A Roth

Phylogeny of the Genus Nocardia Based on Reassessed 16S rRNA Gene Sequences Reveals Underspeciation and Division of Strains Classified as Nocardia asteroides into Three Established Species and Two Unnamed Taxons

ABSTRACT Conventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species Nocardia asteroides, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus Nocardia, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species N. asteroides. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between N. paucivorans and N. brevicatena). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as N. asteroides fell into five distinct phylogenetic groups: the type strain cluster (N. asteroides sensu strictu), N. abscessus, N. cyriacigeorgica, and two clusters closely related to N. carnea or N. flavorosea. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that N. abscessus, N. cyriacigeorgica, and the two Nocardia species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to N. asteroides and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view.

Early Bactericidal Activity of Moxifloxacin in Treatment of Pulmonary Tuberculosis: a Prospective, Randomized Study

ABSTRACT Moxifloxacin is the most active fluoroquinolone against Mycobacterium tuberculosis in vitro. However, data about the efficacy in patients are not available. We enrolled 17 patients with tuberculosis in a prospective, randomized study. After 5 days of monotherapy with either moxifloxacin or isoniazid, we detected significant decreases in mean CFU per milliliter in sputum in both groups. The calculated early bactericidal activities for isoniazid and moxifloxacin were 0.209 and 0.273 log10 CFU per ml of sputum per day, respectively. According to the data from our study, moxifloxacin exhibits an early bactericidal activity that is comparable to that of isoniazid.

The frequency of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC): routine screening data for central Europe from a cohort study

Objectives Owing to novel therapy strategies in epidermal growth factor receptor (EGFR)-mutated patients, molecular analysis of the EGFR and KRAS genome has become crucial for routine diagnostics. Till date these data have been derived mostly from clinical trials, and thus collected in pre-selected populations. We therefore screened ‘allcomers’ with a newly diagnosed non-small cell lung carcinoma (NSCLC) for the frequencies of these mutations. Design A cohort study. Setting Lung cancer centre in a tertiary care hospital. Participants Within 15 months, a total of 552 cases with NSCLC were eligible for analysis. Primary and secondary outcome measures Frequency of scrutinising exons 18, 19 and 21 for the presence of activating EGFR mutation and secondary codon 12 and 13 for activating KRAS mutations. Results Of the 552 patients, 27 (4.9%) showed a mutation of EGFR. 19 of these patients (70%) had deletion E746-A750 in codon 19 or deletion L858R in codon 21. Adenocarcinoma (ACA) was the most frequent histology among patients with EGFR mutations (ACA, 22/254 (8.7%) vs non-ACA, 5/298 (1.7%); p<0.001). Regarding only ACA, the percentage of EGFR mutations was higher in women (16/116 (14%) women vs 6/138 (4.3%) men; p=0.008). Tumours with an activating EGFR mutation were more likely to be from non-smokers (18/27; 67%) rather than smoker (9/27; 33%). KRAS mutation was present in 85 (15%) of all cases. In 73 patients (86%), the mutation was found in exon 12 and in 12 cases (14%) in exon 13. Similarly, ACA had a higher frequency of KRAS mutations than non-ACA (67/254 (26%) vs 18/298 (6.0%); p<0.001). Conclusions We found a lower frequency for EGFR and KRAS mutations in an unselected Caucasian patient cohort as previously published. Taking our results into account, clinical trials may overestimate the mutation frequency for EGFR and KRAS in NSCLC due to important selection biases.

Analytical performance of the Roche LightCycler® Mycobacterium Detection Kit for the diagnosis of clinically important mycobacterial species.

Background The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility. Methodology/Principal Findings Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assays reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent. Significance The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.

Bedaquiline as a potential agent in the treatment of Mycobacterium abscessus infections

Mycobacterium abscessus is increasingly being recognised as a significant human pathogen, especially in patients with cystic fibrosis, and the specific M. abscessus subspecies seems to influence the clinical outcome [1]. The pulmonary manifestation of this nontuberculous mycobacteria (NTM) infection is one of the most difficult to treat forms, leading to substantial morbidity and mortality in this population [1, 2]. M. abscessus strains are highly resistant to most antibacterial drugs [2]. The recent development of liposomal amikacin for inhalation for patients with cystic fibrosis suggested a therapeutic breakthrough. However, even though sputum conversion was improved in a phase 2 study, the primary end-point (change from baseline to day 84 on a semi-quantitative mycobacterial growth scale) was not reached [3]. We analysed the minimal inhibitory concentration (MIC) of another promising new anti-tuberculous drug, bedaquiline, using 20 clinical isolates of M. abscessus. No systematic studies on the distribution of MIC for M. abscessus have been performed previously. Bedaquiline may be a potential agent to treat severe or relapsing Mycobacterium abscessus infection http://ow.ly/3lUp309VZVj

Isoniazid minimal inhibitory concentrations of tuberculosis strains with katG mutation.

The detection of a katG mutation commonly leads to the exclusion of isoniazid (INH) from anti-tuberculosis treatment regimens.1,2 INH is nevertheless one of the most important drugs against Mycobacterium tuberculosis due to its bactericidal activity, especially in the early phase of treatment.3 Most INH-resistant strains show mutations in the inhA or katG region.4,5 katG mutations can be found in 60–70% of resistant strains, and are described to be associated with high levels of INH resistance.2,6,7 The definition of INH resistance remains a matter of debate, as data that link katG mutation (Ser315Thr) to clinical outcome parameters are limited,2 and prospective studies with clinical endpoints such as treatment success are missing. A moderate level of resistance for strains above the minimal inhibitory concentration (MIC) of 1 lg/ml has been described, suggesting that patients with intermediate MICs might still benefit from treatment with INH.6,8–10 With quantitative susceptibility testing, the exact level of INH resistance can be determined in culture. On solid media (Middlebrook 7H10), the common break-point concentration for INH is 0.25 lg/ml. Low-level resistance is considered for MICs from 0.25 to 1 lg/ml, and high-level resistance for MICs . 1 lg/ml.11 We report a retrospective series of 84 clinical M. tuberculosis isolates with katG mutation Ser315Thr, cultured at the Chest Hospital Heckeshorn, Berlin, between January 2005 and July 2015. Clinical copy strains were not included. The majority of the strains originated from patients born in Eastern Europe or former Soviet Union states; only five patients were German. katG mutation Ser315Thr was detected by the low-cost, low-density (LCD) Chip MycoResistQ (Chipron GmbH, Berlin, Germany). The decision to test routinely for this mutation was based on the observation that 75–90% of mutations are located in codon 315.12 The epidemiology of the Ser315Thr mutation varies by region. In Russia, where most of our patients with multidrug-resistant tuberculosis (MDR-TB) come from, it was found in up to 92% of resistant isolates.13 The MICs of all 84 strains were tested on solid media, Middlebrook 7H10 agar, using the proportion method.10,11 MICs between 0.25 and 1 lg/ml were defined as low-level resistant and MICs . 5 lg/ml as high-level resistant.10 Strains with MICs between 1 and 5 lg/ml were defined as moderate-level resistant in this analysis. Of the 84 M. tuberculosis strains, 3/ 84 (4%) showed MICs referring to low-level resistance, 27/84 (32%) moderate-level resistance and 54/ 84 (64%) high-level resistance. A considerable proportion (36%) of our M. tuberculosis strains with a katG Ser315Thr mutation showed low-to-moderate levels of resistance. In the TBNET consensus statement for the management of drug-resistant tuberculosis, Lange et al. recommended that dosing of INH should be adjusted to the MIC test results wherever possible.1 The authors propose that M. tuberculosis strains with MICs between 1 and 5 lg/ ml should be treated with high-dose INH (16–20 mg/ kg) three times per week, if it is safely tolerated. However, pharmacological data show that even standard doses of 300 mg INH lead to peak serum concentrations of 3–6 lg/ml,14 and drug concentrations in the lung are similar to serum concentrations.15 These data suggest that even standard doses of INH could result in efficient drug concentrations that exceed the MICs of moderate-level resistant strains. We conclude that the finding of a katG Ser315Thr mutation alone should not lead to exclusion of INH from the treatment regimen. In our series of M. tuberculosis strains with the katG Ser315Thr mutation, 36% showed low-to-moderate MICs that might be reached at serum level even with standard doses of INH. Our results support the TBNET recommendation to guide INH dosing by MIC results where possible.1 It remains a matter of debate whether highdose INH is necessary to ensure treatment success in low-to-moderate level resistant strains. Nevertheless, therapeutic drug monitoring may be helpful to ensure appropriate dosing in vivo.16 The reasons for the variation of MICs with Ser315Thr mutation in our study are not known and should be investigated. While MIC-based testing may be helpful to guide treatment, it is more timeconsuming than molecular analysis. Rapid molecular testing based on detection of katG mutation is needed for an immediate, but preliminary, treatment decision. Future studies should focus on the correlation between katG mutations and MIC levels in relation to clinical treatment outcome.

Evaluation of IS1245-based PCR for detection of mycobacterium avium bacteraemia in AIDS patients.

A PCR method based on the repetitive IS1245 sequence was evaluated for the detection of Mycobacterium avium bacteraemia in AIDS patients. Two blood preparation methods were applied: lysis of erythrocytes using a hypotonic buffer and Ficoll density centrifugation. Results were compared with culture and PCR amplification of the non-repetitive pMav22 sequence. Fifty-one of 251 tested samples grew M. avium. Bacterial densities lower than 10 cfu/ml blood were frequent, they occurred in 59% of blood samples from patients with mycobacteraemia. Inhibitory substances were detected more frequently with the lysis method (36%) than the Ficoll processed samples (19%). While specificity of PCR was high, sensitivities varied according to the methods used and the load of infection in the bloodstream. False-negative PCR results were attributable to low level bacteraemia and inhibition of PCR. Moreover, 1 of 186 and 9 of 51 M. avium strains investigated lacked the IS1245 and the pMav22 sequence, respectively. Sensitivities of culture, IS1245- and pMav22-based PCR were 88, 64 and 58%, within the lysis processed samples and 95, 86 and 50% using Ficoll prepared samples, respectively. Thus, we conclude that IS1245-based PCR is a rapid and specific method for diagnosing M. avium bacteraemia and shows a higher sensitivity than single copy gene targets, but that the sensitivity is inferior to culture.

Pathogenicity of Mycobacterium kansasii

BACKGROUND In a recent prospective study on pulmonary infections with non-tuberculous mycobacteria (NTM) led by the WATL group, disease rates in patients with M. kansasii infection were found to be 100 %. In the present study we re-evaluated the pathogenicity of M. kansasii infections in a large lung diseases treatment center in Berlin (Lungenklinik Heckeshorn). METHODS All patients in whose respiratory specimen cultures M. kansasii was detected between January 2003 and June 2013 were included. The 2007 ATS diagnostic criteria were applied to differentiate disease from asymptomatic infection. The strains were further investigated by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) region. RESULTS We evaluated 43 consecutive cases. Complete patient data were available in 38 cases. In one patient, no culture results were obtained, in 37 patients M. kansasii was isolated and patient data could be retrieved. In 25/37 patients (68 %) clinical disease was present so that a specific treatment was initiated (underlying diseases were COPD in 8/25 (32 %), bronchiectasis in 5/25 (20 %), TB scar or scar due to prior chest surgery in 3/25 (12 %) and alcohol abuse in 4/25 (16 %)). Twelve out of 37 patients (32 %) were found to be colonized or asymptomatically infected (underlying diseases were COPD in 7/12 (58 %), bronchiectasis in 3/12 (25 %) and TB scar or scar due to prior chest surgery in 3/12 (25 %)). Sequencing results identified 30 strains as genotype I, and 2 strains as genotype II. In 22/30 cases (73 %) genotype I was considered pathogenic. CONCLUSIONS In our cohort, we could not confirm the high M. kansasii pathogenicity of 100 % found in a previous multi-center study; we therefore support the clinical and semiquantitative microbiologic diagnostic criteria also for infection with M. kansasii.

The "COLD-PCR approach" for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer.

Background:Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients’ exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Materials and Methods:Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. Results:COLD-PCR–based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. Conclusions:COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

Frühe bakterizide Aktivität und Wirksamkeit von Moxifloxacin versus Isoniazid in der Behandlung der akuten Lungentuberkulose – eine prospektive, randomisierte Studie

Hintergrund: In in vitro Untersuchungen konnte gezeigt werden, dass Moxifloxacin derzeitig das aktivste Fluorchinolone gegenuber Mycobacterium tuberculosis (Mtb)ist. Allerdings existieren bisher keine ausreichenden Daten zur Wirksamkeit von Moxifloxacin bei Patienten. Wir untersuchten im Rahmen einer prospektiven, offenen, randomisierten, monozentrischen Studie den Abfall der Kolonie bildenden Einheiten pro Milliliter (cfu) von Mtb im Sputum und die fruhe bakterizide Aktivitat (EBA) von Moxifloxacin im Vergleich zu INH. Methoden: 17 immunkompetente Erwachsene mit einer aktiven und Sputum positiven Lungentuberkulose wurden vor Beginn der Standardtherapie fur 5 Tage mit Moxifloxacin (400mg/Tag; MOX, n=8) oder Isoniazid (5mg/kg KG; INH, n=9) behandelt. Die EBA ist definiert als Abfall des dekadischen Logarithmus der Kolonie bildenden Einheiten pro Milliliter im Sammelsputum innerhalb der ersten 5 Behandlungstage. Ergebnisse: Nach 5 Tagen zeigte sich in beiden Gruppen ein signifikanter Abfall der CFU/ml (INH: Tag 0=11.5×106/ml, Tag 5=1.2×106/ml, p=0.015; MOX: Tag 0=14.2×106/ml, Tag 5=0.70×106/ml, p=0.028). Es bestand kein signifikanter Unterschied zwischen den Gruppen bezuglich der fruhen bakteriziden Aktivitat. Die Studienmedikation wurde in beiden Gruppen gut vertragen. Zusammenfassung: Die in vivo Wirksamkeit von Moxifloxacin ist der von INH vergleichbar. Unter Berucksichtigung der noch kleinen Fallzahl mussen weitere Untersuchungen folgen.