Sustained transmission over two decades of a previously unrecognised MPT64 negative strain in Queensland, Australia: a whole genome sequencing study.

Background: MPT64 is a key protein used for (MTB) complex strain identification. We describe protracted transmission of an MPT64 negative MTB strain in Queensland, Australia, and explore genomic factors related to its successful spread.

Methods: All MPT64 negative strains identified between 2002 and 2022 by the Queensland Mycobacteria Reference Laboratory, and an additional 2 isolates from New South Wales (NSW), were whole genome sequenced.

Analytical performance of the Xpert MTB/XDR® assay for tuberculosis and expanded resistance detection.

In a manufacturer-independent laboratory validation study, the Xpert MTB/XDR® assay demonstrated equivalent limit of detection to Xpert MTB/RIF®, detected 100% of tested resistance mutations and showed some utility for resistance detection in strain mixtures. The Xpert MTB/XDR assay is a reliable, sensitive assay for tuberculosis and expanded resistance detection.

Rapid macrolide and amikacin resistance testing for in people with cystic fibrosis.

complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment.

Evaluation of the SpeeDx Carba (beta) multiplex real-time PCR assay for detection of NDM, KPC, OXA-48-like, IMP-4-like and VIM carbapenemase genes.

Background: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM.

Methods: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard.

Identification of carbapenem-resistant Pseudomonas aeruginosa in selected hospitals of the Gulf Cooperation Council States: dominance of high-risk clones in the region.

Purpose: The molecular epidemiology and resistance mechanisms of carbapenem-resistant Pseudomonas aeruginosa (CRPA) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, the United Arab Emirates, Oman, Qatar, Bahrain and Kuwait.

Methodology: Isolates were screened for common carbapenem-resistance genes by PCR. Relatedness between isolates was assessed using previously described genotyping methods: an informative-single nucleotide polymorphism MassARRAY iPLEX assay (iPLEX20SNP) and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay, with selected isolates being subjected to multilocus sequence typing (MLST).

Antibiotic perturbation of mixed-strain Pseudomonas aeruginosa infection in patients with cystic fibrosis.

Background: Pulmonary exacerbations in cystic fibrosis (CF) remain poorly understood and treatment is usually targeted at Pseudomonas aeruginosa. Within Australia a predominant shared P. aeruginosa strain (AUST-02) is associated with greater treatment needs.

A comparison of two informative SNP-based strategies for typing Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

Background: Molecular typing is integral for identifying Pseudomonas aeruginosa strains that may be shared between patients with cystic fibrosis (CF). We conducted a side-by-side comparison of two P. aeruginosa genotyping methods utilising informative-single nucleotide polymorphism (SNP) methods; one targeting 10 P.

High-throughput single-nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform.

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory.

Simple, rapid, and inexpensive detection of Neisseria gonorrhoeae resistance mechanisms using heat-denatured isolates and SYBR green-based real-time PCR.

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without the availability of appropriate public health strategies to combat this problem, gonorrhea could become untreatable. For this reason, surveillance for gonococcal antimicrobial resistance must be optimal both in terms of obtaining a representative sample of gonococcal isolates and in terms of having the appropriate tools to identify resistance.

Rapid genotyping of Pseudomonas aeruginosa isolates harboured by adult and paediatric patients with cystic fibrosis using repetitive-element-based PCR assays.

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome.

Detection and differentiation of Plasmodium species by polymerase chain reaction and colorimetric detection in blood samples of patients with suspected malaria.

Polymerase chain reaction (PCR) is now recognized as a sensitive and specific method for detecting Plasmodium species in blood. In this study, we tested 279 blood samples, from patients with suspected malaria, by a PCR assay utilizing species-specific colorimetric detection, and compared the results to light microscopy. Overall, both assays were in agreement for 270 of the 279 specimens.

A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples.

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus.

Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction.

Background: In recent years polymerase chain reaction (PCR) has proven to be a highly sensitive and specific method for the diagnosis of herpes simplex virus (HSV) infections. The advent of real-time HSV PCR protocols now enables rapid result turnaround times with minimal hands-on time.

Objectives: In this study, we developed a real-time duplex PCR assay (HSVgD-dPCR) comprising of HSV and internal control PCR reactions.

Detection of Neisseria Meningitidis in clinical samples by a duplex real-time PCR targeting the porA and ctrA genes.

Background: In recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused by Neisseria meningitidis.

Study Design: We developed and evaluated a N. meningitidis LightCycler real-time duplex PCR (NM-LCdPCR) capable of simultaneously detecting and distinguishing between two separate genes on the N.

Simultaneous detection and differentiation of human polyomaviruses JC and BK by a rapid and sensitive PCR-ELAHA assay and a survey of the JCV subtypes within an Australian population.

Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV.

Molecular assays for detection of human metapneumovirus.

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe.

Detection of human respiratory syncytial virus in respiratory samples by LightCycler reverse transcriptase PCR.

Laboratory diagnosis of human respiratory syncytial virus (hRSV) infections has traditionally been performed by virus isolation in cell culture and the direct fluorescent-antibody assay (DFA). Reverse transcriptase PCR (RT-PCR) is now recognized as a sensitive and specific alternative for detection of hRSV in respiratory samples. Using the LightCycler instrument, we developed a rapid RT-PCR assay for the detection of hRSV (the LC-RT-PCR) with a pair of hybridization probes that target the hRSV L gene.