Sushil Pandey

Characterisation of pncA mutations in clinical Mycobacterium tuberculosis isolates in New Zealand.

Aims: To describe the molecular basis of pyrazinamide (PZA) resistance among Mycobacterium tuberculosis in New Zealand, and to compare the pncA gene sequence among isolates with phenotypic (Bactec 960 and Waynes assay) evidence of PZA resistance. Methods: The pncA gene of 26 clinical isolates of M. tuberculosis found to be PZA resistant by phenotypic methods was sequenced. Phenotypic and genotypic data were compared. Results: Mutations were detected in 11 isolates, predominantely among isolates with a multi‐drug resistance phenotype. While the mutations were dispersed along the pncA gene, a clustering of mutations were found at amino acid Ser18–His57 and His82–Val128 regions. Seven of the identified mutations have not been described previously. Conclusions: A high diversity of pncA gene mutation was seen among PZA resistant strains of M. tuberculosis. The Bactec 960 phenotypic test may over report PZA resistance.

First Case of Mycobacterium heckeshornense Lymphadenitis

ABSTRACT Mycobacterium heckeshornense is a slow-growing nontuberculous mycobacterium first characterized in 2000. It is reported to cause lung disease and tenosynovitis. We report a case of isolated massive axillary lymphadenopathy in an elderly woman, where histology showed necrotizing granulomata and M. heckeshornense was isolated as the causative organism.

Diversity of Mycobacterium tuberculosis and drug resistance in different provinces of Papua New Guinea

BackgroundPapua New Guinea (PNG) is a high tuberculosis (TB) burden country of the WHO Western Pacific Region, but so far research on drug resistance (DR) and genotypes of Mycobacterium tuberculosis (M. tuberculosis) was only conducted in few provinces in the country. The aim of the present study was to obtain baseline data on the level of drug resistance and the genotypic diversity of circulating M. tuberculosis in additional provinces and to investigate the differences between three selected sites across PNG.ResultsGenotyping of 147 M. tuberculosis clinical isolates collected in Goroka, Eastern Highlands Province, in Alotau, Milne Bay Province and in Madang, Madang Province revealed three main lineages of M. tuberculosis: Lineage 4 (European-American lineage), Lineage 2 (East-Asian lineage) and Lineage 1 (Indo-Oceanic lineage). All three lineages were detected in all three sites, but the individual lineage compositions varied significantly between sites. In Madang Lineage 4 was the most prevalent lineage (76.6%), whereas in Goroka and Alotau Lineage 2 was dominating (60.5% and 84.4%, respectively) (p < 0.001). Overall, phenotypic drug susceptibility testing showed 10.8% resistance to at least one of the first-line drugs tested. Of all resistant strains (23/212) 30.4% were Streptomycin mono-resistant, 17.4% were Isoniazid mono-resistant and 13% were Rifampicin mono-resistant. Multi-drug resistant (MDR) TB was found in 2.8% of all tested cases (6/212). The highest amount of MDR TB was found in Alotau in Milne Bay Province (4.6%).ConclusionA large number of drug resistant TB infections are present in the country and MDR TB has already been detected in all three surveyed regions of PNG, highlighting the importance of monitoring drug resistance and making it a high priority for the National Control Program. Due to the high prevalence of Lineage 2 in Milne Bay Province and given the frequent association of this lineage with drug resistance, monitoring of the latter should especially be scaled up in that province.

A case of infection caused by the basidiomycete Phellinus undulatus.

We present a case of soft tissue infection caused by the basidiomycete Phellinus undulatus. To our knowledge, this is the first reported case of human infection caused by this fungus. Definitive identification was only possible through molecular analysis as the isolate failed to produce any distinct morphological features in vitro.

Comparison of the MGIT TBc immunochromatographic assay with the Accuprobe Gen-Probe TB assay for identification of Mycobacterium tuberculosis complex: results from a low-burden tuberculosis setting.

We compared the BD MPT64 TBc Antigen assay with the Gen-Probe TB assay for identifying Mycobacterium tuberculosis (MTB) from liquid culture vials. The BD TBc Antigen assay was more sensitive than and as specific as the Gen-Probe TB assay, making it a useful alternative for the rapid detection of MTB.

Bacteraemia caused by Catabacter hongkongensis

Catabacter hongkongensis is a recently recognised anaerobic gram positive bacillus. We report the isolation of a C. hongkongensis strain that does not utilise mannose, a novel phenotype, from the blood cultures of a patient with a perforated appendix. The isolate was identified by 16s rRNA gene sequencing. This case highlights the difficulties with phenotypic identification of rare anaerobic isolates. It also illustrates the association of C. hongkongensis bacteraemia with a gastrointestinal source and intestinal perforation.

Multi-clonal evolution of multi-drug-resistant/extensively drug-resistant Mycobacterium tuberculosis in a high-prevalence setting of Papua New Guinea for over three decades

An outbreak of multi-drug resistant (MDR) tuberculosis (TB) has been reported on Daru Island, Papua New Guinea. Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. Whole genome sequencing of 100 of 165 clinical isolates referred from Daru General Hospital to the Supranational reference laboratory, Brisbane, during 2012–2015 revealed that 95 belonged to a single modern Beijing sub-lineage strain. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with potentially enhanced virulence mediated by an mycP1 mutation. The Beijing sub-lineage strain demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2 %) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multi-drug resistance, observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance, and evidence of local transmission of extensively drug resistant (XDR) strains from 2009. These findings underline the importance of whole genome sequencing in informing an effective public health response to MDR/XDR TB.

Non-tuberculous mycobacteria : baseline data from three sites in Papua New Guinea, 2010-2012

OBJECTIVE To determine the proportion of non-tuberculous mycobacteria (NTM) in samples of pulmonary tuberculosis (TB) cases from Papua New Guinea who were diagnosed using acid-fast microscopy. METHODS As part of a case detection study for TB, conducted in three provincial hospitals in Papua New Guinea, sputum samples of suspected tuberculous cases aged 15 years or older were collected from November 2010 to July 2012. Mycobacterial species isolated from sputum and grown in culture were examined to distinguish between NTM and the Mycobacterium tuberculosis complex (MTBC). RESULTS NTM were detected in 4% (9/225) of sputum samples grown in culture. Five (2.2%) of them were identified as NTM only and four (1.8%) were identified as mixed cultures containing both MTBC and NTM. Four different NTM species were identified; M. fortuitum, M. intracellulare, M. terrae and M. avium. DISCUSSION This is the first report from Papua New Guinea identifying NTM in three different locations. As NTM cannot be distinguished from M. tuberculosis through smear microscopy, the presence of NTM can lead to a false-positive diagnosis of tuberculosis. The prevalence of NTM should be determined and a diagnostic algorithm developed to confirm acid-fast bacilli in a smear as M. tuberculosis.

Multi-clonal evolution of MDR/XDR M. tuberculosis in a high prevalence setting in Papua New Guinea over three decades

An outbreak of multi-drug resistant tuberculosis has been reported on Daru Island, Papua New Guinea. The Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. We analyzed 100 isolates using whole genome sequencing and found 95 belonged to a single modern Beijing strain cluster. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with virulence potentially enhanced by a mycP1 mutation. The outbreak cluster demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2%) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multidrug resistance (MDR), observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance; with evidence of local transmission of extensively-drug resistant (XDR) strains from 2009. These findings underscore the importance of whole-genome sequencing in informing an effective public health response to MDR/XDR M. tuberculosis.

Identification of Mycobacterium abscessus complex and M. abscessus subsp. massiliense culture isolates by real-time assays

Recent worldwide increases in the incidence of Mycobacterium abscessus complex (MABSC) infection are of concern given it is recognized as the most pathogenic of the rapidly growing Mycobacterium species (Benwill & Wallace, 2014). MABSC is typically associated with soft-tissue and skin infections, but can also cause potentially fatal pulmonary disease in immunocompromised patients and those with chronic pulmonary disease, including in patients with cystic fibrosis (Harris & Kenna, 2014). Recent taxonomic descriptions based on whole-genome sequencing studies have shown that MABSC comprises three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii and M. abscessus subsp. massiliense (Bryant et al., 2013). Correct identification of MABSC species and subspecies is imperative due to their differing antibiotic treatment regimens (Griffith et al., 2007). Of note, M. abscessus subsp. massiliense is generally more susceptible to current treatments than M. abscessus subsp. abscessus and M. abscessus subsp. bolletii (Roux et al., 2014), particularly as it has a truncated erm41 gene, being a non-functional form of an erythromycin ribosomal methylase gene sequence (Kim et al., 2010). Therefore, rapid and specific identification of M. abscessus subsp. massiliense can be useful to inform treatment. It is in this context that M. abscessus subsp.massiliense has garnered a new level of importance in cystic fibrosis microbiology, given recent reports of crossinfection between patients with cystic fibrosis in the UK and USA (Aitken et al., 2012; Tettelin et al., 2014), as well as increased rates of MABSC infection in our own local cystic fibrosis clinics in Queensland, Australia (unpublished data).