Genomic epidemiology of Lineage 4 Mycobacterium tuberculosis subpopulations in New York City and New Jersey, 1999-2009.

BACKGROUND: Whole genome sequencing (WGS) has rapidly become an important research tool in tuberculosis epidemiology and is likely to replace many existing methods in public health microbiology in the near future. WGS-based methods may be particularly useful in areas with less diverse Mycobacterium tuberculosis populations, such as New York City, where conventional genotyping is often uninformative and field epidemiology often difficult. This study applies four candidate strategies for WGS-based identification of emerging M. tuberculosis subpopulations, employing both phylogenomic and population genetics methods. RESULTS: M. tuberculosis subpopulations in New York City and New Jersey can be distinguished via phylogenomic reconstruction, evidence of demographic expansion and subpopulation-specific signatures of selection, and by determination of subgroup-defining nucleotide substitutions. These methods identified known historical outbreak clusters and previously unidentified subpopulations within relatively monomorphic M. tuberculosis endemic clone groups. Neutrality statistics based on the site frequency spectrum were less useful for identifying M. tuberculosis subpopulations, likely due to the low levels of informative genetic variation in recently diverged isolate groups. In addition, we observed that isolates from New York City endemic clone groups have acquired multiple non-synonymous SNPs in virulence- and growth-associated pathways, and relatively few mutations in drug resistance-associated genes, suggesting that overall pathoadaptive fitness, rather than the acquisition of drug resistance mutations, has played a central role in the evolutionary history and epidemiology of M. tuberculosis subpopulations in New York City. CONCLUSIONS: Our results demonstrate that some but not all WGS-based methods are useful for detection of emerging M. tuberculosis clone groups, and support the use of phylogenomic reconstruction in routine tuberculosis laboratory surveillance, particularly in areas with relatively less diverse M. tuberculosis populations. Our study also supports the use of wider-reaching phylogenomic and population genomic methods in tuberculosis public health practice, which can support tuberculosis control activities by identifying genetic polymorphisms contributing to epidemiological success in local M. tuberculosis populations and possibly explain why certain isolate groups are apparently more successful in specific host populations.

Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia.

Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007-2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the circulation of Genotype IV in the community.

Molecular epidemiology of tuberculosis: current insights.

Molecular epidemiologic studies of tuberculosis (TB) have focused largely on utilizing molecular techniques to address short- and long-term epidemiologic questions, such as in outbreak investigations and in assessing the global dissemination of strains, respectively. This is done primarily by examining the extent of genetic diversity of clinical strains of Mycobacterium tuberculosis. When molecular methods are used in conjunction with classical epidemiology, their utility for TB control has been realized. For instance, molecular epidemiologic studies have added much-needed accuracy and precision in describing transmission dynamics, and they have facilitated investigation of previously unresolved issues, such as estimates of recent-versus-reactive disease and the extent of exogenous reinfection. In addition, there is mounting evidence to suggest that specific strains of M. tuberculosis belonging to discrete phylogenetic clusters (lineages) may differ in virulence, pathogenesis, and epidemiologic characteristics, all of which may significantly impact TB control and vaccine development strategies. Here, we review the current methods, concepts, and applications of molecular approaches used to better understand the epidemiology of TB.

Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers.

Mycobacterium tuberculosis Beijing genotype strains are highly prevalent in Asian countries and in the territory of the former Soviet Union. They are increasingly reported in other areas of the world and are frequently associated with tuberculosis outbreaks and drug resistance. Beijing genotype strains, including W strains, have been characterized by their highly similar multicopy IS6110 restriction fragment length polymorphism (RFLP) patterns, deletion of spacers 1 to 34 in the direct repeat region (Beijing spoligotype), and insertion of IS6110 in the genomic dnaA-dnaN locus. In this study the suitability and comparability of these three genetic markers to identify members of the Beijing lineage were evaluated. In a well-characterized collection of 1,020 M. tuberculosis isolates representative of the IS6110 RFLP genotypes found in The Netherlands, strains of two clades had spoligotypes characteristic of the Beijing lineage. A set of 19 Beijing reference RFLP patterns was selected to retrieve all Beijing strains from the Dutch database. These reference patterns gave a sensitivity of 98.1% and a specificity of 99.7% for identifying Beijing strains (defined by spoligotyping) in an international database of 1,084 strains. The usefulness of the reference patterns was also assessed with large DNA fingerprint databases in two other European countries and for identification strains from the W lineage found in the United States. A standardized definition for the identification of M. tuberculosis strains belonging to the Beijing/W lineage, as described in this work, will facilitate further studies on the spread and characterization of this widespread genotype family of M. tuberculosis strains.

Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

Spoligologos: a bioinformatic approach to displaying and analyzing Mycobacterium tuberculosis data.

Spacer oligonucleotide (spoligotyping) analysis is a rapid polymerase chain reaction-based method of DNA fingerprinting the Mycobacterium tuberculosis complex. We examined spoligotype data using a bioinformatic tool (sequence logo analysis) to elucidate undisclosed phylogenetic relationships and gain insights into the global dissemination of strains of tuberculosis. Logo analysis of spoligotyping data provides a simple way to describe a fingerprint signature and may be useful in categorizing unique spoligotypes patterns as they are discovered. Large databases of DNA fingerprint information, such as those from the U.S. National Tuberculosis Genotyping and Surveillance Network and the European Concerted Action on Tuberculosis, contain information on thousands of strains from diverse regions. The description of related spoligotypes has depended on exhaustive listings of the individual spoligotyping patterns. Logo analysis may become another useful graphic method of visualizing and presenting spoligotyping clusters from these databases.

Global distribution of Mycobacterium tuberculosis spoligotypes.

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11708 patterns from as many clinical isolates originating from more than 90 countries. The 11708 spoligotypes were clustered into 813 shared types. A total of 1300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review.

Strains of the Beijing/W genotype family of Mycobacterium tuberculosis have caused large outbreaks of tuberculosis, sometimes involving multidrug resistance. This genetically highly conserved family of M. tuberculosis strains predominates in some geographic areas. We have conducted a systematic review of the published reports on these strains to determine their worldwide distribution, spread, and association with drug resistance. Sixteen studies reported prevalence of Beijing strains defined by spoligotyping; another 10 used other definitions. Beijing strains were most prevalent in Asia but were found worldwide. Associations with drug resistance varied: in New York, Cuba, Estonia, and Vietnam, Beijing strains were strongly associated with drug resistance, but elsewhere the association was weak or absent. Although few reports have measured trends in prevalence, the ubiquity of the Beijing strains and their frequent association with outbreaks and drug resistance underline their importance.

Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster.

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.

Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains.

A large, genetically related group of Mycobacterium tuberculosis strains, variously called W or Beijing, is distinguished by specific molecular markers and referred to as the W-Beijing family strains. Molecular epidemiological studies suggest that these strains are highly prevalent throughout Asia and the countries of the former Soviet Union and they have also been reported in several other geographical regions, including North America. Although the spread of W-Beijing family strains in diverse populations is well documented, the underlying host-pathogen factors accounting for their continued dissemination and burden of disease have yet to be determined.