220 strains were assigned to the ancient and modern Beijing sublineages, respectively. 24-MIRU-VNTR clustering was higher in modern versus ancient Beijing strains (35.9% vs. 12.1%; P <0.001). After taking into consideration the presence of rpoB and katG mutations, clustering decreased to 15.9% in modern and 0% in ancient strains. The most frequent combination of mutations (rpoB531-TTG and katG315-ACC) was more prevalent in clustered versus non-clustered isolates in the modern sublineage (23/35 vs. 47/185; P <0.0001). To conclude, a combination of the known low-fitness-cost rpoB531-TTG and katG315-ACC mutations likely facilitates the increased transmission ability of MDR strains of the modern but not ancient Beijing sublineage. Accordingly, positive epistasis of major low-cost drug resistance-conferring mutations is influenced by the phylogenetic background of M. tuberculosis strains.

Molecular epidemiology of Mycobacterium tuberculosis complex in Brussels, 2010-2013.

The tuberculosis (TB) incidence rate in Brussels-Capital Region is 3-fold higher than in Belgium as a whole. Eight years after the realization of initial prospective population-based molecular epidemiology investigations in this Region, a similar study over the period 2010-2013 was conducted. TB strains isolated from 945 patients were submitted to genotyping by standardized 24-locus-MIRU-VNTR typing and spoligotyping. The phylogenetic analysis showed that the LAM (16.7%) and Haarlem (15.7%) branches are the two most prevalent TB lineages circulating in Brussels. Analysis of the MDR subgroup showed an association with Beijing strains (39.9%) and patients native of Eastern Europe (40.7%). Genotyping detected 113 clusters involving 321 patients, giving a recent transmission index of 22.9%. Molecular-guided epidemiological investigations and routine surveillance activities revealed family transmission or social contact for patients distributed over 34 clusters. Most of the patients were foreign-born (75.7%). However, cluster analysis revealed only limited trans-national transmission. Comparison with the previous study shows a stable epidemiological situation except for the mean age difference between Belgian-born and foreign-born patients which has disappeared. This study confirms that molecular epidemiology has become an important determinant for TB control programs. However, sufficient financial means need to be available to perform all required epidemiological investigations.

Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population.

A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission-despite opportunities-and challenged some of the advantages associated with Beijing strains.

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

BACKGROUND: In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. METHODS: A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods. RESULTS: Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases. CONCLUSION: We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.

Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays.

We report on a molecular investigation carried out to ascertain the prevalence of drug-resistant tuberculosis (TB) and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH) in Iraq. In total, 110 clinical isolates from category II TB cases from Baghdad (58%) and several Iraqi provinces (42%) were analysed using colorimetric, low-cost and low-density (LCD) microarrays (MYCO-Direct and MYCO-Resist LCD array kits) to identify the point mutations responsible for resistance in Mycobacterium tuberculosis isolates. We found 76 patients (69.1%) had resistant strains, of which 40 (36%) were multidrug-resistant (MDR)-TB. Where mono-resistance was identified, it was found to be predominantly to RIF (83%). The most common mutations were rpoB S531L (50%), inhA C15T (25%) and katG S315T (15%). The most common MDR-TB genotypes were rpoB S531L with inhA C15T (60%) and rpoB S531L with katG S315T (20%). Where phenotypic analysis of clinical isolates was also performed, genotypic data were found to show excellent correlation with phenotypic results. Correlation was found between the MYCO-Resist LCD array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDR-TB in 36% of category II TB cases in Baghdad and surrounding Iraqi provinces, which reflects the World Health Organization findings based on phenotypic studies. Diagnosis of TB and MDR-TB using culture-based tests is a significant impediment to global TB control. The LCD arrays investigated herein are easy to use, sensitive and specific molecular tools for TB resistance profiling in resource-limited laboratory settings.

Transforming the diagnosis of tuberculosis: an editorial board member's opinion at the 15th year of Expert Review of Molecular Diagnostics.

Interview with Professor Madhukar Pai, MD, PhD by Claire Raison (Commissioning Editor). Professor Madhukar Pai did his medical training and community medicine residency in Vellore, India. He completed his PhD in epidemiology at the University of California, Berkeley (CA, USA) and a postdoctoral fellowship at the University of California, San Francisco (CA, USA). He is currently an associate professor of epidemiology at McGill University in Montreal (Canada). He serves as the Director of Global Health Programs, and as an Associate Director of the McGill International Tuberculosis Centre. In addition, he serves as a Consultant for the Bill & Melinda Gates Foundation. He also serves on the Scientific Advisory Committee of the Foundation for Innovative New Diagnostics, Geneva, Switzerland. His research is focused on improving the diagnosis and treatment of tuberculosis, especially in high-burden countries such as India and South Africa. His research is supported by grant funding from the Gates Foundation, Grand Challenges Canada and Canadian Institutes of Health Research. He has more than 200 peer-reviewed publications. He is recipient of the Union Scientific Prize, Chanchlani Global Health Research Award and Stars in Global Health award from Grand Challenges Canada, and is a member of the Royal Society of Canada.

A first assessment of Mycobacterium tuberculosis genetic diversity and drug-resistance patterns in twelve Caribbean territories.

With the exception of some French-speaking islands, data on tuberculosis (TB) in the Caribbean are scarce. In this study, we report a first assessment of genetic diversity of a convenience sample of Mycobacterium tuberculosis strains received from twelve Caribbean territories by spoligotyping and describe their drug-resistance patterns. Of the 480 isolates, 40 (8.3%) isolates showed resistance to at least one anti-TB drug. The proportion of drug-resistant strains was significantly higher in The Bahamas (21.4%; P = 0.02), and Guyana (27.5%; P < 0.0001), while it was significantly lower in Jamaica (2.4%; P = 0.03) than in other countries of the present study. Regarding genetic diversity, 104 distinct spoligotype patterns were observed: 49 corresponded to clustered strains (2 to 93 strains per cluster), while 55 remained unclustered among which 16 patterns were not reported previously. Combining the study results with regional data retrieved from the international SITVIT2 database underlined a connection between frequency of certain M. tuberculosis phylogenetic lineages and the language spoken, suggesting historical (colonial) and ongoing links (trade, tourism, and migratory flows) with European countries with which they shared a common past.

Detection of Mycobacterium tuberculosis complex by PCR in fresh cheese from local markets in Hidalgo, Mexico.

The objective of this study was to identify the presence of Mycobacterium tuberculosis complex bacterial DNA in samples extracted from fresh cheeses; 95 samples of fresh cheese were obtained from municipal markets in the state of Hidalgo, in central Mexico, and were analyzed in triplicate. The exogenous control for the amplification was the mitochondrial gene for cytochrome b (cyt-b). M. tuberculosis complex DNA was detected by nested-PCR amplification of a fragment of the mpb70 gene in six samples, four of which were obtained from regions with enzootic bovine tuberculosis. These results suggest that cheeses prepared with raw milk contaminated with M. bovis are being sold and consumed by humans, which may cause tuberculosis.