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.

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). Ninety-five non-repetitive isolates that were found to be resistant to carbapenems were subjected to further investigation.Results/Key findings. The most prevalent carbapenemase-encoding gene, blaVIM-type, was found in 37/95 (39 %) isolates, while only 1 isolate (from UAE) was found to have blaIMP-type. None of the CRPA were found to have blaNDM-type or blaKPC-type. We found a total of 14 sequence type (ST) clusters, with 4 of these clusters being observed in more than 1 country. Several clusters belonged to the previously recognized internationally disseminated high-risk clones ST357, ST235, ST111, ST233 and ST654. We also found the less predominant ST316, ST308 and ST823 clones, and novel MLST types (ST2010, ST2011, ST2012 and ST2013), in our collection. CONCLUSION: Overall our data show that 'high-risk' CRPA clones are now detected in the region and highlight the need for strategies to limit further spread of such organisms, including enhanced surveillance, infection control precautions and further promotion of antibiotic stewardship programmes.

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. aeruginosa SNPs and using real-time polymerase chain reaction technology (HRM10SNP) and the other targeting 20 SNPs and based on the Sequenom MassARRAY platform (iPLEX20SNP). METHODS: An in-silico analysis of the 20 SNPs used for the iPLEX20SNP method was initially conducted using sequence type (ST) data on the P. aeruginosa PubMLST website. A total of 506 clinical isolates collected from patients attending 11 CF centres throughout Australia were then tested by both the HRM10SNP and iPLEX20SNP assays. Type-ability and discriminatory power of the methods, as well as their ability to identify commonly shared P. aeruginosa strains, were compared. RESULTS: The in-silico analyses showed that the 1401 STs available on the PubMLST website could be divided into 927 different 20-SNP profiles (D-value = 0.999), and that most STs of national or international importance in CF could be distinguished either individually or as belonging to closely related single- or double-locus variant groups. When applied to the 506 clinical isolates, the iPLEX20SNP provided better discrimination over the HRM10SNP method with 147 different 20-SNP and 92 different 10-SNP profiles observed, respectively. For detecting the three most commonly shared Australian P. aeruginosa strains AUST-01, AUST-02 and AUST-06, the two methods were in agreement for 80/81 (98.8%), 48/49 (97.8%) and 11/12 (91.7%) isolates, respectively. CONCLUSIONS: The iPLEX20SNP is a superior new method for broader SNP-based MLST-style investigations of P. aeruginosa. However, because of convenience and availability, the HRM10SNP method remains better suited for clinical microbiology laboratories that only utilise real-time PCR technology and where the main interest is detection of the most highly-prevalent P. aeruginosa CF strains within Australian clinics.

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. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96% agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24%) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

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. To aid with this surveillance, molecular tools are increasingly being used. In the present study, we investigated the use of a simple heat denaturation protocol for isolate DNA preparation combined with SYBR green-based real-time PCR for the identification of mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested by high-resolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics: a penA 345A insertion, ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porB1b, and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter. The results were compared to those obtained by DNA sequencing. Our HRM assays provided the accurate discrimination of heat-treated isolates in which the sequence types differed in GC content, including isolates with the penA 345A insertion and the ponA L421P and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the adenine deletion in the mtrR promoter. Heat-denatured DNA combined with SYBR green-based real-time PCR offers a simple, rapid, and inexpensive means of detecting gonococcal resistance mechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.

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. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38 % of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF.

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. P. vivax was detected in 131 (47.0%) specimens, P. falciparum in 64 (22.9%) specimens, P. ovale in 6 (2.1%) specimens, and P. malariae in 5 (1.8%) specimens. Both P. falciparum and P. vivax were detected in a further 10 (3.6%) specimens, and 54 (19.3%) specimens were negative by both assays. In the remaining nine specimens, microscopy either failed to detect the parasite or incorrectly identified the species present. In summary, the sensitivity, specificity and simplicity of the PCR assay makes it particularly suitable for use in a diagnostic laboratory.

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. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type 1 virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

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. STUDY DESIGN: Using the LightCycler, the HSVgD-dPCR targeted the HSV glycoprotein D gene and HSV typing was performed by melting curve analysis. The internal control PCR reaction targeted sequences of the DNA of the human endogenous retrovirus (ERV-3). In total, 300 swab specimens, from patients with suspected HSV infection, were tested by the HSVgD-dPCR assay. The results were then compared to the results obtained by another HSV LightCycler assay, which utilized published primer and probe sequences targeting the HSV DNA polymerase gene (Dpol-HSV-LCPCR). RESULTS: Overall, 91 (30.3%) specimens were positive and 204 (68.0%) specimens were negative for HSV by both LightCycler assays. In addition, four (1.3%) specimens were positive by Dpol-HSV-LCPCR and negative by HSVgD-dPCR, whereas one (0.3%) specimen was positive by HSVgD-dPCR and negative by Dpol-HSV-LCPCR. The presence of HSV in these five specimens was confirmed by conventional PCR. Melting curve analysis by the HSVgD-dPCR assay enabled all HSV positive specimens to be typed, whereas sequence variation prevented three HSV positive specimens from being typed by the Dpol-HSV-LCPCR. Using the ERV-3 PCR, 5% specimens were found to contain inhibitory substances. CONCLUSIONS: By developing the HSVgD-dPCR we have enhanced the diagnostic utility of real-time detection of HSV by incorporating an internal control reaction and by accurately typing a greater proportion of HSV positive specimens.

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. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol-PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory.

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. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

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. meningitidis genome. METHODS: The NM-LCdPCR was developed on the LightCycler platform (Roche Diagnostics, Castle Hill, NSW, Australia) and comprised two primer pairs and two hybridization probe sets, enabling the detection of both the porA and ctrA genes within the same reaction mix. To distinguish between the fluorescence emitted by each hybridization probe set, each downstream probe was labeled with a different fluorophore (either LC-Red640 or LC-Red705). The results obtained by the NM-LCdPCR were then compared with the results obtained by a mono-specific LightCycler assay targeting the porA gene only (porA-LCPCR). PATIENTS: One-hundred and forty-eight clinical samples from patients with suspected meningococcal infection were evaluated. RESULTS: The results of the NM-LCdPCR and porA-LCPCR gave 100% agreement; N. meningitidis DNA was detected in 25 samples whereas 123 samples were negative by both assays. The breakdown of the NM-LCdPCR results show that both genes were detected in 26 of the 28 positive samples. DISCUSSION: By targeting two separate N. meningitidis genes, the NM-LCdPCR has the potential to prevent the false-positive results which may arise from sequence variation. In addition, the ability to detect and discriminate between the two different N. meningitidis genes within the same reaction mix offers a rapid means for confirming the presence of N. meningitidis DNA in clinical samples, thereby reducing the need for subsequent confirmatory assays to be performed. CONCLUSIONS: The sensitivity and specificity of the NM-LCdPCR assay, combined with its ability to detect and discriminate both the N. meningitidis porA and ctrA genes, make it suitable for the diagnosis of N. meningitidis infections in the routine clinical laboratory.

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. In the present study, 190 nasopharyngeal aspirate samples from patients with clinically recognized respiratory tract infections were examined for hRSV. The results were then compared to the results obtained with a testing algorithm that combined DFA and a culture-augmented DFA (CA-DFA) assay developed in our laboratory. hRSV was detected in 77 (41%) specimens by LC-RT-PCR and in 75 (39%) specimens by the combination of DFA and CA-DFA. All specimens that were positive by the DFA and CA-DFA testing algorithm were positive by the LC-RT-PCR. The presence of hRSV RNA in the two additional LC-RT-PCR-positive specimens was confirmed by a conventional RT-PCR method that targets the hRSV N gene. The sensitivity of LC-RT-PCR was 50 PFU/ml; and this, together with its high specificity and rapid turnaround time, makes the LC-RT-PCR suitable for the detection of hRSV in clinical specimens.