Mycobacterium Tuberculosis Complex Isolates Research Articles

Characterization of mutations in the rpoB gene conferring rifampicin resistance in Mycobacterium tuberculosis complex isolated from lymph nodes of slaughtered cattle from South Africa.

Tuberculosis (TB) is an ongoing public health care, with the state of affairs exacerbated by the growth of anti-TB drug-resistant forms in South Africa. Not much attention is given to zoonotic TB. Thus, this study aimed to determine the presence of rpoB mutations among Mycobacterium tuberculosis complex (MTBC) isolates of lymph nodes from slaughtered cattle. A count of 14,950 carcasses from selected abattoirs were examined for nodular lesions and enlarged lymph nodes; 376 lymph nodes were cultured for MTBC. Positive isolates were tested for drug sensitivity against three anti-TB drugs, rifampicin, isoniazid, and ethambutol, using the Lowenstein-Jensen proportion method. Rifampicin-resistant isolates were sequenced, and spoligotyping was performed for lineage classification. A total of 162 isolates were confirmed as MTBC and 42 isolates were resistant to rifampicin. All rifampicin-resistant isolates carried the H526D rpoB mutation, and almost all of them carried an additional nonsynonymous nucleotide substitution in the hot spot region, in three other codons (510, 516 and 522). In total, 5 different mutations at four codons are reported, including one isolate showing 3 of them which has never been reported in South Africa. In addition, we report 4 different spoligo patterns, with 34 isolates known and 8 unknown spoligotype international types. From the known clades, 5 (11.9%) isolates were identified as Bov_4 caprae lineage, 29 (69%) Beijing, and 8 (19.1%) remaining unknown clades. The detection of MTBC-resistant patterns from cattle lymph nodes (Eastern Cape, South Africa) necessitates the investigation of other possible routes of MTBC transmission.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

ScopeSeveral methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC. MethodsThe EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. RecommendationsThe EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 105 CFU/mL suspension, obtained from a 10−2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 103 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03–0.12 for isoniazid, 0.12–0.5 for levofloxacin and 0.25–1 mg/L for amikacin. ConclusionsThe EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.

Spoligotype and Drug Susceptibility Profiles of Mycobacterium tuberculosis Complex Isolates in Golestan Province, North Iran.

IntroductionDespite the moderate incidence of tuberculosis (TB) in many parts of Iran, Golestan province had a permanently higher TB incidence rate than the national average. Moreover, Golestan province receives immigrants, mainly from TB-endemic areas of Iran and neighbor countries. Here, we aimed to characterize the circulating Mycobacterium tuberculosis complex (MTBC) isolates in terms of the spoligotype and drug resistance patterns, across Golestan province.Materials and MethodsA set of 166 MTBC isolates was collected during July 2014 to July 2015 and subjected to drug susceptibility testing for first- and second-line anti-TB drugs and spoligotyping.ResultsOf 166 MTBC isolates, 139 (83.7%) isolates were assigned to 28 spoligotype international types (SITs). The most frequent SITs were SIT127/Ural-2 (n=25, 15.1%), followed by SIT1/Beijing (n=21, 12.7%) and SIT3427/Ural-2 (n=18, 10.8%). The set of 18 isolates (10.8%) showed resistance to at least one drug, which mainly belonged to SIT1/Beijing (n=7, 38.9%), orphan patterns (n=4, 22.2%) and SIT357/CAS1-Delhi (n=3, 16.7%). In addition, four isolates (2.4%) were resistant to pyrazinamide. The analysis of mutation corresponded to resistance to rifampin and isoniazid showed that two isolates had Ser531Leu substitution in rpoB, four isolates had Ser315Thr substitution in katG and one isolate had [C(−15)T] in inhA locus.ConclusionHigh diversity in spoligotypes of the MTBC isolates and lack of dominant genotype might be due to residence of immigrants in this region and consequent reactivation of latent infection. In addition, due to the presence of extensively drug-resistant (XDR) isolates in Golestan province, it is important to conduct future studies to determine transmission pattern of drug-resistant isolates in this region.

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis

The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is important for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.

A Current Microbiological Picture of Mycobacterium Isolates from Istanbul, Turkey.

Despite advances in diagnosis and treatment, tuberculosis (TB) continues to be one of the essential health problems throughout the world. Turkey is considered to be endemic for TB. In this study, we analyzed the distribution of Mycobacterium species, compare the diagnostic methods, and susceptibilities to anti-tuberculosis drugs of TB isolates. The aim was to document the current status and to provide a frame of reference for future studies. In this study, 278 Mycobacterium species isolated from 7,480 patients between September 2015 and June 2019 were included. Löwenstein-Jensen medium (LJ) and MGIT 960 were used for the isolation of strains. Susceptibility to 1st-line anti-tuberculosis drugs was determined. Positivity rates in clinical samples were as follows: 1.4% for direct microscopic acid-fast bacilli (AFB) detection, 3.4% for growth on the LJ, and 3.7% for growth on MGIT-960. Two hundred thirty-three isolates were identified as Mycobacterium tuberculosis complex (MTBC) and 45 were non-tuberculous mycobacteria (NTMs). Eleven of the NTMs (24.4%) were Mycobacterium fortuitum group isolates, and eight NTMs (17.7%) were Mycobacterium abscessus complex isolates. A number of patients diagnosed with tuberculosis peaked twice between the ages of 20–31 and 60–71. A hundred and eighty-two MTBC isolates (78.1%) were susceptible to all 1st-line anti-tuberculosis drugs, while 51 isolates (21.9%) were resistant to at least one drug tested. The multidrug-resistant tuberculosis rate was 13.7% among resistant strains and 3% in all strains. The liquid cultures were better for detection of both MTBC and NTMs isolates. The data demonstrate that MTBC continues to be challenge for this country and indicates the need for continued surveillance and full-spectrum services of mycobacteriology laboratory and infectious diseases.

Whole Genome Sequencing and Spatial Analysis Identifies Recent Tuberculosis Transmission Hotspots in Ghana.

Whole genome sequencing (WGS) is progressively being used to investigate the transmission dynamics of Mycobacterium tuberculosis complex (MTBC). We used WGS analysis to resolve traditional genotype clusters and explored the spatial distribution of confirmed recent transmission clusters. Bacterial genomes from a total of 452 MTBC isolates belonging to large traditional clusters from a population-based study spanning July 2012 and December 2015 were obtained through short read next-generation sequencing using the illumina HiSeq2500 platform. We performed clustering and spatial analysis using specified R packages and ArcGIS. Of the 452 traditional genotype clustered genomes, 314 (69.5%) were confirmed clusters with a median cluster size of 7.5 genomes and an interquartile range of 4–12. Recent tuberculosis (TB) transmission was estimated as 24.7%. We confirmed the wide spread of a Cameroon sub-lineage clone with a cluster size of 78 genomes predominantly from the Ablekuma sub-district of Accra metropolis. More importantly, we identified a recent transmission cluster associated with isoniazid resistance belonging to the Ghana sub-lineage of lineage 4. WGS was useful in detecting unsuspected outbreaks; hence, we recommend its use not only as a research tool but as a surveillance tool to aid in providing the necessary guided steps to track, monitor, and control TB.

Using of the Sensititre MycoTB Plate Method for the Detection of Anti-tuberculous Drug Susceptibility of Rifampin Resistant Mycobacterium tuberculosis Complex Isolates

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis (TB). Rifampin resistance in the clinical isolates of MTBC is an important indicator for multidrug resistant-TB (MDR-TB) cases. In this study, it was aimed to evaluate whether the Sensititre MycoTB plaque method is suitable for the routine use in determining drug susceptibility of rifampin resistant MTBC strains. Xpert MTBC/ RIF positive rifampin resistant 100 MTBC isolates were included in the study. Xpert MTBC/RIF (Cepheid, USA) test were performed after the samples were processed by homogenization and decontamination and acid-fast staining. Rifampin resistant clinical samples were cultured in automated MGIT/BACTEC 960 (Becton Dickinson, USA) system and acid fast bacteria (AFB) were investigated. The anti-TB drug susceptibility tests of all culture positive AFB and cord factor identified as MTBC by using a cart test (MPB64, Capilla TB-Neo, Tauns Laboratories, Inc., Numazu, Japan) were performed with the Löwenstein-Jensen proportion method (LJPM) and Sensititre MycoTB (Trek Diagnostic Systems, Cleveland, OH, USA) methods. For the comparison of the methods used, the tests were performed simultaneously. The standard LJPM was performed according to the previously described procedures by World Health Organization and the Sensititre MycoTB plate method was performed as defined by the manufacturer. The final concentrations of isoniazide, rifampin, rifabutin, ethambutol, ofloxacin, moxifloxacin amikacin, kanamycin, cycloserine, ethionamide and p-aminosalicylic acid in Löwenstein-Jensen media for LJPM were 0.2 µg/ml, 40.0 µg/ml, 20.0 µg/ml, 2.0 µg/ml, 2.0 µg/ml, 1.0 µg/ml, 4.0 µg/ml, 30.0 µg/ml, 30.0 µg/ml, 40.0 µg/ml, 40.0 µg/ml and 1.0 µg/ ml, respectively. The results were obtained in 14 days for all of the drugs in the Sensititre MycoTB plate method and in 28-42 days in the LJPM. In this study, the sensitivity and specificity percentages of the Sensititre MycoTB method and the categorical agreement between the two methods were calculated. The sensitivity and specificity percentages of the Sensititre MycoTB plate method were between 84.4-100% and 95.6- 100%, respectively. The categorical agreements between the two methods were 92-100% for the drugs tested in the study. Ethambutol was found to have the lowest sensitivity (84.4%) and specificity (95.6%). The sensitivities of isoniazide, ofloxacin, streptomycin, kanamycin, ethionamide and p-aminosalicylic acid were 98.8%, 90.0%, 94.3%, 87.5%, 91.7% and 95.6%, respectively, while rifampicin, rifabutin, moxifloxacin, amikacin, cycloserine were calculated as 100%. The specificities of isoniazid, rifampicin, rifabutin, ofloxacin, moxifloxacin, amikacin, kanamycin, and cycloserine were found to be 100%, streptomycin, ethionamide and p-aminosalicylic specificity were 96.9%, 97.4% and 98.9% respectively. The categorical agreement was 96-100% in all tested drugs except ethambutol (92%). As a result, although the cost is high, owing to the short incubation period, easy to perform, the possibility for evaluating both first and second line anti-TB drugs simultaneously, determination of minimum inhibitory concentration values of the drugs, long shelf life, high sensitivity, specificity and the categorical agreement values, the Sensititre MycoTB method was determined as an effective method that can be used especially in laboratories where the workload and the MDR-TB cases are high.

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings.

Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex (MTBC) isolates, based on 24 loci, is still widely used as the standard for routine molecular surveillance of tuberculosis (TB). QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods regarding MIRU-VNTR typing of MTBC. We aimed to evaluate the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories. A total of 173 DNA were used for the study. Results obtained with QIAxcel were compared to the reference results obtained with an ABI 3730 DNA analyzer. In Albania, the overall agreement with the reference method was 97.92%. A complete agreement result was obtained for 17 loci. In Tunisia, the overall agreement with the reference method was 98.95%. A complete agreement result was obtained for 17 loci. Overall agreement in both centers was 98.43%. In our opinion, use of QIAxcel technology has the potential to be reliable, given an optimized algorithm. Inaccuracies in sizing of long fragments should be solved, especially regarding locus 4052.

The molecular patterns of resistance to anti-tuberculosis drugs: an analysis from Istanbul, Turkey

Resistances to anti-tuberculosis (TB) drugs are related to the mutations in the genome of the Mycobacterium tuberculosis complex (MTBC). To expose the genomic mutations that cause anti-TB drug resistance. The GenoType MTBDRplus and MTBDRsl assays were used to detect genetic mutations. In this study of 1329 MTBC isolates, the sensitivities and specificities of genotypic methods for the detection of isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and multi-drug resistance were 0.77, 0.84, 0.65, 0.89 and 0.99, 0.98, 0.67, 0.94, respectively. MUT3 mutations (S531L) of the rpoB gene for RIF resistance and MUT1 mutations (S315T1 and C15T) of the katG and inhA genes for INH resistance were dominant. The most frequently observed mutations that created resistance to fluoroquinolones (FLQ) were MUT3C (D94G) of the gyrA gene. The predominant mutations of EMB resistance were MUT1B (M306V) of the embB gene. Aminoglycosides/cyclic peptides (AG/CP) resistance was rare. The molecular mechanisms of anti-TB drugs resistance in MTBC strains found in Istanbul are similar to those in the literature.

Genetic Diversity and Drug Resistance of Mycobacterium tuberculosis Complex Isolates and Nontuberculous Mycobacteria Identification from Presumptive Tuberculosis Cases in Oaxaca, Mexico

Tuberculosis (TB) is a re-emerging health problem worldwide. In Mexico, information about genetic diversity and drug resistance of Mycobacterium Tuberculosis complex (MTBc) is limited. Samples from 463 Presumptive TB cases were tested for TB by smear, culture and PCR, from which 19.2% were identify as MTBc and 16% as nontuberculous mycobacteria. MTBc isolates were characterized by Large Sequence Polymorphisms (LSPs), spoligotyping and MIRU-VNTR 24 loci typing methods. Clade designations showed 10 sub-lineages: Haarlem (35.7%), EAI (26.2%), LAM (9.5%), Ghana (4.8%), X (2.4%), New-1 (2.4%), H37Rv-like (7.1%), M. bovis (2.4%), S (7.1%), Uganda I (2.4%). The finding of EAI as one of the principal genotypes may be associated with high migration rates. Drug resistance was found in 35.71% of the isolates: 14.2% were multidrug-resistant (MDR-TB), 14.2% mono-resistant and 7.14% poly-resistant. This study provides the first description of genetic diversity and drug resistance profile of MTBc in Oaxaca, Mexico.

Bovine tuberculosis at the human–livestock–wildlife interface and its control through one health approach in the Ethiopian Somali Pastoralists: A review

Pastoralism is a way of life in which food supply is produced from animals by using a variety of herding practices based on constant or partial herd mobility in the low land areas of Ethiopia. It covers 12% of the total livestock population and 61% of the total area of land in the country. As a result of their mobile lifestyle, pastoralists are almost completely excluded from the available health services. This review article focuses on bovine tuberculosis in the Ethiopian Somali Pastoralist. It describes Mycobacterium bovis in humans, livestock, and wildlife, and how the disease can be controlled by using One Health approach. Bovine tuberculosis is a chronic bacterial disease caused by Mycobacterium bovis. A study done from 2006 to 2008 on the prevalence of BTB in Ethiopian wildlife showed that sera from 20 of 87 animals (23%) were positive for BTB. In Ethiopia there is no comprehensive report about the status of M. bovis in wildlife populations that often share habitat with livestock. A study done on bovine tuberculosis in Somali pastoral livestock showed low prevalence of the disease. An individual animal prevalence of 2.0%, 0.4%, and 0.2% was reported in cattle, camels, and goats, respectively. In a simultaneous human and cattle study in a pastoralist areas of south-eastern Ethiopia, out of 163 human Mycobacterium tuberculosis complex isolates three were M. bovis. Due to the moderate resistance of the etiological agent to the environmental conditions in one hand and the capacity of its survival in acid milk for not less than 15 days on the other and the habitual consumption of unpasteurized milk by humans make this disease a vital zoonosis in Somali pastoralists in Ethiopia. M. bovis is a pathogen at the human-livestock-wildlife interface. Diseases transmitted between humans, livestock, and wildlife are increasingly challenging public and veterinary health systems. Therefore, studies concerning the burden of the diseases in wildlife, livestock and human beings in Somali Pastoralists should be undertaken. A One Health approach that takes the wellbeing of the pastoralists, the health of their livestock and environment into consideration is also necessary for the control of BTB.

Low antimicrobial resistance rates of Mycobacterium tuberculosis complex between 2000 and 2015 in Gipuzkoa, northern Spain

ObjectiveAlthough the incidence of tuberculosis (TB) has declined, TB drug resistance remains a major problem. The TB rate in Gipuzkoa (northern Spain) is higher than the European average. The objective of this study was to determine the antimicrobial susceptibility of 1855 Mycobacterium tuberculosis complex isolates (94.5% of confirmed cases between 2000 and 2015). MethodsSusceptibility testing was performed using the agar proportion method and a commercial broth system (MGIT 960). In isoniazid- or rifampicin-resistant strains, we studied genetic determinants of drug resistance and genotype (MIRU-VNTR). ResultsThe annual mean incidence of TB was 24.5 cases per 100,000 population on average, and tended to decrease. The multidrug-resistant TB rate was 0.5% (9/1855), and no extensively drug-resistant TB strains were detected. Rates of resistance to isoniazid and rifampicin were 3.9% (range, 3.4–4.3%) and 0.6% (range, 0.4–1.4%), respectively. TB resistance was more common among foreign-born individuals and those who had received previous TB treatment. Genotyping of 102 resistant strains showed predominance of the Euro-American lineage, although 4/9 multidrug-resistant strains had Eastern lineages (2 East African-Indian, and 2 East Asian [Beijing]). ConclusionsIn Gipuzkoa, with a moderate incidence of TB, resistance was very low, mostly being detected among individuals who were born abroad or who had a history of TB treatment.

Population structure of drug-resistant Mycobacterium tuberculosis in Central Asia

BackgroundDrug-resistant tuberculosis (TB) is a major public health concern threathing the success of TB control efforts, and this is particularily problematic in Central Asia. Here, we present the first analysis of the population structure of Mycobacterium tuberculosis complex isolates in the Central Asian republics Uzbekistan, Tajikistan, and Kyrgyzstan.MethodsThe study set consisted of 607 isolates with 235 from Uzbekistan, 206 from Tajikistan, and 166 from Kyrgyzstan. 24-loci MIRU-VNTR (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats) typing and spoligotyping were combined for genotyping. In addition, phenotypic drug suceptibility was performed.ResultsThe population structure mainly comprises strains of the Beijing lineage (411/607). 349 of the 411 Beijing isolates formed clusters, compared to only 33 of the 196 isolates from other clades. Beijing 94–32 (n = 145) and 100–32 (n = 70) formed the largest clusters. Beijing isolates were more frequently multidrug-resistant, pre-extensively resistant (pre-XDR)- or XDR-TB than other genotypes.ConclusionsBeijing clusters 94–32 and 100–32 are the dominant MTB genotypes in Central Asia. The relative size of 100–32 compared to previous studies in Kazakhstan and its unequal geographic distribution support the hypothesis of its more recent emergence in Central Asia. The data also demonstrate that clonal spread of resistant TB strains, particularly of the Beijing lineage, is a root of the so far uncontroled MDR-TB epidemic in Central Asia.

Antituberculosis drug resistance in isolates of Mycobacterium tuberculosis complex in southeast Spain.

The aim of this study was to determine resistance to antituberculosis (anti-TB) drugs in Mycobacterium tuberculosis complex isolates from patients diagnosed with Tuberculosis (TB) in southeast Spain and to study related epidemiological factors. This retrospective study analysed 5-year data (2012-2016) obtained in southeast Spain for a total equivalent population of 1 735 608 inhabitants. Clinical samples were examined from 557 patients with suspected pulmonary TB (n=470; 84.4%) or extrapulmonary TB (n=87; 15.6%), taking into account patient age, sex, human immunodeficiency virus (HIV) infection, country of birth and prior anti-TB treatment. TB was found more frequently in men than in women (66.6% vs. 33.4%), and the age group with the most cases (43.7%) was 36-55 years. Among the first-line anti-TB drugs, 7.0% of patients harboured isolates resistant to isoniazid (INH) and 1.6% to rifampicin (RIF); moreover, 1.4% of isolates were multidrug-resistant TB (MDR-TB) and 0.7% were extensively drug-resistant TB. There was a statistically significant relationship (P=0.028) between MDR-TB isolates and non-Spanish-born patients, but not between the latter and INH resistance. Resistance to INH and RIF was observed at levels similar to those published nationwide, with rates of MDR-TB being somewhat lower. Rates of HIV/TB co-infection have decreased considerably between 2012 and 2016.

Retrospective Analysis of Archived Pyrazinamide Resistant Mycobacterium tuberculosis Complex Isolates from Uganda-Evidence of Interspecies Transmission.

The contribution of Mycobacterium bovis to the proportion of tuberculosis cases in humans is unknown. A retrospective study was undertaken on archived Mycobacterium tuberculosis complex (MTBC) isolates from a reference laboratory in Uganda to identify the prevalence of human M. bovis infection. A total of 5676 isolates maintained in this repository were queried and 136 isolates were identified as pyrazinamide resistant, a hallmark phenotype of M. bovis. Of these, 1.5% (n = 2) isolates were confirmed as M. bovis by using regions of difference PCR analysis. The overall size of whole genome sequences (WGSs) of these two M. bovis isolates were ~4.272 Mb (M. bovis Bz_31150 isolated from a captive chimpanzee) and 4.17 Mb (M. bovis B2_7505 from a human patient), respectively. Alignment of these genomes against 15 MTBC genome sequences revealed 7248 single nucleotide polumorphisms (SNPs). Theses SNPs were used for phylogenetic analysis that indicated a strong relationship between M. bovis and the chimpanzee isolate (Bz_31150) while the other M. bovis genome from the human patient (B2_7505) analyzed did not cluster with any M. bovis or M. tuberculosis strains. WGS analysis also revealed multidrug resistance genotypes; these genomes revealed pncA mutations at positions H57D in Bz_31150 and B2_7505. Phenotypically, B2_7505 was an extensively drug-resistant strain and this was confirmed by the presence of mutations in the major resistance-associated proteins for all anti-tuberculosis (TB) drugs, including isoniazid (KatG (S315T) and InhA (S94A)), fluoroquinolones (S95T), streptomycin (rrs (R309C)), and rifampin (D435Y, a rare but disputed mutation in rpoB). The presence of these mutations exclusively in the human M. bovis isolate suggested that these occurred after transmission from cattle. Genome analysis in this study identified M. bovis in humans and great apes, suggesting possible transmission from domesticated ruminants in the area due to a dynamic and changing interface, which has created opportunity for exposure and transmission.

First-line anti-tuberculosis drug resistance trends of Mycobacterium tuberculosis complex isolates. A tertiary hospital study in Turkey.

Tuberculosis (TB) is continuing to be a important public health problem in the undeveloped countries. Drug sensitivity rate should be monitored for the effective treatment and control in the TB. The aim of this study was to determine the rate of resistance to first line TB drugs in the Mycobacterium tuberculosis complex isolates. During one-year period, M. tuberculosis complex was isolated in the 1193 samples from 974 patients in the Mycobacterial Laboratory of Yedikule Chest Diseases and Chest Surgery Education and Research Hospital, Istanbul, Turkey. The majority of samples isolated in the M. tuberculosis complex were sputum (n= 897, 92.1%). Anti-TB drug susceptibility testing was performed with Mycobacterium Growth Indicator Tube 960 system. Two hundred and sixty isolat (26.7%) were resistant to at least one of the four first-line anti-TB drugs tested. One hundred ninety seven isolates were resistances to isoniazid (20.2%); 82 to rifampin (8.4%), 63 to ethambutol (6.5%) and 140 to streptomycin (14.4%). Of the 197 isoniazid-resistant isolates, 89 (45.2%) isolates was only isoniazid-resistance, only rifampin-resistance were found 15.9% (n= 13), ethambutol 7.9% (n= 5) and streptomycin 30.7% (n= 43). There were 48 (4.9%) isolates with two drugresistance, 22 (2.3%) isolates with three drug-resistance, and 42 (4.3%) isolates with four drug-resistance. The multidrug resistance rate was 7% (68 of 974). There was no relationship with between the frequency of TB drug resistance and gender or age. The isoniazid--resistance and streptomycin-resistance were seen to tend to increase if together considered the results of this study with outcomes of previously reported studies from Turkey in the 1998-2003, 2004-2007 and 2008-2010 years. Monitoring of drug susceptibility test results can contribute to the management of TB treatment and increase treatment success. Isoniazid-resistance and streptomycin-resistance tend to increase in Turkey. Further clinical studies are needed to investigate regional and global factors affecting the development of resistance to first-line TB drugs.

WGS more accurately predicts susceptibility of Mycobacterium tuberculosis to first-line drugs than phenotypic testing.

Drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide. DST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT. In total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance. Applying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST.

Demographic risk factors for extra-pulmonary tuberculosis among adolescents and adults in Saudi Arabia.

Despite low infectious potential of extrapulmonary tuberculosis (EPTB), it poses significant clinical challenges in terms of diagnosis and treatment monitoring. Understanding the main demographical risk factors for disease characteristics of EPTB plays a crucial role in speeding up diagnosis process and improving overall clinical experience. The aim of this study was to investigate the main demographical and clinical risk factors for EPTB among adults and adolescents for the first time in Saudi Arabia. A cross-sectional multicenter study was carried out on a collection of 902 extrapulmonary Mycobacterium tuberculosis complex (MTBC) isolates with demographical and clinical data. All isolates were subjected to spoligotyping and 24-loci based MIRU-VNTR typing. The association between two potential variables was assessed using odd ratios (OR) calculations. Independent risk factors for EPTB and diseases characteristics of EPTB were identified using multivariate regression model analyses. Gender was found to be significantly associated with lymph node, gastrointestinal, central nervous system and urogenital TB. Lymph node TB showed statistical association to age group below 25 years, non-Saudis and South East Asian ethnicity. While gastrointestinal TB demonstrated an association with patients above 60 years old, and Saudis. Multivariate analysis showed that gender is an independent risk factor to urogenital TB (p 0.03) and lymph node TB (p 0.005). On the other hands, South Asian (p 0.01) and South East Asian (p 0.03) ethnicities were both identified as independent risk factors significantly associated with EPTB. MTBC lineages, site of infections, gender, HIV and smear positivity showed no significant association. Nationwide qualitative-studies are highly warranted in the future to further understand the main demographic risk factors for disease characteristics of EPTB.

A new oligonucleotide array for the detection of multidrug and extensively drug-resistance tuberculosis

Drug-resistant tuberculosis (TB) is a global crisis and a threat to health security. Since conventional drug susceptibility testing (DST) takes several weeks, we herein described a molecular assay to rapidly identify multidrug-resistant (MDR) and extensively drug-resistant (XDR) and reveal transmission associated-mutations of Mycobacterium tuberculosis complex (MTBC) isolates in 6 to 7 hours. An array was designed with 12 pairs of primers and 60 single nucleotide polymorphisms of 9 genes: rpoB, katG, inhA, ahpC, embB, rpsL, gyrA, rrs and eis. We assessed the performance of the array using 176 clinical MTBC isolates. The results of culture-based DST were used as the gold standard, the GenoType MTBDRplus and MTBDRsl tests were used for parallel comparison, and gene sequencing was performed to resolve the discordance. The sensitivities and specificities of the array are comparable to those of the MTBDRplus test for resistance to isoniazid (INH) (100.0%, 96.7%) and rifampicin (RIF) (99.4%, 96.7%) and of the MTBDRsl test for resistance to fluoroquinolones (FQs) (100%, 100%) and second-line injectable drugs (SLIDs) (98.3%, 100%). The sensitivities of the array for detecting resistance to ethambutol and streptomycin were 79.3% and 64.9%, respectively. The array has potential as a powerful tool for clinical diagnosis and epidemiological investigations.

Low antimicrobial resistance rates of Mycobacterium tuberculosis complex between 2000 and 2015 in Gipuzkoa, northern Spain

ObjectiveAlthough the incidence of tuberculosis (TB) has declined, TB drug resistance remains a major problem. The TB rate in Gipuzkoa (northern Spain) is higher than the European average. The objective of this study was to determine the antimicrobial susceptibility of 1855 Mycobacterium tuberculosis complex isolates (94.5% of confirmed cases between 2000 and 2015). MethodsSusceptibility testing was performed using the agar proportion method and a commercial broth system (MGIT 960). In isoniazid- or rifampicin-resistant strains, we studied genetic determinants of drug resistance and genotype (MIRU-VNTR). ResultsThe annual mean incidence of TB was 24.5 cases per 100,000 population on average, and tended to decrease. The multidrug-resistant TB rate was 0.5% (9/1855), and no extensively drug-resistant TB strains were detected. Rates of resistance to isoniazid and rifampicin were 3.9% (range, 3.4–4.3%) and 0.6% (range, 0.4–1.4%), respectively. TB resistance was more common among foreign-born individuals and those who had received previous TB treatment. Genotyping of 102 resistant strains showed predominance of the Euro-American lineage, although 4/9 multidrug-resistant strains had Eastern lineages (2 East African-Indian, and 2 East Asian [Beijing]). ConclusionsIn Gipuzkoa, with a moderate incidence of TB, resistance was very low, mostly being detected among individuals who were born abroad or who had a history of TB treatment.