Kevin Jacob

Molecular Assays for Detection of Human Metapneumovirus

ABSTRACT 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.

Presence of the newly discovered human polyomaviruses KI and WU in Australian patients with acute respiratory tract infection

Abstract Background Currently, the role of the novel human polyomaviruses, KI (KIV) and WU (WUV) as agents of human disease remains uncertain. Objectives We sought to determine the prevalence of these viruses and their rate of co-detection with other viral respiratory pathogens, in an Australian population. Study design Polymerase chain reaction assays previously described were used to examine the presence of KIV and WUV in 2866 respiratory specimens collected from January to December 2003 from Australian patients with acute respiratory infections. Results KIV and WUV were present in our population with an annual prevalence of 2.6% and 4.5%, respectively. There was no apparent seasonal variation for KIV, but a predominance of infection was detected during late winter to early summer for WUV. The level of co-infection of KIV or WUV with other respiratory viruses was 74.7% and 79.7%, respectively. Both viruses were absent from urine and blood specimens collected from a variety of patient sources. Conclusions KIV and WUV circulate annually in the Australian population. Although there is a strong association with the respiratory tract, more comprehensive studies are required to prove these viruses are agents causing respiratory disease.

Genetic Diversity of Human Metapneumovirus over 4 Consecutive Years in Australia

The molecular epidemiologic profile of human metapneumovirus (hMPV) infection has likely been skewed toward certain genetic subtypes because of assay-design issues, and no comprehensive studies have been conducted to date. Here, reverse-transcription polymerase chain reaction was used to screen 10,319 specimens from patients presenting to hospitals with suspected respiratory tract infections during 2001-2004. After analysis of 727 Australian hMPV strains, 640 were assigned to 1 of 4 previously described subtypes. hMPV was the most common pathogen detected, and subtype B1 was the most common lineage. Concurrent, annual circulation of all 4 hMPV subtypes in our study population was common, with a single, usually different hMPV subtype predominating in each year.

Exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer.

Several different viruses have been proposed to play a role in breast carcinogenesis. The aim of this study was to investigate the prevalence of a subset of viruses in breast cancer tissue. We investigated the prevalence of 12 DNA viruses: EBV and CMV from the Herpesviridae family and SV40, BKV, JCV, MCV, WUV, KIV, LPV, HPyV6, HPyV7, and TSV from the Polyomaviridae family in 54 fresh frozen breast tumour specimens. Relevant clinical data and basic lifestyle data were available for all patients. The tissue samples were DNA extracted and real-time PCR assays were used for viral detection. The highest prevalence, 10% (5/54), was found for EBV. MCV, HPyV6, and HPyV7 were detected in single patient samples (2% each), while WUV, KIV, JCV, BKV, LPV, SV40, TSV and CMV were not detected in the 54 breast cancer specimens analysed here. Further investigations are needed to elucidate the potential role of viruses, and particularly EBV, in breast carcinogenesis.

Screening for H7N9 influenza A by matrix gene-based real-time reverse-transcription PCR.

Rapid detection of novel influenza A strains, including H7N9, is pivotal to ensuring appropriate public health-based responses and real-time reverse-transcription polymerase chain reaction (RT-PCR) methods are used typically for this purpose. However, the utility of such methods can be undermined by ongoing sequence variations, particularly when targeting the variable influenza A haemagglutinin (HA) and neuraminidase (NA) genes. This may often be a source of frustration for clinical laboratories that are implementing methods in preparation for potential pandemics as primers and probe targets may need to be checked regularly and updated. In this study, screening methods were developed for H7N9 influenza A strains based on the highly-conserved influenza A matrix gene. Three assays were developed and evaluated in parallel, and included two methods which simply involved inclusion of a single H7N9 probe sequence into an established influenza A and B multiplex RT-PCR (FluAB-PCR). The detection limits of the methods were compared using ten-fold dilutions of H7N9 RNA, and the specificity of the methods were tested using 32 influenza A RT-PCR-positive samples and a panel of 18 influenza A isolates, including representives of seasonal H3N2, seasonal H1N1, pandemic H1N1, H5N1, H5N3, H9N2 and H7N7. The detection limits of the three methods were the same, and no cross-reactions were observed with sH3N2, sH1N1, pH1N1 or H5N1. However, cross-reactions were observed with H5N3, H9N2 and H7N7. Overall, the results show that the methods are useful for front-line screening for H7N9.

Improved detection of genetic markers of antimicrobial resistance by hybridization probe-based melting curve analysis using primers to mask proximal mutations: Examples include the influenza H275Y substitution

OBJECTIVES Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, the performance of these methods can be undermined by sequence variation in the regions flanking the codon of interest. This is a problem encountered more broadly in microbial diagnostics. METHODS In this study, we developed a modification of hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations in the sequence targets of hybridization probes, so as to limit the potential for sequence variation to interfere with typing. The approach was applied to the H275Y alteration of the influenza A (H1N1) 2009 strain, as well as a Neisseria gonorrhoeae mutation associated with antimicrobial resistance. Assay performances were assessed using influenza A and N. gonorrhoeae strains characterized by DNA sequencing. RESULTS The modified hybridization probe-based approach proved successful in limiting the effects of proximal mutations, with the results of melting curve analyses being 100% consistent with the results of DNA sequencing for all influenza A and N. gonorrhoeae strains tested. Notably, these included influenza A and N. gonorrhoeae strains exhibiting additional mutations in hybridization probe targets. Of particular interest was that the H275Y assay correctly typed influenza A strains harbouring a T822C nucleotide substitution, previously shown to interfere with H275Y typing methods. CONCLUSIONS Overall our modified hybridization probe-based approach provides a simple means of circumventing problems caused by sequence variation, and offers improved detection of the influenza A H275Y alteration and potentially other resistance mechanisms.