Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma / 中华妇产科杂志

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all P<0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all P<0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.

Risk factor analysis of lymph node metastasis in endometrial carcinoma combined with molecular types / 中华妇产科杂志

Objective: To investigate the relationships between molecular types of the cancer genome atlas (TCGA) of patients with endometrial carcinoma (EC) and lymph node metastasis and other clinicopathological features. Methods: The clinical pathological information of 295 patients with EC who underwent initial inpatient surgical treatment and accepted the detection of the molecular types of TCGA with next-generation sequencing technology at Peking University People's Hospital were collected during April 2016 and May 2022. The TCGA molecular typing of EC was divided into four types: POLE-ultramutated (15 cases), high microsatellite instability (MSI-H; 50 cases), copy-number low (CNL; 175 cases), and copy-number high (CNH; 55 cases). The differences of clinical pathological features among different molecular types and the risk factors of lymph node metastasis were analyzed retrospectively. Results: Among 295 patients with EC, the average age was (56.9±0.6) years. (1) There was a statistically significant difference in lymph node metastasis (0, 8.0%, 10.3% and 25.5%) among the four molecular types (χ2=12.524, P=0.006). There were significant differences in age, stage, pathological type, grade (only endometrioid carcinoma), myometrium invasion, lymphatic vascular space infiltration, and estrogen receptor among the EC patients of four molecular types (all P<0.05). Among them, while in the patients with CNH type, the pathological grade was G3, the pathological type was non-endometrioid carcinoma, and the proportion of myographic infiltration depth ≥1/2 were higher (all P<0.05). (2) Univariate analysis suggested that pathological type, grade, myometrium infiltration depth, cervical interstitial infiltration, lymphatic vascular space infiltration, and progesterone receptor were all factors which significantly influence lymph node metastasis (all P<0.01); multivariate analysis suggested that the lymphatic vascular space infiltration was an independent risk factor for lymph node metastasis (OR=5.884, 95%CI: 1.633-21.211; P=0.007). (3) The factors related to lymph node metastasis were different in patients with different molecular types. In the patients with MSI-H, the non-endometrioid carcinoma of pathological type was independent risk factor for lymph node metastasis (OR=29.010, 95%CI: 2.067-407.173; P=0.012). In the patients with CNL, myometrium infiltration depth≥1/2 (OR=4.995, 95%CI: 1.225-20.376; P=0.025), lymphatic vascular space infiltration (OR=14.577, 95%CI: 3.603-58.968; P<0.001) were the independent risk factors for lymph node metastasis. While in the CNH type patients pathological type of non-endometrioid carcinoma (OR=7.451, 95%CI: 1.127-49.281; P=0.037), cervical interstitial infiltration (OR=22.938, 95%CI: 1.207-436.012; P=0.037), lymphatic vascular space infiltration (OR=9.404, 95%CI: 1.609-54.969; P=0.013), were the independent risk factors for lymph node metastasis. Conclusions: POLE-ultramutated EC patients have the lowest risk of lymph node metastasis, and CNH patients have the highest risk of lymph node metastasis. The risk factors of lymph node metastasis of different molecular types are different. According to preoperative pathological and imaging data, lymph node metastasis is more likely to occur in patients with non-endometrioid carcinoma in MSI-H and CNH type patients, and lymph node metastasis is more likely to occur in patients with myometrium infiltration depth ≥1/2 in CNL type patients.

Analysis of epidemic characteristics of anthrax in China from 2017 to 2019 and molecular typing of Bacillus anthracis / 中华预防医学杂志

Objective: To analyze the epidemiological characteristics of anthrax in China from 2017 to 2019 and molecular typing of Bacillus anthracis isolated from some provinces (autonomous regions). Methods: Surveillance data of anthrax cases reported from 2017 to 2019 in the Infectious Disease Surveillance information System of China Disease Prevention and Control and the Public Health Emergency Reporting and Management Information System were collected, and descriptive epidemiological methods were used to analyze the epidemic characteristics, including the temporal, geographic and demographic distribution of this disease. A total of 47 strains of Bacillus anthracis isolated from 2017 to 2019 were analyzed by canSNP and MLVA15. Results: A total of 951 cases of anthrax were reported from 2017 to 2019, of which 938 were cutaneous anthrax, representing 98.63% of the total number reported. It was mainly distributed in the west and northeast of China, and the three provinces with the highest number of cases were Gansu (215), Sichuan (202) and Qinghai (191). Cases had been reported throughout the year, more cases occurred in the summer and autumn, and August was the month with the most cases,66.35% (211/318), 72.32% (243/336) and 68.01% (202/297) of cases were reported during June to September. The age distribution was mainly between 20 and 59 years old, accounting for more than 80% of all cases. The number of male cases was significantly higher than that of female cases, the ratio of male to female was about 3∶1. The occupations were mainly herdsmen and farmers, accounting for 49.70% to 58.18% and 31.45% to 36.70%, respectively. Public health events occurred every year, and 29 events had been reported from 2017 to 2019. canSNP analysis showed that 37 of the 47 strains belonged to the A.Br.001/002 subgroup and 10 belonged to the A.Br.Ames subgroup. MLVA15 analysis showed that there were 17 genotypes, of which 10 genotypes contained only one strain. Conclusion: Cutaneous anthrax was the predominant clinical type in China from 2017 to 2019.The seasonal, geographic and demographic distribution characteristics were evident.Molecular typing methods such as canSNP and MLVA15 can be used to trace the source of infectious diseases and provide technical support for anthrax prevention and control.

Advances on molecular typing methods and evolution of human parainfluenza virus / 中华预防医学杂志

Human parainfluenza viruses (HPIVs) is one of the main causes of acute respiratory tract infections in children. HPIVs have been grouped into four serotypes (HPIV1~HPIV4) according to serological and genetic variation. Different serotypes of HPIVs have diverse clinical disease spectrum, epidemic characteristics and disease burden. Based on the nucleotide variation in structural protein genes, HPIVs can be further divided into distinct genotypes and subtypes with diverse temporal and spatial distribution features. The standard molecular typing methods are helpful to clarify the gene evolution and transmission patterns of HPIVs in the process of population transmission. However, the development of molecular epidemiology of HPIVs has been hindered by the lack of a standardized molecular typing method worldwide. Therefore, this study reviewed the viral characteristics, genome structure, existing genotyping methods and evolution of HPIVs, and screened the reference strains for molecular typing, so as to improve the understanding of gene characteristics and molecular typing of HPIVs, and provide an important scientific basis for the monitoring and research of molecular epidemiology of HPIVs in China.

Advances in Molecular Typing of Small Cell Lung Cancer / 中国肺癌杂志

Small cell lung cancer (SCLC) is a highly aggressive and fatal malignant tumor. It has the characteristics of complex etiology, low differentiation, high malignancy, fast growth, strong invasiveness, early metastasis and acquired drug resistance, resulting in poor prognosis. In recent years, with the gradual deepening understanding on the molecular mechanism of SCLC and multi-omics data, it is proposed that molecular typing can be carried out according to the differential expression of key transcription factors, including SCLC-A, SCLC-N, SCLC-P and SCLC-I subtypes. Molecular typing of SCLC and its clinical application will help doctors to further optimize the detailed diagnosis and treatment plan of SCLC patients, so as to prolong the survival time and improve the quality of life of patients.
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Uso racional de antibióticos y tecnología FilmArray para identificación rápida de bacteriemias en unidad de cuidados intensivos pediátrica / Rational use of antibiotics and FilmArray technology for rapid identification of bacteremias in a pediatric intensive care unit

INTRODUCCIÓN: Las infecciones graves son la principal causa de ingreso a cuidados intensivos pediátricos. El panel FilmArray BCID permite identificar rápidamente a microorganismos causantes de bacteriemias. OBJETIVO: evaluar la eficacia de la identificación rápida de microorganismos asociado a un Programa de Uso Racional de Antibióticos (URA) en reducir los tiempos de terapias antibióticas, en un hospital pediátrico. PACIENTES Y MÉTODO: Estudio retrospectivo, que incluyó 100 pacientes, en su primer episo dio de bacteriemia, divididos en 2 grupos de 50 cada uno: Intervención (FilmArray BCID y programa URA) y Controles históricos pareados para la misma especie del microrganismo identificado (microbiología convencional). Las variables evaluadas fueron los tiempos de identificación microbiana, latencia de la terapia dirigida y de desescalar antibióticos. RESULTADOS: Los grupos fueron comparables en características demográficas, foco de infección y etiología de bacteriemia. El tiempo promedio de identificación de microorganismos fue de 23 h (IC 95% 12,4-26,7) en el grupo intervención, y 70,5 h (IC 95% 65,2-78,6) en el control (p < 0,05), mientras que la latencia de inicio de terapia dirigida fue de 27,9 h (IC 95% 22,3-32,8) y 71,9 h (IC 95% 63,2-77,8) respectivamente (p < 0,05). El tiempo de desescalar o suspender antibióticos fue de 6,4 h (IC 95% 2,76-9,49) y 22 h (IC 95% 6,74-35,6) en los grupos mencionados (p > 0,05). CONCLUSIÓN: El panel FilmArray BCID articulado a un programa URA, contribuye a la identificación de los microorganismos causantes de bacteriemias en menor tiempo que los métodos convencionales, siendo una herramienta que optimiza las terapias antibióti cas en niños críticamente enfermos.

INTRODUCTION: Severe infections are the leading cause of admission to pediatric intensive care. The FilmArray BCID panel quickly identifies microorganisms that cause bacteremia. OBJECTIVE: To evaluate if the rapid identification of the microorganisms that cause bacteremia, along with a Rational Use of Antibio tics (RUA) Program, allows optimizing the time of antibiotic therapy in a pediatric hospital. PATIENTS AND METHOD: Retrospective study which included 100 patients presenting their first episode of bacteremia, divided into 2 groups of 50 each. The first one was Intervention (FilmArray BCID and RUA program) and the second one was Historical Controls (conventional automated ID/AST). The variables evaluated were the time required for microbial identification, duration of appropriate therapy, and antibiotic de-escalation. RESULTS: The groups were comparable in terms of demographic characteristics, focus of infection, and etiology of bacteremia. The average time of microorganisms' identification of the control group was 70.5 hours (IC 95% 65.2-78.6) and 23.0 hours (IC 95% 12.4 -26.7) in the intervention one (p < 0.05). The average time of targeted therapy onset was shorter in the intervention group (27.9 h [IC 95% 22.3-32.8]) than that of the control one (71.9 h [IC 95% 63.2-77.8]) (p < 0.05). Finally, the time to de-escalate or discontinue antibiotics in the intervention group and the control one was 6.4 hours (IC 95% 2.76-9.49) hours and 22.0 hours (IC 95% 6.74-35.6 h) respectively (p > 0.05). CONCLUSION: The FilmArray panel along with the RUA Program allows the identification of the microorganisms causing bacteremia faster than conventional methods, which positions it as a tool that optimizes antibiotic therapy of critical patients.

Diversidad molecular de variantes patogénicas de Vibrio parahaemolyticus en el Perú / Molecular diversity in pathogenic variants of vibrio parahaemolyticus in Peru

RESUMEN Con el objetivo de determinar la diversidad de variantes patogénicas de Vibrio parahaemolyticus en el Perú durante el periodo 1995-2017, se analizaron 102 genomas peruanos (97 clínicos y 5 ambientales) empleando el esquema de tipificación multilocus y BLASTn para la búsqueda de genes de virulencia. Se identificaron 15 tipos de secuencia diferentes, encontrándose que el genotipo ST3, perteneciente al clon pandémico, fue el más abundante, con 52% (n=53); seguido por el ST120, con 23,5% (n=24); y el complejo clonal CC345, con 11,8% (n=12). Un total de 89 cepas analizadas presentaron genes que codifican la isla de patogenicidad VpaI-7 (87,3%), mientras que 96 presentaron el gen tdh (94,1%), y 6, el trh (5,9%). Durante el periodo evaluado, se resalta la predominancia del ST3, causante de un importante brote en el pasado del Perú, además de otros genotipos patógenos que representan un riesgo latente en salud pública asociado al consumo de alimentos marinos.

ABSTRACT During the period from 1995 to 2017, in order to determine the diversity of Vibrio parahaemolyticus pathogenic variants in Peru, 102 Peruvian genomes (97 from a hospital setting and 5 from an out-of-hospital setting) were analyzed using the multilocus typification scheme and BLASTn in the search for virulence genes. Fifteen different sequence types were identified. It was found that the ST3 genotype, which is found in the pandemic clone, was the most abundant, with 52% (n=53); followed by ST120, with 23.5% (n=24); and the CC345 clonal complex, with 11.8% (n=12). A total of 89 analyzed strains presented genes encoding the pathogenicity island VpaI-7 (87.3%), while 96 presented the tdh gene (94.1%), and 6 the trh gene (5.9%). The ST3 genotype was the predominant one during the evaluated period, this genotype was the cause of a major outbreak in Peru's past history. Other pathogenic genotypes found represent a latent public health risk associated with seafood consumption.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Caracterización molecular y detección de genes blaCTX-M grupos 1 y 9 en Klebsiella pneumoniae resistentes a ceftazidima, en un hospital de San José de Cúcuta, Colombia / Molecular characterization and detection of genes blaCTX-M groups 1 and 9 in Klebsiella pneumoniae resistant to ceftazidime, in a hospital in San José de Cúcuta, Colombia

Resumen Introducción: La expresión de β-lactamasas CTX-M pertenecientes a los grupos 1 y 9 en Klebsiella pneumoniae produce grados altos de resistencia a ceftazidima, y presentan una amplia distribución mundial. Objetivo: Identificar y caracterizar los genes blaCTX-M-Grupo1 y blaCTX-M-Grupo9 en aislados de K. pneumoniae resistentes a ceftazidima en un hospital de San José de Cúcuta, Colombia. Material y Método: Se diseñaron partidores para la identificación de K. pneumoniae y los genes blaCTX-M mediante reacción de polimerasa en cadena (RPC). Posteriormente se realizó el análisis de la relación genética de estos aislados por medio de la RPC basada en secuencias repetitivas (RPC-REP). Resultados: Treinta y ocho por ciento de los 24 aislados identificados por RPC como K. pneumoniae presentaron los genes blaCTX-M-3, blaCTX-M-15 y blaCTX-M-32 (Grupo CTX-M-1) y 42% los genes blaCTX-M14, blaCTX-M-24 y blaCTX-M-27 (Grupo CTX-M-9). El análisis filogenético agrupó los aislados de K. pneumoniae en cuatro clusters, mostrando correlación en los clusters I, II y IV, al comparar los perfiles genéticos con el tipo de muestra y grupo de genes. Discusión: Se encontró una frecuencia similar de los genes blaCTX-M-Grupo1 y blaCTX-M-Grupo 9 en aislados de K. pneumoniae resistentes a ceftazidima. La correlación entre la RPC-REP con los grupos de CTX-M y el tipo de muestra reveló la presencia de tres patrones clonales.

Background: The expression of CTX-M β-lactamases belonging to groups 1 and 9 in Klebsiella pneumoniae produces high levels of resistance to ceftazidime, and they have a wide distribution worldwide. Aim: To identify and characterize the blaCTX-M-Group1 and blaCTX-M-Group9 genes in K. pneumoniae isolates resistant to ceftazidime in a hospital in San José de Cúcuta, Colombia. Material and Methods: Primers were designed for the identification of K. pneumoniae and blaCTX-M genes by PCR. Subsequently, the genetic relationship of these isolates was analyzed by REP-PCR. Results: A 38% of the 24 isolates identified by PCR as K. pneumoniae showed blaCTX-M-3. blaCTX-M-15 y blaCTX-M-32 genes (Group CTX-M-1) and 42% blaCTX-M14. blaCTX-M-24 y blaCTX-M-27 genes (Group CTX-M-9). The phylogenetic analysis grouped the K. pneumoniae isolates into 4 clusters, showing correlation in clusters I, II and IV, when comparing the genetic profiles with the type of sample and group of genes. Discussion: We found a similar frequency of blaCTX-M-Group 1 and blaCTX-M-Group 9 genes in isolates of K. pneumoniae resistant to ceftazidime. The correlation between the REP-PCR with the CTX-M groups and the type of sample revealed the presence of three clonal patterns.

Molecular identification and microbiological evaluation of isolates from equipments and food contact surfaces in a hospital Food and Nutrition Unit / Avaliação microbiológica e identificação molecular de isolados de equipamentos e superfícies de contato com alimentos em um hospital Unidade de Alimentação e Nutrição

Abstract The hygienic and sanitary control in Food and Nutrition Units (FNU) is considered a standard procedure to produce adequate meals and reduce the risk of foodborne diseases and hospital infections. This study aimed to evaluate the isolation and identification of bacteria from equipment and food contact surfaces in a hospital FNU as well as to evaluate the sanitary condition. Likewise, it was analyzed the adhesion of the microorganisms on polyethylene cutting boards. The presence of aerobic mesophilic microorganisms, yeasts, molds, coagulase-positive staphylococci, coliform and fecal coliform, and Escherichia coli were analyzed on eating tables, countertop surfaces and cutting boards used for meat or vegetable handling, and equipment such as microwaves and refrigerators. The molecular identification it was done by 16S rRNA gene sequencing. The adhesion of the microorganisms (biofilm formation) on meat and vegetable cutting boards was also evaluated by scanning electron microscopy. The results showed high numbers of all microorganisms, except for E. coli , which was not observed in the samples. The molecular analysis identified species of the Enterobacteriaceae family and species of the Pseudomonadaceae family. Scanning electron microscopy analyses revealed bacterial adhesion on the cutting board surfaces. The results obtained in this study indicated that the hygienic conditions of surfaces like plastic cutting boards and equipment in this hospital FNU were inadequate. The achievement and application of standard operating procedures could positively help in the standardization of sanitary control, reducing the microbial contamination and providing a safe food to hospitalized patients.

Resumo O controle higiênico e sanitário nas Unidades de Alimentação e Nutrição (UAN) é considerado um procedimento padrão para produzir refeições adequadas e reduzir o risco de doenças transmitidas pelos alimentos e infecções hospitalares. Este estudo teve como objetivo isolar e identificar bactérias de equipamentos e superfícies de contato com alimentos em uma UAN hospitalar, bem como avaliar a condição sanitária. Do mesmo modo, analisou-se a adesão dos micro-organismos em tábuas de corte de polietileno. A presença de micro-organismos aeróbios mesófilos, leveduras, fungos, Sthapylococcus coagulase-positivos, coliformes, coliformes fecais e Escherichia coli foi analisadas na superfície de mesas do refeitório, superfícies de bancada e tábuas de corte usadas para manuseio de carne ou vegetais e, em equipamentos como micro-ondas e refrigeradores. A identificação molecular foi feita pelo sequenciamento do gene 16S rRNA. A adesão dos micro-organismos (formação de biofilmes) em tábuas de corte de carne e de vegetais também foi avaliada por microscopia eletrônica de varredura. Os resultados mostraram elevada contagem para todos os micro-organismos analisados, exceto para E. coli, a qual não foi observada nas amostras. A análise molecular identificou espécies da família Enterobacteriaceae e Pseudomonadaceae. A análise de microscopia eletrônica de varredura revelaram adesão bacteriana nas superfícies das placsa de corte. Os resultados obtidos neste estudo indicaram que as condições higiênicas das superfícies e de equipamentos nesta UAN hospitalar estavam inadequadas. A aplicação de procedimentos operacionais padrão poderia auxiliar positivamente na padronização do controle higiênico-sanitário, reduzindo a contaminação microbiana e fornecendo um alimento seguro para pacientes hospitalizados.

Molecular Epidemiology of Staphylococcus aureus in Skin and Soft Tissue Infections and Bone and Joint Infections in Korean Children

Prevalence of cryptococcosis in Atlántico, department of Colombia assessed with an active epidemiological search

Abstract INTRODUCTION Cryptococcosis is the second most frequent cause of opportunistic infections in human immunodeficiency virus (HIV) patients in Colombia. We aimed to determine the prevalence of cryptococcosis in the Colombian department of Atlántico. METHODS An active search for cryptococcosis cases was conducted between 2015 and 2017 in health institutions by distributing surveys to clinicians and characterizing samples phenotypically and genotypically. RESULTS Thirty-eight cryptococcosis cases were identified (81.6% men, 76.3% HIV patients). The calculated annual prevalence was 5.08/1 million inhabitants. Cryptococcus neoformans var. grubii VNI was isolated in 34 cases. CONCLUSIONS These results provide the basis for passive surveillance of cryptococcosis.

Molecular characteristics and virulence gene profiles of Staphylococcus aureus causing bloodstream infection

ABSTRACT Background: The rate of methicillin-resistant Staphylococcus aureus (MRSA) among the total of S. aureus isolates decreased to 35.3% in 2017 in China. It is unclear whether the molecular characteristics of S. aureus isolates have changed as the rate decreased. Objective: This study aimed to investigate the molecular characteristics and virulence genes profile of S. aureus isolates causing bloodstream infection and analyze the correlation between the prevalence rates of the common sequence types and MRSA. Methods: A total of 112 S. aureus strains from eight hospitals of four cities, including 32 MRSA isolates, were identified and evaluated through multilocus sequence typing, spa typing, and determination of virulence genes. Results: Twenty-five STs were identified, of which ST5 (21.4%) was the most prevalent, whereas the prevalence of ST239 correlated with the rate of MRSA among all S. aureus isolates. Forty-six spa types were identified, of which t2460 (14.3%) was the most common. clfa, hla, seb, fnbA and hlb were the prevailing virulence genes. 81.3% MRSA and 45.0% methicillin-sensitive S. aureus (MSSA) isolates harbored six or more tested virulence genes. ST5-t2460, seldom noted in bloodborne S. aureus isolates in China, was the most common clone. The prevalence of harboring six or more virulence genes in ST5-t2460 and ST188-t189 were 93.8% and 8.3%, respectively. Conclusion: ST5-t2460 was the most common clone in S. aureus causing bloodstream infection followed by ST188-t189, which had never been noted in China before. Moreover, ST5-t2460 harbored more virulence genes than ST188-t189, and the prevalence of ST239 clone decreased with the proportion of MRSA among all S. aureus isolates.

First molecular typing of Mycobacterium avium subspecies paratuberculosis identified in animal and human drinking water from dairy goat farms in Brazil

Abstract Mycobacterium avium subspecies paratuberculosis, the etiologic agent of Johne's disease or paratuberculosis, was identified by culture and/or polymerase chain reaction (PCR) in 50% and 30% of water samples for animal and human consumption, respectively, from ten dairy goat farms in Brazil. IS1311 restriction fragment length polymorphism analysis identified the isolates as cattle type C.

Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study / Tipificación molecular de Leishmania (Leishmania) amazonensis y especies del subgénero Viannia asociadas a la leishmaniasis cutánea y la mucosa en Colombia: un estudio de concordancia

Resumen Introduction: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. Objective: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. Materials and methods: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. Results: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Conclusion: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Abstract Introducción. La electroforesis de enzimas multilocus (Multilocus Enzyme Electrophoresis, MLEE) es el estándar de referencia para la tipificación de las especies de Leishmania. La prueba está restringida a laboratorios especializados por su complejidad técnica, sus costos y el tiempo necesario para obtener resultados. La PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) se utiliza para tipificar especies de Leishmania. Objetivo. Establecer la concordancia entre las dos pruebas como métodos de tipificación de las especies circulantes en Colombia. Materiales y métodos. Se seleccionaron 96 aislamientos de pacientes con leishmaniasis cutánea o mucocutánea y se tipificaron mediante MLEE y PCR-RFLP con los blancos moleculares miniexon y hsp70 usados en serie. Las enzimas de restricción aplicadas fueron la HaeIII y la BccI, respectivamente. Se calculó el coeficiente kappa y un intervalo de confianza (IC) de 95 %. Resultados. Se determinó que la concordancia fue "muy buena" al obtener un coeficiente de 0,98 (IC95%: 0,98-1,00). Las especies identificadas fueron: Leishmania Viannia braziliensis, L. (V.) panamensis, L. (V.) guyanensis y L. (L,) amazonensis. De los 96 aislamientos, 80 se enviaron a secuenciación y se confirmaron los resultados obtenidos mediante PCR-RFLP. Conclusión. Dada la concordancia obtenida con la PCR-RFLP amplificando los genes miniexon y hsp70, se propone esta prueba como alternativa para la tipificación de especies de Leishmania circulantes en Colombia.

Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil

Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

Differences in Colistin-resistant Acinetobacter baumannii Clinical Isolates Between Patients With and Without Prior Colistin Treatment

BACKGROUND: The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. METHODS: We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. RESULTS: Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. CONCLUSIONS: Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.

Frequency of Red Blood Cell Antigens According to Parent Ethnicity in Korea Using Molecular Typing

Frequencies of red blood cell (RBC) blood group antigens differ by ethnicity. Since the number of immigrants is increasing in Korea, RBC antigens should be assessed in children/youths with parents of different ethnicities to ensure safe transfusions. We investigated the frequency of RBC antigens, except for ABO and RhD, in 382 children and youths with parents having Korean and non-Korean ethnicities. Subjects were divided into those with ethnically Korean parents (Korean group; N=252) and those with at least one parent of non-Korean ethnicity (non-Korean group; N=130). The 37 RBC antigens were genotyped using the ID CORE XT system (Progenika Biopharma-Grifols, Bizkaia, Spain). The frequencies of the Rh (E, C, e, hr(S), and hr(B)), Duffy (Fy(a)), MNS (Mi(a)), and Cartwright (Yt(b)) antigens differed significantly between the two groups. Eight and 11 subjects in the Korean and non-Korean groups, respectively, exhibited negative expression of high-frequency antigens, whereas 14 subjects in the non-Korean group showed positive expression of low-frequency antigens. The frequency of RBC antigens has altered alongside demographic changes in Korea and might lead to changes in distribution of RBC antibodies that cause acute or delayed hemolytic transfusion reaction.

Molecular characterization and spoligotyping analysis of multi drug resistant mycobacterium tuberculosis from a high TB endemic area of Pakistan

Background: Multidrug resistant tuberculosis caused by Mycobacterium tuberculosis is an infection that is resistant to rifampicin and isoniazid. Management of Multidrug resistant tuberculosis is a serious challenge worldwide

Objectives: To investigate hotspot mutations in rpoB, katG and inhA genes and its possible co-relation with predominant genotypes in Khyber Pakhtunkhwa, Pakistan

Study design, settings and duration: This cross sectional study was conducted after approval from research and ethics committee of Provincial TB Control Program, Khyber Pakhtunkhwa in March 2015

Materials and Methods: A total of 166 clinical isolates were analysed which were collected from programmatic management of drug-resistant tuberculosis units. All samples were characterized by phonotypical drug susceptibility test, genotypic drug resistant test [line probe assay] and spoligotyping analysis using ''TB-SPRINT' micro bead assay

Results: Out of the total 166 samples, 97 strains were resistant to rifampicin [RIF] and 106 strains were resistant to isoniazid [INH]. Most common mutation in rpoB was S531L in 75 [77%] isolates followed by D516V in 10 [10%] and H526Y in 6 [6%] samples respectively. A rare mutation in rpoB gene at codon 522 and deletion of codon 518 was also reported. In 106 INH resistant strains, 97[91%] were associated with mutation in katG gene while resistance in 9 [8.4%] strain was due to mutation in the inhA promoter region. Spoligotyping analysis revealed 55 distinct types of different patterns. Spoligotyping patterns of 146 samples matched with 15 different linage of M.tuberculosis in which 101 [60%] were identified as the predominant CAS1-Delhi linage. The pattern of 20 strains [12%] did not matched to any other pattern in the SITVIT database and were named orphan KP

Conclusion: Molecular characterization of M.tuberculosis is very helpful in the early identification of MDR-TB. As CAS1-Delhi is the predominant type in this region, its association with drug resistance, treatment failure and patient demographic profiles should be investigated

Molecular characterization of Mycobacterium tuberculosis and Mycobacterium bovis isolates by Enterobacterial Repetitive Intergenic Consensus-PCR

Tuberculosis (TB) is an infectious disease in which the molecular typing methods allow to have important information about the dynamics of transmission and to assist properly in disease control. Although the ERIC-PCR (Enterobacterial repetitive intergenic consensus-PCR) assay is fast and easy to perform, scarce studies have reported its use in epidemiological studies in TB outbreaks. In this study, we aimed to genotype Mycobacterium tuberculosis and M. bovis isolates by ERIC-PCR and compare its discriminatory power with two other classically used methods: 12 loci-MIRU (Mycobacterial Interspersed Repetitive Units) and Spoligotyping. The M. tuberculosis isolates studied were from northwestern and southwestern and M. bovis from northwestern Parana, Brazil. ERIC-PCR rendered banding patterns with great diversity (1 to 12 bands) of molecular sizes, ranging from 100 to 1600 bp. ERIC-PCR showed to be fast, simple and affordable to differentiate isolates. ERIC-PCR would be an important tool in the epidemiology of TB as screening in case of outbreak, which demands rapid intervention. However if any doubt persist, as it may occur with the application of only one genotypic method, other genotyping methods should be applied and carefully interpreted, always with additional epidemiological information.