Culture-Independent Detection of Nontuberculous Mycobacteria in Clinical Respiratory Samples.

Culture-based detection of nontuberculous Mycobacteria (NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.

Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus) in South Australia.

This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

A case of severe Mycobacterium thermoresistibile pneumonia.

Mycobacterium thermoresistibile is a thermotolerant nontuberculous mycobacterium which can rarely result in human infection. Although immunosuppression has been identified as a risk factor for infection, it is possible that mycobacterial laboratories may have previously under-recognized M. thermoresistibile as standard mycobacterial incubation temperatures are suboptimal for culture of this organism. Here, we present a case of severe M. thermoresistibile pneumonia associated with achalasia requiring life support in the intensive care unit. We speculated that the interplay between specific host and environmental risk factors contributed to acquisition of infection. Infection with this fastidious organism required prolonged treatment with multiple antimicrobials and adjunctive therapeutic drug monitoring which led to clinical cure despite residual lung injury. We also reviewed literature documenting cases of human infection with M. thermoresistibile. The diagnosis of M. thermoresistibile requires a high degree of clinical suspicion considering its association with immunosuppressive conditions, postulated environmental inoculation and eponymous culture growth characteristics.

Legionella steelei sp. nov., isolated from human respiratory specimens in California, USA, and South Australia.

Legionella-like bacteria were isolated from the respiratory tract of two patients in California, USA, and South Australia, but were not thought to cause disease. These bacteria, strains F2632 and IMVS-3376(T), were found to have identical Legionella macrophage infectivity potentiator (mip) gene sequences and were therefore further characterized to determine their genetic and phenotypic relatedness and properties. Both of these Gram-negative-staining bacterial strains grew on buffered charcoal yeast extract medium, were cysteine auxotrophs and made a characteristic diffusible bright yellow fluorescent pigment, with one strain making a late appearing colony-bound blue-white fluorescent pigment. The optimal in vitro growth temperature was 35 °C, with very poor growth at 37 °C in broth or on solid media. There was no growth in human A549 cells at either 35 or 37 °C, but excellent growth in Acanthamoeba castellani at 30 °C and poorer growth at 35 °C. Phylogenetic analysis of these bacteria was performed by sequence analysis of 16S rRNA, mip, ribonuclease P, ribosomal polymerase B and zinc metalloprotease genes. These studies confirmed that the new strains represented a single novel species of the genus Legionella for which the name Legionella steelei sp. nov. is proposed. The type strain is IMVS-3376(T) ( = IMVS 3113(T) = ATCC BAA-2169(T)).

Revised guidelines for Australian laboratories performing mycobacteriology testing.

Mycobacteriology laboratories play a key role in tuberculosis (TB) control by providing phenotypic and molecular diagnostics, by performing molecular typing to aid contact tracing, and by supporting research and similar laboratories in Australia's neighbouring countries where TB is prevalent. The National Tuberculosis Advisory Committee (NTAC) published a set of laboratory guidelines in 2006 aiming to document the infrastructure, equipment, staffing and work practices required for safe high-quality work in Australian mycobacteriology laboratories. These revised guidelines have the same aims and have been through a similar extensive consultative peer-review process involving the Mycobacterium Reference Laboratory (MRL) network, the Mycobacterium Special Interest Group (SIG) of the Australian Society for Microbiology (ASM), and other relevant national bodies. This revised document contains several significant changes reflecting the publication of new biosafety guidelines and tuberculosis standards by various national and international organisations, technology developments - such as the MPT64-based immunochromatographic tests (ICTs) and the Xpert MTB/RIF assay, and updated work practices in mycobacteriology laboratories. The biosafety recommendations affirm the latest Australian/New Zealand Standard 2243.3: 2010 and promote a biorisk assessment approach that, in addition to the risk categorisation of the organism, also considers the characteristics of the procedure being performed. Using this biorisk assessment approach, limited manipulations, such as Ziehl-Neelsen (ZN) microscopy, MPT64 ICTs, and culture inactivation/DNA extraction for molecular testing, may be performed on a positive TB culture in a PC2 laboratory with additional features and work practices. Other significant changes include recommendations on the integration of MPT64 ICTs and novel molecular tests into TB laboratory workflows to provide rapid accurate results that improve the care of TB patients. This revised document supersedes the original 2006 publication. NTAC will periodically review these guidelines and provide updates as new laboratory technologies become available.

Cost-effective implementation of a custom MALDI-TOF library for the identification of South Australian Nocardia isolates.

Mass spectrometry plays a significant role in the routine identification of micro-organisms and provides the ability to incorporate newly found pathogens into the database in a cost-effective fashion. This work aims to highlight the role of mass spectrometry through improved identification of Nocardia species in a diagnostic clinical microbiology laboratory. Prior to this study we constructed a custom in-house matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) library for Nocardia isolates consisting of isolates identified to the species level. Subsequently over a period of 5 years, we isolated a further 153 Nocardia clinical isolates, of which 91.5% (140/153) were identified correctly with the custom MALDI-TOF library and 8.5% (13/153) needed further molecular sequencing for final identification. We estimate our cost savings to be approximately 9,800 AUD overall with this implementation over the study period. Continued expansion and maintenance of this custom library will eventually result in little or no 16S ribosomal DNA sequencing needed for specific identification of Nocardia isolates.

Mycobacterial inactivation protein extraction protocol for matrix-assisted laser desorption ionization time-of-flight characterization of clinical isolates.

Background: Rapid identification of mycobacteria has been made possible with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) in recent years. Working with high concentrations of mycobacteria in a PC-3 containment facility makes MALDI-TOF cumbersome and costly. Therefore removing the inactivated isolate's protein extract from the PC-3 facility is needed for efficient identification in a routine PC-2 laboratory. Methods: This work describes a novel chemical and mechanical disruption protein extraction method, which provides reliable MALDI-TOF results from solid and liquid media, while ensuring laboratory safety. Results: When compared to sequencing results, 93.9% of the clinical isolates were identified in LJ media and 89% of the clinical isolates were identified in MGIT media. Conclusion: The MIPE protocol produces a high quality protein extract with improved isolate identification without compromising result turn-around-times or laboratory safety.

Evaluation of (SD) MPT64 antigen rapid test, for fast and accurate identification of Mycobacterium tuberculosis complex.

AIM: To evaluate the SD MPT64 assay for rapid, preliminary identification of Mycobacterium tuberculosis complex (MTBC). METHODS: All specimens were processed using a standard methodology and inoculated into an automated liquid culture system (BD MGIT960). All signal positive cultures had a smear prepared and tested using a commercial molecular assay. From a carefully mixed MGIT960 vial, 100  µL of broth was loaded into the sample well, and the result recorded after 15 min. Repeat isolates from patients were excluded as were positive cultures contaminated with non-mycobacteria. RESULTS: Fifty MTBC and 150 non-tuberculous mycobacteria were isolated during the study period. Test sensitivity was 98.04%, specificity (98.68%), positive predictive value (96.15%), and a negative predictive value (99.34%). There were two false positive results: Mycobacterium gastri and Mycobacterium fortuitum which were both identified by 16S rDNA and rpoB sequence analysis. CONCLUSIONS: The SD MPT64 assay showed excellent performance. The major advantages are: (1) simplicity of test procedure, (2) rapid turnaround time, and (3) relatively inexpensive. When used in conjunction with the presence of serpentine cording in a stained smear from culture, a preliminary identification of MTBC may be made with high confidence.