Number Tandem Repeat Typing Research Articles

Validation and implementation of whole-genome sequencing-based analytical methods for molecular surveillance and relatedness analysis of Mycobacterium tuberculosis Complex isolates at a National Reference laboratory

Whole genome sequencing-based methodologies have become extremely relevant for the molecular surveillance of human pathogens and are being increasingly introduced into national reference laboratory services. In this study, we describe the validation and implementation of core-genome Multi-Locus Sequence Typing (cgMLST) and whole genome single-nucleotide polymorphism (wgSNP) analysis at the Irish Mycobacteria Reference Laboratory, as a replacement for Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing. Concordance of clustering, discriminatory power, and ease-of-use of both WGS analytical methods were evaluated. Although wgSNP analysis (MTBseq) was the most discriminatory method (p<0,001), we recommend cgMLST (SeqSphere+), as the first-line approach for molecular typing of Mycobacterium tuberculosis isolates in the context of routine surveillance work due to its ease of use and decreased turnaround time, while reserving wgSNP-analysis for a more in-depth cluster analysis of new isolates that show a distance of ≤ 12 alleles to any other isolate(s) in the cgMLST database.

Molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis in homeless individuals in the Addis Ababa city, Ethiopia

Although homeless segment of the society could be the hotspots for tuberculosis (TB) transmission, there is little data on TB in homeless individuals in Ethiopia. The objective of this study was to investigate the molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis (M. tuberculosis) isolated from homeless individuals in Addis Ababa, Ethiopia. The study was conducted on 59 M. tuberculosis isolates, which were recovered by the clinical screening of 5600 homeless individuals and bacteriological examination of 641 individuals with symptoms of pulmonary tuberculosis (PTB). Region of difference-9 (RD9) based polymerase-chain reaction (PCR), Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing were used for genotyping of the isolates. In addition, drug sensitivity test was performed on the isolates using BD Bactec Mycobacterial Growth Inhibition Tube (MGIT) 960. Fifty-eight of the 59 isolates were positive by spoligotyping and spoligotyping International type (SIT) 53, SIT 37, and SIT 149 were the dominant spoligotypes; each consisting of 19%, 15.5%, and10.3% of the isolates, respectively. The majority of the isolates (89.7%) were members of the Euro-American (EA) major lineage. MIRU-VNTR identified Ethiopia_3, Delhi/CAS, Ethiopia_2, TUR, X-type, Ethiopia_H37Rv-like strain, Haarlem and Latin-American Mediterranean (LAM) sub lineages. The proportion of clustering was 77.6% (45/58) in spoligotyping while it was 39.7% (23/58) in 24-loci MIRU-VNTR typing. Furthermore, the proportion of clustering was significantly lowered to 10.3% (6/58) when a combination of spoligotyping and 24-loci MIRU-VNTRplus was used. The recent transmission index (RTI) recorded by spoligotyping, 24-loci MIRU-VNTR typing, and a combination of the two genotyping methods were 58.6%, 27.6% and 5.2%, respectively. Young age and living in groups were significantly associated with strain clustering (P < 0.05). The drug sensitivity test (DST) result showed 8.9% (4/58) of the isolates were resistant to one or more first line ant-TB drugs; but multidrug resistant isolate was not detected. Clustering and RTI could suggest the transmission of TB in the homeless individuals, which could suggest a similar pattern of transmission between homeless individuals and the general population. Hence, the TB control program should consider homeless individuals during the implementation of TB control program.

Mycobacterial Interspersed Repeat Unit-Variable Number Tandem Repeat Typing of Mycobacterium avium Strains Isolated from the Lymph Nodes of Free-Living Carnivorous Animals in Poland.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms, of which some, especially those of the Mycobacterium avium complex (MAC), may be opportunistic animal and human pathogens. Infection with NTM can interfere with tuberculosis (TB) diagnosis and induce zoonoses, especially in immunocompromised individuals. Diseases caused by NTM have become more readily recognized; however, they are likely still underestimated. In this study, we identified and genotyped Mycobacterium avium strains that were isolated during TB monitoring among free-living carnivorous animals from southeastern Poland. In 2011-2020, lymph node samples from 192 such animals were tested for mycobacteria. A total of 41 isolates of M. avium strains were detected with the use of IS901, IS900, IS1245, and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) identification. Thirty-three were identified as M. avium subsp. avium. These strains were derived from 1 beech marten (Martes foina), 1 common buzzard (Buteo buteo), 2 European badgers (Meles meles), 3 wolves (Canis lupus), and 26 red foxes (Vulpes vulpes). One strain isolated from a wolf was identified as M. avium subsp. hominissuis. The results show the widespread occurrence of MAC bacilli in the studied environment and additionally comprise new data on the molecular characteristics of M. avium subspecies carried by free-living southeastern Polish carnivores.

Investigating Pseudomonas aeruginosa population structure and frequency of cross-infection in UK cystic fibrosis clinics - a reference laboratory perspective

BackgroundWe aimed to describe the UK Pseudomonas aeruginosa population structure amongst people with cystic fibrosis (PWCF), and to examine evidence for cross-infection. MethodsVariable Number Tandem Repeat (VNTR) typing was performed on 4640 isolates from 2619 PWCF received from 55 hospital laboratories between 2017 and 2019. A combination of whole genome sequence (WGS)-based analysis of four clusters from one hospital, and epidemiological analysis of shared strains in twelve hospitals evaluated cross-infection. ResultsOf 2619 PWCF, 1324 (51%) harboured common clusters or known transmissible strains, while 1295 carried unique strains/those shared among small numbers of patients. Of the former, 9.5% (250 patients) harboured the Liverpool epidemic strain (LES), followed in prevalence by clone C (7.8%; 205 patients), cluster A (5%;130 patients), and cluster D (3.6%; 94 patients). WGS analysis of 10 LES isolates, 9 of cluster D and 6 isolates each of cluster A and clone C from one hospital revealed LES formed the tightest cluster (between 7 and 205 SNPs), and cluster D the loosest (between 53 and 1531 SNPs). Hospital-specific shared strains were found in some centres, although cross-infection was largely historical, with few new acquisitions. Fifty-nine PWCF (2.3%) harboured “high-risk” clones; one ST235 isolate carried a blaIMP-1 allele. ConclusionOf 2619 PWCF who had P. aeruginosa isolates submitted for VNTR, 51% harboured either common clusters or known transmissible strains, of which LES was the most common. Limited evidence of recent patient-to-patient strain transmission was found, suggesting cross-infection prevention measures and surveillance effectively reduce transmission.

Genetic Diversity and Population Structure of Mycobacterium bovis at the Human-Animal-Ecosystem Interface in France: "A One Health Approach".

Mycobacterium bovis infects cattle and wildlife, and also causes a small proportion of tuberculosis cases in humans. In most European countries, M. bovis infections in cattle have been drastically reduced, but not eradicated. Here, to determine the M. bovis circulation within and between the human, cattle, and wildlife compartments, we characterized by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing the genetic diversity of M. bovis isolates collected from humans, cattle, and wildlife in France from 2000 to 2010. We also assessed their genetic structure within and among the different host groups, and across time and space. The M. bovis genetic structure and its spatiotemporal variations showed different dynamics in the human and animal compartments. Most genotypes detected in human isolates were absent in cattle and wildlife isolates, possibly because in patients, M. bovis infection was contracted abroad or was the reactivation of an old lesion. Therefore, they did not match the genetic pool present in France during the study period. However, some human-cattle exchanges occurred because some genotypes were common to both compartments. This study provides new elements for understanding M. bovis epidemiology in France, and calls for increased efforts to control this pathogen worldwide.

Identification of Pathogenic Leptospira kirschneri Serogroup Grippotyphosa in Water Voles (Arvicola terrestris) from Ruminant Pastures in Puy-de-Dôme, Central France

Rodents are the primary reservoirs for pathogenic Leptospira species, which cause leptospirosis. Among the key potential carriers are water voles, whose population outbreaks can consequently pose a major threat to human and animal health. We studied the prevalence, prominence, and epidemiology of pathogenic Leptospira species in water voles in central France. First, 46 voles were captured, and DNA was extracted from kidney, lung, liver, blood, and urine and tested for the presence of Leptospira using three molecular methods: PCR, O-antigen typing, and variable number tandem repeat (VNTR) typing. We also attempted to culture leptospires from kidney and urine samples. In addition, we investigated leptospiral antibodies in serum samples from 60 sheep using microscopic agglutination testing. These animals co-occurred with the voles, so we sought to assess their degree of exposure and involvement in pathogen dynamics. The overall prevalence of infection was 76.1% (CI95% [61.2%, 87.4%]). The only strain found was L. kirschneri serogroup Grippotyphosa and a similar VNTR profile was acquired. Leptospires were successfully cultured from kidney and urine samples for four voles. Three sheep had low antibody titers against the Leptospira serogroup Grippotyphosa. Taken together, our results suggest the exclusive carriage of L. kirschneri serogroup Grippotyphosa among water voles in central France. Nevertheless, their ability to act as reservoir hosts that transmit the pathogen to co-occurring livestock remains unclear and merits further research.

Genetic Profiling of Salmonella enteritidis Strains Isolated in the Stavropol Region in 2016–2019

Introduction: Salmonella enteritidis is the most common etiologic agent of salmonellosis in humans in the Stavropol Region, which is in line with the global trend. In this regard, the definition of a serovar provides little information in the course of an epidemiological investigation and necessitates subspecific typing of S. enteritidis isolates. Objective: MLVA typing of S. enteritidis strains isolated in the Stavropol Region in 2016–2019 and the analysis of the genetic structure of the local Salmonella population. Materials and methods: We studied 122 strains of S. enteritidis isolated in 2016–2019 from stool samples of patients with acute intestinal infections in the city of Stavropol and the Caucasian Mineral Waters (CMW) of the Stavropol Region. Multi Locus Variable Number Tandem Repeat (MLVA) typing was performed based on five variable loci. The size of amplified loci was determined by capillary electrophoresis. Results: The studied strains were distinguished by high genetic heterogeneity and belonged to 25 MLVA genotypes. S. enteritidis strains belonging to 24 MLVA types were identified on the territory of Stavropol. The dominant genetic variant in the city was 3-10-5-4-1, which included 40 Salmonella strains (44.4%) isolated in 2016–2019. In some years, the proportion of other genetic variants increased. In the CMW area, strains belonging to seven MLVA genotypes were isolated, most of which belonged to the profile 3-10-5-4-1, while a change in the dominant variant of S. Enteritidis was registered annually. Prevalent regional MLVA types are widespread in the world and have a significant epidemic potential. Conclusions: We obtained new data on S. enteritidis MLVA genotypes in the Stavropol Region, established dominant genetic variants, and noted differences in the ratio of circulating MLVA genotypes of S. enteritidis in 2016–2019. Our findings necessitate constant molecular genetic monitoring to assess real-time dynamic changes in the genetic structure of the Salmonella population. They can also be used in investigations of possible cases (outbreaks) of salmonellosis to determine their source and routes of transmission.

Highly Discriminative Genotyping of Mycobacterium abscessus Complex Using a Set of Variable Number Tandem Repeats in China.

In this study, our aims were to comparatively analyze the power of variable number tandem repeat (VNTR) typing to discriminate isolates within subspecies and to identify a potential genetic marker for better molecular typing of Mycobacterium abscessus complex (MABC) strains. A total of 103 clinical MABC isolates were collected from a nationwide cross-sectional study in China. Eighteen VNTR loci were chosen to genotype the MABC isolates. Of the 103 clinical MABC isolates, there were 76 (73.8%) M. abscessus subsp. abscessus (MAA) and 27 (26.2%) M. abscessus subsp. massiliense (MAM) isolates. Among the patients with MAA lung diseases, the percentage of patients older than 45 years (67.1%) was significantly higher than that of patients with MAM lung diseases [33.3%, adjusted odds ratio (aOR) = 0.36, 95% CI = 0.13–0.98, p = 0.046]. Fifteen VNTR loci were designated as being “highly discriminant” in our sample, except for TR109. The total of 103 MABC isolates were fully discriminated into 103 unique patterns by an 18-locus VNTR set [Hunter–Gaston Discriminatory Index (HGDI) = 1.000], of which the inclusion of the top 12 loci yielded a comparative HGDI value (HGDI = 0.9998). Remarkably, the order of the diversity of the VNTR loci showed significant difference between the MAA and MAM isolates. TR137 and TR2, two loci with high diversity indices for the MAA isolates, only yielded poor discriminatory power for the MAM isolates; the allelic diversity (h) values were 0.0000 and 0.2621, respectively. A detailed analysis of TR137 in combination with the other 17 VNTR loci showed that the combination of TR137–TR2 could fully distinguish MAA from MAM isolates. In conclusion, our data revealed that MAA is more prone to affect elderly patients. Additionally, the population structure of the MABC isolates circulating in China has high diversity. The combined use of the TR137 and TR2 loci provides a simple criterion for the precise identification of MABC to the subspecies level.

Transmission of multidrug-resistant Mycobacterium tuberculosis in Wuhan, China: A retrospective molecular epidemiological study.

How multidrug-resistant tuberculosis (MDR-TB) spreads and expands in Wuhan population is not clear. The study aimed to determine the transmission patterns of MDR-TB in Wuhan city, China, including 149 patients with MDR-TB.Tuberculosis isolates were genotyped by deletion-targeted multiplex polymerase chain reaction, mycobacterial interspersed repetitive unit-variable number tandem repeat typing, and sequencing of drug resistance-associated genes. The risk factors of genomic-clustering were analyzed with logistic regression. The genomic-clustering patients were deeply investigated.The analysis identified 111 unique and 11 clustered genotypes (38 isolates). The clustering rate was 25.50% and the minimum estimate proportion of recent transmission was 18.12%. Two clusters (5 isolates) shared the same mutation, the remain 9 clusters (33 isolates) had different mutation. Logistic regression showed that older than 60 years (adjusted OR 2.360, 95% CI:1.052-5.292) was an independent factor associated with the genomic-clustering of MDR-TB. Among the 38 genomic-clustering cases, 14 cases had epidemiological transmission links. The most common type of transmission link was social contact.The local transmission of MDR-TB in Wuhan was really an issue. The elderly population might be the high-risk groups for transmission of MDR-TB, and the community or public transportation might be the main transmission places.

The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe.

Tuberculosis (TB) is a severe public health challenge in Korea. Of all Mycobacterium tuberculosis (M. tb) strains, the Beijing genotype strain reportedly correlates with hypervirulence and drug resistance. Hence, an early identification of the Beijing genotype strain of M. tb plays a significant role in initial TB treatment. Kogenebiotech® (KoRT-polymerase chain reaction [PCR]) has developed a real-time PCR 17 18 kit to determine the Beijing genotype strain classified as M. tb. To determine the feasibility of the commercially produced KoRT-PCR kit in identifying the M. tb strain. We used 100 clinical isolates of M. tb and 100 non-M. tb samples for the assessment. We evaluated the overall concordance between the KoRT-PCR kit and the mycobacterial interspersed repetitive unite variable number tandem repeat typing kit (GenoScreen, Lille, France). Moreover, we measured the detection limits based on the chromosomal DNA copies for the KoRT-PCR kit. In addition, we determined the reproducibility among individual technicians using the KoRT-PCR. The KoRT-PCR kit successfully discriminated all M. tb (confidence interval [CI]: 96.38%-100.00% for both sensitivity and specificity) and Beijing genotype strain (CI: 95.70%-100.00% for sensitivity and 96.87%-100.00% for specificity). We confirmed no significant deviation in the reproducibility between the technicians. The KoRT-PCR kit displayed sufficient capability of discriminating the Beijing genotype strain, which enabled the rapid identification of the Beijing genotype strain from the M. tb clinical isolates.

RAPID DISCRIMINATION AMONG METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATES USING VARIABLE NUMBER TANDEM REPEAT ANALYSIS IN A SAMPLE OF IRAQI PATIENTS AND

There is so little information available in Iraq about genetic variability in methicillin resistant Staphylococcus aureus (MRSA), so current study aimed to use six tandem repeat loci of multilocus variable number tandem repeat (MLVA) typing to discriminate among MRSA, so to achieve the aim of this study, six loci, clfA, clfB, sdrC, spa, sspa and sav1078 were selected for multiplex PCR. The PCR product were subjected to capillary electrophoresis by using ABI-Genetic analyzer, then the data were analyzed by using GeneMapper™ Software 5. Fragment sizes were converted into repeats number. The total number of repeats are used to generate allelic profile. The allelic profile used to draw the minimum spanning tree and dendrogram , all the 85 MRSA isolates are clustered into 54 MLVA type.

Genetic Diversity and Drug Susceptibility Profiles of Multidrug-Resistant Tuberculosis Strains in Southeast China.

BackgroundMultidrug-resistant tuberculosis (MDR-TB) isolates collected from Fujian province, China were assessed for molecular epidemiological characteristics. Analysis of isolate genotype profiles revealed that the Beijing genotype was associated with especially high drug resistance and community transmission rates.MethodsA total of 119 MDR-TB isolates obtained from TB patients in Fujian province were typed using 24–locus mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and spoligotyping. Drug susceptibility testing of all isolates was conducted using the L-J proportion method, with pyrazinamide (PZA) susceptibility testing conducted using the Mycobacterium Growth Indicator Tube System 960 (MGIT 960).ResultsWe obtained 26 spoligotypes for the 119 isolates examined in this work. Spoligotyping results revealed that 80 (67.2%) isolates possessed the Beijing family genotypic profiles. Patients aged 25–44 years and ≥45 years were most likely to be infected by non-Beijing genotypes. The percentage of clustered cases with both PZA and ofloxacin (OFLX) resistance was significantly greater than the corresponding percentage for non-clustered cases. Of 44 PZA-resistant isolates, 28 isolates (63.6%) harbored pncA mutations, while pncA mutations were only detected in 7 (9.3%) PZA-susceptible isolates.ConclusionOur data demonstrate that the Beijing genotype is the dominant lineage among MDR-TB strains circulating in Fujian. Thus, MDR-TB infections occurring within this province are not likely associated with recent transmission events. PZA and fluoroquinolone resistance profiles were found to be associated with clustered isolates. Mutation of pncA is the main driver of MDR-TB PZA resistance and is associated with mutation sites scattered throughout the entire pncA protein-coding region.

An outbreak of pulmonary tuberculosis and a follow-up investigation of latent tuberculosis in a high school in an eastern city in China, 2016-2019.

In October 2016, a senior high school student was diagnosed with sputum-smear positive [SS(+)] pulmonary tuberculosis (TB). We conducted an investigation of an outbreak in the school, including among students and teachers diagnosed with latent TB, who we followed until July 2019. We defined latent TB infection (LTBI) as a tuberculin skin test (TST) induration of 15mm or larger; probable TB as a chest radiograph indicative of TB plus productive cough/hemoptysis for at least 2 weeks, or TST induration of 15mm or larger; and confirmed TB as two or more positive sputum smears or one positive sputum smear plus a chest radiograph indicative of TB or culture positive with M. tuberculosis. We conducted mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing based on 24 loci in the isolates. Between October 2016 and July 2019, we identified 52 cases, including nine probable, six confirmed, and 37 LTBI cases. The index case-student had attended school continuously despite having TB symptoms for almost three months before being diagnosed with TB. We obtained three isolates from classmates of the index case in 2016; all had identical MIRU-VNTR alleles with the index case. The LTBI rate was lower among students (7.41%, 30/405) than among teachers (26.92%, 7/26) (rate ratio [RR] = 0.28, 95% confidential interval [CI]: 0.13-0.57). Among the 17 students who had latent TB and refused prophylaxis in October 2016, 23.53% (4/17) became probable/confirmed cases by July 2019. None of the six teachers who also refused prophylaxis became probable or confirmed cases. Of the 176 students who were TST(-) in October 2016, 1.70% (3/176) became probable/confirmed cases, and among the 20 teachers who were TST(-), 1 became a probable case. Delayed diagnosis of TB in the index patient may have contributed to the start of this outbreak; lack of post-exposure chemoprophylaxis facilitated spread of the outbreak. Post-exposure prophylaxis is strongly recommended for all TST-positive students; TST-negative students exposed to an SS(+) case should be followed up regularly so that prophylaxis can be started if LTBI is detected.

Bovine Tuberculosis: A Review of Molecular Diagnostic Methods and Impact on Public Health

Bovine tuberculosis (BTB) is a zoonotic infectious disease of cattle, other domesticated animals, and certain wildlife populations. It has been widely distributed throughout the world, and it has been a cause for great economic loss in animal production. In developed countries, the eradication programs have reduced or eliminated TB in cattle. Many factors contribute to the persistence of the disease, such as the limitations of diagnostic tests, larger herd sizes, increase in animal movements and trade, and limited options for control, such as restrictions on whole herd depopulation. The available advanced TB diagnostic techniques can detect and differentiate the causative mycobacterial species, and help an early confirmation of the diagnosis, which is useful to design appropriate control measures in the national BTB control programs. Molecular diagnostics, such as polymerase chain reaction, spoligotyping, restriction fragment length polymorphism (RFLP), variable number tandem repeats typing (VNTR), and polymorphism GC-rich repeat sequence (PGRS) are the techniques used for concurrent detection and typing of Mycobacterium species at the strain level. The molecular epidemiology is also being used to identify the source of contamination, to determine the risk factors of BTB transmissions, to investigate the drug resistance pattern, and to track the geographic distribution and spread of clones of Mycobacteria species. The molecular diagnosis is the tool to check whether active transmission or reactivation of BTB, hence, it is better to adopt these methods for the epidemiological survey of BTB. Since BTB is a major public health problem, at least a single reference laboratory should be available for molecular based diagnosis as part of its control.

Comparing genotypes and antibiotic resistance profiles of Mycobacterium abscessus and Mycobacterium massiliense clinical isolates in China

Abstract This study aimed to investigate differences in the antimicrobial susceptibility of members of the Mycobacterium abscessus complex (MABC): subsp. massiliense and subsp. abscessus, and to identify associations between strain genotypes and antimicrobial resistance phenotypes. A total of 383 clinical MABC isolates (subsp. abscessus: n = 218, 56.9%; subsp. massiliense: n = 163, 42.6%; subsp. bolletii: n = 2, 0.5%) were characterised using multilocus variable number tandem repeat (VNTR) typing and drug susceptibility testing. Most isolates exhibited susceptibility to amikacin, clarithromycin and azithromycin but resistance to cefoxitin and minocycline was statistically more associated with isolates unclustered by VNTR type. The Simpson's diversity indexes of VNTR typing for M. abscessus and M. massiliense isolates were 0.999 and 0.997, respectively. Genotyping of M. abscessus and M. massiliense isolates by VNTR may provide valuable information for predicting resistance phenotype.

Risk factors for recurrent tuberculosis after successful treatment in a high burden setting: a cohort study

BackgroundPeople successfully completing treatment for tuberculosis remain at elevated risk for recurrent disease, either from relapse or reinfection. Identifying risk factors for recurrent tuberculosis may help target post-tuberculosis screening and care.MethodsWe enrolled 500 patients with smear-positive pulmonary tuberculosis in South Africa and collected baseline data on demographics, clinical presentation and sputum mycobacterial cultures for 24-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. We used routinely-collected administrative data to identify recurrent episodes of tuberculosis occurring over a median of six years after successful treatment completion.ResultsOf 500 patients initially enrolled, 333 (79%) successfully completed treatment for tuberculosis. During the follow-up period 35 patients with successful treatment (11%) experienced a bacteriologically confirmed tuberculosis recurrence. In our Cox proportional hazards model, a 3+ AFB sputum smear grade was significantly associated with recurrent tuberculosis with a hazard ratio of 3.33 (95% CI 1.44–7.7). The presence of polyclonal M. tuberculosis infection at baseline had a hazard ratio for recurrence of 1.96 (95% CI 0.86–4.48).ConclusionOur results indicate that AFB smear grade is independently associated with tuberculosis recurrence after successful treatment for an initial episode while the association between polyclonal M. tuberculosis infection and increased risk of recurrence appears possible.

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth Indicator Tube (MGIT 960) culture, respectively. Sensitivity and specificity of GeneXpert was 72.7% and 98.5%, respectively by using MGIT 960 as a gold standard (GeneXpert (n = 20, 6.8%) vs MGIT 960 (n = 22, 7.5%)). All Mtb positive cultures were subjected to 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing, Line probe assay (LPA) and MGIT 960- Drug Susceptibility Testing (DST). The rpoB gene was amplified and sequenced for selected isolates. Among our TBM patients, East African Indian (EAI) lineage (n = 16, 72.7%) was most predominant followed by Beijing (n = 3, 13.6%), S-family (n = 2, 9.1%) and Delhi/CAS (n = 1, 4.5%). Three Mtb strains were found to be Isoniazid (INH) resistant by MGIT 960; however LPA revealed that two strains were INH resistant and one strain was multi drug resistant (MDR) (Resistant to Isoniazid and Rifampicin (RIF)). We identified rifampicin resistant isolate with the mutation D516F in rifampicin resistance-determining region (RRDR) and observed discordant results between LPA, GeneXpert and MGIT 960. In addition, GeneXpert showing false RIF resistance was identified (no mutation in RRDR). We conclude that GeneXpert is useful for the diagnostic confirmation of TBM; however a GeneXpert negative sample should be subjected to MGIT 960 culture or LPA to rule out TBM. EAI lineage was the most predominant among TBM patients in South India and associated with drug resistance. The discordance between GeneXpert, MGIT 960 and LPA with respect to rifampicin resistance has to be ruled out to avoid TB treatment failure or relapse.

Successful Leptospira genotyping strategy on DNA extracted from canine biological samples

Leptospirosis is an emerging worldwide zoonosis with a changing epidemiology responsible for an acute disease in humans and dogs. A better knowledge of the responsible bacterium Leptospira and in particular its various serovars and serogroups prevalence is essential for better diagnosis and prevention of the disease. The gold standard for leptospirosis diagnosis is the Microscopic Agglutination Test (MAT) but it requires long and fastidious laboratory work and sometimes results in controversial data. For these reasons, PCR-based techniques for detection of pathogenic leptospiral DNA in biological samples are currently replacing the MAT. However, these strategies do not provide any information regarding the infecting serovar or serogroup. In this study, an optimized genotyping method is described to allow the identification of Leptospira ssp. directly at serovars level using DNA extracted from canine blood and urine. 16S rDNA, Variable Number Tandem Repeat (VNTR) and Multispacer Sequence Typing (MST) protocols were adapted to biological samples. Eighty-eight DNA samples were analyzed from 72 different European canine clinical cases of leptospirosis confirmed by real-time PCR. 92% of DNA samples with Ct values below 34 were fully typed, and typing success decreased to about 30% for the other samples. Typing failure also showed a specie-specific correlation, with 63% of complete typing for L. interrogans and only 40% for L. kirschneri. Additionally, an exact match was observed between serological and molecular data for the few investigated cases where MAT data were available. This methodology is a suitable alternative to the MAT for determining the infecting serovar when Leptospira DNA from blood or urine is detected at Ct values below 34, contributing to clinical surveillance of leptospirosis.

Genotyping and drug susceptibility testing of Mycobacterium tuberculosis in Iran: a multi-centre study.

Tuberculosis (TB) is a deadly infection and caused 1.4 million deaths in 2018. Assessing the geographic distribution of major lineages of Mycobacterium tuberculosis can contribute greatly to TB control. Mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) typing is commonly used to differentiate various lineages of M. tuberculosis. A total of 2747 clinical specimens were collected consecutively from October 2018 through June 2019. Clinical isolates were identified as M. tuberculosis using standard biochemical tests. The standard 15-locus MIRU-VNTR typing was used for the genotyping of clinical isolates. Drug susceptibility testing was performed using the conventional proportion method. From the collected specimens, 100 were culture positive for M. tuberculosis. Using MIRU-VNTR, 99 different patterns were detected among the 100 isolates. They were distributed in one cluster comprising two strains and 98 unique patterns. Most of our isolates were similar to New-1 and Delhi/CAS strains. Of the M. tuberculosis isolates, 83 (83.0%) were pan-susceptible and 17 (17.0%) were resistant to at least one drug. Our study showed that MIRU-VNTR is a useful method for studying the genetic diversity of M. tuberculosis isolates in different regional settings and will help the health authorities to construct a preventive programme for TB.

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.