PCR múltiplex para el diagnóstico de microorganismos atípicos transmitidos sexualmente / Multiplex PCR testing for the atypical microorganisms diagnosis sexually transmitted

Resumen Antecedentes: Las infecciones de transmisión sexual son un problema de salud pública mundial. El análisis rutinario incluye solo pruebas microbiológicas y serológicas para el diagnóstico de patógenos. Los microorganismos atípicos como Chlamydia trachomatis y micoplasmas no son identificados debido a los requerimientos. Además, no es incluida Gardnerella vaginalis, aunque se asocia a la vaginosis bacteriana. Objetivo: Desarrollar una PCR múltiplex para el diagnóstico de C. trachomatis, micoplasmas y G. vaginalis. Método: Se estandarizó la PCR múltiplex utilizando oligonucleótidos para C. trachomatis (gen ompA, orf6 plasmídico), Mycoplasma/Ureaplasma y G. vaginalis (genes rRNA16s). Resultados: Se estandarizaron pruebas de PCR múltiplex para los microorganismos estudiados, optimizándose las concentraciones y condiciones de las reacciones múltiplex. Se obtuvieron PCR dúplex para C. trachomatis (ompA, orf6), Chlamydia/Gardnerella y Chlamydia/micoplasmas y tríplex para Chlamydia/Mycoplasma/Ureaplasma. También un cuádruplex para Chlamydia/Mycoplasma/Ureaplasma/Gardnerella. Los resultados fueron verificados por PCR e hibridación automática (HybriSpot 12) y análisis in silico. Conclusión: Se desarrollaron pruebas de PCR múltiplex con una alta sensibilidad y especificidad para la identificación de C. trachomatis, micoplasmas y G. vaginalis.

Abstract Background: Sexually transmitted infections are a global public health problem. Routine analysis includes microbiological and serological tests for the diagnosis of pathogens. Atypical microorganisms such as Chlamydia trachomatis and mycoplasmas are not determined due to the requirements for their identification. Furthermore, Gardnerella vaginalis is not included despite being associated with bacterial vaginosis. Objective: To develop a multiplex PCR to diagnose Chlamydia, mycoplasmas, and Gardnerella. Method: Standardization of multiplex PCR tests was carried out using oligonucleotides for the identification of Chlamydia (ompA gene, plasmid orf6), Mycoplasma/Ureaplasma and Gardnerella (rRNA16s genes). Results: Multiplex PCR tests were standardized for the microorganisms studied, optimizing the concentrations and conditions of the multiplex reactions. Duplex PCR was obtained for Chlamydia (ompA, orf6), Chlamydia/Gardnerella, and Chlamydia/mycoplasmas, and triplex PCR for Chlamydia/mycoplasmas. Also, a quadruplex for Chlamydia, Mycoplasma/Ureaplasma and Gardnerella. PCR and automatic hybridization verified the results obtained (HybriSpot 12) and in silico analysis. Conclusion: Multiplex PCR tests with high sensitivity and specificity were developed to identify C. trachomatis, mycoplasmas, and G. vaginalis.

Detección molecular de patógenos en deposiciones en niños consultantes por diarrea aguda en Servicios de Urgencia

Introducción: La diarrea aguda continúa siendo una de las principales causas de morbilidad en niños; sin embargo, el diagnóstico etiológico presenta limitaciones dada la baja sensibilidad de los métodos tradicionales. Objetivo: Describir los microorganismos identificados en niños que acudieron al Servicio de Urgencia (SU) de un hospital universitario en Santiago, Chile, por diarrea aguda y a los que se le solicitó panel molecular gastrointestinal. Métodos: Se revisaron fichas clínicas y resultados de panel gastrointestinal realizados entre junio de 2017 y marzo de 2020. Resultados: Se incluyeron 198 pacientes, edad promedio de 54,5 meses y 60,6% (120/198) de sexo masculino. La positividad del panel fue de 78,8% (156/198) con 35,3% (55/156) de las muestras polimicrobianas. Se identificaron 229 microorganismos, de los cuales 72,9% (167/229) corresponden a bacterias, 25,8% (59/229) a virus y 1,3% (3/229) a parásitos. Destacaron Campylobacter spp. y Escherichia coli enteropatógena (ECEP) como las bacterias más frecuentemente identificadas. Los pacientes con detección de Campylobacter spp. presentaron con mayor frecuencia fiebre (p = 0,00). ECEP se aisló principalmente (82,5%) en muestras polimicrobianas. Discusión: Los resultados enfatizan el potencial que poseen los estudios moleculares para mejorar el diagnóstico etiológico de la diarrea, pero a la vez llevan a cuestionar el rol patogénico de algunos microorganismos identificados.

Background: Acute diarrhea continues to be one of the main causes of morbidity in children, however the etiologica diagnosis presents limitations given the low sensitivity of traditional methods. Aim: To describe the microorganisms identified in children who attended the emergency department (ED) in Santiago, Chile, due to acute diarrhea and to whom a gastrointestinal panel was requested as part of their study. Material and Methods: Clinical records and results of the gastrointestinal panel carried out between June 2017 and March 2020 were reviewed. Results: 198 patients were included, the average age was 54.5 months and 60.6% (120/198) were males. Positivity was 78.8% (156/198) with 35.3% (55/156) of the samples being polymicrobial. 229 microorganisms were identified, of which 72.9% (167/229) corresponded to bacteria, 25.8% (59/229) to viruses, and 1.3% (3/229) to parasites. Campylobacter spp. and enteropathogenic Escherichia coli (EPEC) were the most frequently identified bacteria. Patients with detection of Campylobacter spp. presented a higher frequency of fever (p = 0.00). EPEC was isolated in 82.5% of the cases in polymicrobial samples. Discussion: The results emphasize the potential of molecular studies to improve the etiological diagnosis of diarrhea and at the same time lead to question the pathogenic role of some microorganisms.

Molecular Detection of Plasmid-Mediated Quinolone Resistance Genes (qnrA and aac(6?)-Ib-cr) in Drug Resistant Escherichia coli, Sudan

Background: The quinolone group, a synthetic antimicrobial, is widely used worldwide to treat many diseases, including those caused by Gram-negative bacteria. Escherichia coli and others are among the bacteria that produce quinolone resistance genes (qnr) such as qnrA and aac(6?)-Ib-cr. Objective: The present study aimed to the isolate Escherichia coli from patients attending some Hospitals in Wad Medani city, identification of drug resistance patterns and detection of the frequency of quinolones resistance genes; qnrA and aac(6?)-Ib-cr among isolated strains. Methods: A cross-sectional descriptive, hospital-based study included 119 Escherichia coli strains was conducted. A designed questionnaire used for demographic data collection and the attitude toward antimicrobials usage. Clinical specimens were processed for aerobic bacterial isolation and identification. Antimicrobial sensitivity performed by Kirby Bauer disc diffusion technique according to the CLSI guidelines. Presence of qnrA and aac(6?)-Ib-cr genes was assessed by multiplex PCR. Results: Most strains of Escherichia coli originated from urine 53.8% (64/119) and wounds 42.9% (51/119) specimens. Meropenem had the best effect against tested strains with susceptibility of 85% (101/119). Multiplex PCR assay, using specific primers, demonstrated that 41.2% (49/119) and 37.8% (45/119) of isolated Escherichia coli possessed qnrA and aac(6?)-Ib-cr genes respectively. Conclusion: The high rate of qnrA and aac (6)-Ib-cr genes among Escherichia coli necessitate the usage of molecular tools in detecting the genetic determinants of drug resistance microorganisms in countries such as Sudan.

Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA)signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3–0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics.

Etiología de las infecciones de transmisión sexual (ITS) diagnosticadas por la técnica de PCR múltiple-hibridación en población colombiana de la ciudad de Medellín atendida en el Laboratorio Clínico VID / Etiology of sexually transmitted infections (STIs) diagnosed by multiplex PCR-hibridization assay technique in a Colombian population attending the VID Clinical Laboratory in Medellin

Introducción. Las infecciones de transmisión sexual (ITS) son y seguirán siendo un serio problema de salud pública en todo el mundo según los datos de la OMS, con el agravante que la mayoría de los casos son asintomáticos y, además, no existe otro reservorio distinto al humano. El diagnóstico se puede realizar con pruebas tradicionales y moleculares, estas últimas incluyen la reacción en cadena de la polimerasa (PCR), de las cuales existen varios tipos, entre ellas, la PCR múltiple que tiene la capacidad de detectar ITS polimicrobianas a partir de una sola muestra. El objetivo de este estudio fue establecer cuáles fueron las infecciones de transmisión sexual más frecuentes en diferentes grupos de pacientes, así como determinar la utilidad del uso de la técnica de PCR múltiple en el diagnóstico de las ITS. Metodología. Se trata de un estudio observacional de corte transversal realizado entre los años 2021 y 2022 con pacientes que acudieron al servicio de diagnóstico del Laboratorio Clínico VID por sospecha de ITS. Las muestras recolectadas fueron evaluadas utilizando una prueba comercial basada en la técnica de PCR múltiple e hibridación. Las muestras procesadas fueron: orina e hisopados de endocérvix, uretra, recto, faringe y úlceras. Resultados. Se estudiaron 1.027 pacientes, de estos, 228 (22,2 %) fueron positivos para diferentes agentes de trasmisión sexual, distribuidos así: 50 (21,9 %) mujeres, 129 (56,6 %) hombres heterosexuales y 49 (21,5 %) hombres que tenían sexo con hombres (HSH). La edad promedio de las mujeres fue 30 años, y la de ambos grupos de hombres fue 36 años. Los microorganismos más frecuentemente identificados en mujeres fueron: C. trachomatis (A-K) en 28,6 %, seguido de virus herpes simplex tipo 2 (VHS-2) en 26,8 % y N. gonorrhoeae en 17,9 %. En hombres heterosexuales fueron C. trachomatis (A-K) en 37,5 %, N. gonorrhoeae en 21,5 % y VHS-2 en 18,7 %. En HSH fueron C. trachomatis (L1-L3) en 32,7 %, seguido de N. gonorrhoeae en 27,6 %, y de C. trachomatis (A-K) y VHS-2, ambos en 13,8 %. En 11 hombres heterosexuales, 8 HSH y en 6 mujeres, se identificó infección polimicrobiana. Conclusiones. C. trachomatis (A-K) fue el microorganismo más prevalente causante de ITS, seguido de N. gonorrhoeae en ambos grupos de hombres, y de VHS-2 en las mujeres, muy similar a lo reportado a nivel mundial. La prueba de PCR múltiple permite la detección de infecciones polimicrobianas comúnmente asociadas a ITS y el diagnóstico es preciso y confiable, incluso en pacientes asintomáticos

Sexually transmitted infections (STIs) are and will continue to be a serious public health problem throughout the world according to WHO data, with the aggravating factor that most cases are asymptomatic and, furthermore, there is no other reservoir other than humans. The diagnosis can be made with traditional and molecular tests, the latter include the polymerase chain reaction (PCR), of which there are several types, among them, multiplex PCR that has the capacity to detect polymicrobial STIs from a single sample. The objective of this study was to establish which were the most frequent sexually transmitted infections in different groups of patients, as well as to determine the usefulness of the multiplex PCR technique in the diagnosis of STIs. Methodology. This is an observational, cross-sectional study carried out between 2021 and 2022 with patients who attended the VID Clinical Laboratory for suspected STIs. The collected samples were evaluated using a commercial test based on the multiplex PCR technique and hybridization. The samples processed were: urine and swabs from endocervix, urethra, rectum, pharynx, and ulcers. Results. The study included 1,027 patients, of these, 228 (22.2%) were positive for different sexually transmitted agents, distributed as follows: 50 (21.9%) women, 129 (56.6%) heterosexual men and 49 (21.5%) men who had sex with men (MSM). The average age of the women was 30 years, and that of both groups of men was 36 years. The microorganisms most frequently identified in women were: C. trachomatis (A-K) in 28.6%, followed by herpes simplex virus type 2 (HSV-2) in 26.8% and N. gonorrhoeae in 17.9%. In heterosexual men they were C. trachomatis (A-K) in 37.5%, N. gonorrhoeae in 21.5% and HSV-2 in 18.7%. In MSM they were C. trachomatis (L1-L3) in 32.7%, followed by N. gonorrhoeae in 27.6%, and C. trachomatis (A-K) and HSV-2, both in 13.8%. Polymicrobial infection was identified in 11 heterosexual men, 8 MSM, and 6 women. Conclusions. C. trachomatis (A-K) was the most prevalent STI-causing microorganism, followed by N. gonorrhoeae in both groups of men, and HSV-2 in women, very similar to that reported worldwide. The multiplex PCR test allows the detection of polymicrobial infections commonly associated with STIs and the diagnosis is accurate and reliable, even in asymptomatic patients

Utilidad del panel de PCR multiplex en el diagnóstico microbiológico temprano y adecuación antimicrobiana en pacientes críticos con neumonía / Usefulness of the multiplex PCR panel in the early microbiological diagnosis and antimicrobial suitability in critical patients with pneumonia / Utilidade do painel de PCR multiplex no diagnóstico microbiológico precoce e adequação antimicrobiana em pacientes críticos com pneumonia

Resumen: Introducción: el inicio temprano de la antibioticoterapia adecuada en infecciones graves se asocia con reducción de la mortalidad. La identificación precoz del microorganismo es fundamental para realizar un tratamiento dirigido y disminuir la terapéutica inicial inapropiada. Objetivo: valorar la utilidad de una técnica de biología molecular por amplificación de ácidos nucleicos mediante reacción en cadena de polimerasa en tiempo real para diagnóstico microbiológico temprano y adecuación de la antibioticoterapia en pacientes con neumonías graves. Metodología: estudio retrospectivo observacional llevado a cabo en la unidad de cuidados intensivos del Hospital Maciel. Se analizaron muestras respiratorias de pacientes con diagnóstico o sospecha de neumonía. Se compararon los resultados microbiológicos obtenidos por técnicas convencionales y por biología molecular multiplex (panel neumonía). Resultados: se incluyeron 53 muestras obtenidas de 51 pacientes. El multiplex detectó al menos un microorganismo en 38 (71,7%) muestras frente a 30 (56.6%) desarrollos en cultivos tradicionales. La mayoría de las muestras se obtuvieron bajo antibioticoterapia previa (86.8%). El panel neumonía mostró un porcentaje de concordancia positiva combinado de 100% y un porcentaje de concordancia negativa del 94% para la identificación bacteriana en comparación con los métodos microbiológicos tradicionales. En 27 (51%) casos el resultado del panel de neumonía determinó un cambio en la conducta terapéutica. Conclusiones: la técnica de PCR permite la identificación temprana de microorganismos causantes de neumonía optimizando la terapéutica empírica inicial y racionalizando el uso de antimicrobianos. Un panel negativo aleja el planteo de infección respiratoria a gérmenes habituales y permite considerar diagnósticos diferenciales en cuanto a foco y/o etiología.

Summary: Introduction: the early initiation of the adequate antibiotic therapy in severe infections is associated to a reduction in mortality. Early identification of the microorganism is essential to define directed therapy and decrease the initial inadequate treatment. Objective: to assess usefulness of a molecular biology technique by nucleic acid amplification through a polymerase chain reaction in real time for an early microbiological diagnosis and correction of the antibiotic therapy in patients with severe pneumonias. Method: retrospective, observational study conducted in the intensive care unit of Maciel Hospital. The respiratory samples of patients with a diagnosis of pneumonia or suspicious to have pneumonia were analyzed. The microbiological results obtained were compared using conventional techniques and multiplex molecular biology (pneumonia panel). Results: 53 samples obtained from 51 patients were included in the study. Multiplex detected at least one microorganism in 38 (71.7%) samples compared to 30 (56.6%) in traditional cultures. Most samples were obtained under the previous antibiotic therapy (86.8%). The pneumonia panel showed a combined positive agreement percentage of 100% and a negative agreement of 94% for the identification of bacteria when compared to the traditional microbiological methods. In 27 cases (51%) the pneumonia panel results determined changing the therapeutic behavior. Conclusions: the PCR technique allows for the early identification of microorganisms causing pneumonia, thus optimizing initial empirical therapy and rationalizing the use of antibiotics. A negative panel reduces the suspicion of a respiratory infection caused by the usual germs and enables considering differential diagnosis in terms of etiology or cause.

Resumo: Introdução: o início precoce da antibioticoterapia adequada em infecções graves está associado à redução da mortalidade. A identificação precoce do microrganismo é essencial para realizar o tratamento dirigido e reduzir o uso inicial inadequado de antimicrobianos. Objetivo: avaliar a utilidade de uma técnica de biologia molecular para amplificação de ácidos nucleicos por reação em cadeia da polimerase em tempo real para diagnóstico microbiológico precoce e adequação da antibioticoterapia em pacientes com pneumonia grave. Metodologia: estudo observacional retrospectivo realizado na unidade de terapia intensiva do Hospital Maciel. Amostras respiratórias de pacientes com diagnóstico ou suspeita de pneumonia foram analisadas. Os resultados microbiológicos obtidos por técnicas convencionais e por biologia molecular multiplex (painel de pneumonia) foram comparados. Resultados: foram incluídas 53 amostras obtidas de 51 pacientes. O multiplex detectou pelo menos um microrganismo em 38 (71,7%) amostras em comparação com 30 (56,6%) usando culturas tradicionais. A maioria das amostras foi obtida com antibioticoterapia prévia (86,8%). O painel de pneumonia mostrou uma concordância percentual positiva combinada de 100% e uma concordância percentual negativa de 94% para identificação bacteriana em comparação com métodos microbiológicos tradicionais. Em 27 (51%) casos, o resultado do painel de pneumonia determinou mudança no comportamento terapêutico. Conclusões: a técnica de PCR permite a identificação precoce de microrganismos causadores de pneumonia, otimizando a terapia empírica inicial e racionalizando o uso de antimicrobianos. Um painel negativo afasta a suspeita de infecção respiratória pelos germes usuais e permite considerar diagnósticos diferenciais em termos de foco e/ou etiologia.

A Multiplex PCR assay for a differential diagnostic of rickettsiosis, Lyme disease and scrub typhus

Background & objectives: Coexistence of tick-borne diseases in some regions in Latin America makes the diagnosis difficult due to shared initial signs and symptoms. Rickettsiosis, Lyme disease and recently, scrub typhus are gaining more importance. The objective of this study is to develop a multiplex-PCR assay for a differential diagnosis of rickettsiosis, Lyme disease and scrub typhus. Methods: By using bibliographic and bioinformatic analysis, we identify candidate regions to perform the multiplexPCR assay for Rickettsia sp., Borrelia burgdorferi and Orientia tsutsugamushi as well as identify optimal melting temperature and sensibility analysis. Results: We identified specific primer pairs for Rickettsia sp, Borrelia burgdorferi and Orientia tsutsugamushi with different PCR fragment length but a common melting temperature, 58°C. Interpretation & conclusion: We successfully developed a Multiplex PCR assay for differential diagnosis of rickettsiosis, Lyme disease and scrub typhus that could be a rapid and easy option in clinical and epidemiological practice.Laboratorio de Enfermedades Infecciosas y Parasitarias 1. Unidad Interinstitucional de Investigación Clínica y Epidemiológica, Facultad de Medicina, Universidad Autónoma de Yucatán. Yucatán, Mexico

Laboratorio de Enfermedades Emergentes y Re-emergentes. Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”. Universidad Autonoma de Yucatan. Yucatán, Mexico

First report of invasive Pomacea snails in Shandong Province / 中国血吸虫病防治杂志

Objective To characterize the species of invasive Pomacea snails that were discovered for the first time in Shandong Province. Methods Pomacea snails samples were collected in the field of Jining City, Shandong Province on October 2021 for morphological identification. Pomacea snails were randomly sampled and genomic DNA was extracted from foot muscle tissues of Pomacea snails for multiplex PCR amplification. The PCR amplification product was sequenced. Then, the sequence was aligned and a phylogenetic tree was created using the software MegAlign 7.1.0. In addition, Angiostongylus cantonensis infection was detected in Pomacea snails with the lung microscopy. Results A total of 104 living Pomacea snails were collected, and all were characterized as Pomacea spp. based on morphological features. Of 12 randomly selected adult Pomacea snails, multiplex PCR assay and sequencing identified eleven snails as P. canaliculata and one as P. maculata. No A. cantonensis infection was detected in 104 Pomacea snails. Conclusion This is the first report of invasive Pomacea snails in Shandong Province, where P. canaliculata and P. maculata are found.

Interpretation of results obtained using real-time fluorescence PCR based on multiplex PCR fragment analysis for ABCB1 tri-allele / 四川精神卫生

ObjectiveTo resolve the problems related to the abnormal interpretations of real-time fluorescence polymerase chain reaction （PCR） results for tri-allele， to formulate the interpretation methods of real-time fluorescence PCR by referring to multiplex PCR fragment analysis， so as to obtain an accurate， simple and cheap detection method for ABCB1 tri-allele. MethodsA total of 2 794 DNA samples were collected from Xi'an Mental Health Center from March 2020 to March 2021， and 5% of which were selected as experiments. Real-time fluorescence PCR method and multiplex PCR fragment analysis method were performed respectively. According to the comparison of Ct values of PCR curves and the comparison of base peak intensity in multiplex PCR fragment analysis， comparison and analysis were conducted on the interpretation results of the two methods， and samples with different interpretation results were verified， thereafter， PCR interpretation method was formulated. ResultsA total of 139 samples were collected， of which 120 alleles and 19 tri-allele were detected. The results of allele detection by two methods were absolutely consistent. In combination with the results of multiplex PCR fragment analysis， a method for the interpretation of real-time fluorescence PCR for 19 cases of tri-allele was developed as follows： when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ in amplification curve was less than 3， the interpretation results were the combination of the base pairs with small Ct values； when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ was greater than or equal to 3， the interpretation results were homozygotes from the base pairs with minimum Ct values. According to the interpretation method， the results of real-time fluorescence PCR were revised， and it showed 1 case of G/G， 1 case of A/A， 4 cases of T/G， 5 cases of T/A and 8 cases of T/T， which were consistent with the results of multiplex PCR fragment analysis. ConclusionReferring to the multiplex PCR fragment analysis method， the interpretation of ABCB1 tri-allele by real-time fluorescence PCR is developed， and the two interpretation methods are in good agreement.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning / 法医学杂志

OBJECTIVES@#To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning.@*METHODS@#Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated.@*RESULTS@#The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05).@*CONCLUSIONS@#MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Étiologies des méningites et méningoencéphalites dans les services de réanimation polyvalente à Libreville de 2020 à 2021 / Etiologies of meningitis and meningoencephalitis in intensive care units of Libreville from 2020 to 2021

Introduction. Les méningites/méningo-encéphalites sont des urgences médicales d'étiologies variées. La technique de diagnostic Multiplex Polymerase Chain Reaction (PCR) permet de détecter la présence de bactéries et de virus dans le liquide céphalorachidien (LCR) avec une spécificité et une sensibilité ≥ 90%. L'objectif de cette étude était d'identifier en utilisant cette technique, les principaux germes responsables des méningites et méningo-encéphalites en réanimation à Libreville. Patients et méthodes. Nous avons mené une étude transversale allant d'octobre 2020 à septembre 2021. Les critères d'inclusion étaient : être admis en réanimation au CHUL et à l'HIAOBO pour suspicion de méningite ou méningo-encéphalite, obtenir l'accord des familles pour l'analyse du liquide céphalorachidien (LCR) par multiplex PCR. Les variables étudiées étaient : la fréquence, les données sociodémographiques, les aspects cliniques et paracliniques. Résultats. Soixante et onze patients ont répondu aux critères d'inclusion. L'âge moyen était de 21,1 ± 10,4 ans et le sex ratio de 1,2. Les motifs d'admission étaient l'altération de l'état de conscience (77%) et l'état de mal épileptique (21%). Plasmodium falciparum a été retrouvé seul chez 38 patients (53,5%) et associé à Listeria monocytogenes chez 4 patients (1,4%). Les méningo-encéphalites à Herpès simplex virus ont été observées chez 4 patients (1,4%) dont l'âge variait entre 40 ans et moins de 50 ans. Un patient (1,4%) présentait une coinfection à S. épidermidis, flavivirus et alphavirus. Des méningo-encéphalites sans germes ont été observées chez 5 patients (%). Conclusion. Le principal germe responsable de méningoencéphalite en réanimation à Libreville est P. falciparum. Des virus tels que le flavivirus et l'alphavirus non détectés par les méthodes usuelles ont aussi été mis en évidence grâce au multiplex PCR.

Introduction. Meningitis/meningoencephalitis are medical emergencies of various etiologies. The Multiplex Polymerase Chain Reaction (PCR) technique allows the detection of the presence of bacteria and viruses in the cerebrospinal fluid (CSF) with a specificity and sensibility of above 90%. The aim of this study was to identify the most common germs responsible for meningitis and meningoencephalitis in the intensive care units of Libreville using this technique,. Patients and methods. We conducted a transversal study from October 2020 to September 2021. Inclusion criteria were: being admitted to intensive care unit of CHUL and HIAOBO for suspicion of meningitis or meningoencephalitis and having the parent's approval for multiplex PCR analysis of CSF. Variables studied included frequency, sociodemographic data, clinical and paraclinical aspects. Results. Seventy one patients were included. Mean age was 21.1 ± 10.4 years and the sex ratio was 1.2. Reasons for admission were altered consciousness (77%) and epilepsy (21%). Plasmodium (P) faciparum was detected alone in 38 cases (53.5%) and associated to Listeria monocytogenes in 4 patients (5.6%). Herpex simplex viral meningoencephalitis was observed in 4 patients (5.6%) aged between 40 and less than 50 years. One patient (1.4%) had co-infection with S. epidermidis, flavivirus and alphavirus. Meningoencephalitis with no germs was found in 5 patients (7%). Conclusion. The main etiology of meningoencephalitis in intensive care units of Libreville is P. falciparum. Viruses not detected by usual methods like flavivirus and alphavirus were detected by multiplex PCR.

Comparing multiplex and multiplex real-time polymerase chain reaction with traditional blood culture in bacterial detection among patients with septicemia

Aims@#This study was aimed to test the specificity of primers and probes with target genes by using multiplex PCR and multiplex real-time PCR methods. These methods were compared with traditional blood culture methods in detecting five bacteria causing sepsis, including Acinetorbacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.@*Methodology and results@#A total of 587 blood samples from patients diagnosed with sepsis and septic shock were collected at Thanh Nhan Hospital, Hanoi, Vietnam. Each sample was divided into three parts for bacterial culture, multiplex PCR and multiplex real-time PCR to detect the similarity of the two PCR methods with the bacterial culture method. Conditions in multiplex PCR and multiplex real-time PCR were optimized to ensure the successful amplification of target genes. Results showed that the primers and probes were tested completely specific to the target genes and using multiplex PCR and multiplex real-time PCR techniques could detect five pathogens causing sepsis, including A. baumannii, K. pneumoniae, P. aeruginosa, E. coli and S. aureus.@*Conclusion, significance and impact of study@#Both multiplex PCR and multiplex real-time PCR methods have high similarities with the culture method, showing potential in the application of bacteria detection in sepsis.

A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation

@#Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/μl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Establishment and evaluation of a genome-wide multiplex PCR sequencing method for severe fever with thrombocytopenia syndrome virus based on high-throughput sequencing technology / 中华微生物学和免疫学杂志

Objective:To establish a sequencing method for the genome of severe fever with thrombocytopenia syndrome virus (SFTSV) based on next-generation sequencing (NGS).Methods:SFTSV RNA was extracted from serum samples of patients with severe fever with thrombocytopenia syndrome. SFTSV-specific primers were designed using Primer 5.0 software. A multiplex PCR method was constructed and used to amplify the nucleotide sequence of SFTSV. Whole-genome sequencing was performed on the NGS platform.Results:The whole genes of SFTSV isolates in 28 serum samples were amplified by the multiplex PCR with a coverage over 94%. Sequencing and phylogenetic analysis of those strains revealed that the predominant strains ( n=20) belonged to genotype A, followed by genotypes B ( n=4) and E ( n=3). Conclusions:A high-throughput sequencing method for SFTSV based on multiplex PCR was established in this study. This method was characterized by high specificity and good quality and could improve the sequencing efficiency.

One-step multiplex nested real-time RT-PCR assay for 2019-nCoV and Influenza A/B viruses detection / 中华检验医学杂志

Objective:To develop a single-tube one-step multiplex nested real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the simultaneous detection of 2019-nCoV, influenza A virus, influenza B virus and internal-control with human-derived gene.Methods:This study included 30 positive specimens for 2019-nCoV nucleic acid detection and 336 screening specimens collected from the arrivals at Guangzhou Baiyun Airport between February 2020 and February 2022. Sixty-four positive specimens of other respiratory pathogens were also collected from the arrivals at Guangzhou Baiyun Airport during the three-year period before the occurrence of COVID19 outbreak in 2020, and 7 positive viral strains of respiratory pathogens were provided by collaborative laboratories. In order to establish a set of multiplex nested real-time RT-PCR assay, a group of primers and probe combinations for a multiplex nested real-time RT-PCR was designed and screened according to a selection of nucleotide conserved regions of the ORF and N genes of 2019-nCoV and the M gene of influenza A and B viruses, while nested amplification primers and probe for the internal-control with human-derived gene were introduced. Then the prepared pseudovirus-positive quality control and sample discs were applied to evaluate the sensitivity and specificity. Clinical specimens were performed to validate the applicability of the method.Results:The results show that the established one-step multiplex nested real-time RT-PCR assay can specifically detect 2019-nCoV and influenza A and B viruses, with the limit-of-detection of about 125 copies/ml for 2019-nCoV and about 250 copies/ml for influenza A and B viruses. Totally 101 positive samples of various respiratory pathogens were detected, showing that the detection sensitivities of 2019-nCoV and influenza A and B viruses were 96.67%, 92.86% and 96.15%, respectively, with the specificity of 100%. No false-positive detection was found in the applied detection of more than 300 clinical samples.Conclusions:A one-step multiplex nested real-time RT-PCR assay for 2019-nCoV, influenza A and B viruses and human-derived gene internal-control was developed. The assay has good sensitivity and specificity and can be used for rapid screening of 2019-nCoV and influenza A and B viruses in high-volume samples.

Molecular biological characteristics of the 2019 novel Coronavirus in Shijiazhuang / 中华检验医学杂志

Objective:To analyze the molecular epidemiological characteristics of the Corona virus disease 2019 (COVID-19) cases in Shijiazhuang, which can reveal the origin of the outbreak and provide a scientific basis for COVID-19 prevention and control.Methods:From January 2 to January 8, 2021, a total of 404 samples from 170 COVID-19 cases were collected from the Shijiazhuang Fifth Hospital. The consensus sequence of 2019 novel Coronavirus(2019-nCoV) was obtained through multiplex polymerase chain reaction-based sequencing. The sequences of 170 COVID-19 cases were analyzed by the PANGOLIN, and the data were statistically analyzed by T-test.Results:Among the 404 COVID-19 samples, a total of 356 samples obtained high quality genome sequences (>95%,100×sequencing depth). The whole genome sequences of 170 COVID-19 cases were obtained by eliminating repeated samples. All 170 sequences were recognized as lineage B1.1 using PANGOLIN. The number of single nucleotide polymorphism arrange from 18-22 and most of the single nucleotide polymorphism were synonymous variants. All of 170 genomes could be classified into 48 sub-groups and most of the genomes were classified into 2 sub-groups (66 and 31, respectively).Conclusions:All cases in this study are likely originated from one imported case. The viruses have spread in the community for a long time and have mutated during the community transmission.

Estudio multicéntrico argentino sobre la utilidad del panel BCID2 del Sistema FilmArrayTM en la detección de bacteriemias / Argentine multicenter study on the usefulness of the BCID2 panel of the FilmArray™ System in the detection of bacteremia

Resumen El panel BCID2 de BioFire® (BioFire, Salt Lake City, EE.UU.) utiliza un análisis de PCR múltiple a partir de hemocultivos positivos con resultados en una hora. El objetivo de este estudio fue determinar el desempeño del método a partir de hemocultivos positivos de pacientes sépticos en 5 hospitales de la Argentina. Se incluyeron 121 pacientes y 124 episodios. Con respecto a la identificación microbiana, la sensibilidad global y la correspondiente a los microorganismos incluidos en la base de datos fue del 94% y 97% respectivamente. La sensibilidad del BCID2 para detectar CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B fue del 100% y la especificidad fue del 99% para NDM y VIM y del 100% para el resto. Esto llevó a cambios en el tratamiento antimicrobiano en 57/98 episodios (58%). El panel BCID2 es una herramienta importante para la adecuación del tratamiento antimicrobiano de pacientes con sepsis.

Abstract The BioFire® BCID2 panel (BioFire, Salt Lake City, UT) uses multiplex PCR analysis from positive blood cultures with results within one hour. The objective of this study was to determine the performance of the method from positive blood cultures of septic patients in 5 hospitals in Argentina. A total of 121 patients and 124 episodes were included. With regard to microbial identification, the global sensitivity and that corresponding to the microorganisms included in the database was 94% and 97%, respectively. The sensitivity of BCID2 to detect CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B was 100% and the specificity was 99% for NDM and VIM and 100% for the rest. This led to changes in antimicrobial treatment in 57/98 episodes (58%). The BCID2 panel is an important tool for the adequacy of antimicrobial treatment of patients with sepsis.

Resumo Estudo multicêntrico argentino sobre a utilidade do painel BCID2 do Sistema FilmArray™ na detecção de bacteremia O painel BCID2 de BioFire® B (BioFire, Salt Lake City, EUA) utiliza uma análise de PCR múltipla de hemoculturas positivas com resultados em uma hora. O objetivo deste estudo foi determinar o desempenho do método a partir de hemoculturas positivas de pacientes sépticos em 5 hospitais da Argentina. Cento e vinte e um pacientes e 124 episódios foram incluídos. No que se refere à identificação microbiana, a sensibilidade global e correspondente aos microrganismos incluídos na base de dados foi de 94% e 97%, respectivamente. A sensibilidade do BCID2 para detectar CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B foi de 100% e a especificidade foi de 99% para NDM e VIM e 100% para o resto. Isso levou a mudanças no tratamento antimicrobiano em 57/98 episódios (58%). O painel BCID2 é uma ferramenta importante para a adequação do tratamento antimicrobiano de pacientes com sepse.

El diagnóstico de Infecciones de Transmisión Sexual por la técnica de biología molecular es la mejor estrategia para su diagnóstico oportuno y específico. Un caso clínico / The diagnosis of Sexually Transmitted Infections by the molecular biology technique is the best strategy for its timely and specific diagnosis. A clinical case

Resumen Se presenta el caso de un paciente a quien se le diagnosticó una Infección de Transmisión Sexual (ITS) por la técnica de PCR múltiple y en quién se logró por esta técnica, detectar cuatro agentes diferentes simultáneamente: Neisseria gonorreae, Mycoplasma hominis, Ureaplasma urealyticum/parvum y Trichomonas vaginalis, situación esta, que no hubiera sido posible utilizando el procedimiento estándar.

Summary Here we report the case of a patient with a Sexually Transmitted Disease (STI) in whom four different agents were detected by a multiple PCR technique: Neisseria gonorreae, Mycoplasma hominis, Ureaplasma urealyticum / parvum and Trichomonas vaginalis. This detection of multiple agents would not have been possible using conventional procedures.

Utilidad del sistema FilmArrayTM en la optimización del tratamiento antimicrobiano de pacientes con bacteriemia / FilmArrayTM BCID panel usefulness for the optimization of the antimicrobial treatment in patients with bacteremia / Utilidade do sistema FilmArrayTM na otimização do tratamento antimicrobiano de pacientes com bacteremia

Resumen La bacteriemia es una de las principales causas de muerte en todo el mundo y se asocia con alto costo. Los resultados rápidos obtenidos desde los hemocultivos positivos son una herramienta importante para la optimización temprana del tratamiento antimicrobiano. Los objetivos de este trabajo fueron evaluar el rendimiento del panel BCID del sistema FilmArrayTM y determinar su impacto en la adecuación del tratamiento antimicrobiano. Se analizaron 127 episodios de bacteriemia. Un porcentaje significativo de los tratamientos (45,8%) fueron cambiados en base a este resultado. La identificación global correcta fue del 89,2% y del 97,2% para los microorganismos incluidos en la base de datos, en tanto que la sensibilidad para la detección de los genes mecA y KPC fue del 100%. El panel BCID de FilmArrayTM es un método rápido y confiable para la detección de los microorganismos relacionados a bacteriemia y de alto impacto en la decisión terapéutica.

Abstract Bacteremia is one of the leading causes of death worldwide and is associated with high cost. The rapid results obtained from positive blood cultures are an important tool for early optimization of antimicrobial therapy. The objectives of this work were to evaluate the performance of the BCID panel of the FilmArrayTM system and determine its impact on the adequacy of the antimicrobial treatment. One hundred and twenty seven episodes of bacteremia were analyzed. A significant percentage of the treatments (45.8%) were changed based on this result. The correct global identification was 89.2% and 97.2% for the microorganisms included in the database, while the sensitivity for the detection of the mecA and KPC genes was 100%. The FilmArrayTM BCID panel is a fast and reliable method for the detection of microorganisms related to bacteremia and has a high impact on the therapeutic decision.

Resumo A bacteremia é uma das principais causas de morte no mundo e está associada a alto custo. Resultados rápidos obtidos de hemoculturas positivas são uma ferramenta importante para a otimização precoce da terapia antimicrobiana. Os objetivos deste trabalho foram avaliar o desempenho do painel BCID do sistema FilmArrayTM e determinar seu impacto na adequação do tratamento antimicrobiano. Foram analisados 127 episódios de bacteremia. Percentual significativo dos tratamentos (45,8%) foi alterado com base nesse resultado. A correta identificação global foi de 89,2% e 97,2% para os microrganismos incluídos na base de dados, enquanto a sensibilidade para detecção dos genes mecA e KPC foi de 100%. O painel FilmArrayTM BCID é um método rápido e confiável para a detecção de microrganismos relacionados à bacteremia e tem alto impacto na decisão terapêutica.

Estudio colaborativo sobre la utilidad del panel Meningitis/Encephalitis FilmArray (Biofire) en el diagnóstico de pacientes con sospecha de meningitis o encefalitis / Collaborative study on the usefulness of the Meningitis/Encephalitis FilmArray (Biofire) panel in the diagnosis of patients with suspected meningitis or encephalitis / Estudo colaborativo sobre a utilidade do painel Meningitis/Encephalitis FilmArray (Biofire) no diagnóstico de pacientes com suspeita de meningite ou encefalite

Resumen El tratamiento inicial para pacientes con sospecha de meningitis bacteriana aguda depende de una evaluación diagnóstica rápida y una terapia antimicrobiana adecuada. El panel de Meningitis/Encephalitis FilmArray (ME) (BioFire Diagnostics, Salt Lake City, EE.UU.) utiliza análisis de PCR múltiple e incluye 14 microorganismos; los resultados se obtienen directamente del LCR en 1 h. Los objetivos de este estudio fueron: a) determinar el rendimiento del ME y b) establecer el impacto del resultado obtenido en el tratamiento antimicrobiano de pacientes con meningoencefalitis. Se incluyeron 112 pacientes y 26 episodios de meningitis. Del total de resultados positivos con el panel para bacterias y hongos, 4/13 no se aislaron de cultivos y correspondieron a pacientes con tratamiento antimicrobiano. De los 26 episodios, los resultados condujeron a cambios terapéuticos en 21 casos. El ME es una herramienta rápida y útil para adecuar un tratamiento antimicrobiano en pacientes con meningoencefalitis.

Abstract The initial treatment for patients with suspected acute bacterial meningitis depends on a rapid diagnostic evaluation and adequate antimicrobial therapy. The Meningitis/Encephalitis FilmArray panel (ME) (BioFire Diagnostics, Salt Lake City, USA) uses multiplex PCR analysis and includes 14 microorganisms; the results are obtained directly from the CSF in 1 h. The objectives of this study were: a) to determine the performance of ME and b) to establish the impact of the result obtained on the antimicrobial treatment of patients with meningoencephalitis. A number of 112 patients and 26 episodes of meningitis were included. Of the total positive results for bacteria and fungi with the panel, 4/13 were not isolated from cultures and corresponded to patients with antimicrobial treatment. Of the 26 episodes, the results led to therapeutic changes in 21 cases. ME is a quick and useful tool to adapt antimicrobial treatment in patients with meningoencephalitis.

Resumo O tratamento inicial para pacientes com suspeita de meningite bacteriana aguda depende de uma avaliação diagnóstica rápida e terapia antimicrobiana adequada. O painel de Meningitis/Encephalitis FilmArray (ME) (BioFire Diagnostics, Salt Lake City, EUA) usa análise PCR multiplex e inclui 14 microrganismos; os resultados são obtidos diretamente do LCR em 1 h. Os objetivos deste estudo foram: a) determinar o desempenho do ME e b) estabelecer o impacto do resultado obtido no tratamento antimicrobiano de pacientes com meningoencefalite. 112 pacientes e 26 episódios de meningite foram incluídos. Do total de resultados positivos com o painel para bactérias e fungos, 4/13 não foram isolados das culturas e corresponderam a pacientes em tratamento antimicrobiano. Dos 26 episódios, os resultados levaram a alterações terapêuticas em 21 casos. ME é uma ferramenta rápida e útil para adaptar o tratamento antimicrobiano em pacientes com meningoencefalite.