Spoligotyping Of Mycobacterium Tuberculosis Research Articles

Single-tube protocol for culture-independent spoligotyping of Mycobacterium tuberculosis based on MeltArray.

Spacer oligonucleotide typing (spoligotyping), the first-line genotyping assay for Mycobacterium tuberculosis (MTB), plays a fundamental role in the investigation of its epidemiology and evolution. In this study, we established a single-tube spoligotyping assay using MeltArray, a highly multiplex polymerase chain reaction (PCR) approach that runs on a real-time PCR thermocycler. The MeltArray protocol included an internal positive control, gyrB, to indicate the abundance of MTB via the quantification cycle and 43 spacers to identify the spoligotype via melting curve analysis. The entire protocol was completed in a single step within 2.5 hours. The lowest detectable copy number for the tested strains was 20 copies/reaction and thus sufficient for analyzing both culture and sputum samples. We conclude that MeltArray-based spoligotyping could be used immediately in low- and middle-income countries with a high tuberculosis burden, given its easy access, improved throughput, and potential applicability to clinical samples.

Spoligotyping of Mycobacterium tuberculosis – Comparing in vitro and in silico approaches

Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare the spoligoprofiles of M. tuberculosis clinical isolates, obtained using in vitro and in silico approaches.The study included 230 M. tuberculosis isolates, recovered from Poland and Lithuania between 2018 and 2021. Spoligotyping in vitro was performed with a commercially available kit. Whole genome sequencing (WGS) was done with Illumina NovaSeq 6000 sequencer. Spoligotype International Types (SITs) were assigned according to the SITVIT2 database or using three different in silico tools, and based on WGS data, namely SpoTyping, SpolPred, and lorikeet.Upon in vitro spoligotyping, the isolates produced 65 different spoligotypes. Spoligotypes inferred from the WGS data were congruent with in vitro generated patterns in 81.7% (188/230) for lorikeet and 81.3% (187/230) for SpolPred and SpoTyping. Spacers 18 and 31 produced the highest ratio of discrepant results between in vitro and in silico approaches, with their signals discordantly assigned for 15 (6.5%) and 9 (3.9%) isolates, respectively.All three in silico approaches used were similarly efficient for M. tuberculosis spoligotype prediction. However, only SpoTyping could predict spoligotypes without a need for manual curation. Thus, we consider it as the most accurate tool. Its use is further advocated by the shortest time of analysis. A relatively high (ca. 20%) discordance between in vitro and in silico spoligotyping results was observed. While we discourage comparing conventional spoligotyping with in silico equivalents, we advise the use of the latter, as it improves the accuracy of spoligopatterns, and thus depicts the relatedness between the isolates more reliably.

Retraction Note: Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype.

The original article can be found online.

Differential RD-1-specific IFN-γ host responses to diverse Mycobacterium tuberculosis strains in HIV-uninfected persons may be explained by genotypic variation in the ESX-1 region

ObjectivesBetween-person variability in T-cell-specific interferon-gamma release assay (IGRA) responses and discordance between IGRA test formats are poorly understood. MethodsWe evaluated the IFN-γ responses (QuantiFERON-TB Gold-In-Tube [QFT-GIT] and TSPOT-TB) stratified according to the Mycobacterium tuberculosis spoligotype of the culture isolate obtained from the same patients with confirmed active tuberculosis (n = 91). We further analysed differences within the RD-1-encoding ESX-1 region between the different strain types using whole genome sequencing. ResultsIn HIV-uninfected patients, TSPOT.TB and QFT-GIT IFN-γ responses were 5-fold (p < 0.01) and 2-fold higher (p < 0.05) for those infected with family 33 compared to the LAM strain (additionally, TSPOT.TB responses were 5.6-fold [p < 0.05] and 2.6-fold higher [p < 0.05] for the patients infected with the family 33 versus the X strain and Beijing versus the LAM strain, respectively). Multivariate analysis revealed that strain type (determined by spoligotyping) was independently associated with the magnitude of the IGRA response (varied by IGRA test type) and this is likely explained by variability in the ESX-1 region of Mycobacteriumtuberculosis (determined by next-generation sequencing). ConclusionsThese data have implications for the understanding of between-person heterogeneity in IGRA responses, Mycobateriumtuberculosis-specific host immunity, and the discordance between different IGRA test formats.

Usefulness of 3'- 5' IS6110-RFLP genotyping and spoligotyping of Mycobacterium tuberculosis isolated in a tertiary hospital: a retrospective study detecting unsuspected epidemiological events.

ABSTRACTA drug resistance survey involving Mycobacterium tuberculosis isolated from patients of a tertiary Hospital in the Rio de Janeiro city (RJ), Brazil, between the years 1996 and 1998 revealed a high frequency of isoniazid (HR) resistance. These isolates were revisited and genotyped. Patients came from different RJ neighborhoods and municipalities, and 70% were outpatients. Applying the 3’ and 5’ IS 6110 -RFLP and the Spoligotype genotyping methods, the clonal structure of this population was investigated obtaining a snapshot of past epidemiological events. The 3’ clusters were subsequently 5’ IS 6110 -RFLP typed. Spoligotyping was analyzed in the SITVIT2 database. Epidemiological relationships were investigated. The major lineage was T (54.4%), and SIT 53/T1 and SIT 535/T1 were the most frequent. The T1 sublineage comprises 12.8% of resistant strains and SIT 535 were assigned for 31.8% of them. Orphan patterns corresponded to 12% and 73.3% and belonged to the T lineage. One pattern was unlisted in the SITVIT2. The 5’ IS 6110 -RFLP did not confirm 3/12 of the 3’ IS 6110 -RFLP clusters. A combination of all methods decreased the number of clusters to three. Nosocomial transmission was associated with one cluster involving a hospital cupbearer. This event was suspected in a multidrug resistant-TB inpatient caregiver who harbored a mixed infection. The 3’ IS 6110 clusters were associated with HR (p=0.046). These genotypic retrospective data may reflect a fraction of more extensive recent transmission in different communities that may be corroborated by the concentration of HR patients, and may serve as a database for further evolutionary and characterization evaluation of circulating strains and together with epidemiological data favors a more effective transmission control.

Microbead-based spoligotyping of Mycobacterium tuberculosis from Ziehl-Neelsen-stained microscopy preparations in Ethiopia

The worldwide dissemination of Mycobacterium tuberculosis strains has led to the study of their genetic diversity. One of the most used genotyping methods is spoligotyping, based on the detection of spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) locus. This study assessed the performance of a microbead-based spoligotyping assay using samples extracted from Ziehl-Neelsen-stained smear-microscopy preparations and described the genetic diversity of Mycobacterium tuberculosis among new TB patients in Southern Nations, Nationalities and Peoples’ Region (SNNPR) in Ethiopia. Among the 91 samples analysed, 59 (64.8%) generated spoligotyping patterns. Fifty (84.7%) samples were classified into 12 clusters (mostly Lineage 4 or 3) comprising 2–11 samples and nine had unique spoligotyping patterns. Among the 59 spoligotyping patterns, 25 belonged to the T1 sublineage, 11 to the T3-ETH, 5 to the URAL, 4 to the H3 and 14 to other L4 sublineages. There was a remarkable variation in genetic distribution in SNNPR compared to other regions of the country. Microbead-based spoligotyping is an easy-to-perform, high-throughput assay that can generate genotyping information using material obtained from smear microscopy preparations. The method provides an opportunity to obtain data of the M. tuberculosis genetic epidemiology in settings with limited laboratory resources.

Molecular epidemiology of Mycobacterium tuberculosis strains from prison populations in Santa Catarina, Southern Brazil

The Tuberculosis (TB) notification rates are 5 to 81 times higher in prisons worldwide when compared to the general population. The state of Santa Catarina (SC) has few epidemiological data regarding TB in prisons. The aim of this study was to evaluate the molecular epidemiology of circulating strains in prisons of SC. The study comprised 95 clinical samples from six prisons. Among the cases included, all subjects were male, predominantly caucasians, and young adults, with low education level. The positive smear in the TB diagnosis comprised 62.0% of cases. About 50% of subjects had some condition associated with TB. The Spoligotyping results showed that the most frequent lineages were LAM (50.7%), T (22.2%) and S (11.6%). The 12-loci MIRU generated 62 different genotypes. The MSTs showed evolutionary relationships between Mycobacterium tuberculosis spoligotypes from SC and evolutionary relationships between the prison isolates and studied parameters. This first study on TB in prison units of SC highlighted the predominance of SIT216/LAM5, and SIT34/S. Interestingly, his profile was found to be different from that observed in a previous study performed with the state's general population. This data shows the need for continued surveillance of episodes of TB occurring among prison inmates in an emerging country like Brazil.

Comparative Mycobacterium tuberculosis Spoligotype Distribution in Mexico

In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico.

RETRACTED ARTICLE: Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype

An animal keeper was diagnosed with pulmonary tuberculosis (TB) after bi-annual screening for latent TB infection in zoo employees. In the same period, several bonobos of the zoo were suffering from TB as well. The Mycobacterium tuberculosis strains from both the animal keeper and the bonobos appeared identical. We provide evidence that the animals infected their keeper.

Label-free and multiplexed electrochemical detection of PCR fragments on screen-printed electrode arrays. Application to spoligotyping of Mycobacterium tuberculosis

Label-free and multiplexed electrochemical detection of PCR fragments on screen-printed electrode arrays. Application to spoligotyping of Mycobacterium tuberculosis

Mycobacterium tuberculosis spoligotypes circulating in the Syrian population: A retrospective study

ObjectivesTo characterize by spoligotyping clinical isolates of Mycobacterium tuberculosis (MTB) collected between July 2003 and October 2005 from all Syrian provinces (muhafazat). MethodsAll isolates (n=96) were cultured and identified by biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. ResultsTwelve patterns were identified: 46.8% of the strains belonged to T 1 family; 20.8% to LAM 9; 10.4% to CAS; 9.3% to Haarlem 3; 4.1% to Haarlem 1; 2.1% to Family 34; and 1% to each of Family 36, EAI 5, LAM 1, LAM 8, T 3, and X 3 families. The noticeable absence of the Beijing family was not consistent with the patterns reported in most neighboring countries. ConclusionA more inclusive study of the Syrian population is necessary to more accurately identify most of the prevailing families in the country.

Use of Luminex MagPlex Magnetic Microspheres for High-Throughput Spoligotyping of Mycobacterium tuberculosis Isolates in Port-au-Prince, Haiti

Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct "spacers" separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.

Mycobacterium tuberculosis spoligotypes circulating in the Lebanese population: a retrospective study

Genotyping Mycobacterium tuberculosis in Lebanon on the national level may be beneficial for assessing patients and monitoring the therapeutic response to DOTS. This study aimed to characterize the spoligotypes of clinical isolates of M. tuberculosis patients collected between April 2004 and October 2005 from all Lebanese provinces. Isolates (n = 60) were cultured and identified by their biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. Thirteen (13) patterns of M. tuberculosis complex family strains were identified: 41.6% of the strains belonged to the T 1 family, 25.0% to LAM 9, 10.0% to Haarlem 3, 3.3% to each of CAS, LAM 8, BCG and Family 36 and 1.7% to each of Haarlem 1, LAM 10, S, M. africanum, X 1 and T 3 families. The noticeable absence of Beijing and East African Indian families was not consistent with the patterns reported in neighbouring countries. A more inclusive study of the Lebanese population is necessary to accurately identify most of the prevailing families in the country.

MIRU–VNTR profiles of three major Mycobacterium tuberculosis spoligotypes found in western India

We performed 12 loci MIRU-VNTR on 327 Mycobacterium tuberculosis (Mtb) isolates belonging to three major spoligotypes MANU1, CAS1_Delhi and Beijing from Mumbai, western India and two proximal rural locations. Complete allele and drug susceptibility data was available for 232 isolates. These included 143 MANU1 (ST100), 65 CAS1_Delhi (ST26) and 24 Beijing (ST1) isolates. Of the 232 isolates, 26 were rural consisting 6 CAS1_Delhi and 20 MANU1 isolates. Using eBURST multi-locus sequence typing (MLST), cluster analyses was performed for each of the spoligotypes and drug susceptibility profiles. MANU1 MLST consisted of 90 related isolates (clustered and grouped) and 53 singletons; CAS1_Delhi MLST consisted of 44 related isolates and 21 singletons; Beijing MLST consisted of 10 related isolates and 14 singletons. Although the number of related isolates were different in MANU1 (63%), CAS1_Delhi (68%) and Beijing (42%) clusters, it was not statistically significant. Furthermore, it was observed that while MANU1 and CAS1_Delhi singletons (n=74) had only 12 (16%) MDR isolates, the Beijing MLST had 8/14 (57%) MDR singleton isolates. Phylogenetic ananlysis using minimum spanning tree (MST) and a UPGMA radial tree revealed MANU1 had the largest number of nodes as compared to the CAS1_Delhi and Beijing spoligotypes. Additionally the CAS isolates were more homogeneous than the MANU1 isolates. The 12 loci MIRU-VNTR was used to provide greater discrimination than spoligotyping, but 6 of the 12 loci provided less than 50% discriminatory power. The highest discrimination was achieved using locus 26 (80%). Our results concur with recent reports that the most discriminatory MIRU-VNTR combination varied across different lineages. The results also highlight the need for more robust genetic markers for studying the transmission of Mtb in endemic regions like India.

Mycobacterium tuberculosis spoligotypes that may derive from mixed strain infections are revealed by a novel computational approach

Global control of tuberculosis is increasingly dependent on rapid and accurate genetic typing of Mycobacteriumtuberculosis. Spoligotyping is a first-line genotypic fingerprinting method for M.tuberculosis isolates. An international online database (SpolDB4) of spoligotype patterns has been established wherein a clustered pattern (shared by ⩾2 isolates) is designated a shared international type (SIT). Dual infections of single patients by distinct strains of M. tuberculosis is increasingly reported in high tuberculosis incidence areas, raising the possibility of false composite spoligotype patterns if performed upon mixed strain samples. A computational approach was applied to SpolDB4 and found that of the reported 1939 SITs, 54% could be a composite of two other SITs. Although many of the spoligotypes listed in SpolDB4 may be the product of admixing, the majority of patterns were reported with a corresponding low case frequency and so the effect of misclassification upon database integrity with these is likely minimal. Phylogenetic analysis of the five SITs most prone to be a composite demonstrated that these patterns designate nodes from which the ramifications of large families T, MANU, LAM, and EAI emerged. We illustrate how geographic context may indicate when an observed pattern could be the product of mixed infection. Importantly, when one of the most composite-prone SITs is obtained, further genetic testing by alternate methods is prudent to rule-out mixed infection, especially in high tuberculosis prevalence areas. These findings have broad practical implications for tuberculosis control and surveillance, as well as highlight the utility of a computational approach in providing solutions to biological questions in which the information can be digitalized.

Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda

BackgroundDetermination of the prevalence and drug susceptibility of the M. tuberculosis strains is important in tuberculosis control. We determined the genetic diversity and susceptibility profiles of mycobacteria isolated from tuberculosis patients in Mbarara, South Western Uganda.MethodsWe enrolled, consecutively; all newly diagnosed and previously treated smear-positive TB patients aged ≥ 18 years. The isolates were characterized using regions of difference (RD) analysis and spoligotyping. Drug resistance against rifampicin and isoniazid were tested using the Genotype® MDRTBplus assay and the indirect proportion method on Lowenstein-Jensen media. HIV-1 testing was performed using two rapid HIV tests.ResultsA total of 125 isolates from 167 TB suspects (60% males) with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analyzed. Majority (92.8%) were new cases while only 7.2% were retreatment cases. All the 125 isolates were identified as M. tuberculosis strict sense with the majority (92.8%) of the isolates being modern strains while seven (7.2%) isolates were ancestral strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty two (49.6%) of the isolates formed 16 clusters consisting of 2-15 isolates each. A majority (59.2%) of the isolates belong to the Uganda genotype group of strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 15 isolates and SIT 128 (T2) with 3 isolates. Sixty nine (87%) of the 79 patterns had not yet been defined in the SpolDB4.0.database. Resistance mutations to either RIF or INH were detected in 6.4% of the isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpoβ gene mutations seen in the sample were D516V, S531L, H526Y H526D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations only while one strain showed the inhA promoter gene mutation.ConclusionThe present study shows that the TB epidemic in Mbarara is caused by modern M. tuberculosis strains mainly belonging to the Uganda genotype and anti-TB drug resistance rate in the region is low.

Bulgarian specificity and controversial phylogeography ofMycobacterium tuberculosisspoligotype ST125_BGR

The local specificity of bacterial clones may be explained by long-term presence or recent importation/fast dissemination in an area. Mycobacterium tuberculosis spoligotype ST125, noticeably prevalent among Bulgaria-specific spoligotypes, has a characteristically 'abridged' profile and an uncertain clade position [Latin-American-Mediterranean (LAM)/S]. A comparison with the SITVIT2 database (Institut Pasteur de Guadeloupe) demonstrated its high gradient in Bulgaria (14.3%) compared with the negligible presence in the rest of the world. Further typing of all available Bulgarian ST125 strains revealed that they: (i) monophyletically clustered in 21-mycobacterial interspersed repetitive units (MIRU)-loci tree of all Bulgarian strains; (ii) grouped closely with the ST34 spoligotype, a prototype of the S family; and (iii) did not harbor a LAM-specific IS6110 insertion. Comparison of the 21-MIRU-based network with geographic data revealed a complex dissemination pattern of ST125 in Bulgaria. Interestingly, this variable number of tandem repeats (VNTR) network remarkably corroborated with a recent hypothesis of single repeat loss as the primary mode of evolution of VNTR loci in M. tuberculosis. In conclusion, M. tuberculosis spoligotype ST125 is phylogeographically specific for Bulgaria. This spoligotype was not associated with drug resistance or increased transmissibility; its prevalence in Bulgaria can rather be attributed to the historical circulation in the country, having led, speculatively, to adaptation to the local human population.

Beijing genotype and other predominant Mycobacterium tuberculosis spoligotypes observed in Mashhad city, Iran

Beijing genotype and other predominant Mycobacterium tuberculosis spoligotypes observed in Mashhad city, Iran

Mycobacterium tuberculosisspoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in peri-urban Kampala, Uganda

BackgroundThe poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated in the increase of tuberculosis (TB). Presence of different lineages of Mycobacterium tuberculosis has been described in different parts of the world. This study determined the predominant strain lineages that cause TB in Rubaga division, Kampala, Uganda, and the prevalence of resistance to key anti-tuberculosis drugs in this community.MethodsThis was a cross-sectional study of newly diagnosed sputum smear-positive patients aged ≥ 18 years. A total of 344 isolates were genotyped by standard spoligotyping and the strains were compared with those in the international spoligotype database (SpolDB4). HIV testing and anti-tuberculosis drug susceptibility assays for isoniazid and rifampicin were performed and association with the most predominant spoligotypes determined.ResultsA total of 33 clusters were obtained from 57 spoligotype patterns. According to the SpolDB4 database, 241 (70%) of the isolates were of the T2 family, while CAS1-Kili (3.5%), LAM9 (2.6%), CAS1-Delhi (2.6%) were the other significant spoligotypes. Furthermore, a major spoligotype pattern of 17 (4.5%) strains characterized by lack of spacers 15–17 and 19–43 was not identified in SpolDB4. A total of 92 (26.7%) of the patients were HIV sero-positive, 176 (51.2%) sero-negative, while 76 (22.1%) of the patients did not consent to HIV testing. Resistance to isoniazid was found in 8.1% of strains, while all 15 (4.4%) strains resistant to rifampicin were multi-drug resistant. Additionally, there was no association between any strain types in the sample with either drug resistance or HIV sero-status of the patients.ConclusionThe TB epidemic in Kampala is localized, mainly caused by the T2 family of strains. Strain types were neither associated with drug resistance nor HIV sero-status.

IS6110-RFLP and spoligotyping of Mycobacterium tuberculosis isolates in Iran.

To evaluate and compare the usefulness of IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping in the epidemiology of tuberculosis in Iran, Mycobacterium tuberculosis complex strains, isolated in 2 different areas of Iran, were subjected to RFLP and spoligotyping. The average number of IS6110 copies per strain was 11 and ranged from 5 to 18 among the M. tuberculosis strains. In total, among the 62 isolates, 56 different patterns were observed. 50 strains had unique RFLP patterns (89%) and 12 (11%) revealed patterns that were found among at least 1 other isolate. Spoligotyping of 97 isolates resulted in 42 different patterns, of which 72% were found in 15 clusters. 14 (29%) out of 48 investigated isolates were resistant to 1 or more antituberculosis drugs and 57% of the resistant isolates were isolated from Afghan immigrants. Ten percent of the isolates represented the Beijing genotype, including 4 of the 14 (36%) resistant strains. Three of these resistant Beijing strains were isolated from Afghan patients. IS6110-RFLP typing could be useful for studying the epidemiology of tuberculosis in Iran. IS6110 patterns were polymorphic and the average IS6110 copy number was high.