Outbreak of Intermediate Species Leptospira venezuelensis Spread by Rodents to Cows and Humans in L. interrogans-Endemic Region, Venezuela.

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.

Performance evaluation of core genome multilocus sequence typing for genotyping of Mycobacterium tuberculosis strains in China: based on multicenter, population-based collection.

PURPOSE: To evaluate the performance of core genome multilocus sequence typing (cgMLST) for genotyping Mycobacterium tuberculosis (M.tuberculosis) Strains in regions where the lineage 2 strains predominate. METHODS: We compared clustering by whole-genome SNP typing with cgMLST clustering in the analysis of WGS data of 6240 strains from five regions of China. Using both the receiver operating characteristic (ROC) curve and epidemiological investigation to determine the optimal threshold for defining genomic clustering by cgMLST. The performance of cgMLST was evaluated by quantifying the sensitivity, specificity and concordance of clustering between two methods. Logistic regression was used to gauge the impact of strain genetic diversity and lineage on cgMLST clustering. RESULTS: The optimal threshold for cgMLST to define genomic clustering was determined to be ≤ 10 allelic differences between strains. The overall sensitivity and specificity of cgMLST averaged 99.6% and 96.3%, respectively; the concordance of clustering between two methods averaged 97.1%. Concordance was significantly correlated with strain genetic diversity and was 3.99 times (95% CI, 2.94-5.42) higher in regions with high genetic diversity (π > 1.55 × 10-4) compared to regions with low genetic diversity. The difference missed statistical significance, while concordance for lineage 2 strains (96.8%) was less than that for lineage 4 strains (98.3%). CONCLUSION : cgMLST showed a discriminatory power comparable to whole-genome SNP typing and could be used to genotype clinical M.tuberculosis strains in different regions of China. The discriminative power of cgMLST was significantly correlated with strain genetic diversity and was slightly lower with strains from regions with low genetic diversity.

Local adaptation of Mycobacterium tuberculosis on the Tibetan Plateau.

During its global dispersal, Mycobacterium tuberculosis (Mtb) has encountered varied geographic environments and host populations. Although local adaptation seems to be a plausible model for describing long-term host-pathogen interactions, genetic evidence for this model is lacking. Here, we analyzed 576 whole-genome sequences of Mtb strains sampled from different regions of high-altitude Tibet. Our results show that, after sequential introduction of a few ancestral strains, the Tibetan Mtb population diversified locally while maintaining strict separation from the Mtb populations on the lower altitude plain regions of China. The current population structure and estimated past population dynamics suggest that the modern Beijing sublineage strains, which expanded over most of China and other global regions, did not show an expansion advantage in Tibet. The mutations in the Tibetan strains showed a higher proportion of A > G/T > C transitions than strains from the plain regions, and genes encoding DNA repair enzymes showed evidence of positive selection. Moreover, the long-term Tibetan exclusive selection for truncating mutations in the thiol-oxidoreductase encoding sseA gene suggests that Mtb was subjected to local selective pressures associated with oxidative stress. Collectively, the population genomics of Mtb strains in the relatively isolated population of Tibet provides genetic evidence that Mtb has adapted to local environments.

Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

Evaluation of the Cepheid 3-gene host response blood test for tuberculosis diagnosis and treatment response monitoring in a primary-level clinic in rural China.

A rapid, accurate, non-sputum-based triage test for diagnosing tuberculosis (TB) is a high-priority need. Cepheid developed a novel prototype blood test, Xpert Mycobacterium tuberculosis Host Response (Xpert-MTB-HR), which generates a TB score based on the mRNA expression of three genes. We conducted a case-control study with prospective recruitment to evaluate its accuracy in the clinic of the Wusheng County Centers for Disease Prevention and Control in China. We enrolled 149 TB patients, 248 other respiratory diseases (ORD) patients, and 193 healthy controls. In addition, whole-blood samples taken from TB patients after 2, 5, and 6 months of treatment were tested with Xpert-MTB-HR to evaluate its ability to monitor treatment response. Xpert-MTB-HR discriminated between TB and healthy controls with an area under the curve (AUC) of 0.912 (95% CI, 0.878-0.945). With the specificity of 70% envisioned for a triage test, its sensitivity was 90.1% (84.9%-94.6%). Xpert-MTB-HR discriminated between TB and ORD with an AUC of 0.798 (0.750-0.847), and at specificity of 70%, the sensitivity was only 75.8% (68.5%-82.8%). In patients determined by Ultra to have medium or high sputum bacillary loads, with specificity of 70%, the sensitivity for discriminating patients with TB from healthy controls was 100.0% (100.0-100.0) and from patients with ORD, 95.1% (89.8-100.0). The TB scores generally increased by 2 months of treatment and then remained stable. Xpert-MTB-HR met the criteria for a triage test to discriminate between TB and healthy controls, but not between TB and ORD, except when limited to patients with high sputum bacillary loads. Xpert-MTB-HR showed promise for monitoring response to treatment but needs to be further evaluated in larger prospective studies.

Lesion Heterogeneity and Long-Term Heteroresistance in Multidrug-Resistant Tuberculosis.

Tuberculosis heteroresistance, in which only a fraction of the bacteria in a patient with tuberculosis contains drug-resistant mutations, has been a rising concern. However, its origins and prevalence remain elusive. Here, whole-genome sequencing was performed on 83 serial isolates from 31 patients with multidrug-resistant tuberculosis, and heteroresistance was detected in isolates from 21 patients (67.74%). Heteroresistance persisted in the host for long periods, spanning months to years, and was associated with having multiple tubercular lesions. Our findings indicate that heteroresistance is common and persistent in patients with multidrug-resistant tuberculosis and may affect the success of their treatment regimens.

Citywide Transmission of Multidrug-resistant Tuberculosis Under China's Rapid Urbanization: A Retrospective Population-based Genomic Spatial Epidemiological Study.

BACKGROUND: Population movement could extend multidrug-resistant tuberculosis (MDR-TB) transmission and complicate its global prevalence. We sought to identify the high-risk populations and geographic sites of MDR-TB transmission in Shenzhen, the most common destination for internal migrants in China. METHODS: We performed a population-based, retrospective study in patients diagnosed with MDR-TB in Shenzhen during 2013-2017. By defining genomic clusters with a threshold of 12-single-nucleotide polymorphism distance based on whole-genome sequencing of their clinical strains, the clustering rate was calculated to evaluate the level of recent transmission. Risk factors were identified by multivariable logistic regression. To further delineate the epidemiological links, we invited the genomic-clustered patients to an in-depth social network investigation. RESULTS: In total, 105 (25.2%) of the 417 enrolled patients with MDR-TB were grouped into 40 genome clusters, suggesting recent transmission of MDR strains. The adjusted risk for student to have a clustered strain was 4.05 (95% confidence interval, 1.06-17.0) times greater than other patients. The majority (70%, 28/40) of the genomic clusters involved patients who lived in different districts, with residences separated by an average of 8.76 kilometers. Other than household members, confirmed epidemiological links were also identified among classmates and workplace colleagues. CONCLUSIONS: These findings demonstrate that local transmission of MDR-TB is a serious problem in Shenzhen. While most transmission occurred between people who lived distant from each other, there was clear evidence that transmission occurred in schools and workplaces, which should be included as targeted sites for active case finding.The average residential distance between genomic-clustered cases was more than 8 kilometers, while schools and workplaces, identified as sites of transmission in this study, deserve increased vigilance for targeted case finding of multidrug-resistant tuberculosis.

Fitness Cost and Compensatory Evolution in Levofloxacin-Resistant Mycobacterium aurum.

We isolated spontaneous levofloxacin-resistant strains of Mycobacterium aurum to study the fitness cost and compensatory evolution of fluoroquinolone resistance in mycobacteria. Five of six mutant strains with substantial growth defects showed restored fitness after being serially passaged for 18 growth cycles, along with increased cellular ATP level. Whole-genome sequencing identified putative compensatory mutations in the glgC gene that restored the fitness of the resistant strains, presumably by altering the bacterial energy metabolism.

Combination of Xpert MTB/RIF and MTBDRplus for Diagnosing Tuberculosis in a Chinese District.

BACKGROUND The incidence of tuberculosis (TB) remains high in many countries, including some middle- and high-income countries without financial constraints for diagnosis and treatment. The implementation of an improved algorithm for diagnosis using 2 rapid molecular tests should help reduce the TB burden. MATERIAL AND METHODS Between April 2018 and March 2019, sputum samples from 711 patients suspected of TB in Nanshan, Shenzhen, China, were included in this prospective study. All sputum samples were examined by smear microscopy, Mycobacterium Growth Indicator Tube (MGIT) 960 culture, and Xpert MTB/RIF. The sputum remnants of Xpert MTB/RIF were used for MTBDRplus to confirm the Xpert results both for the presence of TB bacilli and for resistance to rifampicin (RIF), and also to diagnose multidrug-resistant tuberculosis (MDR-TB). RESULTS In total, 200 (28.1%) of the 711 sputa were positive for TB by Xpert MTB/RIF, and the sputum remnants were used for MTBDRplus. The simultaneous use of Xpert MTB/RIF and MTBDRplus directly on sputum samples permitted accurate bacteriologic confirmation of TB in 64% (119/187) of cases and detection of 70% (7/10) of strains that were MDR. CONCLUSIONS The implementation of 2 rapid nucleic acid-based tests on sputum samples could facilitate the prompt and appropriate treatment of most TB cases.

Leptospira venezuelensis sp. nov., a new member of the intermediate group isolated from rodents, cattle and humans.

Three strains, CLM-U50T, CLM-R50 and IVIC-Bov1, belonging to the genus Leptospira, were isolated in Venezuela from a patient with leptospirosis, a domestic rat (Rattus norvegicus) and a cow (Bos taurus), respectively. The initial characterisation of these strains based on the rrs gene (16S rRNA) suggested their designation as a novel species within the 'intermediates' group of the genus Leptospira. Further phylogenomic characterisation based on single copy core genes was consistent with their separation into a novel species. The average nucleotide identity between these three strains was >99 %, but below 89 % with respect to any previously described leptospiral species, also supporting their designation as a novel species. Given this evidence, these three isolates were considered to represent a novel species, for which the name Leptospiravenezuelensis sp. nov. is proposed, with CLM-U50T (=CIP 111407T=DSM 105752T) as the type strain.

Use of GeneXpert Remnants for Drug Resistance Profiling and Molecular Epidemiology of Tuberculosis in Libreville, Gabon.

Multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis pose major problems for global health. The GeneXpert MTB/RIF (Xpert) assay rapidly detects resistance to rifampin (RIFr), but for detection of the additional resistance that defines MDR-TB (MDR tuberculosis) and XDR-TB, and for molecular epidemiology, specimen cultures and a biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for the Xpert assay could be used directly to find mutations associated with drug resistance and to study molecular epidemiology, thus providing precise characterization of MDR-TB cases in countries lacking biosafety level 3 (BSL3) facilities for M. tuberculosis cultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for the Xpert assay were used for PCR amplification and sequencing or for a line probe assay to detect mutations associated with resistance to additional drugs, as well as for molecular epidemiology with spoligotyping and selective mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Of 130 sputum samples from Gabon tested with the Xpert assay, 124 yielded interpretable results; 21 (17%) of these were determined to be RIFr Amplification and sequencing or a line probe assay of the Xpert remnants confirmed 18/21 samples as MDR, corresponding to 12/116 (9.5%) new and 6/8 (75%) previously treated TB patients. Spoligotyping and MIRU typing with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for the Xpert assay can be used in PCRs to find mutations associated with the resistance to the additional drugs that defines MDR and XDR-TB and to study molecular epidemiology without the need for culturing or a biosafe infrastructure.

Molecular characterization of KPC-producing Klebsiella pneumoniae isolated from patients in a Public Hospital in Caracas, Venezuela.

INTRODUCTION: Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are amongst the most important causative agents of nosocomial infections worldwide. Isolates of this bacterium have been identified in Venezuela but little is known about their local spread. The aim of this study was to perform the molecular characterization of KPC-producing strains isolated from 2012 to 2013 in a public hospital in Caracas, Venezuela. METHODS: Twenty-two K. pneumoniae clinical isolates phenotypically classified as KPC producing were subjected to PCR screening for the presence of blaKPC genes and their location within transposon Tn4401. The blaKPC PCR product was sequenced to identify the KPC alleles. Genotypic analysis was performed by means of repetitive extragenic palindromic PCR (rep-PCR) and Multi Locus Sequence Typing (MLST). Finally, conjugation and electroporation assays were used to determine whether the blaKPC genes were found in plasmids. RESULTS: All isolates contained the blaKPC-2 variant, and 21 of the 22 were associated with the Tn4401b isoform. The strains were distributed in 8 sequence types (ST), three of which were new. Conjugation and electroporation assays indicated that 95.5% (n=21/22) of the isolates contained the blaKPC gene in plasmids. CONCLUSIONS: This study on circulating bacterial strains and the identification of KPC alleles may help to understand the routes of dissemination and control their spread within this hospital.

Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolated from patients in two public hospitals in Carabobo and Zulia states, Venezuela. / Epidemiología molecular de aislados clínicos de Klebsiella pneumoniae productores de carbapenemasas tipo KPC provenientes de dos hospitales públicos en los estados Carabobo y Zulia, Venezuela.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria (K. pneumonia carbapenemase) are the most important causative agents of nosocomial infections worldwide. These isolates have been identified in Venezuela, but little is known about their local spread. The aim of this study was to perform molecular epidemiology of KPC-producing K. pneumoniae isolated from two public hospitals in the Carabobo and Zulia states of Venezuela. Thirty-two K. pneumoniaei solates, phenotypically classified as KPC producers were subjected to PCR to detect the presence of blaKPC genes and their location within transposon Tn4401, and the blaKPC product was sequenced to identify the KPC allele. Genotypic analysis was performed using repeated extragenic palindromic PCR (rep-PCR) and Multi Locus Sequence Typing (MLST). Finally, a conjugation assay determined whether the blaKPC genes were carried on transferable plasmids. The results indicate that the 32 isolates contained the blaKPC-2 variant associated with isoform Tn4401b, and were distributed in nine sequence types (ST), one of which was new. Conjugation assays indicate that 87.5% of the isolates contain the gene blaKPC on mobilizable plasmids. In these hospitals, the blaKPC-2 gene is spreading through the plasmids carrying the transposon Tn4401b. The most common ST belongs to Clonal Complexes CC258 and CC147, which play an important role in the dispersion of resistance to carbapenems worldwide.

A new dehydratase conferring innate resistance to thiacetazone and intra-amoebal survival of Mycobacterium smegmatis.

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.

A cluster of KPC-2 and VIM-2-producing Klebsiella pneumoniae ST833 isolates from the pediatric service of a Venezuelan Hospital.

BACKGROUND: Klebsiella pneumoniae is a bacterial pathogen that has developed resistance to multiple antibiotics and is a major cause of nosocomial infections worldwide. Carbapenemase-producing Klebsiella pneumoniae have been isolated in many hospitals in Venezuela, but they have not been well-studied. The aim of this study was to characterize carbapenem-resistant Klebsiella pneumoniae isolates from the pediatric service of a hospital located in Anzoategui State, in the eastern part of Venezuela. METHODS: Nineteen Klebsiella pneumoniae strains isolated in the hospital from April to July 2014 were evaluated phenotypically and molecularly for the presence of carbapenemases blaKPC, blaIMP and blaVIM. Molecular epidemiology was performed with Repetitive Extragenic Palindromic-PCR (REP-PCR) and Multilocus Sequence Typing (MLST). They were also studied for phenotypic and molecular resistance to a quaternary ammonium compound (QAC) disinfectant. RESULTS: All 19 isolates contained both bla VIM-2 and bla KPC-2 genes, and the bla KPC-2 gene was associated with Tn4401b. All isolates were phenotypically sensitive to QACs and contained qacΔE and addA2 genes typical of class 1 integrons. Analysis by REP-PCR and MLST showed that all isolates had identical profiles characteristic of sequence type ST833. CONCLUSION: All 19 strains are bla VIM-2 and bla KPC-2-producing ST833 K. pneumoniae sensitive to QACs. This analysis may help to understand the routes of dissemination and confirms that QAC disinfectants can be used to help control their spread.

[Evaluation of discriminatory power of molecular epidemiology techniques in Mycobacterium tuberculosis Venezuelan isolates]. / Evaluación del poder discriminatorio de técnicas de epidemiología molecular en aislados Venezolanos de Mycobacterium tuberculosis.

The techniques of spoligotyping and mycobacterial interspersed repetitive unit and variable-number tandem repeat typing with 24 loci (MIRU-VNTR-24), have been used to study the molecular epidemiology of tuberculosis. The aim of this study was: to evaluate the discriminative power of MIRU-VNTR 24 loci alone and in association with spoligotyping in clinical isolates of M tuberculosis in Venezuela; the allelic diversity of the 24 loci; and the discriminative power for the combination of 24 and 15 loci, 12 traditional loci (12t), those with higher allelic diversity and a new combination named 12inv. We analyzed one set of 104 strains of different lineages and a second set of 431 strains belonging to the Latin-America and Mediterranean lineage (LAM) that is predominant in Venezuela. The determination of allelic diversity showed that 4052, 2163b, 424 y 2996 are highly discriminative. Clustering rates of MIRU-VNTR 24 loci, spoligotyping and MIRU-VNTR combined with spoligotyping for 104 isolates were 18.27%, 71.15% and 14.4%, respectively, whereas with the 431 LAM strains the values were 43.2 %, 95.8% and 37.4%. MIRU-VNTR combinations of 15, 12inv and 4 loci were more discriminatory than 12t. Clustering rates for MIRU-VNTR 15 and 12inv loci coupled with spoligotyping in the 104 isolated was 21% and 23%, while for LAM strains was 52% and 46% respectively. The number of different genetics patterns for 15 and 12inv loci were similar. In conclusion, we propose the use of a small number of informative loci MIRU-VNTR coupled to spoligotyping to investigate the transmission of tuberculosis in Venezuela.

Frequency of common CFTR gene mutations in Venezuelan patients with cystic fibrosis.

Mutations in the CFTR gene in Cystic Fibrosis (CF) patients have geographic differences and there is scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in these patients. We amplified and sequenced exons 7, 10, 11, 19, 20 and 21, which contain the most common CFTR mutations, from 105 Venezuelan patients in the National CF Program. Eleven different mutations were identified, four with frequencies greater than 1%: p.Phe508del (26,17%), p.Gly542X (3,33%), p.Arg334Trp (1,43%) and p.Arg1162X (1.43%). No mutations were found in 63.3% of patients. This report represents the largest group of Venezuelan CF patients ever examined and includes a wider mutation panel than has been previously studied in this population. Southern European CFTR mutations predominate in the Venezuelan population, but a high percentage of the causative alleles remain unidentified.

Mycobacterium tuberculosis population structure and molecular epidemiological analysis in Sucre municipality, Miranda state, Venezuela.

Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Venezuela that consistently has more cases of tuberculosis than other municipalities in the country. To identify the neighborhoods in the municipality with the highest prevalence of tuberculosis, and determine whether the Mycobacterium tuberculosis strain distribution in this municipality is different from that previously found in the western part of Caracas and the rest of Venezuela, we collected data on all tuberculosis cases in the municipality diagnosed in 2005-6. We performed two separate molecular epidemiological studies, spoligotyping 44 strains in a first study, and spoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolates in the second. With spoligotyping, the most common patterns were Shared International Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), a distribution similar to other parts of Venezuela, except that SIT42 and SIT20 were more common. MIRU-VNTR 15 showed that six of seven SIT17 strains examined belonged to a large cluster previously found circulating in Venezuela, but all of the SIT42 strains were related to a cluster centered in the neighborhoods of Unión and Maca, with a MIRU-VNTR pattern not previously seen in Venezuela. It appears that a large percentage of the tuberculosis in the Sucre municipality is caused by the active transmission of two strain families centered within distinct neighborhoods, one reflecting communication with the rest of the country, and the other suggesting the insular, isolated nature of some sectors.

Internal migrants as drivers of long-distance cross-regional transmission of tuberculosis in China.

OBJECTIVES: Internal migrants in China frequently travel between their hometowns and the cities where they work, creating ample opportunities for cross-regional transmission of tuberculosis (TB). The aim of this study was to explore the role of internal migrants in transmitting TB across different regions and the contribution of cross-region transmission to China's TB burden. METHODS: The study included a total of 8664 patients with TB and their Mycobacterium tuberculosis isolates, collected from two large cities and three rural regions. Genomic clusters were defined as having a genomic distance of ≤12-single nucleotide polymorphisms. Cross-regional clusters were defined as clusters containing patients from at least two regions, indicative of cross-regional transmission. RESULTS: A total of 2403 clustered cases (27.7%) were grouped into 845 clusters, of which 142 (16.8%) were cross-regional. An increased risk for cross-regional transmission was found for internal migrants (adjusted OR (aOR), 1.45; 95% CI, 1.13-1.87), individuals aged <55 years (aOR, 2.73; 95% CI, 1.81-4.13), and housekeepers/factory workers (aOR, 1.16; 95% CI, 0.90-1.50). Among 200 cross-regional transmission events identified by transmission inference, 96 occurred between urban patients, 98 between urban and rural patients, and only six between rural patients. Notably, 93.5% (187/200) of cross-regional transmission events involved internal migrants. Epidemiological data showed that just 5.5% of cross-regional transmission events involved patients from the same township or neighbouring counties, where the transmission likely occurred. DISCUSSION: The mobility of the internal migrant population appears to be responsible for most cross-regional transmission of TB in China. The magnitude and dynamics of cross-regional transmission should be addressed in future strategies to reduce the incidence of TB in China.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.