A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Clinical Application of Matrix-Assisted Laser-Resolved Ionization Time-Of-Flight Mass Spectrometry for Rapid Detection of Blood-Borne Pathogens.

Objective: To evaluate the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical pathogenic microorganisms. Methods: Blood culture-positive specimens were collected from inpatients in our hospital from March to December 2022 and identified using VITEK 2XL (biochemical), VITEK MS (colony), VITEK MS (bacterial membrane) and VITEK MS (separating gel) methods, respectively, to compare the compliance rate and identification values of the four methods. Results: A total of 280 strains were included in the analysis, including 155 (55.36%) Gram-negative and 125 (44.64%) Gram-positive strains. 279 (99.64%) of the 280 strains were identified by VITEK 2XL (biochemical), including 154 (99.35%) Gram-negative and 125 (100%) Gram-positive strains. VITEK MS (colony) identified 278 (99.29%) strains, including 153 (98.71%) Gram-negative and 125 (100%) Gram-positive. 261 (93.21%) strains were identified in VITEK MS (bacterial membrane), including 148 (95.48%) Gram-negative and 113 (90.40%) Gram-positive strains. VITEK MS (separating gel) identified 232 (82.86%) strains, including 136 (87.74%) Gram-negative and 96 (76.80%) Gram-positive strains. Conclusion: MALDI-TOF MS findings are highly consistent with traditional culture identification methods in terms of identification accuracy, and the VITEK MS (bacterial membrane) and VITEK MS (separating gel) identification methods significantly reduce the turnaround time for identification in the laboratory.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay.

Human immunodeficiency virus 1 (HIV-1) attacks the immune system, making people susceptible to various diseases, thus increasing their risk of death. Comprehensive detection of major HIV-1 strains circulating in China is vital for effective HIV-1 infection prevention and treatment. HIV-1 nucleic acid detection is considered effective for HIV-1 diagnosis since traditional immunological testing may fail to detect HIV-1 infection during the window period. This work demonstrates a one-pot two-stage amplification assay (RT-RAP), a combination of reverse transcription recombinase (RT- RAA), and quantitative real-time polymerase chain reaction (qRT-PCR). The turn-around time of the assay is only 50 min and can be performed with commonly available laboratory equipment, the qPCR devices. The RT-RAP assay could detect approximately 5 and 14 copies/reaction of HIV-1 DNA and RNA using recombinant plasmids and standard reference strains, respectively. Additionally, we found that the clinical performance of RT-RAP (detected 169 samples out of 170 specimens) was consistent with that of qRT-PCR. The sensitivity and specificity of RT-RAP were 100.00% (99/99) and 98.59% (70/71), respectively, while its positive and negative predictive values were 99.00% (99/100) and 100.00% (70/70), respectively. The total coincidence rate of the RT-RAP was 99.41% (169/170), with a kappa value of 0.988 (p < 0.05). We demonstrated that RT-RAP could rapidly detect the common HIV-1 subtypes commonly circulating in China with comparable sensitivity and specificity to qRT-PCR.

Simultaneous detection of 9 respiratory pathogens using a newly developed multiplex real-time PCR panel based on an automatic molecular detection and analysis system.

Timely identification of respiratory pathogens guides specific treatment, reduces hospital costs and minimizes the excessive use of antibiotics. A new multiplex real-time PCR panel was developed based on an automatic molecular detection and analysis system (AutoMolec system), consisting of three separate internally controlled assays. Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, human metapneumovirus, influenza B virus, respiratory syncytial virus and human parainfluenza virus 1-3 may be directly detected in original samples. The system's clinical performance was evaluated by comparison with an approved commercial kit, using 517 clinical samples. The limit of detection of the AutoMolec mRT-PCR panel ranged from 4 × 10-4 ∼3.3 TCID50/mL and no cross-reaction with common respiratory pathogens was observed. The AutoMolec mRT-PCR panel had 99.09% sensitivity and 100.0% specificity and overall detection consistency was 99.61%, making it comparable to that of the commercial kit. Therefore, the AutoMolec mRT-PCR panel has great potential for routine screening of respiratory infection in China.

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay.

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein-protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection.

Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypes.

BACKGROUND: The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoVs) (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) still circulate worldwide. The early clinical symptoms of SARS-CoV-2 and seasonal HCoV infections are similar, so rapid and accurate identification of the subtypes of HCoVs is crucial for early diagnosis, early treatment, prevention and control of these infections. However, current multiplex molecular diagnostic techniques for HCoV subtypes including SARS-CoV-2 are limited. METHODS: We designed primers and probes specific for the S and N genes of SARS-CoV-2, the N gene of severe acute respiratory syndrome coronavirus (SARS-CoV), and the ORF1ab gene of four seasonal HCoVs, as well as the human B2M gene product. We developed and optimized a quadruple quantitative real-time PCR assay (qq-PCR) for simultaneous detection of SARS-CoV-2, SARS-CoV and four seasonal HCoVs. This assay was further tested for specificity and sensitivity, and validated using 184 clinical samples. RESULTS: The limit of detection of the qq-PCR assay was in the range 2.5 × 101 to 6.5 × 101 copies/µL for each gene and no cross-reactivity with other common respiratory viruses was observed. The intra-assay and inter-assay coefficients of variation were 0.5-2%. The qq-PCR assay had a 91.9% sensitivity and 100.0% specificity for SARS-CoV-2 and a 95.7% sensitivity and 100% specificity for seasonal HCoVs, using the approved commercial kits as the reference. Compared to the commercial kits, total detection consistency was 98.4% (181/184) for SARS-CoV-2 and 98.6% (142/144) for seasonal HCoVs. CONCLUSION: With the advantages of sensitivity, specificity, rapid detection, cost-effectiveness, and convenience, this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-CoV-2, SARS-CoV and seasonal HCoVs.

Development and evaluation of a sensitive recombinase aided amplification assay for rapid detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is a widely distributed pathogen in the coastal areas, which causes food poisoning and leads to gastroenteritis and sepsis. Therefore, developing a simple, sensitive, and rapid detection method for V. parahaemolyticus is a major concern globally. This study established a sensitive and rapid technique based on recombinase aided amplification (RAA) to detect V. parahaemolyticus. The RAA reaction was carried out successfully at 39 °C within 30 min. The sensitivity of the RAA assay was 101 copies/µL using the recombinant plasmid and 10-3 ng/µL using the V. parahaemolyticus strain. In addition, RAA directly detected 7 × 103 CFU/mL of simulated fecal samples and 0.1 CFU/mL after enrichment for 4 h. The sensitivity and specificity of the RAA assay using fecal and fish samples were 100% similar to that of the real-time PCR. We conclude that the RAA assay is an ideal screening method for detecting V. parahaemolyticus due to its rapidity, high accuracy, and simplicity in operation.

A highly sensitive one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis using the LNA technique.

OBJECTIVES: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity. METHODS: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison. RESULTS: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%. CONCLUSIONS: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.

Development and evaluation of a panel of multiplex one-tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions.

Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses.

Molecular and clinical characterization of human adenovirus associated with acute respiratory tract infection in hospitalized children.

BACKGROUND: Human adenovirus (HAdV) is a common pathogen in children that can cause acute respiratory tract infection (ARTI), but the molecular epidemiological and clinical information relating to HAdV among hospitalized children with ARTI are few reported in China. OBJECTIVES: To evaluate the epidemiological, clinical, and molecular characteristics of HAdV infections among hospitalized children with ARTI in Hebei, Northern China from June 2017 to May 2018. STUDY DESIGN: A 12-month longitudinal, retrospective study on HAdV, typed by nested polymerase chain reaction targeting the hexon gene's hypervariable region (typing was merely performed by sequencing of the hexon neutralization epitope and thus genotypes could not be identified unequivocally), associated with ARTI was performed. The epidemiological and clinical data of different types of HAdV were analyzed using statistical product and service solutions (SPSS) 21.0 software. RESULTS: HAdV was detected in 330 (3.71%) of the 8906 specimens, with most (88.48%, 292/330) HAdV-positives cases detected among children < 3 years old. HAdV were detected throughout the year with a higher prevalence in spring. 11 types were identified, with HAdV-2 (33.33%, 110/330) as the predominant type, followed by HAdV-3 (21.21%, 70/330) and HAdV-7 (13.94%, 46/330). Of the 330 HAdV-positive specimens, 247 (74.85%) were co-detected with other respiratory pathogens, most commonly rhinovirus (HRV) (58.7%, 145/247). Additionally, patients with HAdV-7 positive had longer duration of fever than HAdV-2 or -3 positive patients. CONCLUSIONS: During the study period, HAdV-2, HAdV-3 and HAdV-7 were the predominant types identified from children with ARTI in Hebei Province. Pediatric patients with HAdV-7 positive may not present more severe clinical outcome except a longer duration of fever.

Risk factors of 90-day rehospitalization following discharge of pediatric patients hospitalized with mycoplasma Pneumoniae pneumonia.

BACKGROUND: Among pediatric patients hospitalized for Mycoplasma pneumoniae pneumonia (MPP), the risk factors for 90-day readmission after discharge is undefined. METHODS: We conducted a retrospective observational study of patients <14 years of age who were discharged with a diagnosis of MPP between January 2016 and February 2017. We collected clinical, laboratory and radiographic variables at the time of initial admission. We assessed pneumonia-related readmission within 90-day after discharge. Risk factors independently associated with rehospitalization were identified using multiple logistic regression models. RESULTS: Of the 424 MPP hospitalizations, 48 (11.3%) were readmitted within 90 days and were mainly diagnosed with pneumonia. Patients with younger age or coinfection with influenza A were more likely to be readmitted. In addition, compared with children without readmission, the readmission ones showed different clinical and laboratory characteristics at the index hospital admission. Multiple logistic regression analysis identified age (OR 0.815, 95%CI 0.706-0.940) and body temperature (OR 0.659, 95%CI 0.518-0.839) were significantly associated with lower risk of 90-day readmission. Coinfection with influenza was independently associated with a greater likelihood of 90-day readmission (OR 4.746, 95%CI 1.191-18.913). CONCLUSIONS: Readmission after MPP are common and is related to patients' age, body temperature and influenza A coinfection during initial hospital stay, indicating potential targets could be noticed to reduce the rehospitalization after pediatric MPP.

Impact and clinical profiles of Mycoplasma pneumoniae co-detection in childhood community-acquired pneumonia.

BACKGROUND: Increasing number of hospitalized children with community acquired pneumonia (CAP) is co-detected with Mycoplasma pneumoniae (Mp). The clinical characteristics and impact of Mp co-detected with other bacterial and/or viral pathogens remain poorly understood. The purpose of this study was to evaluate the demographic and clinical features of CAP children with Mp mono-detection and Mp co-detection. METHODS: A total of 4148 hospitalized children with CAP were recruited from January to December 2017 at the Children's Hospital of Hebei Province, affiliated to Hebei Medical University. A variety of respiratory viruses, bacteria and Mp were detected using multiple modalities. The demographic and clinical features of CAP children with Mp mono-detection and Mp co-detection were recorded and analyzed. RESULTS: Among the 110 CAP children with Mp positive, 42 (38.18%) of them were co-detected with at least one other pathogen. Co-detection was more common among children aged ≤3 years. No significant differences were found in most clinical symptoms, complications, underlying conditions and disease severity parameters among various etiological groups, with the following exceptions. First, prolonged duration of fever, lack of appetite and runny nose were more prevalent among CAP children with Mp-virus co-detection. Second, Mp-virus (excluding HRV) co-detected patients were more likely to present with prolonged duration of fever. Third, patients co-detected with Mp-bacteria were more likely to have abnormal blood gases. Additionally, CAP children with Mp-HRV co-detection were significantly more likely to report severe runny nose compared to those with Mp mono-detection. CONCLUSION: Mp co-detection with viral and/or bacterial pathogens is common in clinical practice. However, there are no apparent differences between Mp mono-detection and Mp co-detections in terms of clinical features and disease severity.

A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling.

OBJECTIVES: Pertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis. The purpose of this study was to develop a rapid, simple and sensitive diagnostic test for detecting this pathogen. METHODS: Here we present a recombinase aided amplification (RAA) assay incorporating competitive internal amplification control (IAC) to detect Bordetella pertussis using the DNA obtained by boiling. This assay was performed in a single closed tube at 39°C within 30min. A total of 115 clinical samples suspected of pertussis were collected and tested by the internally controlled RAA assay using both extracted DNA with the commercial kit and the DNA obtained by boiling. For comparison, the real-time PCR (RT-PCR) was also performed with DNA extraction in parallel. RESULTS: The sensitivity of the internally controlled RAA assay was 101 copies or 10CFU/ml per reaction in detecting plasmid DNA or B. pertussis strain. The optimum concentration of the IAC plasmid was determined to be 100 copies, and the introduction of IAC effectively reduced the occurrence of false negatives. Compared to the RT-PCR, RAA results with DNA extraction obtained 100% sensitivity and specificity, and the RAA results with heat-treated DNA showed 85.96% sensitivity and 100% specificity. CONCLUSION: With the advantages of 45min turn-around time and simple steps of DNA purification, this assay could become a useful diagnostic tool for Bordetella pertussis detection and is potentially suitable for point-of-care identification to guide prompt clinical treatment.

Comparing the yield of oropharyngeal swabs and sputum for detection of 11 common pathogens in hospitalized children with lower respiratory tract infection.

BACKGROUND: Advances in molecular laboratory techniques are changing the prospects for the diagnosis of viral infectious diseases. Multiplex polymerase chain reaction assay (multiplex-PCR) can detect dozens of pathogens simultaneously, greatly reducing turnaround time (TAT) and improving detection sensitivity. But as a double-edged sword, due to the high sensitivity of PCR, the type of respiratory specimens is critical to diagnosis. In this work, we performed a head-to-head comparison to evaluate the multiplex-PCR yields between two samples, sputum and flocked oropharyngeal swabs (OPS). METHODS: Eleven common respiratory pathogens were tested in hospitalized children< 13 years of age who met the criteria for lower respiratory tract infection by GeXP-based multiplex-PCR of paired OPS and sputum. RESULTS: From January to June 2018, 440 children with paired OPS and sputum were tested. The positive rate was 84% (369/440) for OPS and 88% (386/440) for sputum (p = .007). The frequency of detection of HRV, RSV, Influenza A virus, HMPV, parainfluenza virus, adenovirus, M. pneumoniae, coronavirus, bocavirus and C. pneumoniae in sputa was higher than that of OPSs (all p < .001). Both types of specimens had similarly very good kappa values for most of pathogens, except for Mycoplasma pneumonia (κ = 0.61) and Chlamydia pneumoniae (κ = 0.24). Additionally, 79.3% (349/440) of cases showed consistent results between the two types of samples, and they were significantly younger than patients with inconsistent results (p = .002). CONCLUSIONS: Flocked oropharyngeal swabs and sputum performed similarly for the detection of common respiratory pathogens in hospitalized children by multiplex-PCR, except for Mycoplasma pneumoniae and Chlamydia pneumoniae. Young patients are likely to have consistent results between the two specimens.

A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction.

BACKGROUND: Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. METHODS: Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). RESULTS: A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. CONCLUSIONS: We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid--based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02 × 10-1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.

A case control study on the prevalence of enterovirus in children samples and its association with diarrhea.

Some serotypes of enterovirus (EV) may lead to transient and symptomatic gastrointestinal infections while others are commensal residents of the human gut. To determine whether certain EV types are more often associated with diarrhea, we conducted a preliminary study on the prevalence of EV serotypes and common diarrhea viruses in fecal samples of diarrhea children and healthy controls. EV was tested with one step nest polymerase chain reaction and typed by direct sequencing while common causative diarrhea viruses rotavirus (RV), norovirus (NoV), adenovirus (AdV), bocavirus (HBoV), and astrovirus (AstV) were screened with multiplex PCR assays. Human Rhinovirus (HRV) and human EVs that were present in both groups were further quantified and their odds ratios (OR) were calculated. Enteric pathogens were detected in 89 (32.6%) of 273 children with diarrhea and included human EVs (51, 18.68%), HRV (32, 11.72%), RV (38, 13.92%), AdV (24, 8.79%), NoVGII (16, 8.79%), HBoV (8, 2.93%) and AstV (3, 1.09%). Potential enteric pathogens were found in 25 (6.93%) of 361 healthy controls and included human EV (59, 16.34%), HRV (8, 2.22%), RV (1, 0.28%), NoVGII (5, 1.39%), AstV (2, 0.55%), AdV (16, 4.43%) and HBoV (1, 0.28%). In addition, EV71, echovirus 3,9,14,25 and coxsackievirus A14 existed in healthy controls only, while HRV, echovirus11,18, coxsackievirus A2,4,6 and B2,4 were found in both patients and healthy controls. OR assessment confirmed a strong association of HRV (P < 0.001) and a weak one for echovirus 11 and coxsackievirus A6 with diarrhea (P > 0.05). Our results indicate the diversity of EV serotypes in diarrhea and healthy control groups varies, and the potential etiological role of HRV in diarrhea.

A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus.

BACKGROUND: Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. RESULTS: A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. CONCLUSION: mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.