Large Sequence Polymorphisms Research Articles

Molecular Detection of Mycobacterium avium paratuberculosis by Large Sequence Polymorphisms (LSPs)

Background: Mycobacterium avium subspecies paratuberculosis is a significant pathogen causing Johne’s disease. Rational evaluation of the prevalence of this organism in animals has been restricted by the lack of accurate diagnostic techniques. DNA microarray-based and computational study of Mycobacterium avium subsp. paratuberculosis strain K10 discovered 17 distinct large sequence polymorphisms present. Of these 17 LSPPs, 7 were with a specificity of greater than 98% and in some cases, sensitivity of up to 95% suggesting that the LSPs are ideal for diagnosing M. avium subsp. paratuberculosis. Methods: A total of 218 fecal samples were collected from cattle (n=180) and buffaloes (n=38) for detection of M. avium subsp. paratuberculosis (MAP) using Large Sequence Polymorphisms (LSPs) and IS900 PCR. The fecal samples were subjected to Ziehl-Neelsen staining, isolation and PCR. A total of 90 fecal samples were found ZN staining positive and were subjected to isolation of MAP, growths were seen in two fecal samples after 16 weeks of incubation. Result: Out of 218 fecal samples tested, 7 samples were positive by both LSP PCR viz; LSPP2 (MAP0284), LSPP4 (MAP0865), LSPP11(MAP2154) LSPP12 (MAP2182c), LSPP12 (MAP2188c), LSPP15 (MAP3774), LSPP16 (MAP3815) and IS900 sequence with an amplicon size of 600, 597, 375, 430, 719, 621, 611 and 229 bp. DNA was also extracted from the isolates (n=2) and were confirmed by PCR targeting Large Sequence Polymorphism (LSPs) and IS900. Detection of MAP in all the 7 fecal samples both by IS900 and by 7 LSPPs indicates the detection potential of selected 7 LSPPs (LSPP2, LSPP4, LSPP11, LSPP12 (MAP2182c), LSPP12 (MAP2188c), LSPP15, LSPP16) in fecal samples of cattle and buffaloes suspected for Johne’s disease.

Molecular Sexing of Nothobranchius furzeri Embryos and Larvae.

Differences between the sexes are of increasing interest in basic and applied research with regard to development, behavior, aging, and diseases. Although the African turquoise killifish Nothobranchius furzeri, a model for aging research well known for its remarkably short life span, develops strong sexual dimorphism in adulthood, there is no visible indicator of its sex in embryonic and juvenile stages. To address this issue, we developed a molecular sexing assay exploiting two large sequence polymorphisms in the minimal sex-determining region (SDR) of N. furzeri These polymorphisms are sequence deletions on the Y chromosome that involve the lack or truncation of one or multiple microsatellites. The simple polymerase chain reaction (PCR) readout of the assays described here allows the sexing of N. furzeri embryos and larvae in a medium- to high-throughput and cost-efficient manner.

Identification of region of difference and H37Rv-related deletion in Mycobacterium tuberculosis complex by structural variant detection and genome assembly.

Mycobacterium tuberculosis complex (MTBC), the main cause of TB in humans and animals, is an extreme example of genetic homogeneity, whereas it is still nevertheless separated into various lineages by numerous typing methods, which differ in phenotype, virulence, geographic distribution, and host preference. The large sequence polymorphism (LSP), incorporating region of difference (RD) and H37Rv-related deletion (RvD), is considered to be a powerful means of constructing phylogenetic relationships within MTBC. Although there have been many studies on LSP already, focusing on the distribution of RDs in MTBC and their impact on MTB phenotypes, a crumb of new lineages or sub-lineages have been excluded and RvDs have received less attention. We, therefore, sampled a dataset of 1,495 strains, containing 113 lineages from the laboratory collection, to screen for RDs and RvDs by structural variant detection and genome assembly, and examined the distribution of RvDs in MTBC, including RvD2, RvD5, and cobF region. Consistent with genealogical delineation by single nucleotide polymorphism (SNP), we identified 125 RDs and 5 RvDs at the species, lineage, or sub-lineage levels. The specificities of RDs and RvDs were further investigated in the remaining 10,218 strains, suggesting that most of them were highly specific to distinct phylogenetic groups, could be used as stable genetic markers in genotyping. More importantly, we identified 34 new lineage or evolutionary branch specific RDs and 2 RvDs, also demonstrated the distribution of known RDs and RvDs in MTBC. This study provides novel details about deletion events that have occurred in distinct phylogenetic groups and may help to understand the genealogical differentiation.

Investigation on the genetic-inconsistent paternity cases using the MiSeq FGx system

Mutations might challenge the paternity index calculation in forensic identification. While many studies have focussed on the autosomal short tandem repeats (A-STR), the mutation status of sex chromosomes and single nucleotide polymorphism (SNP) remain blank. Next generation sequencing (NGS), known as high throughput and large sequence polymorphism, is a promising tool for forensic genetics. To describe the mutation landscapes in the paternity cases with genetic inconsistencies, a total of 63 parentage confirmed paternity cases contained at least one mismatched locus have been collected. The mutations were subsequently evaluated using Verogen’s MPS ForenSeqTM DNA Signature Kit and a microsatellite instability (MSI) detection kit. The result showed 98.41% (62/63) of the cases had no additional autosomal mutations even when the number of A-STRs increased to 27. As for the sex chromosomes, about 11.11% (7/63) of the cases exhibited either X-STR or Y-STR mutations. D2S1338, FGA and Penta E were the most frequent altered STRs, which suggested they might be the mutation hotspots. In addition, a male with sex chromosome abnormality was observed accidently, whose genotype might be 47, XXY, rather than MSI. Nearly 56.90% of the STR loci possessed isoalleles, which might result in higher STR polymorphisms. No Mendelian incompatibility was detected among the SNP markers, which indicated that SNP was a more reliable genetic marker in the genetic-inconsistent paternity cases. Supplemental data for this article is available online at https://doi.org/10.1080/20961790.2021.2009631 .

Molecular epidemiology and multidrug resistance of Mycobacterium tuberculosis complex from pulmonary tuberculosis patients in the Eastern region of Ghana

BackgroundTuberculosis (TB) and drug-resistant TB (DR-TB) continue to persist as a serious public health challenges in Ghana. Although several research has evaluated the drug resistance of Mycobacterium tuberculosis complex (MTBc) strains across the country, there is a paucity of data on its magnitude as well as the various lineages circulating in the Eastern region of Ghana. ObjectiveThis study therefore evaluated the distribution of the various lineages of MTBc in the Eastern region of the country and the associated drug resistance. Materials and methodsOne hundred and forty-three (143) patients with pulmonary TB attending the Eastern Regional Hospital, Koforidua/Ghana were included in the study. The BACTEC MGIT 960 tube media was used for both sputum culture and drug susceptibility of streptomycin (STR), isoniazid (INH), rifampicin (RIF) and Ethambutol (ETH). Isolates were initially typed using IS6110, followed by large sequence polymorphisms analysis and spoligotyping. ResultsThe majority [108 (75.5%)] of the 143 patients were male gender and the 45–54 years [46 (32.2%)] age range had the highest frequency. Forty-one (28.7%) of the 143 isolates were IS6110 negative. Of the 102 spoligotyped isolates, the main sub-lineages included 45 (44.1%) Cameroon and 23 (22.5%) Ghana. SITs 61 and 53 represented the major cluster with 22/102 (21.6%) and 13/102 (12.7%) isolates respectively, while 59/65 (90.8%) isolates belonged to Lineage 4 with 27/65 (41.5%) LAM10_CAM. MDR-TB occurred in 26/79 (32.9%) isolates, and was not associated with neither gender [20/58 (34.5%) male vs 6/21 (28.6%) female, OR = 1.31; 95%CI, 0.44–3.92; p = 0.624)] nor age. No association was found between MDR-TB and the major sub-lineages [8/25 (32%) Cameroon (OR = 0.94; 95%CI, 0.34–2.59; p = 0.920) and 5/11 (45.5%) Ghana (OR = 1.87; 95%CI, 0.51–6.80; p = 0.489)], or previously treated [8/23 (34.8%), OR = 0.89; 95%CI, 0.32–2.48; p = 0.823)] patients. ConclusionDespite the serious threat posed by MDR in the study area, no sub-lineage was shown to be associated with drug resistance. Nonetheless, a sustained surveillance of drug resistance pattern is advocated. A lower proportion of M. africanum was observed in the Eastern region of Ghana and will require further evaluation.

A Comprehensive Map of Mycobacterium tuberculosis Complex Regions of Difference.

ABSTRACTMycobacterium tuberculosis complex (MTBC) species are classic examples of genetically monomorphic microorganisms due to their low genetic variability. Whole-genome sequencing made it possible to describe both the main species within the complex and M. tuberculosis lineages and sublineages. This differentiation is based on single nucleotide polymorphisms (SNPs) and large sequence polymorphisms in the so-called regions of difference (RDs). Although a number of studies have been performed to elucidate RD localizations, their distribution among MTBC species, and their role in the bacterial life cycle, there are some inconsistencies and ambiguities in the localization of RDs in different members of the complex. To address this issue, we conducted a thorough search for all possible deletions in the WGS data collection comprising 721 samples representing the full MTBC diversity. Discovered deletions were compared with a list of all previously described RDs. As with the SNP-based analysis, we confirmed the specificities of 79 regions at the species, lineage, or sublineage level, 17 of which are described for the first time. We also present RDscan (https://github.com/dbespiatykh/RDscan), an open-source workflow, which detects deletions from short-read sequencing data and correlates the results with high-specificity RDs, curated in this study. Testing of the workflow on a collection comprising ∼7,000 samples showed a high specificity of the found RDs. This study provides novel details that can contribute to a better understanding of the species differentiation within the MTBC and can help to determine how individual clusters evolve within various MTBC species.IMPORTANCE Reductive genome evolution is one of the most important and intriguing adaptation strategies of different living organisms to their environment. Mycobacterium offers several notorious examples of either naturally reduced (Mycobacterium leprae) or laboratory-reduced (Mycobacterium bovis BCG) genomes. Mycobacterium tuberculosis complex has its phylogeny unambiguously framed by large sequence polymorphisms that present unidirectional unique event changes. In the present study, we curated all known regions of difference and analyzed both Mycobacterium tuberculosis and animal-adapted MTBC species. For 79 loci, we have shown a relationship with phylogenetic units, which can serve as a marker for diagnosing or studying biological effects. Moreover, intersections were found for some loci, which may indicate the nonrandomness of these processes and the involvement of these regions in the adaptation of bacteria to external conditions.

Mapping Mycobacterium tuberculosis genetic diversity and associated drug resistance in the gulf of guinea – africa

Background: Tuberculosis (TB) worldwide re-emergence constitutes a major public health problem with multi-drug resistant (MDR) strains becoming a greater threat. Genotyping and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are important in order to improve the control of the disease. This study sought to determine drug susceptibility and genetic diversity of MTBC in three West/Central African countries, namely Cameroon, Nigeria and Ghana located in the Gulf of Guinea. Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from culture using Lowenstein–Jensen medium and Middlebrook 7H9 Broth were included in the study and some isolates tested for drug susceptibility using the proportion method or Xpert MTB/RIF. Isolates were further subjected to IS6110 typing, large sequence polymorphisms and spoligotyping. Results: The Cameroon sub-lineage was the most prevalent genetic family [150/202 (74.3%) in Nigeria, 45/102 (44.1%) in Ghana and 54/150 (36%) in Cameroon] and SIT 61 the largest cluster with 111/202 (55%), 43/150 (28.6%) and 22/102 (21.6%) isolates in Nigeria, Cameroon and Ghana respectively. MDR was observed in 29/202 (14.4%) cases in Nigeria, and HIV positivity [37/202 (18.3%) patients] was associated with rifampicin (RIF) [9/37 (24.3%)] resistance (p=0.012), as well as gender (p=0.009). Equally, gender (p=0.038) and age (p=0.015) had significant associations with the LAM10_CAM family in Nigeria. In Cameroon, 13/147 (8.8 %) isolates were resistant to RIF, and 6 (35.3%) of the 17 UgandaI sub-lineage were RIF resistant (OR, 9.58; 95%CI, 2.74-33.55, p=0.001).). RIF resistance was equally associated (OR, 0.18, 95%CI, 0.05-0.69; p=0.023) with previously treated patients [(4/14 (28.6%)] compared to new ones [9/133 (6.8%)]. No sub-lineage was associated with MDR in Ghana Other sub-lineages such as Haarlem, Ghana, West African 1 were recorded in the three countries. Conclusion: RIF resistance in TB/HIV co-infected patients and its association with a sub-lineage calls for a reinforced surveillance strategy to curb drug resistance development in the Gulf of Guinea.

Mycobacterium tuberculosis strain diversity and non Tuberculous Mycobacteria isolates in the West and Central Africa

Aims and objectives: The prevalence of pulmonary tuberculosis as well as Non Tuberculous Mycobacteria (NTM), has been on the increase worldwide. In order to improve the control of the disease, genotyping and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are important. This study sought to determine drug susceptibility and genetic diversity of MTBC as well as NTM in three West/Central African countries. Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from culture using either Lowenstein–Jensen medium, Middlebrook 7H9 Broth and/or Xpert MTB/RIF were included in the study and some isolates tested for drug susceptibility using the proportion method, BACTEC MGIT 960 or Line Probe Assay (LPA). Isolates were further subjected to IS6110 typing, large sequence polymorphisms, and spoligotyping. All IS6110-negative strains suspected as NTM were tested using hsp65 gene amplification, DNA sequencing and blast analysis. Results: The Cameroon family was the most prevalent genetic strain [128/202 (63.4%) in Nigeria, 54/150 (36%) in Cameroon and 27/119 (22.7%) in Ghana] and SIT 61 the largest cluster with 111/202 (55%) and 43/150 (27%) isolates in Nigeria and Cameroon respectively. Multidrug resistance (MDR) was observed in 29/202 (14.4%) cases in Nigeria, and HIV positivity [37/202 (18.3%) patients] was associated with rifampicin 9/37 (24.3%) resistance (p=0.012), as well as gender (p=0.009). In Cameroon, 13/147 (8.8 %) were resistant to rifampicin (RIF), with 6 (46.2%) of the 13 RIF resistant isolates associated with UgandaI sublineage (p=0.0007). Other sublineages such as Haarlem, Ghana, West African 1 were recorded in the three countries. NTMs isolates included M. colombiense, M. engbaekii, M. intracellulare strain, M. gordonae, M. fortuitum, M. paraense and Nocardia veteran. Conclusion: Neglected NTM timely diagnosis should be reinforced in the study area. Drug resistance in TB/HIV co-infected patients and its association with a sublineage calls for a surveillance strategy to curb its expansion in the west/central Africa.

Complete Genome Sequence of Ovine Mycobacterium avium subsp. paratuberculosis Strain JIII-386 (MAP-S/type III) and Its Comparison to MAP-S/type I, MAP-C, and M. avium Complex Genomes.

Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) is a worldwide-distributed obligate pathogen in ruminants causing Johne’s disease. Due to a lack of complete subtype III genome sequences, there is not yet conclusive information about genetic differences between strains of cattle (MAP-C, type II) and sheep (MAP-S) type, and especially between MAP-S subtypes I, and III. Here we present the complete, circular genome of MAP-S/type III strain JIII-386 (DE) closed by Nanopore-technology and its comparison with MAP-S/type I closed genome of strain Telford (AUS), MAP-S/type III draft genome of strain S397 (U.S.), twelve closed MAP-C strains, and eight closed M.-a.-complex-strains. Structural comparative alignments revealed clearly the mosaic nature of MAP, emphasized differences between the subtypes and the higher diversity of MAP-S genomes. The comparison of various genomic elements including transposases and genomic islands provide new insights in MAP genomics. MAP type specific phenotypic features may be attributed to genes of known large sequence polymorphisms (LSPSs) regions I–IV and deletions #1 and #2, confirmed here, but could also result from identified frameshifts or interruptions of various virulence-associated genes (e.g., mbtC in MAP-S). Comprehensive core and pan genome analysis uncovered unique genes (e.g., cytochromes) and genes probably acquired by horizontal gene transfer in different MAP-types and subtypes, but also emphasized the highly conserved and close relationship, and the complex evolution of M.-a.-strains.

Characterization of Mycobacterium tuberculosis genotypes and their correlation to multidrug resistance in Lusaka, Zambia

ObjectivesThe burden of multidrug-resistant tuberculosis (MDR-TB) has been reported to be increasing in Zambia. The reasons for the increase are still unclear. This study determined the diversity of Mycobacterium tuberculosis genotypes among isolates in Lusaka, the capital city, and investigated their association with MDR-TB. MethodsSpoligotyping, large sequence polymorphism (LSP) analysis, and sequencing of MDR associated genes were performed on a total of 274 M. tuberculosis clinical isolates stored at the University Teaching Hospital from 2013 to 2017. Of these, 134 were MDR-TB while 126 were pan-susceptible. ResultsSpoligotyping showed the LAM family as the most predominant genotype (149/274, 54.4%) followed by the CAS family (44/274, 16.1%), T family (39/274, 14.2%), and minor proportions of X, S, Harleem, EAI and Beijing spoligofamilies were identified. Three M. bovis isolates were also observed. Among those, CAS1-Kili (SIT 21) and LAM1 (SIT 20) subfamilies showed a propensity for MDR-TB with p = 0.0001 and p = 0.001, respectively. ConclusionsThis phenomenon might explain the future increase in the MDR-TB burden caused by specific lineages in Zambia. Therefore, it is recommended that the National TB control program in the country complements conventional control strategies with molecular analysis for monitoring and surveillance of MDR-TB epidemiology.

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.

Genotyping of Mycobacterium tuberculosis spreading in Hanoi, Vietnam using conventional and whole genome sequencing methods

Hanoi is the capital of Vietnam, one of the 30 countries with a high tuberculosis (TB) burden. Fundamental data on the molecular epidemiology of the disease is required for future TB management.To identify lineages and genotypes of Mycobacterium tuberculosis (Mtb), conventional genotyping data from clinical isolates of the Hanoi area was compared with whole genome sequencing (WGS) analysis from 332 of 470 samples. It was obtained from lineage-specific single nucleotide variants (SNVs), large sequence polymorphisms, spoligotyping, and variable number of tandem repeats (VNTR) analysis using mycobacterial interspersed repetitive unit (MIRU) and Japan anti-tuberculosis association (JATA) locus sets. This information was directly compared with results obtained from WGS. Mini-satellite repeat unit variants were identified using BLAST search against concatenated short read sequences, the RepUnitTyping tool.WGS analysis revealed that the Mtb strains tested are diverse and classified into lineage (L) 1, 2 and 4 (24.7, 57.2 and 18.1% respectively). The majority of the L2 strains were further divided into ancient and modern Beijing genotypes, and most of the L1 group were EAI4_VNM strains. Although conventional PCR-based genotyping results were mostly consistent with information obtained through WGS analysis, in-depth analysis identified aberrant deletions and spacers that may cause discordance. JATA-VNTR sets, including hypervariable loci, separated large Beijing genotypic clusters generated by MIRU15 into smaller groups. The distribution of repeat unit variants observed within 33 VNTR loci showed clear variation depending on the three lineages. WGS-based pairwise-SNV differences within VNTR-defined genotypic clusters were greater in L1 than in L2 and L4 (P = .001).Direct comparisons between results of PCR-based genotyping and in silico analysis of WGS data would bridge a gap between classical and modern technologies during this transition period, and provide further information on Mtb genotypes in specific geographical areas.

Genetic diversity of Mycobacterium tuberculosis clinical isolates in Blantyre, Malawi

Despite the high burden of tuberculosis (TB) worldwide, specific factors influencing disease transmission remain elusive. Long term epidemiological studies and in vitro experimental models provide evidence of variable relative fitness of Mycobacterium tuberculosis (Mtb) strains but few such studies are available. Large sequence polymorphisms (LSP) are a robust molecular marker and are feasible as an epidemiological investigative tool. Few Mtb molecular epidemiological studies have been reported in Malawi owing to lack of laboratories with molecular tools. We characterized the genetic diversity of Mtb clinical isolates amongst TB patients in Blantyre, Malawi. We genotyped 64 Mtb clinical isolates using LSP-PCR, assigned specific lineages and confirmed 18 of the isolates using SMRT sequencing. The 64 isolates clustered into 4 lineages (L1-L4) with L4 predominating. There were 10/64 (16%) isolates belonging to L1, 6/64 (9%) belonging to L2, 2/64 (3%) belonging to L3 and 46/64 (72%) belonging to L4. Comparison with a previous study done in Karonga revealed concordance in L1 and L4 but discodance in L2 and L3. The phylogenetic tree constructed, comprised of 3/4 lineages present in Blantyre with 3/18 belonging to L1, 3/18 belonging to L2 and 12/18 belonging to L4. Four Mtb lineages were present in Blantyre with L4 predominating. Larger studies are needed to understand the molecular epidemiology of TB in Blantyre in light of increased bi-directional migration with South Africa.

Insight into genetic diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka reveals predominance of the Euro-American lineage

ObjectiveSri Lanka is a country where the molecular epidemiology of Mycobacterium tuberculosis (MTB) is poorly explored. Therefore, this study was performed to identify circulating lineages/sub-lineages of MTB and their transmission patterns. MethodsDNA was extracted from 89 isolates of MTB collected during 2012 and 2013 from new pulmonary tuberculosis patients in Kandy, Sri Lanka and analyzed by spoligotyping, large sequence polymorphism (LSP), mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing, and drug resistance-associated gene sequencing. ResultsThe predominant lineage was lineage 4 (Euro-American, 45.9%), followed by lineage 1 (Indo-Oceanic, 29.4%), lineage 2 (East-Asian, 23.5%), and lineage 3 (Central-Asian, 1.2%). Among 26 spoligotype patterns, eight were undesignated or new types and seven of these belonged to lineage 4. Undesignated lineage 4/SIT124 (n=2/8) and SIT3234 (n=8/8) clustered together based on 24-locus MIRU-VNTR typing. The dominant sub-lineage was Beijing/SIT1 (n=19), with the isoniazid resistance katG G944C mutation (Ser315Thr) detected in two of them. ConclusionsThe population structure of MTB in Kandy, Sri Lanka was different from that in the South Asian region. The clonal expansion of locally evolved lineage 4/SIT3234 and detection of the pre-multidrug resistant Beijing isolates from new tuberculosis patients is alarming and will require continuous monitoring.

Characteristics of distribution of Mycobacterium tuberculosis lineages in China.

The genotyping methods of Mycobacterium tuberculosis would dramatically improve our understanding of the molecular epidemiology of tuberculosis. 3,929 isolates, from a National Survey of Drug-Resistant Tuberculosis in 2007 in China, were successfully genotyped by large sequence polymorphisms and 15 loci variable number tandem repeats. We found that 2,905 (2,905/3,929, 73.9%) cases belonged to Lineage 2, dominated in the east and central regions, 975 cases (975/3,929, 24.8%) were Lineage 4, highly prevailed in the west regions, and 36 and 13 cases were Lineage 3 and Lineage 1, respectively. We also explored the associations between lineages (Lineage 2 vs. Lineage 4) and clinical characteristics by logistic regression. For Lineage 2, the risk factors were Han-ethnicity population and fever. However, for Lineage 4, they were occupation (farmer), and degree of education (non-literate). Fully understanding of the distribution of Mycobacterium tuberculosis lineage and its risk factors would play a critical role in tuberculosis prevention, control, and treatment.

Impact of Euro-American sublineages of Mycobacterium tuberculosis on new infections among named contacts

The impact of demographic, clinical, and bacterial factors on new infection by Euro-American lineage Mycobacterium tuberculosis among contacts of patients with tuberculosis (TB) has not been evaluated. To describe the risk factors for new infection by Euro-American M. tuberculosis sublineages in San Francisco, California. We included contacts of patients with TB due to Euro-American M. tuberculosis. Sublineages were determined by large-sequence polymorphisms. We used tuberculin skin testing or QuantiFERON®-TB Gold In-Tube to identify contacts with new infection. Regression models with generalized estimating equations were used to determine the risk factors for new infection. We included 1488 contacts from 134 patients with TB. There were 79 (5.3%) contacts with new infection. In adjusted analyses, contacts of patients with TB due to region of difference 219 M. tuberculosis sublineage were less likely to have new infection (OR 0.23, 95%CI 0.06-0.84) than those with other sublineages. Other risk factors for new infection were contacts exposed to more than one patient with TB, contacts exposed for 30 days, or contacts with a history of smoking or excessive alcohol consumption. In addition to well-known exposure and clinical characteristics, bacterial characteristics independently contribute to the transmissibility of TB in San Francisco.

Mycobacterium tuberculosis Lineage Distribution in Xinjiang and Gansu Provinces, China

Mycobacterium tuberculosis (M. tuberculosis) genotyping has dramatically improved the understanding of the epidemiology of tuberculosis (TB). In this study, 187 M. tuberculosis isolates from Xinjiang Uygur Autonomous Region (Xinjiang) and Gansu province in China were genotyped using large sequence polymorphisms (LSPs) and variable number tandem repeats (VNTR). Ten isolates, which represent major nodes of VNTR-based minimum spanning tree, were selected and subsequently subjected to multi-locus sequence analyses (MLSA) that include 82 genes. Based on a robust lineage assignment, we tested the association between lineages and clinical characteristics by logistic regression. There are three major lineages of M. tuberculosis prevalent in Xinjiang, viz. the East Asian Lineage 2 (42.1%; 56/133), the Euro-American Lineage 4 (33.1%; 44/133), and the Indian and East African Lineage 3 (24.8%; 33/133); two lineages prevalent in Gansu province, which are the Lineage 2 (87%; 47/54) and the Lineage 4 (13%; 7/54). The topological structures of the MLSA-based phylogeny support the LSP-based identification of M. tuberculosis lineages. The statistical results suggest an association between the Lineage 2 and the hemoptysis/bloody sputum symptom, fever in Uygur patients. The pathogenicity of the Lineage 2 remains to be further investigated.

Spatio-Temporal Distribution of Mycobacterium tuberculosis Complex Strains in Ghana

BackgroundThere is a perception that genomic differences in the species/lineages of the nine species making the Mycobacterium tuberculosis complex (MTBC) may affect the efficacy of distinct control tools in certain geographical areas. We therefore analyzed the prevalence and spatial distribution of MTBC species and lineages among isolates from pulmonary TB cases over an 8-year period, 2007–2014.MethodologyMycobacterial species isolated by culture from consecutively recruited pulmonary tuberculosis patients presenting at selected district/sub-district health facilities were confirmed as MTBC by IS6110 and rpoß PCR and further assigned lineages and sub lineages by spoligotyping and large sequence polymorphism PCR (RDs 4, 9, 12, 702, 711) assays. Patient characteristics, residency, and risks were obtained with a structured questionnaire. We used SaTScan and ArcMap analyses to identify significantly clustered MTBC lineages within selected districts and spatial display, respectively.ResultsAmong 2,551 isolates, 2,019 (79.1%), 516 (20.2%) and 16 (0.6%) were identified as M. tuberculosis sensu stricto (MTBss), M. africanum (Maf), 15 M. bovis and 1 M. caprae, respectively. The proportions of MTBss and Maf were fairly constant within the study period. Maf spoligotypes were dominated by Spoligotype International Type (SIT) 331 (25.42%), SIT 326 (15.25%) and SIT 181 (14.12%). We found M. bovis to be significantly higher in Northern Ghana (1.9% of 212) than Southern Ghana (0.5% of 2339) (p = 0.020). Using the purely spatial and space-time analysis, seven significant MTBC lineage clusters (p< 0.05) were identified. Notable among the clusters were Ghana and Cameroon sub-lineages found to be associated with north and south, respectively.ConclusionThis study demonstrated that overall, 79.1% of TB in Ghana is caused by MTBss and 20% by M. africanum. Unlike some West African Countries, we did not observe a decline of Maf prevalence in Ghana.

Genomic characterization of Mycobacterium tuberculosis lineage 7 and a proposed name: 'Aethiops vetus'.

Lineage 7 of the Mycobacterium tuberculosis complex has recently been identified among strains originating from Ethiopia. Using different DNA typing techniques, this study provides additional information on the genetic heterogeneity of five lineage 7 strains collected in the Amhara Region of Ethiopia. It also confirms the phylogenetic positioning of these strains between the ancient lineage 1 and TbD1-deleted, modern lineages 2, 3 and 4 of Mycobacterium tuberculosis. Four newly identified large sequence polymorphisms characteristic of the Amhara Region lineage 7 strains are described. While lineage 7 strains have been previously identified in the Woldiya area, we show that lineage 7 strains circulate in other parts of the Amhara Region and also among foreign-born individuals from Eritrea and Somalia in The Netherlands. For ease of documenting future identification of these strains in other geographical locations and recognizing the place of origin, we propose to assign lineage 7 strains the lineage name ‘Aethiops vetus’.

First Report of Meloidogyne haplanaria Infecting Mi-Resistant Tomato Plants in Florida and Its Molecular Diagnosis Based on Mitochondrial Haplotype.

Meloidogyne haplanaria is a species originally found infesting peanut in Texas and, more recently, in Arkansas. In this study, we confirmed the presence of M. haplanaria in Florida based on morphological and molecular characterization. This species was identified from a sample submitted for diagnosis collected from Mi-resistant tomato rootstock grown in Naples, FL. The major diagnostic criteria to distinguish M. haplanaria from other closely related root-knot nematode (RKN) species are based on morphological differences and host range tests, which are time consuming and labor intensive and require living or well-preserved specimens. In our study, we provide an easy diagnostic strategy to distinguish M. haplanaria from other RKN species based on amplification of two mitochondrial DNA regions. These regions span the intergenic spacer and part of the adjacent large subunit ribosomal RNA gene (lrDNA) and sequence polymorphisms in lrDNA revealed by the restriction pattern following digestion with the restriction enzymes HinfI and MnlI. A unique haplotype pattern, which has not been observed in any of the RKN species described thus far, was observed in M. haplanaria. The outcome of molecular analysis of M. haplanaria aligned with morphological measurement and characteristics as well as perineal pattern originally described for M. haplanaria.