Correlation between prevalence of selected enteropathogens and diarrhea in children: a case-control study in China

Abstract Background The application of nucleic acid detection methods improves the ability of laboratories to detect diarrheal pathogens, but it also poses new challenges for the interpretation of the results. It is often difficult to attribute a diarrhea episode to the detected pathogens. Here we investigated the prevalence of 19 enteropathogens among diarrheal and non-diarrheal children and provided support for understanding the clinical significance of the pathogens. Methods A total of 710 fecal samples were collected from children under 5 years old in two different regions of China from May 2017 to March 2018, comprising 383 mild to moderate diarrheal cases and 327 non-diarrheal controls. The enteropathogens were detected using real-time polymerase chain reaction (PCR) or real-time reverse transcription PCR (RT-PCR). Results Enteropathogens were detected in 68.9% of the cases and 41.3% of the controls. Rotavirus A (adjusted OR [aOR], 9.91; 95% CI, 4.99–19.67), norovirus GI and GII (aOR, 3.82; 95% CI, 2.12–6.89), and Campylobacter jejuni (aOR, 20.12; 95% CI, 2.57–157.38) were significantly associated with diarrhea (p < 0.05). Adenovirus, norovirus GII, rotavirus A, and enteroaggregative Escherichia coli (pCVD432) gave lower cycle threshold (Ct) values in the cases than in the controls (p < 0.05). Rotavirus A and norovirus GII were associated with diarrhea when the Ct value were ≤ 30 and ≤ 25, respectively. Conclusions The types and loads of enteropathogens are likely to influence the interpretation of the clinical significance of positive results.

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Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

PLoS ONE ◽

10.1371/journal.pone.0250942 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0250942

Author(s):

Huseyin Tombuloglu ◽

Hussein Sabit ◽

Ebtesam Al-Suhaimi ◽

Reem Al Jindan ◽

Khaled R. Alkharsah

Keyword(s):

Real Time ◽

Single Gene ◽

False Negative ◽

Diagnostic System ◽

Virus Detection ◽

Current Method ◽

Pcr Method ◽

Polymerase Chain ◽

Positive Results ◽

Good Agreement

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.

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Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

Veterinary Sciences ◽

10.3390/vetsci6010002 ◽

2018 ◽

Vol 6 (1) ◽

pp. 2 ◽

Cited By ~ 2

Author(s):

David De la Torre ◽

Claudete Astolfi-Ferreira ◽

Ruy Chacon ◽

Antonio Piantino Ferreira

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Sybr Green ◽

Molecular Techniques ◽

Economic Losses ◽

Rt Pcr ◽

Chain Reaction ◽

Rotavirus A ◽

Avian Nephritis Virus ◽

Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

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A Clinical Epidemiology and Molecular Attribution Evaluation of Adenoviruses in Pediatric Acute Gastroenteritis: a Case-Control Study

Journal of Clinical Microbiology ◽

10.1128/jcm.02287-20 ◽

2020 ◽

Vol 59 (1) ◽

pp. e02287-20

Author(s):

Kanti Pabbaraju ◽

Raymond Tellier ◽

Xiao-Li Pang ◽

Jianling Xie ◽

Bonita E. Lee ◽

...

Keyword(s):

Real Time ◽

Clinical Epidemiology ◽

Human Adenovirus ◽

Asymptomatic Infection ◽

Case Control ◽

Real Time Quantitative Pcr ◽

Reverse Transcription Pcr ◽

Stool Samples ◽

Control Study

ABSTRACTThe objective of this study was to characterize the etiological role of human adenovirus (HAdV) serotypes in pediatric gastroenteritis. Using a case-control design, we compared the frequencies of HAdV serotypes between children with ≥3 episodes of vomiting or diarrhea within 24 h and <7 days of symptoms (i.e., cases) and those with no infectious symptoms (i.e., controls). Stool samples and/or rectal swabs underwent molecular serotyping with cycle threshold (Ct) values provided by multiplex real-time reverse transcription-PCR testing. Cases without respiratory symptoms were analyzed to calculate the proportion of disease attributed to individual HAdV serotypes (i.e., attributable fraction). Between December 2014 and August 2018, adenoviruses were detected in 18.8% (629/3,347) of cases and 7.2% (97/1,355) of controls, a difference of 11.6% (95% confidence interval [CI], 9.6%, 13.5%). In 96% (95% CI, 92 to 98%) of HAdV F40/41 detections, the symptoms could be attributed to the identified serotype; when serotypes C1, C2, C5, and C6 were detected, they were responsible for symptoms in 52% (95% CI, 12 to 73%). Ct values were lower among cases than among controls (P < 0.001). HAdV F40/41, C2, and C1 accounted for 59.7% (279/467), 17.6% (82/467), and 12.0% (56/467) of all typed cases, respectively. Among cases, Ct values were lower for F40/41 serotypes than for non-F40/41 serotypes (P < 0.001). HAdV F40/41 serotypes account for the majority of HAdV-positive gastroenteritis cases, and when detected, disease is almost always attributed to infection with these pathogens. Non-F40/41 HAdV species have a higher frequency of asymptomatic infection and may not necessarily explain gastroenteritis symptoms. Real-time quantitative PCR may be useful in differentiating asymptomatic shedding from active infection.

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Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870501700609 ◽

2005 ◽

Vol 17 (6) ◽

pp. 574-578 ◽

Cited By ~ 41

Author(s):

Ming Y. Deng ◽

He Wang ◽

Gordon B. Ward ◽

Tammy R. Beckham ◽

Thomas S. McKenna

Keyword(s):

Real Time ◽

Classical Swine Fever Virus ◽

Rna Extraction ◽

Classical Swine Fever ◽

Extraction Methods ◽

Rt Pcr ◽

Total Rna ◽

Reverse Transcription Pcr ◽

Rna Extraction Methods ◽

Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

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Expression and clinical significance of syndecan-1 mRNA and HPA-1 mRNA in colorectal cancer detected with real-time fluorescent quantitative polymerase chain reaction

Chinese Journal of Cancer ◽

10.5732/cjc.009.10502 ◽

2010 ◽

Vol 29 (3) ◽

pp. 288-293 ◽

Cited By ~ 3

Author(s):

He Wang ◽

Jun-Li Si ◽

Xiu-Zhen Zhang ◽

Yu-Qin Qi ◽

Zi-Yu Niu ◽

...

Keyword(s):

Colorectal Cancer ◽

Polymerase Chain Reaction ◽

Real Time ◽

Clinical Significance ◽

Quantitative Polymerase Chain Reaction ◽

Chain Reaction ◽

Polymerase Chain

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IL-6 gene rs12700386 Polymorphism is Associated with Increased Risk of Knee Osteoarthritis Development in Chinese Han Population: A Case-Control Study

10.21203/rs.3.rs-32756/v1 ◽

2020 ◽

Author(s):

Hui Yang ◽

Xindie Zhou ◽

Dongmei Xu ◽

Gang Chen

Keyword(s):

Knee Osteoarthritis ◽

Case Control Study ◽

Case Control ◽

Chinese Han ◽

Knee Oa ◽

Reverse Transcription Pcr ◽

Increased Risk ◽

Pcr Rflp ◽

Polymerase Chain ◽

Control Study

Abstract Background: There is an association between Interleukin-6 (IL-6) polymorphism and knee osteoarthritis (OA) risk. The case-control study aims at exploring how IL-6 rs12700386 polymorphism affects the knee OA risk in Chinese Han individuals.Methods: We extracted the DNA from 763 participants, thereinto, 352 were OA patients and 411 were healthy controls. The polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assisted in genotyping the IL-6 gene polymorphism. The relative expression exhibited by IL-6 in blood samples of knee OA patients was determined via a quantitative reverse transcription PCR (qRT-PCR). Results: We found that IL-6 rs12700386 enhanced the knee OA susceptibility. Based on a subgroup analysis, the loci magnified the knee OA risk in smokers, drinkers, and subjects ≥ 55 years old or with BMI ≥ 25 kg/m2. The combination of smoking and drinking and rs12700386 genotype led to an increase in the knee OA risk, indicating an underlying interaction between gene and environment. Additionally, the rs12700386 was found to be related to increased IL-6 gene levels. Conclusion: These data indicate that rs12700386 polymorphism of IL-6 gene led to an increase in the knee OA risk specific to Chinese Han individuals.

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The Effect of Sample Site, Illness Duration, and the Presence of Pneumonia on the Detection of SARS-CoV-2 by Real-time Reverse Transcription PCR

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa335 ◽

2020 ◽

Vol 7 (9) ◽

Cited By ~ 4

Author(s):

Stephanie Sutjipto ◽

Pei Hua Lee ◽

Jun Yang Tay ◽

Shehara M Mendis ◽

Mohammad Yazid Abdad ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Sampling Site ◽

Upper Respiratory Tract ◽

Clinical Sensitivity ◽

Illness Onset ◽

Reverse Transcription Pcr ◽

Upper Respiratory ◽

Polymerase Chain ◽

Respiratory Specimens

Abstract Background The performance of real-time reverse transcription polymerase chain reaction (rRT-PCR) for SARS-CoV-2 varies with sampling site(s), illness stage, and infection site. Methods Unilateral nasopharyngeal, nasal midturbinate, throat swabs, and saliva were simultaneously sampled for SARS-CoV-2 rRT-PCR from suspected or confirmed cases of COVID-19. True positives were defined as patients with at least 1 SARS-CoV-2 detected by rRT-PCR from any site on the evaluation day or at any time point thereafter, until discharge. Diagnostic performance was assessed and extrapolated for site combinations. Results We evaluated 105 patients; 73 had active SARS-CoV-2 infection. Overall, nasopharyngeal specimens had the highest clinical sensitivity at 85%, followed by throat, 80%, midturbinate, 62%, and saliva, 38%–52%. Clinical sensitivity for nasopharyngeal, throat, midturbinate, and saliva was 95%, 88%, 72%, and 44%–56%, respectively, if taken ≤7 days from onset of illness, and 70%, 67%, 47%, 28%–44% if >7 days of illness. Comparing patients with upper respiratory tract infection (URTI) vs pneumonia, clinical sensitivity for nasopharyngeal, throat, midturbinate, and saliva was 92% vs 70%, 88% vs 61%, 70% vs 44%, 43%–54% vs 26%–45%, respectively. A combination of nasopharyngeal plus throat or midturbinate plus throat specimen afforded overall clinical sensitivities of 89%–92%; this rose to 96% for persons with URTI and 98% for persons ≤7 days from illness onset. Conclusions Nasopharyngeal specimens, followed by throat specimens, offer the highest clinical sensitivity for COVID-19 diagnosis in early illness. Clinical sensitivity improves and is similar when either midturbinate or nasopharyngeal specimens are combined with throat specimens. Upper respiratory specimens perform poorly if taken after the first week of illness or if there is pneumonia.

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A Single Nucleotide Polymorphism at the TaqMan Probe-Binding Site Impedes Real-Time Reverse Transcription-PCR-Based Detection of Norovirus GII.4 Sydney

Journal of Clinical Microbiology ◽

10.1128/jcm.01044-15 ◽

2015 ◽

Vol 53 (10) ◽

pp. 3353-3354 ◽

Cited By ~ 4

Author(s):

Ran Zhuo ◽

Maria Eloisa Hasing ◽

Xiaoli Pang ◽

Keyword(s):

Single Nucleotide Polymorphism ◽

Binding Site ◽

Real Time ◽

Reverse Transcription ◽

Taqman Probe ◽

Nucleotide Polymorphism ◽

Genotype 4 ◽

Single Nucleotide ◽

Reverse Transcription Pcr ◽

Norovirus Gii

A single nucleotide polymorphism (SNP) in the TaqMan probe-binding site resulted in decreased sensitivity of real-time reverse transcription-PCR (RT-rtPCR) for detection of norovirus genogroup II genotype 4 (GII.4) Sydney. A new degenerate probe was designed that improved the sensitivity of the detection while not interfering with the detection of other GII and GI strains.

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