Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the "gold standard" for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10(-6) dilution of 4 x 10(6) 50% tissue culture infective doses/ml when 5 microl was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was
Six-hundred and twenty-six stool specimens collected from children with diarrhea over a 12-month period were tested for rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly-conserved region of non-structural protein 3 (NSP3) in rotavirus genome was amplified by a single-step RT-PCR protocol in a closed-tube system. Rotavirus was detected in 123 samples (20%) with the real time RT-PCR assay, 113 samples (18%) with the nested-PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT-PCR with conventional RT-PCR and conventional nested PCR assays. Real time RT-PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT-PCR assay is about half the time required for the conventional nested-PCR. The real time RT-PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross-contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G(1) was the predominant type (89%), followed by G(2) (10%), and G(4) (1%). No rotavirus of G(3), G(8), and G(9) types were found. The peak season for rotavirus infection was January to May in northern Alberta.
Conventional reverse transcription-polymerase chain reaction (Con RT-PCR) assay to detect norovirus is a complex multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the results.
To develop and evaluate a multiplex real time RT-PCR (Mrt RT-PCR) assay that detect and quantify norovirus GI and GII with a single amplification and detection step.
The primers and TaqMan probes for the Mrt RT-PCR were selected from the ORF1-ORF2 junction region. A total of 97 stools from 41 gastroenteritis outbreaks and 726 stools from children with sporadic diarrhoea were used for this study.
For the 97 outbreak samples, norovirus were detected in 61 of the 69 previously tested positive and 11 of the 28 previously tested negative samples. Eight samples that tested positive for GII by Con RT-PCR but negative by the Mrt RT-PCR also tested negative by a Light Cycler RT-PCR assay. Eighty-two GII and two GI were detected in the 726 sporadic samples. Random primers were more sensitive than specific primers in the cDNA synthesis. The two-step assay using the random primers in RT reaction was 100 times more sensitive than the one-step assay. The Mrt RT-PCR had the same sensitivity as that using two real time RT-PCR for separate detection of GI and GII. A wide dynamic range was obtained with the two-step assay, detecting from 3000 to 3x10(11) of copies RNA/g stool. Very good precision was observed with no cross-reaction with other enteric viruses. The new assay is able to detect both GI and GII in one reaction and brings a cost reduction of approximately 40% compared to separate reactions for GI and GII.
The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test.
Although nucleic acid amplification testing (NAAT) for West Nile virus (WNV) is useful in screening blood donors, such methods have not been studied in symptomatic patients. For diagnosis of WNV infection, 1.0 mL of plasma was tested by NAAT, and WNV-specific immunoglobulin M was assayed. Of 276 WNV cases, 191 were tested by both serology and NAAT. Of these, 86 (45.0%), 111 (58.1%), and 180 (94.2%) were detected by NAAT, serology, and combined NAAT and serology, respectively. NAAT-based screening was most useful within 8 days of the onset of symptoms. Viremia is common in early symptomatic WNV infection, and NAAT enhances diagnostic yield.
Rapid HIV testing has the potential to improve medical care and reduce the transmission of infection. In this study, rapid HIV testing was performed on serum samples in acute care settings in five hospitals from urban and rural regions using the INSTI™ HIV-1/HIV-2 Rapid Antibody Test (bioLytical Laboratories, Richmond, British Columbia). Parallel standard HIV antibody tests were performed at the provincial reference laboratory. Patient demographics, indication for testing and risk behaviours were collected. From April 30, 2007 and November 23, 2009, 1708 individuals were tested: 875 (50.3%) tests in pregnant women, 730 (42%) in source individuals in blood and body fluid exposures and 119 (5.8%) in acutely ill persons. Twenty-five (1.4%) samples were reactive by rapid HIV testing, of which 13 were reactive previously and 1 was a false reactive. Sensitivity of the rapid HIV test compared to standard HIV testing was 100%, specificity was 99.9%, the positive predictive value was 96% and the negative predictive value was 100%. The median time from specimen collection to availability of the rapid HIV result varied by site and ranged from 54 min to 1h 42 min. In this study, the INSTI™ HIV-1 Rapid Antibody test identified reactive and non-reactive samples with similar accuracy to the conventional testing algorithm and provided a reliable way to perform rapid HIV testing in acute care settings.
Quantitative HIV RNA viral load (QVL) assays (Roche Diagnostics) were sensitive and specific when used to diagnose HIV infection in (i) HIV-exposed infants (sensitivity of 100% [63.1 to 100%] and specificity of 100% [97.9 to 100%]) and (ii) suspected acute HIV infection patients with a negative/indeterminate Western blot (sensitivity of 97.6% [91.6 to 99.7%] and specificity of 100% [96.1 to 100%]). No false-positive QVL results were identified.
Cites: J Acquir Immune Defic Syndr. 2000 Aug 15;24(5):401-711035610
Cites: AIDS. 2000 Oct 20;14(15):2333-911089621
Cites: Ann Intern Med. 2001 Jan 2;134(1):25-911187417
Cites: AIDS. 2002 May 24;16(8):1119-2912004270
Cites: AIDS. 2002 May 24;16(8):1189-9112004281
Cites: J Acquir Immune Defic Syndr. 2003 Feb 1;32(2):192-512571529