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![Sequence alignment of the primer-probe sets with nonspecific viruses. (A) Pairwise alignment of nucleotides between the SW (j) (7) primer-probe set and the HA gene of a swine H1N2 strain (A/swine/Hainan/1/2005 [GenBank accession number EF556203]). (B) Pairwise alignment of nucleotides between the SW (c) (31) primer-probe set and the HA gene of a human H1N1 strain (A/Puerto Rico/8/1934 [GenBank accession number EF556203]).](publication/49805784/figure/fig1/AS:11431281221175157@1706709260051/Sequence-alignment-of-the-primer-probe-sets-with-nonspecific-viruses-A-Pairwise.jpeg)
Sequence alignment of the primer-probe sets with nonspecific viruses. (A) Pairwise alignment of nucleotides between the SW (j) (7) primer-probe set and the HA gene of a swine H1N2 strain (A/swine/Hainan/1/2005 [GenBank accession number EF556203]). (B) Pairwise alignment of nucleotides between the SW (c) (31) primer-probe set and the HA gene of a human H1N1 strain (A/Puerto Rico/8/1934 [GenBank accession number EF556203]).
Source publication
Real-time reverse transcriptase PCR (rRT-PCR) assays have greatly contributed to the detection, control, and prevention of the pandemic influenza A/H1N1 2009 virus. To improve the rRT-PCR assays for detection of pandemic influenza A/H1N1 2009 virus, we evaluated the sensitivity, specificity, and performance of 12 rRT-PCR primer-probe sets [SW (a) t...
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Citations
... Moreover, excessive antibiotic prescribing can lead to inadequate treatment [3]. The 2009-H1N1 pandemic serves as an example, highlighting the importance of accurate viral species diagnosis through PCR testing to determine appropriate treatment options [4]. Currently, multiplex PCR tests are considered the most valuable diagnostic tool for VRTIs [5,6]. ...
Introduction:
Prior to the emergence of COVID-19, when influenza was the predominant cause of viral respiratory tract infections (VRTIs), this study aimed to analyze the distinct biological abnormalities associated with influenza in outpatient settings.
Methods:
A multicenter retrospective study was conducted among outpatients, with the majority seeking consultation at the emergency department, who tested positive for VRTIs using RT-PCR between 2016 and 2018. Patient characteristics were compared between influenza (A and B types) and non-influenza viruses, and predictors of influenza were identified using two different models focusing on absolute eosinopenia (0/mm3) and lymphocyte count <800/mm3.
Results:
Among 590 VRTIs, 116 (19.7%) were identified as outpatients, including 88 cases of influenza. Multivariable logistic regression analysis revealed the following predictors of influenza: in the first model, winter season (adjusted odds ratio [aOR] 7.1, 95% confidence interval [CI] 1.12-45.08) and absolute eosinopenia (aOR 6.16, 95% CI 1.14-33.24); in the second model, winter season (aOR 9.08, 95% CI 1.49-55.40) and lymphocyte count <800/mm3 (aOR 7.37, 95% CI 1.86-29.20). Absolute eosinopenia exhibited the highest specificity and positive predictive value (92% and 92.3%, respectively).
Conclusion:
During the winter season, specific biological abnormalities can aid physicians in identifying influenza cases and guide the appropriate use of antiviral therapy when rapid molecular tests are not readily available.
... [9] and Plasmodium spp. [10] Additionally, real-time RT-PCR (qRT-PCR) was made for the detection of Zika virus [11], Chikungunya virus [12], influenza B virus and influenza A virus [13]. All the tests were made in duplicate, with a negative control from the extraction and a negative control from the RT-PCR process. ...
... All the tests were made in duplicate, with a negative control from the extraction and a negative control from the RT-PCR process. Primers used for these experiments were designed and standardized elsewhere [9][10][11][12][13]. Influenza A infection was confirmed in the lung tissue, in two different experiments, with an average Cycle Threshold CT of 35 (cut-off point of 38.5), and the negative controls showed no amplification curve. ...
Dengue is still an important cause of disease and mortality in tropical countries, as is influenza A virus, which is also a cause of epidemics all over the globe. In this article, we present the case of a 31-year-old woman who was in her second trimester of pregnancy and presented with severe dengue with hematological and neurological complications, and premature labor. She was misdiagnosed with bacterial infection and received antibiotic treatment with no improvement of the clinical manifestations and previous to death, she was diagnosed with dengue infection. She died from cardiorespiratory arrest. In the postmortem evaluation, influenza A co-infection was confirmed and characterization of the tissue damage and immune response in lung, liver, kidney, heart, spleen, and brain was determined, finding a severe inflammatory response in lung with T cells and macrophages infiltrating the tissue. This case report highlights the risks of accepting a single diagnosis, especially in endemic countries to multiple tropical diseases, which can lead to delay in appropriate treatment that could reduce morbidity and mortality.
... The primers and probes targeted the conserved regions of the HA genes of the pandemic viruses. In addition, many assays that were based on the qRT-PCR technology or the WHO-recommended systems have been developed and studied Gunson R, et al., 2010;Jiang T, et al., 2010;Klungthong C, et al., 2010;Lorusso A, et al., 2010;Pabbaraju K, et al., 2009;Schulze M, et al., 2010;Yang Y, et al., 2011). These assays using the oligonucleotides have greatly facilitated the detection and monitoring of the infections associated with the pandemic H1N1/2009 virus. ...
... For example, previous analyses revealed 1-2 mismatches in the probe region, 1 mismatch in the forward primer, and 1-3 mismatches in the reverse primer region (Fig. 1). These mismatches potentially affect the properties of the WHO qRT-PCR assay such as the sensitivity and specificity (Klungthong C, et al., 2010;Yang Y, et al., 2011). Our analyses of the WHO primers that were used as the reference method in our study suggest that one mismatch was found in the probe region at the 16th nucleotide (G to A), and three mismatches in the reverse primer region at the 12th nucleotide (A to G), the 15th nucleotide (G to A), and the 19th nucleotide (A to G), respectively (Fig. 1). ...
A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.
... Yang et al. compared the performance of 12 rRT-PCR primer-probe sets designed for detecting the hemagglutinin (HA) or the neuraminidase (NA) gene of the pandemic influenza A/H1N1 2009 virus, using the primer-probe set developed at the CDC as reference. They found that although all primer-probe sets had specificity levels as high as 98.4 % to 100 %, some of the primer-probe sets had better specificity, sensitivity, and amplification efficiency than others, and that a combination of primer-probe sets targeted to the HA and NA genes had higher detection sensitivity than those targeting HA or NA individually [37]. In another study, Lam et al. showed that although rRT-PCR assays can be 10-fold more sensitive than cRT-PCR, newly developed cRT-PCR assays targeting the HA gene are a reliable alternative for laboratories where a rRT-PCR machine is not available [38]. ...
A potentially fatal complication of influenza infection is the development of pneumonia, caused either directly by the influenza virus, or by secondary bacterial infection. Pneumonia related to the 2009 influenza A pandemic was found to be underestimated by commonly used pneumonia severity scores in many cases, and to be rapidly progressive, leading to respiratory failure. Confirmation of etiology by laboratory testing is warranted in such cases. Rapid antigen and immunofluorescence testing are useful screening tests, but have limited sensitivity. Confirmation of pandemic H1N1 influenza A infection can only be made by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) or viral culture. The most effective preventive measure is annual influenza vaccination in selected individuals. Decisions to administer antiviral medications for influenza treatment or chemoprophylaxis should be based upon clinical and epidemiological factors, and should not be delayed by confirmatory laboratory testing results. Neuraminidase inhibitors (NI) are the agents of choice.
The objectives of this study were to develop a user-friendly, gel element microarray test for influenza virus detection, subtyping, and neuraminidase inhibitor resistance detection, assess the performance characteristics of the assay, and perform a clinical evaluation on retrospective nasopharyngeal swab specimens. A streamlined microarray workflow enabled a single user to run up to 24 tests in an 8hour shift. The most sensitive components of the test were the primers and probes targeting the A/H1pdm09 HA gene with an analytical limit of detection (LoD) <100 gene copies (gc) per reaction. LoDs for all targets in nasopharyngeal swab samples were ≤ 1000 gc, with the exception of one target in the seasonal A/H1N1 subtype. Seasonal H275Y variants were detectable in a mixed population when present at>5% with wild type virus, while the 2009 pandemic H1N1 H275Y variant was detectable at ≤ 1% in a mixture with pandemic wild type virus. Influenza typing and sub typing results concurred with those obtained with real-time RT-PCR assays on more than 97% of the samples tested. The results demonstrate that a large panel of single-plex, real-time RT-PCR tests can be translated to an easy-to-use, sensitive, and specific microarray test for potential diagnostic use.
Active surveillance and diagnosis of the influenza pandemic (H1N1) 2009 (pH1N1) have played a critical role in the effective control and prevention of the pandemic in China. Although several commercially available real-time PCR kits for pH1N1 virus have been used in diagnostic laboratories in Beijing, little has been known about the performance of these kits for detecting pH1N1 virus. In this study, the performance of two commercial real-time PCR kits in Beijing was evaluated. Analysis of clinical samples showed that the positive detection rate for the AgPath-ID™ kit (38.2%) was significantly higher than that for the Da An H1N1 kit (30.0%) (McNemar's chi-square test, P=0.000). The limit of detection (LOD) of the AgPath-ID™ kit was 10(2), 10(2), and 10(3) copies/reaction for the Influenza A (set 1), H1N1 Influenza A (set 2) and H1N1 Influenza A Sub H1 (set 3) genes, respectively, whereas the LOD of the Da An kit was 10(3) copies/reaction for both H1 and N1 genes. Although the AgPath-ID™ kit exhibited a significantly higher detection rate for pH1N1 than the Da An kit, cross-reactivity to A/PR8/34 was found for the AgPath-ID™ kit for H1N1 Influenza A (set 2).
