James T. Douglas

rpoB Genotypes of Mycobacterium tuberculosis Beijing Family Isolates from East Asian Countries

ABSTRACT The 81-bp region of the rpoB gene in 66 RifrMycobacterium tuberculosis isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the rpoB gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.

Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

ABSTRACT Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.

Characterization of the Manila Family of Mycobacterium tuberculosis

ABSTRACT Forty-eight Mycobacterium tuberculosis strains were obtained from patients living in metropolitan Manila, Republic of the Philippines. Three molecular typing methods, IS6110 restriction fragment length polymorphism, spoligotyping, and DNA sequencing of the oxyR, gyrA, and katG loci, established that these strains have restricted diversity and are members of a related genetic group of organisms. Comparison of the DNA fingerprint patterns with those in international databases confirmed the uniqueness of this group of isolates, which we designate the Manila family of M. tuberculosis.

The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan

Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers > or = 1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high sensitivity of these assays. The strong reactivity in the LFA observed for the majority (92.1%) of the samples from the patients with culture-confirmed brucellosis together with the user-friendliness of the assay procedure makes the LFA ideal for the confirmation of brucellosis in endemic areas in Kazakhstan. The Rose Bengal test lacked sensitivity in particular for patients with chronic brucellosis therefore limiting its value as a quick screening assay. The study emphasizes the importance of the LFA as a useful, rapid, and easy-to-perform tool in the diagnostic testing of brucellosis.

Rapid Deletion-Based Subtyping System for the Manila Family of Mycobacterium tuberculosis

ABSTRACT The Manila family of Mycobacterium tuberculosis is a group of clonal isolates seen throughout the Pacific Basin. Commonly used rapid molecular typing methods often leave large groups of Manila family isolates clustered together. Here we describe a simple deletion-based PCR method that improves the discrimination for Manila family isolates, with or without the use of mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) classification, and that is both rapid and affordable. Twenty-eight Manila family isolates, classified by spoligotyping, were collected from around the Pacific Basin from 1995 to 2003 and were tested for known genomic deletions. Nine of 15 regions of difference tested were identified as potentially discriminatory, with 18 distinct patterns; of these 9, 5 were selected for optimal discrimination using 61 Manila family isolates collected from California in 2009. For this geographically limited sample, the single large cluster was reduced to 14 distinct patterns. When the isolates were tested by spoligotyping and MIRU-VNTR, the addition of deletion analysis increased the number of distinct patterns from 43 to 56. In summary, the two study groups, which together form a single group of 89 isolates by spoligotyping, were segregated into 17 subgroups by our deletion-based subtyping system.

Genomic analyses of the ancestral Manila family of Mycobacterium tuberculosis

With its airborne transmission and prolonged latency period, Mycobacterium tuberculosis spreads worldwide as one of the most successful bacterial pathogens and continues to kill millions of people every year. M. tuberculosis lineage 1 is inferred to originate ancestrally based on the presence of the 52-bp TbD1 sequence and analysis of single nucleotide polymorphisms. Previously, we briefly reported the complete genome sequencing of M. tuberculosis strains 96121 and 96075, which belong to the ancient Manila family and modern Beijing family respectively. Here we present the comprehensive genomic analyses of the Manila family in lineage 1 compared to complete genomes in lineages 2–4. Principal component analysis of the presence and absence of CRISPR spacers suggests that Manila isolate 96121 is distinctly distant from lineages 2–4. We further identify a truncated whiB5 gene and a putative operon consisting of genes encoding a putative serine/threonine kinase PknH and a putative ABC transporter, which are only found in the genomes of Manila family isolates. Six single nucleotide polymorphisms are uniquely conserved in 38 Manila strains. Moreover, when compared to M. tuberculosis H37Rv, 59 proteins are under positive selection in Manila family isolate 96121 but not in Beijing family isolate 96075. The unique features further serve as biomarkers for Manila strains and may shed light on the limited transmission of this ancestral lineage outside of its Filipino host population.

Immunization with hybrid recombinant Mycobacterium tuberculosis H37Rv proteins increases the TH1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria

The aim of this research was to identify subunit immunogens that can generate enhanced CD8 T cell and TH1 responses against Mycobacterium tuberculosis. A genomic comparison of the M. tuberculosis H(37)R(V) and M. bovis BCG identified 61 proteins that are unique to H(37)R(V). Further screening of these 61 proteins using in silico analyses mimicking proteasomal digestion, transporter-associated antigen processing and H-2 antigen presentation identified 13 proteins with high densities of predicted MHC class I epitopes. Two native proteins, Rv1986c and Rv3875, were selected on the basis of their secreted or transmembrane characteristics and relatively lower frequencies of predicted MHC class II epitopes. To further enhance the CD8 T cell and TH1 responses, a hybrid protein, H32, was constructed by combining the nucleotide sequences encoding the MHC class I antigen-rich segment of Rv1986c and the entire Rv3875 sequence. The two native proteins and the hybrid were used to immunize C57BL/6 and Balb/c mice, which was followed by pulmonary instillation with irradiated M. tuberculosis H(37)R(V). All three proteins elicited elevated IFN-gamma responses, with the hybrid showing significant increases over the native proteins in both mice. This strategy of immunogen selection might be used to improve the current subunit vaccines against M. tuberculosis as well as other intra-cellular pathogens.

Whole genome SNP analysis suggests unique virulence factor differences of the Beijing and Manila families of Mycobacterium tuberculosis found in Hawaii

While tuberculosis (TB) remains a global disease, the WHO estimates that 62% of the incident TB cases in 2016 occurred in the WHO South-East Asia and Western Pacific regions. TB in the Pacific is composed predominantly of two genetic families of Mycobacterium tuberculosis (Mtb): Beijing and Manila. The Manila family is historically under-studied relative to the families that comprise the majority of TB in Europe and North America (e.g. lineage 4), and it remains unclear why this lineage has persisted in Filipino populations despite the predominance of more globally successful Mtb lineages in most of the world. The Beijing family is of particular interest as it is increasingly associated with drug resistance throughout the world. Both of these lineages are important to the State of Hawaii, where they comprise over two-thirds of TB cases. Here, we performed whole genome sequencing on 82 Beijing family, Manila family, and outgroup clinical Mtb isolates from Hawaii to identify lineage-specific SNPs (SNPs found in all isolates from their respective families, and exclusively in those families) in established virulence factor genes. Six non-silent lineage-specific virulence factor SNPs were found in the Beijing family, including mutations in alternative sigma factor sigG and polyketide synthases pks5 and pks7. The Manila family displayed more than eleven non-silent lineage-specific and characteristic virulence factor mutations, including in genes coding for MCE-family protein Mce1B, two mutations in fatty-acid-AMP ligase FadD26, and virulence-regulating transcriptional regulator VirS. This study further identified an ancient clade that shared some virulence factor mutations with the Manila family, and investigated the relationship of those and other “Manila-like” spoligotypes to the Manila family with this SNP dataset. This work identified a set of virulence genes that are worth pursuing to determine potential differences in transmission or virulence displayed by these two Mtb families.