Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries.

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

Pyrosequencing for rapid detection of extensively drug-resistant Mycobacterium tuberculosis in clinical isolates and clinical specimens.

Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.

Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations.

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.

Evaluation of the BD Bactec MGIT 320 system for detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis.

Two different laboratories evaluated growth and detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis by the BD Bactec MGIT 320 using the BD Bactec MGIT 960 (BD Diagnostics, Sparks, MD) as a reference method. Out of 359 processed sputum specimens for detection of mycobacteria, 99.7% were in agreement between the MGIT 320 and MGIT 960. Streptomycin (STR), isoniazid (INH), rifampin (RIF), ethambutol (EMB) (collectively known as SIRE), and pyrazinamide (PZA) drug susceptibility testing was performed on 89 clinical strains, prepared from both liquid and solid inocula. The results of SIRE and PZA were 100% reproducible between the two instruments tested at both laboratories.

Diagnostic accuracy and reproducibility of WHO-endorsed phenotypic drug susceptibility testing methods for first-line and second-line antituberculosis drugs.

In an effort to update and clarify policies on tuberculosis drug susceptibility testing (DST), the World Health Organization (WHO) commissioned a systematic review evaluating WHO-endorsed diagnostic tests. We report the results of this systematic review and meta-analysis of the diagnostic accuracy and reproducibility of phenotypic DST for first-line and second-line antituberculosis drugs. This review provides support for recommended critical concentrations for isoniazid and rifampin in commercial broth-based systems. Further studies are needed to evaluate critical concentrations for ethambutol and streptomycin that accurately detect susceptibility to these drugs. Evidence is limited on the performance of DST for pyrazinamide and second-line drugs.

Lipoprotein processing is essential for resistance of Mycobacterium tuberculosis to malachite green.

Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

Multicenter evaluation of Bactec MGIT 960 system for second-line drug susceptibility testing of Mycobacterium tuberculosis complex.

The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethionamide. The critical concentration established for levofloxacin and amikacin was 1.5 microg/ml, that established for capreomycin was 3.0 microg/ml, and that established for ethionamide was 5.0 microg/ml. The overall level of agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin, and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the participating laboratories was 99.5%.

Bergeyella cardium: Clinical Characteristics and Draft Genome of an Emerging Pathogen in Native and Prosthetic Valve Endocarditis.

Bergeyella cardium is a new species in the family Flavobacteriaceae that was recently described in 3 cases of native valve infective endocarditis. We report the first case of B. cardium prosthetic valve endocarditis, provide the first draft genome of this species, and review the microbiologic characteristics of this emerging pathogen.

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.

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.