The Relative Positioning of Genotyping and Phenotyping for Tuberculosis Resistance Screening in Two EU National Reference Laboratories in 2023.

The routine use of whole genome sequencing (WGS) as a reference typing technique for Mycobacterium tuberculosis epidemiology combined with the catalogued and extensive knowledge base of resistance-associated mutations means an initial susceptibility prediction can be derived from all cultured isolates in our laboratories based on WGS data alone. Preliminary work has confirmed, in our low-burden settings, these predictions are for first-line drugs, reproducible, robust with an accuracy similar to phenotypic drug susceptibility testing (pDST) and in many cases able to also predict the level of resistance (MIC). Routine screening for drug resistance by WGS results in approximately 80% of the isolates received being predicted as fully susceptible to the first-line drugs. Parallel testing with both WGS and pDST has demonstrated that routine pDST of genotypically fully susceptible isolates yields minimal additional information. Thus, rather than re-confirming all fully sensitive WGS-based predictions, we suggest that a more efficient use of available mycobacterial culture capacity in our setting is the development of a more extensive and detailed pDST targeted at any mono or multi-drug-resistant isolates identified by WGS screening. Phenotypic susceptibility retains a key role in the determination of an extended susceptibility profile for mono/multi-drugresistant isolates identified by WGS screening. The pDST information collected is also needed to support the development of future catalogues of resistance-associated mutations.

WGS more accurately predicts susceptibility of Mycobacterium tuberculosis to first-line drugs than phenotypic testing.

BACKGROUND: Drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide. METHODS: DST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT. RESULTS: In total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance. CONCLUSIONS: Applying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST.

Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia.

BACKGROUND: The ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region. METHODS: Phenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates. RESULTS: Out of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94-32 and 100-32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region. CONCLUSION: In this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

Utility of rpoB gene sequencing for identification of nontuberculous mycobacteria in the Netherlands.

In the Netherlands, clinical isolation of nontuberculous mycobacteria (NTM) has increased over the past decade. Proper identification of isolates is important, as NTM species differ strongly in clinical relevance. Most of the currently applied identification methods cannot distinguish between all different Mycobacterium species and complexes within species. rpoB gene sequencing exhibits a promising level of discrimination among rapidly and slowly growing mycobacteria, including the Mycobacterium avium complex. In this study, we prospectively compared rpoB gene sequencing with our routine algorithm of reverse line blot identification combined with partial 16S rRNA gene sequencing of 455 NTM isolates. rpoB gene sequencing identified 403 isolates to species level as 45 different known species and identified 44 isolates to complex level, and eight isolates remained unidentifiable to species level. In contrast, our reference reverse line blot assay with adjunctive 16S rRNA gene sequencing identified 390 isolates to species level (30 distinct species) and identified 56 isolates to complex level, and nine isolates remained unidentified. The higher discriminatory power of rpoB gene sequencing results largely from the distinction of separate species within complexes and subspecies. Also, Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium interjectum were separated into multiple groupings with relatively low sequence similarity (98 to 94%), suggesting that these are complexes of closely related species. We conclude that rpoB gene sequencing is a more discriminative identification technique than the combination of reverse line blot and 16S rRNA gene sequencing and could introduce a major improvement in clinical care of NTM disease and the research on the epidemiology and clinical relevance of NTM.

Combined species identification, genotyping, and drug resistance detection of Mycobacterium tuberculosis cultures by MLPA on a bead-based array.

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates.

The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug- and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 microg/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 microg/ml for amikacin, linezolid, and clofazimine, 0.5 microg/ml for moxifloxacin and rifabutin, and 2.5 microg/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.

Clinical relevance of Mycobacterium chelonae-abscessus group isolation in 95 patients.

OBJECTIVES: To determine the clinical relevance of Mycobacterium chelonae-abscessus group isolation from clinical samples. METHODS: We retrospectively reviewed medical files of all patients from whom these mycobacteria were isolated between January 1999 and January 2005 and re-identified the isolates by rpoB sequencing. We applied the American Thoracic Society (ATS) diagnostic criteria to establish clinical relevance. RESULTS: Ninety-five patients were traced (56 M. chelonae, 25 Mycobacterium abscessus, 8 Mycobacterium massiliense, 6 Mycobacterium bolletii). Most isolates were cultured from pulmonary samples in patients with pre-existing pulmonary disease. Among patients with pulmonary isolates, 27% (20/74) meets ATS criteria; M. abscessus is most relevant (50%; 9/18), followed by M. massiliense (29%; 2/7), M. bolletii (20%; 1/5) and M. chelonae (18%; 8/44). Extrapulmonary disease presented as disseminated skin disease, eye disease specific for M. chelonae and otomastoiditis for M. abscessus. Treatment, especially for pulmonary M. abscessus disease, yielded limited results. CONCLUSIONS: One-fourth of the patients with pulmonary M. chelonae-abscessus group isolates met the ATS criteria; this percentage differs by species. Species distribution and clinical relevance differ from other regions. M. abscessus isolation in cystic fibrosis patients warrants special attention. Current ATS criteria might be too lenient to diagnose M. chelonae-abscessus group disease.

Region of difference 1 in nontuberculous Mycobacterium species adds a phylogenetic and taxonomical character.

The esat-6 and cfp-10 genes are essential for virulence in Mycobacterium tuberculosis. Among nontuberculous mycobacteria, we found these genes only in M. kansasii, M. szulgai, M. marinum, and M. riyadhense, with unique sequences. This adds a phylogenetic and taxonomical characteristic and may represent a virulence factor for nontuberculous mycobacteria.

Mycobacterium mantenii sp. nov., a pathogenic, slowly growing, scotochromogenic species.

Slowly growing, scotochromogenic bacteria of a novel Mycobacterium species were isolated from lymph node samples in two children and pulmonary samples in two elderly patients from different regions in the Netherlands as well as from a surface water sample in Zambia. Its 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences are unique in comparison with other mycobacteria. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that these micro-organisms are most closely related to Mycobacterium scrofulaceum ATCC 19981(T) (8 differences; 0.6 % divergence). The hsp65 sequence shows 96 % similarity to that of Mycobacterium saskatchewanense MB54784 and the rpoB sequence shows 95 % similarity to that of Mycobacterium chimaera CIP 107892(T). The 16S-23S ITS sequence places these micro-organisms within the Mycobacterium avium complex, as a novel ITS sequevar. This is not supported by analysis of the 16S rRNA, hsp65 or rpoB gene sequences. Their scotochromogenicity, combined with mostly positive urease, positive semiquantitative catalase and negative tellurite reduction tests, set these isolates apart from related species. The mycolic acid patterns, obtained by HPLC, are similar to that of Mycobacterium scrofulaceum, though the peak heights and distribution present minor differences. We propose the name Mycobacterium mantenii sp. nov. for this novel species. The type strain, isolated from a lymph node biopsy sample, is strain 04-1474(T) (=NLA000401474(T) =CIP 109863(T) =DSM 45255(T)).

Mycobacterium riyadhense sp. nov., a non-tuberculous species identified as Mycobacterium tuberculosis complex by a commercial line-probe assay.

A non-chromogenic, slowly growing Mycobacterium strain was isolated from a maxillary sinus lavage from a symptomatic patient in Riyadh, Saudi Arabia. It was initially identified as a member of the Mycobacterium tuberculosis complex by a commercial line-probe assay. Its 16S rRNA, hsp65 and rpoB gene and 16S-23S internal transcribed spacer sequences were unique; phylogenetic analysis based on the 16S rRNA gene sequence groups this organism close to Mycobacterium szulgai and Mycobacterium malmoense. Its unique biochemical properties and mycolic acid profile support separate species status. We propose the name Mycobacterium riyadhense sp. nov. to accommodate this strain. The type strain is NLA000201958(T) (=CIP 109808(T) =DSM 45176(T)).