Distinguishing between Artemisia stolonifera and A. argyi by specific PCR of leaves and non-glandular trichomes / 中国中药杂志

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification / 生物医学与环境科学（英文）

Objective@#To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .@*Methods@#We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).@*Results@#CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.@*Conclusion@#The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Isothermal amplification technology based on microfluidic chip / 生物工程学报

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus / 生物医学与环境科学（英文）

OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Detection of drug resistance genes of mycobacterium tuberculosis by rolling circle amplification technique with multicolor fluorescent probes / 中华预防医学杂志

The aim of this study was to construct a simple, rapid and ultra-sensitive optical biosensing technique based on rolling circle amplification (RCA), and to apply it to multiple detection of drug-resistant genes of mycobacterium tuberculosis. The common mutation sites of isoniazid, rifampicin and streptomycin resistance genes are katG315 (AGC➝ACC), rpoB531 (CAC➝TAC) and rpsL43 (AAG➝AGG). For these three gene sites, from February 2020 to May 2021, in the Department of Laboratory Medicine of the First Affiliated Hospital of Army Military Medical University, the padlock probe (PLP), primers and capture probes were designed. And a solid-phase RCA constant temperature amplification reaction system based on magnetic beads was constructed and the experimental parameters were optimized. The RCA products were accurately captured by the multicolor fluorescent probes (Cy3/Cy5/ROX), and the single-tube multiple detection of three mutation genes was realized. The sensitivity, specificity and linear range of this method were further verified. The results showed that the response range of katG315 in the same reaction system ranged from 1.0 pmol/L to 0.1 nmol/L. The response range of rpoB531 and rpsL43 ranged from 1.0 pmol/L to 50.0 pmol/L and 1.0 pmol/L to 20.0 pmol/L, and the method had good specificity and sensitivity, and could accurately identify single base mutations in mixed targets, with the minimum detection limit as low as 1.0 pmol/L. The recoveries of simulated serum samples were 95.0%-105.2%. In conclusion, the constant temperature amplification multiple detection method constructed in this study can quickly realize the single-tube multiple detection of three drug resistance mutation sites. This technology is low-cost, simple and rapid, and does not rely on large equipment, providing a new analysis method for pathogen drug resistance gene detection.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection / 生物医学与环境科学（英文）

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride / Acompanhamento de bovinos leiteiros naturalmente infectados com Trypanosoma vivax após tratamento com cloreto de isometamidium

Abstract Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

Resumo Em bovinos, infecções por Trypanosoma vivax geram sinais clínicos inespecíficos que, associados a intervalos aparasitêmicos, faz com que o diagnóstico da enfermidade seja desafiador. No Brasil, somente aceturato de diaminazeno e cloridrato de isometamidum (ISM) estão disponíveis para o tratamento da tripanossomose bovina. Este trabalho teve como objetivo acompanhar bovinos leiteiros naturalmente infectados por T. vivax, após o tratamento com ISM por meio de técnicas moleculares e sorológica. Foram utilizados 30 bovinos naturalmente infectados com T. vivax, sendo estes tratados com duas aplicações de ISM, na dosagem de 1,0 mg/kg por via intramuscular profunda, nos dias 0 e 150. Foram avaliadas, para diagnóstico de T. vivax, amostras de sangue acrescido de EDTA e soro, colhidas nos 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 e 240 dias após os tratamentos pela reação em cadeia da polimerase (PCR), amplificação circular isotérmica do DNA (LAMP) e ensaio de imunoabsorção enzimático (ELISA). Verificou-se a presença de animais com persistência na detecção de DNA de T. vivax pela PCR e LAMP, bem como detecção contínua de anticorpos IgG anti-T. vivax pelo método de ELISA, sugerindo a presença de resistência de T. vivax ao ISM. A combinação dos testes LAMP e ELISA pode evitar falsos diagnósticos da eliminação do parasita nos bovinos tratados, contribuindo para um melhor controle da doença. Este é o primeiro experimento que demonstra infecção persistente do T. vivax em rebanho naturalmente infectado, tratado com ISM, e evidencia possível resistência ao quimioterápico no Brasil.

Evaluación de una reacción en cadena de la polimerasa en tiempo real simple y rápida para la cuantificación del ADN del virus de la hepatitis B / Evaluation of a simple and fast real time polymerase chain reaction assay for quantification of hepatitis B virus DNA

Introducción: Los ensayos para cuantificar el ADN del virus de la hepatitis B (VHB) o carga viral son imprescindibles en el diagnóstico y en el seguimiento de los pacientes con hepatitis B crónica; de ahí que estén disponibles estuches diagnósticos para esta función. En el presente estudio se muestra la validación de SUMASIGNAL VHB (un paso), el cual es un sistema de reacción en cadena de la polimerasa en tiempo real (RCP-TR) para la cuantificación del genoma del VHB, propuesto por el Centro de Inmunoensayo. Objetivo: Evaluar el desempeño analítico de SUMASIGNAL VHB (un paso). Métodos: Se utilizó un panel de 80 muestras de suero bien caracterizadas y el Tercer Estándar Internacional de la OMS para las técnicas de amplificación de ácidos nucleicos del virus de la hepatitis B. Se determinaron las características del ensayo como especificidad clínica, especificidad analítica (reactividad cruzada), rango lineal o linealidad y exactitud, precisión intraensayo y comparación con un ensayo de referencia. Resultados: La especificidad analítica y clínica fue del 100 por ciento. Al evaluar la linealidad y exactitud con un estándar de referencia de la OMS, se obtuvo que la totalidad de las diferencias entre los Log10 del valor obtenido y el de referencia resultaron inferiores a 0,5 Log10 (r= 0,9977 y r2= 0,9954). Además, se obtuvieron bajos coeficientes de variación intraensayo. La evaluación comparativa con el estuche comercial Artus HBV RG PCR kit mostró una correlación fuerte (r= 0,8882). Conclusiones: SUMASIGNAL VHB (un paso) es un ensayo fácil de realizar manualmente, es rápido e incluye reactivos de extracción de ácidos nucleicos. Teniendo en cuenta la validez del método para el uso previsto, puede ser recomendado para su introducción en el diagnóstico, la vigilancia y la indicación de tratamiento en los pacientes con hepatitis B crónica(AU)

Introduction: Assays to quantify hepatitis B virus (HBV) DNA or viral load are indispensable for the diagnosis and follow-up of patients with chronic hepatitis B, hence the availability of diagnostic kits for this purpose. The present study deals with the validation of HBV SUMASIGNAL (one step), a real time polymerase chain reaction (RT-PCR) system for quantification of the HBV genome proposed by the Immunoassay Center. Objective: Evaluate the analytical performance of HBV SUMASIGNAL (one step). Methods: Use was made of a panel of 80 well characterized serum samples and the Third WHO International Standard for hepatitis B virus nucleic acid amplification techniques. Determination was performed of assay characteristics such as clinical specificity, analytical specificity (cross-reactivity), linear range or linearity and accuracy, intra-assay precision and comparison with a reference assay. Results: Analytical and clinical specificity was 100 percent. Evaluation of linearity and accuracy with a WHO reference standard revealed that all the differences between the log10 of the value obtained and the reference value were lower than 0.5 log10 (r= 0.9977 and r2= 0.9954). The intra-assay variation coefficients obtained were low. Comparative evaluation with the commercial Artus HBV RG PCR kit showed a strong correlation (r= 0.8882). Conclusions: The assay HBV SUMASIGNAL (one step) is easy to conduct manually, fast and includes reagents for nucleic acid extraction. Based on the validity of the method for the use in mind, it may be recommended for incorporation into the diagnosis, surveillance and treatment of patients with chronic hepatitis B(AU)

Diagnóstico de la Tricomonas vaginalis en la mujer / Diagnosis of Trichomonas vaginalis in women

OBJETIVO: revisar los diferentes métodos de diagnóstico de la tricomoniasis vaginal disponibles hasta el presente. MATERIALES Y MÉTODOS: se revisó la bibliografía latinoamericano e internacional a través de los sitios electrónicos de Pub-Med y Scielo. RESULTADOS: la Tricomonas vaginalis es considera como la enfermedad de transmisión sexual no viral, curable más frecuente y prevalente en el mundo. Se revisan los diferentes de métodos para diagnosticar la presencia de la tricomonas vaginalis en pacientes femeninos con síntomas y signos de la infección producida por el protozoario flagelado. CONCLUSIONES: se revisaron los diferentes métodos de diagnostico de la infección producida por la Tricomonas vaginalis en pacientes femeninas, desde los clásicos hasta los más actuales que emplean alta tecnología.

OBJECTIVE: to review the different diagnostic methods of Trichomonas vaginal available at the present time. MATERIAL AND METHOD: it was reviewed the Latin-American and international bibliography using the Pub-Med and Scielo web sites. RESULTS: Trichomonas vaginalis is considered the most common and prevalent sexual transmitted disease curable and non-viral worldwide. It was reviewed the different methods to diagnose the presence of Trichomonas vaginalis in female patients with symptoms and signs of infection produces by the flagellate protozoa. CONCLUSION: Different methods of diagnosis of the infection produced by Trichomonas vaginalis, since the classics to the most current methods that use high technology, were reviewed.

Probable Association Between Oral Lichen Planus and presence of Helicobacter Pylori: a Preliminary Study in a Chilean Population / Probable Asociación entre Liquen plano Oral y la presencia de Helicobacter pylori: estudio Preliminar en Chile

ABSTRACT: Oral Lichen planus (OLP) is one of the main inflammatory diseases of the oral mucosa that is considered as a potentially malignant disorder. The exact pathogenesis of OLP remains to be completely understood. However, presence of bacteria has been associated to the inflammatory response observed in OLP. Particularly, Helicobacter pylori a major etiological agent of gastrointestinal inflammatory diseases and risk factor for gastric cancer, has been associated to Lichen planus. Here we studied a group of Chilean patients if there is any association between the presence of Helicobacter pylori and the clinical manifestation of OLP. We found a significant difference between the patients positive for H. pylori and the age of OLP diagnosis, suggesting that oral H. pylori might induce the disease at an earlier age. However, we could not confirm a statistically significance between the presence of the bacteria and OLP.

RESUMEN: Liquen Plano Oral (LPO) es una enfermedad inflamatoria de la mucosa oral considerada como desorden potencialmente maligno. La patogénesis exacta de LPO es desconocida. Sin embargo, se ha asociado la presencia de bacterias como responsables de la inflamación observada en LPO. Particularmente, Helicobacter pylori (H. pylori), agente etiológico principal de enfermedades inflamatorias gastrointestinales y factor de riesgo de cáncer gástrico, ha sido asociado con LPO. Se estudió la posible asociación entre H. pylori y manifestaciones clínicas de LPO en un grupo de pacientes Chilenos. Se encontró diferencia significativa entre los pacientes positivos para H. pylori y la edad de diagnóstico de LPO, sugiriendo que H. pylori podría inducir la enfermedad a temprana edad. Sin embargo, no se pudo confirmar significancia estadística entre la presencia de esta bacteria y la presencia de displasia en LPO.

Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39 / Development of sequence characterized amplified región markers for identification of Azospirillum brasilense Az39

Resumen Azospirillum brasilense Az39 es utilizada por empresas productoras de inoculantespara la formulación de bioinsumos en América del Sur desde hace más de 30 a˜nos. Esta cepapuede promover el crecimiento, desarrollo, así como la capacidad de tolerar diferentes tiposde estrés en las plantas inoculadas, lo que determina un aumento de la productividad de culti-vos de interés agronómico. En la actualidad, no existen protocolos en Argentina que permitanconfirmar la identidad de Az39 en productos comerciales a nivel de laboratorios de control decalidad de inoculantes. Por ello, el objetivo de este trabajo fue desarrollar una metodología enbase molecular que permita la identificación certera de A. brasilense Az39. Con la secuenciacompleta del genoma y mediante herramientas bioinformáticas, se pudieron reconocer frag-mentos de ADN presentes únicamente en el genoma de Az39. Se dise˜naron cebadores dirigidosa amplificar por PCR dichas secuencias. Como resultado se observaron los productos específicosúnicamente en la presencia de la cepa de interés. La reacción pudo detectar un título mínimode 105UFC/ml (4,5 ng/l ADN) o de 102UFC/ml (0,88 ng/l ADN) o una concentración mínimade 0,098 ng/l ADN, dependiendo del método de extracción utilizado. Los cebadores fueronevaluados en el análisis de productos comerciales obtenidos del mercado nacional, arrojandoresultados positivos, tanto en muestras directas como así también en pruebas confirmatoriasa partir de colonias aisladas de tales productos. La metodología desarrollada en este trabajo,permite la detección certera de A. brasilense Az39 en cultivos puros o mezclas complejas demicroorganismos.

Abstract Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/pl ADN) or 102 CFU/ml (0.88 ng/pl DNA) or in a minimal concentration of 0.098 ng/pl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

Management of a colon cancer patient infected with corona virus disease 2019 / 浙江大学学报·医学版

OBJECTIVE@#To explore the feasibility of surgical treatment for cancer patients complicated with corona virus disease 2019 (COVID-19).@*METHODS@#The management and clinical outcome of a sigmoid cancer patient with COVID-19 were analyzed.@*RESULTS@#The inflammation indicators and fever of this patient were effectively controlled and the lung lesions remained stable after active anti-viral treatment, then the radical colorectomy was performed after the viral negative conversion for twice.@*CONCLUSIONS@#The case indicates that it may feasible to undergo radical tumor surgery for cancer patients with COVID-19 after the virus nucleic acid testing turns negative and more studies are needed to confirm this conclusion.

Site-directed mutagenesis of long gene by partial amplification combining with double fragments ligation / 生物工程学报

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Standardized sputum collection increases sputum sample collection rate for novel coronavirus nucleic acid detection / 浙江大学学报·医学版

OBJECTIVE@#To evaluate the effect of standardized health education on the sputum specimen collection rate for nucleic acid detection of coronavirus disease 2019 (COVID-19).@*METHODS@#Two hundred and twenty-seven patients in fever clinics and isolation wards of Sir Run Run Shaw Hospital of Zhejiang University and 307 migrant workers returning to 5 enterprises in Shanghai from February 3 to March 14, 2020 were enrolled in the study. Through clarifying the procedures of collecting sputum specimens, making graphic/video health education materials, standardizing the contents and methods of health education, we conducted education to the subjects. The subject expectorated spontaneously or with medical assistance. For patients, the number of sampling attempts and sputum acquisition times were documented before and after the implementation of the standardized expectoration method; for the returning migrant employees in the enterprises, only the number of collected samples after the implementation of the standardized expectoration method were recorded.@*RESULTS@#A total of 378 sputum samples were collected from 227 patients. The sputum sampling rates before and after the implementation of health education were 40.9%and 58.4%, respectively (@*CONCLUSIONS@#The education for standardized sputum sample collection method can effectively increase the sputum collection rate.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

The use of Gene-Xpert MTB RIF in the diagnosis of extrapulmonary tuberculosis in childhood and adolescence

Abstract INTRODUCTION: Gene-Xpert MTB RIF (Xpert) is based on nucleic acid amplification by real-time polymerase chain reaction, which allows for the identification of Mycobacterium tuberculosis and rifampin resistance. We describe the use of Xpert for extrapulmonary tuberculosis (EPTB) in children and adolescents. METHODS: A case series of two reference centers in Rio de Janeiro from 2014-2019. RESULTS: The final diagnosis of EPTB was established in 11/36 (31%) patients, with five cases detectable by Xpert. For lymph node evaluation (9/11), diagnosis by Xpert occurred in 5/9 patients, all with caseous aspects. CONCLUSIONS: Xpert can facilitate the rapid diagnosis of lymph node tuberculosis.

Molecular and serological characterization of occult hepatitis B among blood donors in Maputo, Mozambique

BACKGROUND Occult hepatitis B virus (HBV) - characterized by the absence of detectable HBsAg in the presence of HBV DNA - represents a potential threat for blood safety. OBJECTIVES This study was conducted with the aim to investigate the serological and molecular characterization of occult HBV infection (OBI) among blood donors in Mozambique. METHODS 1,502 blood donors were tested for HBsAg. All HBsAg-negative individuals were tested for HBV DNA. Antibodies against HBV core, surface and HBe antigen (anti-HBc, anti-HBs, HBeAg) were measured in HBV DNA positive individuals. FINDINGS 1435 serum samples were HBsAg negative and 16 positive for HBV DNA, 14 confirmed to have OBI, corresponding to a frequency of 0.98%. Of the 14 OBI infections identified, 13/14 (92.8%) were positive for anti-HBc, 4/14 (28.5%) for anti-HBs, and no samples were reactive for HBeAg. Of the 14 OBI cases, nine samples (64.2%) were sequenced for the S/P region. Eight samples (88.9%) belonged to genotype A1 and one (11.1%) to genotype E. One escape mutation (T123A) associated with OBI and various amino acid substitutions for genotype A1 and E were observed. MAIN CONCLUSIONS Our results show the importance of using nucleic acid amplification test to detect occult hepatitis B infection in blood donors in Mozambique.

Active human herpesvirus infections in adults with systemic lupus erythematosus and correlation with the SLEDAI score

Abstract Background: Human herpesviruses (HHVs) are responsible for a significant number of clinical manifestations in systemic lupus erythematous (SLE) patients. The aim of this study was to determine the frequency of active HHV infections in SLE patients and correlating them with disease activity. Methods: Serum samples were collected from 71 SLE patients and their DNAs were extracted and analyzed to detect HHV-DNA viruses using the nucleic acid amplification technique. Results: Fifteen out of the 71 (21.1%) patients tested positive for the HHV-DNA virus. Of them, 11/15 HHV-DNA-positive patients (73.3%) had SLE activity index (SLEDAI - Systemic Lupus Erythematosus Disease Activity Index) ≥8 (p = 0.0001). Active HCMV infection was the mostly frequently observed infection, occurring in 6/15 patients (40%). The frequencies of other active viral infections were 22% for HSV-1, 16.7% for HHV-7, and 5.5% for HSV-2. Viral coinfection (two or more viruses detected in the same sample) occurred in three patients (16.7%). Active HHV infections in SLE patients are more frequent in those with active SLE (≥8), who is at high risk of HHV reactivation and HCMV disease. Conclusion: Viral surveillance is important to identify active HHV infections that can cause clinical symptoms and other complication in SLE patients.

Agentes etiológicos en pacientes con enfermedad diarreica aguda detectados por PCR en niños de 0 a 14 años de edad en el Hospital Metropolitano de Quito / Etiological agents in patients with acute diarrheal disease detected by PCR test in children from 0 to 14 years old in Hospital Metropolitano of Quito

Abstract: Objective: Determine the prevalence of gastrointestinal infections in children from 0 to 14 years old at Hospital Metropolitano of Quito from August 2017 to May 2018. Methods: A cross sectional study was carried out to determine the prevalence of gastrointestinal infections in children from 0 to 14 years old at Hospital Metropolitano of Quito. There were 58 patients with gastrointestinal infection from August 2017 to May 2018. We used the results from PCR gastrointestinal panel to determine the etiology of the disease. Results: We studied 58 patients with gastrointestinal infection between 0 to 14 years old. We found 79% of bacterial and 21% viral etiology. The most frequent agent was Clostridium difficile (15%) followed by enteropathogenic E. coli (14%), enteroagregative E. coli (12%). The most important virus was norovirus followed by rotavirus, and Giardia lamblia as a parasite. The most frequent coinfections were Clostridium difficile-Campylobacter, Clostridium difficile-enteropathogenic E. coli, enteropathogenic E. coli-enterotoxigenic E. coli. The months of the year where the greatest number of infections occurred were April and May. Conclusions: The etiology of gastrointestinal infections is a very important issue to study and manage because of its high incidence at the pediatric population, so a timely diagnosis and comprehensive management must be made.

Resumen: Objetivo: determinar la prevalencia de infecciones gastrointestinales en niños de 0 a 14 años que acudieron al Hospital Metropolitano de Quito durante el período comprendido entre agosto de 2017 y mayo de 2018. Método: estudio descriptivo transversal para determinar la prevalencia de infecciones gastrointestinales en niños de 0 a 14 años de edad en el Hospital Metropolitano de Quito. Se presentaron 58 casos de niños diagnosticados de infección gastrointestinal o gastroenteritis, durante un lapso de 10 meses desde agosto de 2017 hasta mayo de 2018. Para el diagnóstico etiológico se utilizaron los resultados del panel gastrointestinal por PCR. Resultados: se obtuvo 79% de infecciones bacterianas y 21% virales. La bacteria más frecuente fue el Clostridium difficile (15%) seguido por E. coli enteropatógena (14%) y E. coli enteroagregativa (12%). De los virus, el más frecuente fue el norovirus seguido por el rotavirus. De los parásitos, la Giardia lamblia. Las coinfecciones más frecuentes fueron causadas por Clostridium difficile-Campylobacter, Clostridium difficile-E. coli enteropatógena, E. coli enteropatógena-E. coli enterotoxigénica. La época del año de mayor incidencia de infecciones gastrointestinales abarcó abril y mayo. Conclusiones: la etiología de las infecciones gastrointestinales es un tema muy importante por su elevada incidencia en la población pediátrica, lo que motiva a realizar el diagnóstico y manejo integral oportunos