Clin Infect Dis

Background.Rotavirus is a common cause of severe pediatric acute gastroenteritis. Two vaccines are licensed in the United States and have demonstrated high effectiveness against moderate to severe disease. However, fewer data are available on rotavirus vaccine effectiveness (VE) against milder disease.Methods.We leveraged active surveillance data from Kaiser Permanente Northwest to calculate rotavirus VE against medically attended rotavirus illness among age-eligible children. We utilized a test-negative case-control design and applied 4 distinct case definitions based on reverse transcription\u2013quantitative real-time PCR (qRT-PCR) assay and enzyme immunoassay (EIA) test results. VE was calculated as 100 7 (1 \u2013 odds ratio), and models were adjusted for age group.Results.The VE analysis population comprised 842 children, 799 (95%) of whom had mild disease requiring at most a clinic visit and 698 (83%) of whom were fully vaccinated against rotavirus. Age-adjusted VE was 70% (95% confidence interval [CI], 37\u201386%) against disease defined solely by qRT-PCR results, 72% (95% CI, 31\u201389%) against disease as defined by qRT-PCR with a quantification cycle (Cq) value <27, 73% (95% CI, 32\u201390%) against disease that was qRT-PCR positive but EIA negative, and 62% (95% CI, 1220\u201388%) against disease defined solely by EIA. Results were similar when restricting to disease resulting in at most an ambulatory clinic or emergency department visit.Conclusions.These results support the effectiveness of rotavirus vaccination in protecting US children from mild to moderate and severe disease. Our findings are also useful to show the effectiveness of rotavirus vaccination against qRT-PCR\u2013defined illness.CC999999/ImCDC/Intramural CDC HHSUnited States/2022-06-01T00:00:00Z32322882PMC813000311425vault:3707

Clin Infect Dis

Background.Acute gastroenteritis (AGE) burden, etiology, and severity in adults is not well characterized. We implemented a multisite AGE surveillance platform in 4 Veterans Affairs Medical Centers (Atlanta, Georgia; Bronx, New York; Houston, Texas; and Los Angeles, California), collectively serving >320 000 patients annually.Methods.From 1 July 2016 to 30 June 2018, we actively identified inpatient AGE case patients and non-AGE inpatient controls through prospective screening of admitted patients and passively identified outpatients with AGE through stool samples submitted for clinical diagnostics. We abstracted medical charts and tested stool samples for 22 pathogens by means of multiplex gastrointestinal polymerase chain reaction panel followed by genotyping of norovirus- and rotavirus-positive samples. We determined pathogen-specific prevalence, incidence, and modified Vesikari severity scores.Results.We enrolled 724 inpatients with AGE, 394 non-AGE inpatient controls, and 506 outpatients with AGE. Clostridioides difficile and norovirus were most frequently detected among inpatients (for AGE case patients vs controls: C. difficile, 18.8% vs 8.4%; norovirus, 5.1% vs 1.5%; P < .01 for both) and outpatients (norovirus, 10.7%; C. difficile, 10.5%). The incidence per 100 000 population was highest among outpatients (AGE, 2715; C. difficile, 285; norovirus, 291) and inpatients 6565 years old (AGE, 459; C. difficile, 91; norovirus, 26). Clinical severity scores were highest for inpatient norovirus, rotavirus, and Shigella/enteroinvasive Escherichia coli cases. Overall, 12% of inpatients with AGE had intensive care unit stays, and 2% died; 3 deaths were associated with C. difficile and 1 with norovirus. C. difficile and norovirus were detected year-round with a fall/winter predominance.Conclusions.C. difficile and norovirus were leading AGE pathogens in outpatient and hospitalized US veterans, resulting in severe disease. Clinicians should remain vigilant for bacterial and viral causes of AGE year-round.20212022-06-14T00:00:00ZCC/CDC HHSUnited States/the Emory Center for AIDS Research/Atlanta VAMC/32584956PMC91954961151

多重PCR技术研究进展

多重PCR (Multiplex polymerase chain reaction,MPCR)是指通过一次PCR反应同时对多个靶标进行扩增,结合一定的检测手段对扩增产物进行检测从而实现对多个靶标进行诊断的技术。MPCR具有高效率、高通量、低成本的特性而被深入研究。目前MPCR技术已被广泛应用于科学研究和疾病诊断等领域,文中从MPCR扩增与检测两方面概述了MPCR技术发展及其应用,讨论了MPCR技术的优点及存在的问题,并且提出将反应体系分隔成小液滴或结合管式对流PCR(Capillary convective PCR,CCPCR)技术的方式有望提高固相载体表面的扩增效率,从而开发出扩增效率高、一致性好、体系稳定、检测重数高的多重PCR技术

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains

Environmental fate of RNA interference biopesticides

Emerging agricultural biotechnology uses RNA interference (RNAi) to protect crops from pests. RNAi is a cellular mechanism in which double-stranded RNA (dsRNA) directs the degradation of the homologous messenger RNA (mRNA), leading to gene silencing and preventing the synthesis of essential proteins. As applied to agriculture, dsRNA biopesticides have been developed that can trigger RNAi inside of the pest tissues to cause reduced pest growth and/or increased pest mortality. When dsRNA biopesticides are released to receiving environments (e.g., agricultural soils and surface water), they pose potential ecological risks to non-target organisms. This dissertation aims to advance the understanding of the environmental fate of dsRNA to enable accurate ecological risk assessments of dsRNA biopesticides.The first objective of this dissertation was to establish a new method using quantitative reverse transcription polymerase chain reaction (RT-qPCR) to measure dsRNA in agricultural soils at environmentally relevant concentrations. Because dsRNA readily adsorbs to soil particles, a method was developed to efficiently transfer dsRNA from soil particles to solutions for RT-qPCR analysis. Furthermore, organic matter was removed using a validated cleanup protocol to prevent inhibition of RT-qPCR. Ultimately, the method was able to quantify dsRNA at orders of magnitude lower concentration than the estimated environmental concentration of dsRNA, indicating the adequate sensitivity of the method. Applying this new method, ≥75% of dsRNA was found to degrade in agricultural soils within 8 hours. The second objective was to characterize the chemical stability of dsRNA in aqueous solutions by examining the impacts of the duplex structure of dsRNA on alkaline hydrolysis. Although dsRNA shares the same primary structure as single-stranded RNA (ssRNA), dsRNA was found to undergo orders-of-magnitude slower alkaline hydrolysis than ssRNA. Furthermore, dsRNA remained intact for multiple months at neutral pH, challenging a prior assumption in dsRNA biopesticide risk assessment that dsRNA is chemically unstable. In systems enabling both enzymatic degradation and alkaline hydrolysis of dsRNA, increasing pH effectively attenuated enzymatic degradation without inducing alkaline hydrolysis that was observed for ssRNA. The third objective was to elucidate a novel abiotic reaction by which RNA, but not DNA, degraded upon adsorption to surfaces of goethite, a ubiquitous mineral in soils and sediments, under environmentally relevant physicochemical conditions. Upon adsorption to goethite, both ssRNA and dsRNA hydrolyzed on the timescale of hours. The reaction products were consistent with iron present in goethite acting as a Lewis acid to accelerate the hydroxide-catalyzed hydrolysis of random phosphodiester bonds comprising the RNA backbone. In contrast to well-established acid or base-catalyzed RNA hydrolysis in solution, mineral-catalyzed hydrolysis was fastest at circumneutral pH, which allowed for both sufficient RNA adsorption and hydroxide concentration. Contact of RNA with the mineral surface was necessary for hydrolysis to occur by demonstrating that RNA degradation was inhibited by compact RNA conformation at elevated ionic strength or competitive adsorption with orthophosphate and organic matter. In addition to goethite, hematite also catalyzed RNA hydrolysis, whereas aluminum-containing minerals (e.g., montmorillonite) did not. Overall, this work (1) contributes to the first method able to quantify dsRNA biopesticides at an environmentally relevant concentration, (2) demonstrates, for the first time, that key degradation pathways of dsRNA significantly differed from those of ssRNA, and (3) reports the first abiotic pathway to contribute to RNA degradation on environmentally relevant timescales. This study advances our understanding of the fate of dsRNA biopesticides in receiving environments and contributes to the ecological risk assessment of dsRNA biopesticides