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.

Molecular diagnosis of tuberculosis and drug resistance.

Molecular drug susceptibility testing (MDST) provides rapid diagnosis of tuberculosis (TB) and detection of drug resistance with commendable sensitivity and specificity. MDST reduces unnecessary isolation or treatment, when a negative result for TB is obtained. Because of the possibility of false detection of rifampin resistance by probe-based MDST, confirmation by sequencing is recommended, especially in regions where the prevalence of resistance is low. Revealing mutation identity by sequencing offers opportunities to study drug minimum inhibitory concentrations for each mutation. Such information enables prediction of resistance levels, and may be helpful in formulating optimal regimens, when treatment options are limited.

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.

Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin.

Levofloxacin, the active l-isomer of the quinolone ofloxacin, is now widely accepted for treatment of multidrug-resistant tuberculosis. Because the drug is now widely used, we sought to establish susceptibility test conditions for Mycobacterium tuberculosis against levofloxacin by the traditional reference method, agar proportion (AP), the commonly used BACTEC 460 radiometric system, and the newer BACTEC MGIT 960 method. To determine the stability of levofloxacin in the two newer test systems (BACTEC 460 and BACTEC MGIT 960), media containing subinhibitory levels of levofloxacin were prepared and stored at 4 and 37 degrees C for 14 days. The stored media were inoculated with H37Rv, and the drug activity was compared to freshly prepared media. Results show that levofloxacin is stable over the course of testing. Next, optimum levofloxacin test concentrations were determined for AP, BACTEC 460, and BACTEC MGIT 960 methods. MICs were determined for 32 pan-susceptible isolates of M. tuberculosis obtained from presumably untreated patients and 14 quinolone-resistant isolates. The levofloxacin-resistant strains either were isolated from patients who remained culture-positive despite treatment with a quinolone agent (six strains) or contained known mutations in gyrA (eight strains). Levofloxacin MICs resulted in a bimodal pattern with values for resistant strains consistently higher than those for pan-susceptible strains. Results show that levofloxacin concentrations of 2 microg/ml (BACTEC 460 and BACTEC MGIT 960) and 1 microg/ml (AP) inhibited the growth of all pan-susceptible strains while permitting the growth of all levofloxacin-resistant strains. Confirmatory tests with a subset of pan-susceptible and levofloxacin-resistant isolates validated the selected test concentrations.

Genomic analysis distinguishes Mycobacterium africanum.

Mycobacterium africanum is thought to comprise a unique species within the Mycobacterium tuberculosis complex. M. africanum has traditionally been identified by phenotypic criteria, occupying an intermediate position between M. tuberculosis and M. bovis according to biochemical characteristics. Although M. africanum isolates present near-identical sequence homology to other species of the M. tuberculosis complex, several studies have uncovered large genomic regions variably deleted from certain M. africanum isolates. To further investigate the genomic characteristics of organisms characterized as M. africanum, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of M. tuberculosis deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that M. africanum subtype II isolates are situated among strains of "modern" M. tuberculosis. In contrast, other M. africanum isolates (subtype I) constitute two distinct evolutionary branches within the M. tuberculosis complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of "M. africanum-like" isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the M. africanum subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for M. tuberculosis complex members, challenging the traditional approach to M. tuberculosis complex speciation. Furthermore, the genomic results suggest a rational framework for defining M. africanum and provide tools to accurately assess its prevalence in clinical specimens.

A prospective, multicenter study of laboratory cross-contamination of Mycobacterium tuberculosis cultures.

A prospective study of false-positive cultures of Mycobacterium tuberculosis that resulted from laboratory cross-contamination was conducted at three laboratories in California. Laboratory cross-contamination accounted for 2% of the positive cultures. Cross-contamination should be a concern when an isolate matches the genotype of another sample processed during the same period.