Utilidad de la implementación de un método molecular automatizado en el diagnóstico de agentes causales de infecciones de transmisión sexual / Usefulness of the implementation of an automated molecular method in the diagnosis of causal agents of sexually transmitted infections / Utilidade da implementação de um método molecular automatizado no diagnóstico de agentes causais de infecções sexualmente transmissíveis

Resumen Se presentan los resultados obtenidos en los primeros meses desde la implementación del diagnóstico de agentes causales de infecciones de transmisión sexual (ITS) por PCR en tiempo real en un sistema automatizado. Se estudiaron 46 muestras endocervicales y 39 muestras de uretra masculina, por examen microscópico en fresco, coloración de Gram, cultivo en agar sangre, agar Thayer Martin, galerías miniaturizadas para investigar Ureaplasma spp. y Mycoplasma hominis (Mycoplasma IST3, bioMérieux, Francia) y PCR múltiple para Neisseria gonorrhoeae, Chlamydia trachomatis . Trichomonas vaginalis (BDMaxTM System, Becton Dickinson, EE.UU.). Mediante PCR múltiple se detectó un agente vinculado a ITS en el 48,7% de las muestras uretrales y en el 21,7% de las muestras endocervicales, además de 5 casos de coinfección. El 18,8% de las infecciones se diagnosticaron sólo por PCR. Estos datos demuestran que mediante PCR se optimizó el diagnóstico de ITS en personas de ambos sexos.

Abstract The results obtained in the first months after the implementation of the diagnosis of causative agents of sexually transmitted infections (STIs) by real- time PCR in an automated system are presented. Forty-six endocervical samples and 39 male urethral samples were studied by fresh microscopic examination, Gram staining, blood agar culture, Thayer Martin agar, miniaturised galleries to investigate Ureaplasma spp. and Mycoplasma hominis (Mycoplasma IST3, bioMérieux, France), and multiplex PCR for Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis (BDMaxTM System, Becton Dickinson, USA). Using multiplex PCR, an agent linked to STIs was detected in 48.7% of the urethral samples and in 21.7% of the endocervical samples, in addition to 5 cases of coinfection. A total of 18.8% of the infections were diagnosed only by PCR. These data show that PCR optimised the diagnosis of STIs in people of both sexes.

Resumo São apresentados os resultados obtidos nos primeiros meses a partir da implementação do diagnóstico de agentes causadores de infecções sexualmente transmissíveis (ISTs) por PCR em tempo real em um sistema automatizado. Quarenta e seis amostras endocervicais e 39 amostras uretrais masculinas foram estudadas por exame microscópico fresco, coloração de Gram, cultura de ágar sangue, ágar Thayer Martin, galerias miniaturizadas para investigar Ureaplasma spp. e Mycoplasma hominis (Mycoplasma IST3, bioMérieux, França) e PCR múltiplo para Neisseria gonorrhoeae, Chlamydia trachomatis e Trichomonas vaginalis (BDMaxTM System, Becton Dickinson, EUA). Utilizando PCR múltiplo, um agente ligado a IST foi detectado em 48,7% das amostras uretrais e em 21,7% das amostras endocervicais, além de 5 casos de coinfecção; 18,8% das infecções foram diagnosticadas apenas por PCR. Esses dados mostram que através do PCR foi otimizado o diagnóstico de IST em pessoas de ambos os sexos.

Experiencia de dos años con el uso de panel de RPC múltiple de meningitis-encefalitis en pacientes pediátricos en un hospital de tercer nivel en Argentina

Introducción: La meningitis bacteriana aguda (MBA) y la encefalitis son infecciones graves y el retraso en el tratamiento determina mayor morbimortalidad. En 2015 la FDA. aprobó un panel de RPC múltiple, BioFire® Filmarray® meningitis-encefalitis (FA-ME), que desde el 2019 se encuentra disponible en nuestro hospital. Objetivos: Estimar número de determinaciones positivas mediante FA-ME, evaluar concordancia con cultivo convencional (CC) y describir si FA-ME permitió realizar cambios en el tratamiento. Material y Métodos: Estudio retrospectivo, descriptivo, realizado durante 2019-2021 en el Hospital de Niños Pedro Elizalde. Se revisaron reportes de niños con meningitis, encefalitis y meningoencefalitis y líquido-cefalorraquídeo patológico a quienes se les realizó FA-ME. Resultados: Se incluyó a 32 niños, edad promedio: 48 meses. Fueron positivas 13 determinaciones de FA-ME: siete bacterias y seis virus. En dos MBA obtuvo desarrollo mediante CC. Con FA-ME se ajustó el tratamiento en dos MBA y se acortó el tratamiento intravenoso (IV). Discusión: Nuestro trabajo permitió conocer la etiología de cinco MBA con cultivo negativo, de las cuales dos habían recibido antimicrobianos, administrar quimioprofilaxis a contactos epidemiológicos, acortar el tratamiento IV y suministrar menos dosis de aciclovir; en concordancia con la literatura médica. Conclusiones: FA-ME permitió identificar la etiología en cinco MBA que no desarrollaron en CC, ajustar tratamientos empíricos inadecuados y acortar duración del tratamiento parenteral.

Background: Bacterial meningitis and encephalitis are life-threatening infections, a delay in its treatment is associated with high mortality. In 2015, FDA approved the Multiplex PCR FilmArray™ meningitis/encephalitis syndromic panel (FA-MEP), and it is available in our hospital since 2019. Aim: To estimate the number of positive FA-MEP, to evaluate the correlation to conventional culture (CC) results and to describe if the FA-MEP technology allowed changes in the treatment. Methods: Retrospective analysis of children with meningitis, encephalitis and meningoencephalitis and pathological cerebrospinal fluid analysis between 2019-2021, who were subject to FA-MEP testing at the Pedro Elizalde Children's Hospital. Results: 32 children, mean age: 48 months. 11 patients had positive FA-ME tests: 7 bacterial, 6 viral. 2 patients correlated with CC. Based on the FAMEP results, treatment was adjusted in 2 bacterial meningitis and the duration of intravenous treatment was shortened. Discussion: Our study allowed to establish the etiology of 5 culture negative bacterial meningitis, (2 had prior antibiotics), administer chemoprophylaxis to close contacts, and to administer fewer doses of acyclovir. Conclusions: The FA-MEP allowed us to identify 5 bacterial meningitis that tested negative by CC and early adjustment of inappropriate empirical antibiotics and to shorten the duration of parenteral treatments.

Implementación del método de FilmArrayTM en pacientes pediátricos con infecciones severas / Implementation of the FilmArrayTM method in pediatric patients with severe infections / Implementação do método de matriz do FilmArrayTM em pacientes pediátricos com infecções graves

Se evaluó la implementación del método de PCR múltiple FilmArrayTM (Biofire Diagnostics, LLC, EE.UU.) en 21 niños con infección respiratoria aguda baja, 3 con meningoencefalitis, y un caso de sepsis. Se registraron el tiempo de demora hasta obtener el resultado y adecuar el tratamiento, los días de internación, los patógenos detectados y el costo de la incorporación de esta metodología. En los niños estudiados con FilmArrayTM el resultado estuvo disponible a los 90 minutos desde la toma de la muestra. Se detectaron patógenos no demostrados por los métodos disponibles, como Rhinovirus, además de diagnosticar coinfección viral; el tiempo promedio de estadía resultó 5 días. Se estimó reducir un 40% el costo global de internación. La implementación de FilmArrayTM resultó sencilla y se pudo incorporar a la sistemática de trabajo. Si bien esta experiencia incluyó un bajo número de pacientes, aportó información que demuestra el potencial de esta metodología para un mejor manejo del paciente crítico.

The implementation of multiple PCR FilmArrayTM (Biofire Diagnostics, LLC, USA) for 21 children with low acute respiratory infection, 3 with meningoencephalitis, and 1 case of sepsis was evaluated. Delay time until the result was obtained and the treatment adapted, hospitalization days, pathogens detected and the cost of incorporating this methodology were all recorded. In the children studied with FilmArrayTM the result was available 90 minutes after the sample was taken. Pathogens were not demonstrated by the available methods, such as Rhinovirus, apart from diagnosing viral coinfection, the average length of stay was 5 days. It was estimated to reduce the overall cost of hospitalization by 40%. The implementation of FilmArrayTM was simple and could be incorporated into the work system. Although this experience included a low number of patients, it provided information that demonstrates the potential of this methodology for better management of the critical patient.

Foi avaliada a implementação do método de PCR múltiplo FilmArrayTM (Biofire Diagnostics, LLC, EUA) em 21 crianças com infecção respiratória aguda baixa, 3 com meningoencefalite e um caso de sepse. O tempo de atraso foi registrado até a obtenção do resultado e a adaptação do tratamento, dias de internação, patógenos detectados e o custo da incorporação dessa metodologia. Nas crianças estudadas com o FilmArrayTM, o resultado ficou disponível 90 minutos após a coleta da amostra; foram detectados os patógenos não demonstrados pelos métodos disponíveis, como o Rinovírus, além de diagnosticar a coinfecção viral; o tempo médio de permanência foi de 5 dias. Foi estimado reduzir o custo total da hospitalização em 40%. A implementação do FilmArrayTM foi simples e pôde ser incorporada à sistemática de trabalho. Embora essa experiência tenha incluído um número de pacientes baixo, forneceu informações que demonstram o potencial dessa metodologia para um melhor gerenciamento do paciente crítico.

Development of a DNA-based microarray for detection of nine pathogens causing rash and fever illness / 军事医学

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71, Varicella zoster virus,Dengue fever virus,Human small FDNA virus B19,Coxsackie virus type A16,A-βStreptococcus pyogenes (hemolytic streptococcus)and Salmonella typhi.Methods Primers and probes were designed based on the specific sequence in the conserved region of genomes of the nine pathogens.The nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method.The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration to identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imaging,the specificity,sensitivity and reproducibility of the chip were evaluated.The serial diluted nucleic acid of Enterovirus type 71 was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Results Nine specific primers and eleven specific probes were selected.The microarray demonstrated high sensitivity and specificity.The minimum detection limit of plasmid DNA and in vitro transcribed RNAs was 3 ×103 copies per reaction.The detection sensitivity of this microarray was 10 percent of that by the real-time PCR method.The rate of sensitivity and specificity of clinical sample detection was 95% and 85.7% respectively,and the rate of accuracy was 93.2%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for clinical diagnosis and epidemiological investigation of rash and fever illnesses.

Comparison of Classical and Molecular Typing Methods for Streptococcus Pneumoniae / 现代检验医学杂志

Objective To compare classical and molecular typing methods for Streptococcus pneumoniae,in order to provide fasty and accurate method for classification.Methods Selected the 150 strains of Streptococcus pneumoniae which isolated from the First People’s Hospital of Zhaoqing from October 2013 to February 2015 for the research objects,using the classi-cal quellung classification method and molecular typing of multiple PCR method for classification.Compared the result and the rate of the classification,used the non parametric test of two independent samples for statistical analysis several higer rate of serotypes,and calculated the total coincidence rate,sensitivity and specificity.Results The typing rate of Streptococ-cus pneumonia capsular was 53.3%,and the main serum group were 19(32.7%),6(7.3%)and 23(4%)respectively.The serotype of 19F(23.3%),19A(2.7%),6C(2%)and 23F(2.7%)respectively by single factor serum.The typing rate of the multiple PCR method was 76.0%,and the main serum group were 19(41.3%),6(10.7%)and 23(10.7%).The serotype were mainly 19F(32.7%),19A(8.0%),6(10.7%)and 23F(9.3%).Used the 2 Independent Sample Test for statistical a-nalysis,compared with the serotypes 19F,19A and 23F(P>0.05).There was no statistically significant difference in the distribution of the types of the two methods.The total coincidence rate was 74.6%.To verify the classic quellung method by multiple PCR method,sensitivity was 70.2%,and specificity was 100%.That showed the capsular swelling method was less sensitivity than the molecular typing method.Conclusion Compared with the traditional serological typing techniques,mo-lecular typing technique have the advantages of fast,accurate,sensitive and high resolution,and become the classification method which can be rely on.

Gene chip technique in detection of vancomycin-resistant Enterococcus gene / 军事医学

Objective To develop a detection method based on the technology of gene chips which can quickly distinguish genes of Enterococcus faecalis, E.faecium and vancomycin resistance.Methods Based on the specific gene ( ddl) sequences of two types of Enterococcus from GenBank, oligonucleotide probes which could detect and distinguish special genes and drug resistance genes ( vanA,vanB) of Enterococcus were designed and compounded.Then,the probes were dotted to modified slide.The target DNA fragments of vancomycin-resistant Enterococcus ( VRE) were labeled with biotin by multiple PCR amplification, and then hybridized with oligonucleotide probes on slide.The results were analyzed by portable imager.The multiple PCR system, hybridization reaction and condition of the chemiluminescence method were optimized before the specificity, sensitivity and reproducibility of the chip were evaluated.Results One universal primer, four specific primers, one universal probe and four specific probes were selected.This gene chip was demonstrated of high specificity and repeatability.The detection sensitivity was 103 CFU/ml.The gene chip detection results of 10 clinical samples were basically consistent with the drug sensitivity test ( 8/10 ) .Conclusion A gene chip technique for the detection of VRE is established successfully.It is possible to distinguish the type of VRE and detect the genetic phenotypes of drug resistance by gene chip technique.

Development of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae non-O1/O139 serogroups / 中华流行病学杂志

Objective To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V.cholerae) serogroups non-O1 and non-O139.Methods The outer membrane protein gene (ompW) specific for V.cholerae,as well as O antigen rfb genes specific for both O1 and O139,were used for the design of the PCR primers.Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139.Specific rfb genes and ompW were developed to evaluate their specificity,limit of detection,reproducibility and consistency.Results We established multiple PCR and real-time SYBR green PCR methods.According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR),both methods could specifically detect the non-O1,non-O139 V.cholerae,and to differentiate them from O1,O139 V.cholerae,other five Vibrios and 3 intestinal bacteria.The detection limits were 7 × 104 cfu/ml (multiple PCR) and 7 × 102 cfu/ml (real-time SYBR green PCR),with statistically significant difference seen (P＜0.05).For the reproducibility of real-time SYBR green PCR,the external coefficient variation ranging from 0.22％ to 0.92％ while the internal coefficient variation ranging from 0.27％ to 1.41％.370 strains of non-O1,non-O139 V.cholerae,were detected,with both consistency rates as 100％.Conclusion Both multiple PCR and real-time SYBR green PCR could detect non-O1,non-O139 V.cholerae,rapidly,specifically,and reproducibly,that could all be used for the detection and identification of non-O 1,non-O 139 under different conditions.

Identification and characteristic of Testudinis carapax et Plastrum and other turtle species by multiple PCR / 中国药学杂志

OBJECTIVE: To explore the characteristic of Chinese herbal medicine Testudinis carapax et Plastrum by the multiple PCR technique. METHODS: The salting out method was modified to extract mitochondrial DNA (mtDNA) from Testudinis carapax et Plastrum and their counterfeits. The sequences of cytochrome b (Cyt b) and cytochrome c oxidase subunit I (Col) genes were downloaded from the GenBank database, and two specific pairs of primers were designed with Premier 5.0. The genes of Testudinis carapax et Plastrum were amplified with multiple PCR and sequenced. RESULTS: The size of mtDNA got by salting out method was 16.6×103 bp, and two bright stripes between 300-500 bp were shown in agarose gel electrophoresis of authentic Testudinis carapax et Plastrum, only one or no stripe appeared for the counterfeits. CONCLUSION: The multiple PCR technique is specific, simple, and accurate for differentiation of authentic Testudinis carapax et Plastrum from their counterfeits.

De genes de toxinas pirogénicas y toxinas exfoliativas en aislamientos clínicos de staphylococcus aureus en colombia

Introducción: staphylococcus aureus está asociado con graves enfermedades sistémicas causadas por superantígenos (toxinas pirogénicas y exfoliativas). Métodos: 100 aislamientos clínicos de S. aureus se identificaron por método automatizado y PCR, la prevalencia de genes de superantígenos por PCR múltiple y las correlaciones mediante la prueba exacta de Fischer. Resultados: en 38 aislamientos se observó que la prevalencia de los genes de enterotoxinas, toxina del síndrome de choque tóxico y toxinas exfoliativas fue 44%, 7% y 4%, respectivamente. La única correlación significativa (p = 0,045) fue entre la presencia de los genes de superantígenos y los aislamientos hospitalarios. Conclusiones: existe una alta prevalencia de genes de enterotoxinas y una baja de genes de toxinas exfoliativas y del síndrome de choque tóxico en aislamientos de S. aureus en esta población. Esta es la primera investigación que presenta datos de prevalencia de superantígenos en Colombia, y proporciona nueva información para América Latina.

Introduction: staphylococcus aureus is associated with serious systematic diseases caused by super antigens (pyogenic and exfoliating toxins). Methods: 100 clinical isolations for S. Aureus were identified by automatic methods and PCR, the prevalence of super antigen genes by multiple PCR and the correlations thru the Fischer exact test. Results: 38 isolation cases were observed and the prevalence of the enterotoxin genes, toxins of the syndrome of toxic shock and exfoliating toxins was 44%, 7% and 4% respectively. The only significant correlation found (p=0.045) was between the presence of the super antigen genes and the clinical isolations. Conclussion: there is a high prevalence of enterotoxin genes and a low prevalence of exfoliating genes and the syndrome of toxic shock in isolation of S.Aureus in this population. This is the first investigation that yields data of prevalence of super antigens in Colombia and provides new information for Latin America.

Introdução: staphylococcus aureus está associado a graves enfermidades sistêmicas causadas por superantígenos (toxinas pirogênicas e esfoliativas). Métodos: cem isolamentos clínicos de S. aureus foram identificados por método automatizado e PCR, a prevalência de penas de superantígenos por PCR múltiplos e correlações mediante a prova exata de Fischer. Resultados: em 38 isolamentos observou-se que a prevalência dos genes de en-terotoxinas, toxina da síndrome de choque tóxico e toxinas esfoliativas foi 44%, 7% e 4%, respectivamente. A única correlação significativa (p = 0,045) foi entre a presença dos genes de superantígenos e os isolamentos hospitalares. Conclusões: existe uma alta prevalência de genes de enterotoxinas e uma baixa de genes de toxinas esfoliativas e da síndrome do choque tóxico em isolamento de S. aureus nessa população. Esta é a primeira pesquisa que apresenta dados de prevalência de superantígenos na Colômbia e propicia nova informação para América Latina.

Detection of Herpes simplex Virus,Treponema pallidum and Haemophilus ducreyi by Multiple PCR / 中华医院感染学杂志

OBJECTIVE To establish multiple PCR(M-PCR) assay for simultaneous detection of Treponema pallidum(TP) and herpes simplex virus(HSV),Haemophilus ducreyi(HD).METHODS According to specific gene sequence of the three agents:HSV DNA polymerse gene,TP tpp47 gene,HD 6s rRNA gene;three sets of specific primers were designed and a multiple PCR assay was developed to detect 3 agents in one test.RESULTS The target DNA of 3 agents were specifically amplified by their specific primers,the appropriate size of four DNA fragments was 202bp,252bp,152bp for TP,HSV and HD,respectively.The DNA of other several common microbes of genital tract and human genome could not be amplified by three sets of primers.CONCLUSIONS Primary study indicated that the M-PCR assay can simultaneously,specifically and rapidly detect out HSV,TP and HD.

Detection of hMSH2 gene mutations in gynecologic tumor population by multiple PCR-SSCP / 中国癌症杂志

Purpose:To set up a new method to screen hMSH2 gene mutations, to detect hMSH2 gene mutations in gynecologic tumor population and to find molecular biomarkers of tumor. Methods:The basic data and blood samples from 42 gynecologic tumor patients werre collected. The genetic mutations of the sixth exon and seventh exon of hMSH2 gene were investigated by multiple polymerase chain reaction single strand conformation polymorphism (PCR SSCP),followed by DNA sequencing. Results:The successes in setting up the multiple PCR SSCP method made it possible to detect hMSH2 gene mutations more quickly and more economically. Two samples of the seventh exon mutation was detected in the tumor population. The mutation is Leu→Phe missense mutation. No mutation of the sixth exon was detected by SSCP.Conclusions:The multiple PCR SSCP method is effective in detecting genetic mutations. There is a mutation site in the seventh exon of hMSH2 gene. It is probably a biomarker of gynecologic tumor. This discovery might offer the basis for further investigation of mutations in large amount population and studies of the function of the mutation.