Further questions regarding the role of mosaic penA sequences in conferring reduced susceptibility to ceftriaxone in Neisseria gonorrhoeae.
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Amino Acid Sequence↗
Biomedical subjects
David M Whiley
Publications and source records attributed to David M Whiley.
Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target.
AIMS: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. METHODS: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. RESULTS: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. CONCLUSIONS: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.
Diagnostic Techniques, Urological↗
Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge.
Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review.
Cross Reactions↗
A 5'-nuclease real-time reverse transcriptase-polymerase chain reaction assay for the detection of a broad range of influenza A subtypes, including H5N1.
A 5'-nuclease real-time reverse transcriptase-polymerase chain reaction assay was developed for the detection of influenza type A and was validated using a range of influenza A subtypes, including avian strains, and 126 nasopharyngeal aspirate samples. The results show the assay is suitable for screening for influenza A infections, particularly in regions where avian strains may be circulating.
Animals↗
Sequence variation can affect the performance of minor groove binder TaqMan probes in viral diagnostic assays.
A minor groove binder (MGB) TaqMan real-time PCR assay was developed for the detection of respiratory syncytial virus (RSV) in clinical specimens. Upon evaluation of the assay, notable differences were observed in the overall fluorescent response obtained from RSV positive specimens, with some linear amplification curves deviating only slightly from baseline fluorescence. Sequencing of the probes targets in these RSV strains revealed single base mismatches with the MGB TaqMan probe. Overall, these results highlight the usefulness of MGB TaqMan probes for the detection of mismatches, but suggest that MGB Taqman probes have limitations for routine screening for uncharacterised viral strains.
Base Pair Mismatch↗
A real-time PCR assay for the detection of Neisseria gonorrhoeae in genital and extragenital specimens.
A Neisseria gonorrhoeae LightCycler (NGpapLC) assay targeting the porA pseudogene was compared with bacterial culture for detection of N. gonorrhoeae in 636 clinical specimens (216 cervical, 185 urethral, 196 throat, and 39 rectal swab specimens). The specificity of the NGpapLC assay was further investigated by testing a bacterial reference panel comprising several Neisseria species. Overall, 19 (3.0%) specimens were positive and 613 (96.4%) specimens were negative by both methods. Four (0.6%) specimens were positive by the NGpapLC assay only. For the cervical and urethral swabs, the NGpapLC provided 100% sensitivity and 100% specificity compared with bacterial culture. Following discrepant analysis, the clinical sensitivity and specificity of the NGpapLC for throat and rectal swabs was also 100%. For the bacterial panel, only N. gonorrhoeae isolates provided positive results. The results show the NGpapLC assay is suitable for use on a range of clinical specimens and could improve detection of pharyngeal N. gonorrhoeae.
Culture Media↗
Sequence variation in primer targets affects the accuracy of viral quantitative PCR.
This study was designed to assess the effect of sequence mismatches between primers and their targets on viral quantitative PCR. Numerous primers were constructed incorporating various mismatches with a target sequence on the BKV T antigen gene. When using these primers in standard Taqman two-step cycling conditions, as few as two mismatches in a single primer increased cycle threshold values and significantly influenced the calculation of viral load.
BK Virus↗
Melting curve analysis using hybridisation probes: limitations in microbial molecular diagnostics.
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Animals↗
Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies.
AIMS: To develop and evaluate multiplex PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae, using real-time and conventional PCR detection methodologies. METHODS: Two real-time multiplex PCR assays, using nuclease (TaqMan-ABI7500) and hybridisation (LightCycler) probe formats, and a third assay using conventional PCR with solid-phase hybridisation and colour detection, were developed. The porA pseudogene was targeted for N. gonorrhoeae, and the major outer membrane protein gene for C. trachomatis. A total of 145 urogenital specimens were tested in all assays, and the results were compared with the Roche Cobas Amplicor assay. RESULTS: There was little difference in clinical sensitivity and specificity, result discrimination and test cost for the three in-house assays. Our results showed that competitive inhibition of the PCR occurred in some samples that were positive for both organisms. CONCLUSIONS: These results highlight the suitability and versatility of three multiplex PCR methods for the detection of C. trachomatis and N. gonorrhoeae.
Adolescent↗
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.
Adolescent↗
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.
Animals↗
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.
Adolescent↗
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.
Adolescent↗
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.
Bacterial Proteins↗
Co-detection and discrimination of six human herpesviruses by multiplex PCR-ELAHA.
BACKGROUND: Herpesviruses are a significant cause of human morbidity. Traditional approaches to the identification of these viruses require infectious or at least antigenic virus. Multiplex PCR (mPCR) is capable of simultaneously amplifying a range of targets from a single preparation of nucleic acids and when combined with a suitable detection assay, it is capable of discriminating each of the amplicons. OBJECTIVES: Several methods have been described in the literature, however, they lack one or more significant design features required to suitably control a routinely applied nucleic acid amplification assay. We aimed to design a multiplex herpesvirus PCR that could co-amplify eight human herpesvirus targets plus an internal control (IC) molecule in a single tube. STUDY DESIGN: Primers were designed to target the DNA polymerase genes of each of the human herpesviruses. Synthetic controls were developed to act as templates for the evaluation of assay sensitivity and specificity and for development of an in-house competitive quantitative PCR. Amplicon was discriminated using a simplified enzyme linked amplicon hybridisation assay (ELAHA). RESULTS AND CONCLUSIONS: For routine diagnostic use we reduced the number of herpesviral targets from 8 to 6 in order to maintain adequate clinical sensitivity. The ELAHA proved more sensitive than agarose gel electrophoresis. Additionally, 36 cytomegalovirus positive patients were examined with an in-house quantitative PCR-ELAHA which was developed to confirm that that the mPCR's co-detection limit of 10(2) copy of synthetic template per millilitre was relevant for use in detecting virus from clinical samples. The mPCR-ELAHA was then applied to the screening of 174 patient specimens resulting in a specificity of 98% and a sensitivity of 93%. This preliminary study demonstrated that the mPCR-ELAHA was a complete approach to the detection of herpesviruses from a range of clinical samples and disease states.
DNA Primers↗
Detection of Neisseria meningitidis by LightCycler PCR.
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DNA, Bacterial↗
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.
DNA Primers↗
A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler.
The detection of Neisseria gonorrhoeae by the polymerase chain reaction (PCR) is now recognized as a sensitive and specific method of diagnosing infection by the organism. In this Study 152 urine specimens were examined for N. gonorrhoeae by a real-time PCR method using the LightCycler platform and results were compared to an "in-house" PCR assay using an ELISA-based detection method. N. gonorrhoeae DNA was detected in 29 (19%) specimens by LightCycler PCR (LC-PCR) and in 31 (20%) specimens by the "in house" PCR method. The LightCycler assay proved to be specific and 94% sensitive when compared to the "in house" PCR method. These features combined with the rapid turn-around time for results makes the LC-PCR particularly suitable for the detection of N. gonorrhoeae in a routine clinical laboratory.
DNA, Bacterial↗