Buenas Prácticas de Manufactura de carnicerías y prevalencia de Escherichia coli productor de toxina Shiga y Salmonella spp. aisladas en carne molida de carnicerías de Asunción, Paraguay

La inocuidad de la carne comercializada debe estar garantizada en la cadena de producción, para evitar enfermedades transmitidas por alimentos (ETA). Escherichia coli productor de toxina Shiga (STEC) y Salmonella spp. pueden encontrarse en el tracto gastrointestinal de los bovinos y contaminar la carne de consumo humano, pudiendo causar enfermedades en el hombre. Este trabajo tuvo como objetivo evaluar las condiciones higiénico-sanitarias de 52 carnicerías localizadas en Asunción y detectar la frecuencia de STEC y Salmonella spp. en muestras de carne molida. Las condiciones higiénico-sanitarias se evaluaron mediante la estimación del riesgo, utilizando una escala de clasificación por categorías. La detección de STEC y Salmonella spp. se realizó por PCR en tiempo real. En la evaluación inicial, se clasificaron a 33% de las carnicerías como de alto y moderado riesgo. Se detectó STEC no-O157 en un 50% (130/258) de las muestras y Salmonella spp. en un 11% (29/258). Se realizaron acciones de mejora. En la etapa post-intervención, no se detectaron carnicerías de alto riesgo. En el muestreo de seguimiento se detectó un 29% (66/237) de muestras positivas para STEC no-O157 y 7% (16 /237) para Salmonella spp. Este estudio permitió realizar recomendaciones específicas y detalladas a cada carnicería, lo que tuvo un efecto significativo en la mejora de sus condiciones. Esta situación resalta la importancia de continuar fortaleciendo la vigilancia multisectorial y multidisciplinaria. Es imperativo que los establecimientos que se dedican al rubro, implementen las Buenas Prácticas de Manufactura (BPM) como una medida para reducir los riesgos asociados.

The safety of marketed meat must be guaranteed in the production chain, to avoid foodborne illness. Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp. can be found in the gastrointestinal tract of cattle and contaminate meat for human consumption, potentially causing diseases in humans. This work aimed to evaluate the hygienic-sanitary conditions of 52 butcher shops located in Asunción and detect the frequency of STEC and Salmonella spp. in ground beef samples. Hygienic-sanitary conditions were evaluated by estimating risk, using a categorical classification scale. The detection of STEC and Salmonella spp. was performed by real-time PCR. In the initial evaluation, 33% of the butcher shops were classified as high and moderate risk. STEC non-O157 was detected in 50% (130/258) of the samples and Salmonella spp. in 11% (29/258). Improvement actions were carried out. In the post-intervention stage, no high-risk butcher shops were detected. In the follow-up sampling, 29% (66/237) of positive samples were detected for STEC non-O157 and 7% (16/237) for Salmonella spp. This study allowed specific and detailed recommendations to be made to each butcher shop, which had a significant effect on improving their conditions. This situation highlights the importance of continuing to strengthen multisectoral and multidisciplinary surveillance. It is imperative that establishments dedicated to the sector implement Good Manufacturing Practices (GMP) as a measure to reduce associated risks.

First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing Molecular characterization of Escherichia coli O157 isolates from Paraguay / Primera caracterización molecular de aislamientos de Escherichia coli O157:H7 provenientes de muestras clínicas en Paraguay utilizando secuenciación de genoma completo

Abstract Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Resumen Escherichia coli O157:H7 es un patógeno transmitido por alimentos implicado en numerosos brotes en todo el mundo y es capaz de causar complicaciones extraintestinales en humanos. La sección de «Enteropatógenos¼ del Laboratorio Central de Salud Pública trabaja en mejorar la caracterización genómica de STEC, de modo de potenciar la vigilancia laboratorial y la investigación de brotes de enfermedades transmitidas por alimentos. La secuenciación de genoma completo (WGS, por sus siglas en inglés) se propone a nivel mundial como una herramienta de alta resolución para ser utilizada en el laboratorio de rutina, ya que permite obtener todos los resultados en un único proceso. El objetivo de este trabajo fue llevar a cabo, por primera vez, la caracterización genómica por WGS de nueve cepas STEC O157:H7 aisladas en Paraguay a partir de muestras de origen humano. Pudimos identificar los factores de virulencia, los mecanismos de resistencia, el subtipo MLST, e incluso pudimos establecer la relación filogenética entre los aislamientos. Además, detectamos que la mayoría de las cepas pertenecían al clado hipervirulento 8.

First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing.

Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Mecanismos plasmídicos de resistencia a quinolonas, betalactámicos y colistina en Salmonella enterica. Paraguay 2020-2021

Las bacterias son capaces de desarrollar mecanismos de resistencia a los antimicrobianos, aquellos adquiridos y transmisibles son los más significativos debido al potencial de diseminación. La aparición de Salmonella enterica con resistencia a C3aG, quinolonas y a colistina representa una amenaza progresiva. El objetivo fue determinar la resistencia a los antimicrobianos y la presencia de los mecanismos de resistencia plasmídicos a quinolonas, ß-lactámicos y colistina en aislados de Salmonella provenientes de la vigilancia integrada de enteropatógenos. Fueron estudiadas 501 cepas de Salmonella spp. colectadas entre los años 2020 y 2021, por la red de enteropatógenos del Laboratorio Central de Salud Pública. Se investigó la resistencia a las C3aG, quinolonas y colistina, en aislamientos de humanos, alimentos, animales de consumo y ambiente. Las cepas estudiadas exhibieron resistencia a tetraciclina (32,5%), ácido nalidíxico (29%), ampicilina (13,2%), nitrofurantoína (11,6%), C3aG (7,2%), cotrimoxazol (5,8%), ciprofloxacina (2,2%). El 18% (90/501) presentaron resistencia trasferible por plásmidos, fueron detectados 111 genes (71 cepas con un gen, 17 cepas dos genes y 2 cepas tres genes diferentes). Qnr B: 41,1% (37/90), mcr-1: 38,9% (35/90), CMY: 23,3% (21/90), CTX-M: 16,7% (15/90) y Qnr S: 3,3% (3/90). Heidelberg fue el serovar predominante en muestras de pollo y el mayor portador de genes de resistencia de tipo CMY y mcr-1. La detección de genes en alimentos y animales de consumo, que pueden transmitirse fácilmente al ser humano es motivo de alerta y resalta la importancia de continuar fortaleciendo la vigilancia multisectorial y multidisciplinaria.

Bacteria can develop antimicrobial resistance mechanisms, those acquired and transmissible being the most significant due to the potential for dissemination. The emergence of Salmonella enterica with resistance to third-generation cephalosporins, quinolones, and colistin represents a progressive threat. The objective was to determine antimicrobial resistance and the presence of plasmid resistance mechanisms to quinolones, ß-lactams, and colistin in Salmonella isolates from integrated surveillance of enteropathogens. Five hundred and one strains of Salmonella spp. collected between 2020 and 2021 were studied by the enteropathogen network of the Laboratorio Central de Salud Publica (Central Public Health Laboratory). Research was conducted on the resistance to third-generation cephalosporins, quinolones, and colistin, isolated from humans, foodstuffs, animals for consumption, and the environment. The strains studied exhibited resistance to tetracycline (32.5%), nalidixic acid (29%), ampicillin (13.2%), nitrofurantoin (11.6%), third-generation cephalosporins (7.2%), cotrimoxazole (5.8%), and ciprofloxacin (2.2%). Eighteen percent (90/501) presented plasmid-transferable resistance, 111 genes were detected (71 strains with one gene, 17 strains with two genes, and 2 strains with three different genes). Qnr B: 41.1% (37/90), mcr-1: 38.9% (35/90), CMY: 23.3% (21/90), CTX-M: 16.7% (15/90), and Qnr S: 3.3% (3/90). Heidelberg was the predominant serovar in chicken samples and the largest carrier of CMY and mcr-1 resistance genes. The detection of genes in foodstuffs and animals for consumption, which can be easily transmitted to humans, is a cause for alarm and highlights the importance of continuing to strengthen multisectoral and multidisciplinary surveillance.

Caracterización epidemio-molecular de la infección por Clostridioides difficile en pacientes hospitalizados con cuadro diarreico en instituciones de salud. 2019

La infección por Clostridioides difficile es una de las principales causas de diarrea nosocomial en hospitales del mundo, asociada a antibióticos de amplio espectro. Estudio descriptivo, retrospectivo, de corte transverso, de tipo censal. Se estudiaron 281 muestras de pacientes hospitalizados con la infección, se analizaron características socio-demográficas, clínicas y se caracterizó molecularmente al patógeno. Como resultado, se obtuvieron pacientes con la infección con una mediana de edad de 64 años siendo el 61,5 % del sexo masculino. El promedio de presentación del cuadro diarreico fue de 5 días, con tratamiento antimicrobiano de 8 días e internación de 15 días. El 94% tuvo tratamiento antimicrobiano previo, y el 6% estuvo expuesto a algún factor de riesgo. Los antimicrobianos más utilizados solos o en combinación fueron betalactámicos, fluoroquinolonas, vancomicina y carbapenémicos. Las áreas de internación con mayor frecuencia de presentación de la infección fueron las unidades de Clínica Médica, Traumatología, Geriatría y Unidad de Terapia Intensiva. La prevalencia de C. difficile toxigénico fue de 14%, y de esta frecuencia el 100% presentó toxinas TcdA y TcdB, con ausencia de toxinas binarias y deleción del gen tcdC. Se constató la presencia grupos clonales en la misma unidad de internación y misma institución de salud. El 100% de las cepas resultaron susceptibles a los antibióticos de elección para la infección. La prevalencia de la infección y presencia de perfiles clonales detectadas revelan la necesidad de un mejoramiento en el sistema de control de infecciones, así como del fortalecimiento y vigilancia de la resistencia antimicrobiana.

Clostridioides difficile infection is one of the main causes of nosocomial diarrhea in hospitals worldwide, associated with broad-spectrum antibiotics. Descriptive, retrospective, cross-sectional, census-type study. Two hundred eighty-one samples from patients hospitalized with the infection were studied, sociodemographic and clinical characteristics were analyzed and the pathogen was characterized molecularly. As a result, patients with the infection had a median age of 64 years and 61.5% male. The average presentation of diarrhea was 5 days, antimicrobial treatment 8 days and hospitalization 15 days. Ninety four percent had previous antimicrobial treatment, and 6% were exposed to some risk factor. The most commonly used antimicrobials alone or in combination were beta-lactams, fluoroquinolones, vancomycin, and carbapenems. The hospitalization areas with the highest frequency of presentation of the infection were the Clinical Medicine, Traumatology, Geriatrics and Intensive Care Unit units. The prevalence of toxigenic C. difficile was 14%, and of this frequency, 100% presented TcdA and TcdB toxins, with the absence of binary toxins and deletion of the tcdC gene. The presence of clonal groups was verified in the same hospitalization unit and the same health institution. All the strains were susceptible to the antibiotics of choice for the infection. The prevalence of infection and the presence of clonal profiles detected reveal the need for improvement of the infection control system, as well as of the strengthening and surveillance of antimicrobial resistance.

Resistencia antimicrobiana en Escherichia coli de muestras cecales de bovinos para carne faenados en frigoríficos de la zona del arroyo Mburicao, Asunción-Paraguay. Año 2021

La resistencia a los antimicrobianos es una de las preocupaciones más importantes a nivel mundial, ya que determina un aumento de la morbimortalidad de los pacientes y el incremento de los costos de la atención de salud; problema cuyo abordaje debe ser realizado bajo el enfoque de Una Salud. El objetivo principal de este trabajo fue evaluar la resistencia antimicrobiana en Escherichia coli provenientes de muestras cecales de bovinos de carne faenados en frigoríficos de la zona del Arroyo Mburicao, Asunción-Paraguay, de setiembre a noviembre de 2021. Fueron colectadas 181 muestras de ganados provenientes de la Región Oriental y Occidental, y cultivadas en agar cromogénico suplementados con ciprofloxacina, colistina, cefotaxima y meropenem. Las colonias aisladas fueron identificadas, genotipificadas (PCR convencional) y sometidas a pruebas de susceptibilidad. El principal hallazgo fue la confirmación de la resistencia a las fluoroquinolonas en un 8,3% debida a la portación de los genes Qnr S, Qnr B y aac-6´-Ib-cr, involucrados en el mecanismo plasmídico de resistencia a quinolonas. Además, la resistencia acompañante encontrada en los aislamientos resistentes del 100% a tetraciclina y 53% a trimetoprim/sulfametoxazol. No se encontraron aislamientos con resistencia a cefotaxima, colistina y meropenem. Los resultados obtenidos son de suma relevancia para la generacion de conocimiento sobre los perfiles de resistencia de microorganismos en el sector veterinario, pudiendo contribuir a la elaboración de guías nacionales para el uso prudente de antimicrobianos y apoyando al trabajo multisectorial en la lucha para la contención de la resistencia antimicrobiana.

Antimicrobial resistance is one of the most critical concerns worldwide since it determines an increase in patient morbidity and mortality and the increase in healthcare costs, a problem whose approach must be carried out under the One Health approach. The main objective of this work was to evaluate antimicrobial resistance in Escherichia coli from cecal samples of cattle for meat slaughtered in meat processing plants in the Arroyo Mburicao area, Asunción-Paraguay, from September to November 2021. One hundred eighty one samples of cattle from the Eastern and Western Region were collected, and cultured on chromogenic agar supplemented with ciprofloxacin, colistin, cefotaxime, and meropenem. Isolated colonies were identified, genotyped (conventional PCR) and subjected to susceptibility tests. The main finding was the confirmation of resistance to fluoroquinolones in 8.3% due to the carrying of genes Qnr S, Qnr B and aac-6'-Ib-cr, involved in the plasmid mechanism of resistance to quinolones. In addition, the accompanying resistance found in the isolates was 100% resistant to tetracycline and 53% to trimethoprim/sulfamethoxazole. Isolates with resistance to cefotaxime, colistin and meropenem were not found. The results obtained are highly relevant for the generation of knowledge about the resistance profiles of microorganisms in the veterinary sector. It can contribute to the development of national therapeutic guidelines for the prudent use of antimicrobials, and supporting multisectoral work in the fight to contain antimicrobial resistance.

Vigilancia de la resistencia del Mycobacterium tuberculosis a los fármacos en Paraguay, 2014 a 2017 / Surveillance of drug resistance of Mycobacterium tuberculosis in Paraguay, 2014 to 2017

El objetivo del estudio fue identificar la resistencia del Mycobacterium tuberculosis a los fármacos en Paraguay, 2014 a 2017. Se realizó un estudio observacional retrospectivo. Se utilizaron los datos del Programa Nacional de Tuberculosis del Paraguay comprendidos entre los años 2014 a 2017. Se incluyeron todos los pacientes con diagnóstico de Tuberculosis que se realizaron un test de resistencia. Se extrajeron los datos en Excel y fueron analizados con Stata 17.0. Se incluyeron 3429 pacientes con tuberculosis que contaban con resultado de al menos una prueba de sensibilidad. La resistencia se encontró en 2.1% de los pacientes. La resistencia a la Rifampicina estuvo presente en el 0.3% de los casos mientras que a la Izionazida en el 0.6% de los casos. La prevalencia de resistencia fue más alta en hombres 3.4 (IC 95% 2.2 - 4.8) p=0.003, que residían en el chaco 6.0 (IC 95% 3.4 - 9.7) p=0.000, previamente tratados 2.7 (IC 95% 1.1 - 5.1) p=0.010. En el modelo se pudo observar que un paciente previamente tratado tiene mayores posibilidades de tener resistencia OR 2.62 (IC 95% 1.1 - 6.24). La prevalencia de resistencia del Mycobacterium tuberculosis a fármacos estuvo relacionada con haber sido previamente tratado

The objective of the study was to identify the resistance of Mycobacterium tuberculosis to drugs in Paraguay, 2014 to 2017. A retrospective observational study was carried out. The data from the National Tuberculosis Program of Paraguay between the years 2014 to 2017 were used. All patients with a diagnosis of Tuberculosis who underwent a resistance test were included. Data were extracted in Excel and analyzed with Stata 17.0. 3429 tuberculosis patients who had a result of at least one sensitivity test were included. Resistance was found in 2.1% of patients. Resistance to Rifampicin was present in 0.3% of cases while to Izionazide in 0.6% of cases. The prevalence of resistance was higher in men 3.4 (95% CI 2.2 - 4.8) p = 0.003, who resided in the Chaco 6.0 (95% CI 3.4 - 9.7) p = 0.000, previously treated 2.7 (95% CI 1.1 - 5.1) p = 0.010. In the model, it was observed that a previously treated patient has a greater chance of having resistance OR 2.62 (95% CI 1.1 - 6.24). The prevalence of resistance of Mycobacterium tuberculosis to drugs was related to having been previously treated

Factores asociados a la mortalidad por tuberculosis en indígenas en Paraguay, 2014 a 2019

Introducción: La tasa de mortalidad estimada de tuberculosis (TBC) en Paraguay en 2019 fue de 3,9 por 100.000 habs. Las comunidades indígenas presentan un elevado riesgo de padecer TBC. Objetivo: Determinar los factores asociados a la mortalidad en personas de origen indígena con diagnóstico de TBC en Paraguay, 2014-2019. Métodos : Se realizó un estudio observacional retrospectivo. Se utilizaron los datos del Programa Nacional de Control de la TBC del Paraguay comprendidos entre los años 2014 y 2019. Se registraron los datos sociodemográficos y factores de riesgo. Resultados: Se incluyeron 2.210 personas de origen indígena con diagnóstico de TBC, el 53,8% fue de sexo masculino, entre 20 y 39 años (32,3%) y 0 a 19 años de edad (30,2%), la localización de la TBC fue mayoritariamente pulmonar (92,3%). Durante el 2014 a 2019 se observaron 217 muertes (9,8%). Los factores asociados a mortalidad en pacientes con TBC fueron la edad (adOR = 13,95; CI: 7,07-27,55 mayor a 80 años), (adOR = 4,20; CI: 2,59-6,82 mayor a 60 años) y (adOR = 3,30; CI: 2,06-5,28 para 40 a 59 años), la co-infección VIH (adOR =), y la localización de la TBC (adOR = 3,60; CI: 1,88-6,90 para TBC diseminada). Conclusión: La mayor edad, el diagnóstico de co-infección VIH y localización de la TBC diseminada, están asociados a un mayor riesgo de muerte en población indígena con TBC.

Background: The estimated tuberculosis (TB) mortality rate in Paraguay in 2019 was 3.9 per 100,000 people. Indigenous communities are at high risk for TB. Aim: To determine the factors associated with mortality in indigenous people with a diagnosis of TB in Paraguay, 2014-2019. Methods: A retrospective observational study was done. sociodemographic data and risk factors data from the National TB Program of Paraguay between the years 2014 to 2019 were used. Results: A total of 2,210 indigenous people with a diagnosis of TB were included, 53,8% were male, between 20 to 39 (32.3%) and 0 to 19 years old (30.2%), the localization of TBC was majority pulmonary (92.3%). During 2014 to 2019 the were 217 deaths (9.8%). The factors associated to mortality in TB patients were the age (adOR = 13.95; CI: 7.07-27.55 older than 80 years), (adOR = 4.20; CI: 2.59-6.82 older than 60 years), and (adOR = 3.30; CI: 2.06 - 5.2840 to 59 years), HIV co-infection (adOR = 7.07; CI 3.74-13.87), and localization of TB (adOR = 3.60; CI: 1.88-6.90 for disseminated TB). Conclusion: Older age, HIV co-infection and disseminated localization of TBC are associated with a higher risk of death in indigenous people with TB.

RMplex: An efficient method for analyzing 30 Y-STRs with high mutation rates.

Y-chromosomal short tandem repeats (Y-STRs) with high mutation rates are recognized as valuable genetic markers for differentiating paternally related men, who typically cannot be separated with standard Y-STRs, and were shown to provide paternal lineage differentiation on a higher resolution level than standard Y-STRs. Both features make Y-STRs with high mutation rates relevant in criminal casework, particularly in sexual assault cases involving highly unbalanced male-female DNA mixtures that often fail autosomal forensic STR profiling for the male donor. Previously, the number of known Y-STRs with mutation rates higher than 10-2 per locus per generation termed rapidly mutating Y-STRs (RM Y-STRs) was limited to 13, which has recently been overcome by the discovery and characterization of 12 additional RM Y-STRs. Here, we present the development and validation of RMplex, an efficient genotyping system for analyzing 30 Y-STRs with high mutation rates, including all currently known RM Y-STRs, using multiplex PCR with capillary electrophoresis (CE) or massively parallel sequencing (MPS), overall targeting a total of 44 male-specific loci. If previously unavailable, repeat number assignations were provided based on newly generated MPS data. Validation tests based on the CE method demonstrated that the results were both repeatable and reproducible, full profiles were achieved with minimal input DNA of 250 pg for RMplex 1 and 100 pg for RMplex 2, and in the presence of inhibitors, or with a surplus of female DNA, the assays performed reasonably well. Application of RMplex to differentiate between paternally related men was exemplified in 32 males belonging to five different paternal pedigrees. Given further successful forensic validation testing, we envision the future application of RMplex in criminal cases where it is suspected, or cannot be excluded, that the crime scene trace originated from a male relatives of the suspect who is highlighted with standard Y-STR matching. Other applications of RMplex are in criminal cases without known suspects to differentiate between male relatives highlighted in familial searching based on standard Y-STR matching.

Resistencia a múltiples antibióticos en serovariedades de Salmonella aisladas de muestras clínicas y alimentos / Resistance to multiple antibiotics in serovarities of Salmonella isolated from clinical samples and foods

Salmonella enterica es un patógeno transmitido por alimentos y agente etiológico de brotes alimentarios de gran impacto en la salud humana. El aumento de la resistencia bacteriana constituye una amenaza a la salud pública, la aparición de cepas de Salmonella con resistencia a múltiples antimicrobianos (MDR) fue descrita en humanos, alimentos y animales para consumo; por ello se considera muy importante conocer la situación epidemiológica local. El objetivo de este trabajo fue generar información sobre los serotipos circulantes, resistencia a los antibióticos y presencia de resistencia simultánea a múltiples fármacos en Salmonella provenientes de muestras clínicas humanas y muestras de alimentos en el periodo desde 2017 a 2019. Fueron analizadas un total de 668 cepas de Salmonella aisladas en los años 2017, 2018 y 2019 a partir de muestras clínicas humanas y de alimentos, en el Laboratorio Central de Salud Pública y/o remitidas por Laboratorios de la Red de Enteropatógenos. Se observaron serotipos muy diversos con prevalencia del serovar Heidelberg en alimentos y Typhimurium en muestras de humanos. Se encontró que el 45,4% de las cepas fueron sensibles a todos los antibióticos (ATB), el 35,6% fueron resistentes de 1 a 6 ATB y el 19% con sensibilidad intermedia; observándose mayor resistencia a Tetraciclina, Ác. Nalidíxico, Ampicilina y Nitrofurantoína, en menor grado se evidenció resistencia a cefalosporinas (C3ªG) y a ciprofloxacina. El 16.9% de las cepas presentaron resistencia múltiple (3 o más antibióticos) con 37 fenotipos distintos. Las serovariedades que presentaron mayor resistencia a los antimicrobianos fueron Heidelberg, Schwarzengrund y Typhimurium

Salmonella enterica is a foodborne pathogen and etiological agent of food outbreaks with a great impact on human health. The increase in bacterial resistance constitutes a threat to public health. The appearance of Salmonella strains with resistance to multiple antimicrobials (MDR) has already been described in humans, food and animals for consumption; for this reason, it is considered very important to know the local epidemiological situation. The target of this work was to generate information on circulating serotypes, antibiotic resistance and the presence of simultaneous resistance to multiple drugs in Salmonella from human clinical samples and food samples in the period from 2017 to 2019. A total of 668 Salmonella strains isolated in the years 2017, 2018 and 2019 were analyzed from human and food clinical samples, at the Central Public Health Laboratory and / or sent by Laboratories of the Enteropathogens Network. Very diverse serotypes were observed with prevalence of Heidelberg serovar in food and Typhimurium in human samples .It was found that 45,4% of the strains were sensitive to all antibiotics (ATB), 35,6% were resistant from 1 to 6 ATB and 19% with intermediate sensitivity; observing greater resistance to Tetracycline, Ác. Nalidixic, Ampicillin and Nitrofurantoin, to a lesser degree resistance to cephalosporins (C3ªG) and ciprofloxacin was evidenced. The 16.9% the strains presented multiple resistance (3 or more antibiotics) with 37 different phenotypes. The serovars with the highest antimicrobial resistance were Heidelberg, Schwarzengrund and Typhimurium

Detección de Clostridioides difficile toxigénico a partir de muestras diarreicas por reacción en cadena de la polimerasa, en pacientes hospitalizados en Paraguay. Periodo 2016-2018 / Detection of toxigenic Clostridioides difficile from diarrheic samples by polymerase chain reaction in hospitalized patients in Paraguay. Period 2016-2018

La infección por Clostridioides difficile (ICD) se considera la principal enfermedad diarreica asociada a pacientes internados en instituciones de salud, generalmente mayores de 61 años y al uso de antimicrobianos de espectro extendido. Es un bacilo grampositivo anaerobio estricto, esporulado. La alteración de la microbiota colónica por el tratamiento antimicrobiano permite la colonización e infección por este microorganismo, cuya manifestación clínica, se basa en la presentación de cuadro diarreico. El objetivo de este estudio fue detectar C. difficile toxigénico a partir de muestras diarreicas por reacción en cadena de la polimerasa en pacientes hospitalizados. Estudio descriptivo de corte transverso, prospectivo que utilizó como un instrumento de medición una ficha epidemiológica conteniendo las variables de estudio y consentimiento informado. En 901 muestras diarreicas, se detectaron las toxinas tcdA, tcdB, ctdA, ctdB y tcdC y del gen de especie. La prevalencia de C. difficile toxigénico fue de 19,7% (n=178) de las muestras que dieron positivas para una o ambas toxinas (toxinas A y B); el 98% presentó ambas toxinas. Se observó mayor presentación de ICD en pacientes con una mediana de 68 años, y en el sexo masculino en un 52%. Se evaluó el tratamiento antimicrobiano y el uso de los antimicrobianos, donde, el uso de clindamicina, cefalosporinas, fluoroquinolonas y vancomicina, presentó valores estadísticamente significativos. Los resultados obtenidos permitieron caracterizar epidemiológicamente la infección por este patógeno. Es de gran importancia realizar en forma temprana el diagnóstico y diseñar e implementar estrategias para evitar la emergencia de este patógeno

Clostridioides difficile infection is considered the main diarrheal disease associated with patients hospitalized in health institutions, older than 61 years and the use of extended spectrum antimicrobials. It is a strict anaerobic, sporulated gram-positive bacillus. The alteration of the colonic microbiota by antimicrobial treatment allows colonization and infection by this microorganism, whose clinical manifestation is based on the presentation of the diarrheal syndrome. The objective of this study was to detect toxigenic C. difficile from diarrheal samples by polymerase chain reaction in hospitalized patients. This was a descriptive, cross-sectional, prospective study in which an epidemiological record containing the study variables and informed consent were used. In 901 diarrheal samples, tcdA, tcdB, ctdA, ctdB and tcdC toxins and the species gene were detected. The prevalence of toxigenic C. difficile was 19.7% (n=178) of the samples, positives for one or both toxins (toxins A and B) while 98% presented both toxins. A higher frequency of ICD was observed in male patients (52%) who had a median age of 68 years. Antimicrobial treatment and use of antimicrobials were evaluated, where the use of clindamycin, cephalosporins, fluoroquinolones and vancomycin had statistically significant values. The results allowed infection by this pathogen to be epidemiologically characterized. It is very important to make early diagnosis and design and implement strategies to prevent the emergence of this pathogen

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA.

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer's recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

Prevalencia de patógenos causantes de enfermedad diarreica aguda en el área Metropolitana de Asunción y Central

Introducción: Las enfermedades diarreicas agudas (EDA) constituyen un problema de salud pública y son una de las causas más importantes de mortalidad y morbilidad en niños a nivel mundial. Objetivo: Determinar la prevalencia de enteropatógenos causantes de EDA en el área metropolitana de Asunción y Central. Materiales y métodos: Se realizó un estudio observacional, descriptivo de corte transverso. Se analizaron 743 muestras de heces diarreicas, en las cuales se investigó la presencia de Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli diarreigénicas y Rotavirus, utilizando técnicas de referencia. Resultados: En el 31,2% (232/743) de las muestras fue posible identificar al menos uno de los patógenos entéricos investigados, siendo las E. coli diarreigénicas fueron las bacterias identificadas con mayor frecuencia, seguido por Rotavirus, Campylobacter spp., Shigella spp. y en último lugar, Salmonella spp. Conclusión: La población más afectada corresponde a niños menores de 5 años. El principal patógeno identificado como agente causal de diarreas fueron las E. coli diarreigénicas en primer lugar, seguido por Rotavirus, Campylobacter spp., Shigella spp. y Salmonella spp. En algunas muestras se detectaron más de un patógeno entérico, encontrando incluso casos de coinfección con hasta cuatro patógenos diferentes.

Introduction: Acute diarrheal diseases (ADD) constitute a public health problem and are one of the most important causes of mortality and morbidity in children worldwide. Objective: To determine the prevalence of enteropathogens causing ADD in the metropolitan area of ​​Asunción and Central. Materials and methods: An observational, descriptive cross-sectional study was conducted. 743 samples of diarrheic feces were analyzed, in which the presence of Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli diarreigenic and Rotavirus was investigated, using reference techniques. Results: In 31.2% (232/743) of the samples it was possible to identify at least one of the enteric pathogens investigated, being the diarrhenetic E. coli were the most frequently identified bacteria, followed by Rotavirus, Campylobacter spp., Shigella spp. and lastly, Salmonella spp. Conclusion: The most affected population corresponds to children under 5 years of age. The main pathogen identified as the causative agent of diarrhea was diarrigenic E. coli, followed by Rotavirus, Campylobacter spp., Shigella spp. and Salmonella spp. In some samples more than one enteric pathogen was detected, even finding cases of coinfection with up to four different pathogens.

Perfiles genéticos bacterianos y análisis de brotes de las Enfermedades Transmitidas por Alimentos empleando Electroforesis de Campo Pulsado como herramienta para la vigilancia epidemiológica molecular / Bacterial genetic profiles and analysis of outbreaks of foodborne diseases using pulsed field gel electrophoresis as a tool for molecular epidemiological surveillance

Las Enfermedades de Transmisión Alimentaria (ETA) son un problema de salud pública con altos índices de morbilidad y mortalidad a nivel global. La vigilancia y estudio de brotes de las ETA a través de Electroforesis de Campo Pulsado (PFGE) constituye un soporte fundamental para la investigación epidemiológica. El objetivo del estudio es presentar la base de datos de perfiles genéticos bacterianos y analizar brotes de enfermedades transmitidas por alimentos empleando Electroforesis de Campo Pulsado. Estudio descriptivo observacional de carácter retrospectivo, muestreo por conveniencia en el que fueron estudiados 778 aislamientos bacterianos causantes de ETA. La Base de Datos Nacional (BDN) quedó conformada por los siguientes patógenos entéricos; Salmonella spp., Shigella sonnei, Vibrio cholerae, Campylobacter spp., Escherichia coli O157:H7 y Escherichia coli no O157 caracterizados por una diversidad de patrones únicos, clusters y brotes. La BDN de Salmonella spp., quedó representada por un total de 558 cepas con 248 PUN, de las cuales 22,6% (126 cepas) corresponden a Salmonella enterica ser. Typhimurium, 20,6% (115 cepas) a Salmonella enterica ser. Enteritidis, 9,1% (51 cepas) a Salmonella enterica ser. Newport, 1,6% (9 cepas) a Salmonella enterica ser. Muenchen, que al mismo tiempo son los serotipos que están asociados a brotes. Fueron confirmados un total de 13 brotes causados por Salmonella spp.; Shigella sonnei con 113 cepas estudiadas, 57 patrones únicos y 19 clusters detectados. Se identificaron 3 patrones PYJ16X01.0012, PYJ16X01.0034 y PYJ16X01.0014 como los predominantes. Vibrio cholerae con 18 cepas estudiadas, 9 patrones únicos y 4 clusters detectados. Se pudo establecer una relación genética del 100% entre cepas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa productora de toxinas ctxA y tcpA aislada del caso índice del brote de cólera. Campylobacter spp., con 62 cepas estudiadas, 42 patrones únicos y 10 clusters detectados. La BDN de E. coli productor de toxina shiga O157 y no O157, con 9 y 20 cepas de origen humano respectivamente, caracterizadas según sus factores de virulencia y subtipos. Se reconocieron 8 patrones electroforéticos PUN y 1 cluster para E. coli productor de toxina shiga O157, y 18 PUN y 1 clúster para E. coli productor de toxina shiga no O157.La disponibilidad de una Base de Datos Nacional de patógenos bacterianos transmitidos por alimentos constituye un importante avance para la salud pública, con un gran aporte en la vigilancia y epidemiología del país permitiendo la confirmación y detección de brotes discriminando aislamientos relacionados genéticamente y por consiguiente el estudio de relaciones clonales y probable origen(AU)

Foodborne diseases (FBD) are a problem of public health with high indexes of morbidity and mortality at global level. The surveillance and study of outbreaks of the FBD through pulsed field gel electrophoresis (PFGE) is a fundamental support for epidemiological research. The aim of the study is to present the database of bacterial genetic profiles and analyze outbreaks of FBD using PFGE. This was an observational descriptive retrospective study with convenience sampling in which 778 bacterial isolates causing FBD were studied. The National Database (NDB) was made up of the following enteric pathogens causing FBD: Salmonella spp., Shigella sonnei, Vibrio cholerae, Campylobacter spp., Escherichia coli O157: H7 and Escherichia coli no O157. Each of them was characterized by a diversity of unique patterns, clusters and outbreaks. The NDB of Salmonella spp. was represented by a total of 558 strains with 248 PUN, of which 22.6% (126 strains) correspond to Salmonella enterica ser. Typhimurium, 20.6% (115 strains) to Salmonella enterica ser. Enteritidis, 9.1% (51 strains) to Salmonella enterica ser. Newport, 1.6% (9 strains) to Salmonella enterica ser. Muenchen, which at the same time are the serotypes associated with outbreaks. A total of thirteen outbreaks caused by Salmonella spp., Shigella sonnei with 113 strains studied, 57 unique patterns and 19 clusters detected were confirmed. Three patterns PYJ16X01.0012, PYJ16X01.0034 and PYJ16X01.0014 were identified as the predominant. Vibrio cholerae with 18 strains studied, 9 unique patterns and 4 clusters were detected. A genetic relationship of 100% was established between strains of Vibrio cholerae O1 biotype El Tor serotype Ogawa toxin producer ctxA and tcpA isolated from the index case of the cholera outbreak. Campylobacter spp., with 62 strains studied, 42 unique patterns and 10 clusters were detected. The NDB of O157 and non-O157 Shiga toxin producing E. coli O157, with 9 and 20 strains of human origin respectively, were characterized according to their virulence factors and subtypes. We recognized 8 PUN electrophoretic patterns and 1 cluster for O157 Shiga toxin producing E. coli, and 18 PUN and 1 cluster for non-O157 Shiga toxin producing E. coli. The availability of a National Database of bacterial pathogens transmitted by food constitutes an important advance for public health, with a great contribution to the surveillance and epidemiology of the country allowing the confirmation and detection of outbreaks discriminating genetically related isolates and therefore, the study of clonal relationships and probable origin(AU)

Primeros resultados de la vigilancia integrada de la resistencia antimicrobiana de patógenos transmitidos por alimentos, campylobacter spp y salmonella spp en tres poblaciones distintas. Paraguay. 2011-2012 / First results of the comprehensive surveillance of the antimicrobial resistance of foodborne pathogens, campylobacter spp and salmonella spp in three different populations. Paraguay, 2011-2012

La infección causada por Salmonella spp. y por Campylobacter spp. son las enfermedades transmitidas por alimentos (ETA) reportadas más frecuentemente en el mundo, siendo la carne de pollo uno de los vehículos alimentarios más importantes para ambas. Se presenta los primeros resultados de la vigilancia antimicrobiana integrada de las ETA de Salmonella spp. y Campylobacter spp. en tres poblaciones. En este estudio descriptivo de corte transverso, de casos consecutivos, se recolectaron muestras de diversos orígenes de carne de pollo y distintas poblaciones para su aislamiento, caracterización y perfil de resistencia. Se observó una prevalencia de Campylobacter spp. del 13% en alimentos, 20% en muestras clínicas y 55% en heces cloacales de aves, con alta prevalencia de Campylobacter jejuni en las tres poblaciones; de Salmonella spp fue 6% en alimentos, 13% en muestras clínicas y 3% en heces cloacales de aves, con predominio del serotipo Salmonella ser. Enteritidis en las muestras clínicas y heces cloacales de aves. La resistencia a ciprofloxacina de Campylobacter spp., entre 59-81% se destacó en las tres poblaciones estudiadas. Para Salmonella spp. se observó una resistencia a nitrofurantoina del 73% en heces cloacales de aves, 55% en alimentos y 19,4% en humanos; a tetraciclina, 42% en alimentos, 5% en muestras clínicas y 9% en heces cloacales; para el ácido nalidíxico la resistencia fue del 72% en animales y 53% en muestras clínicas. Es importante fortalecer la vigilancia integrada de la resistencia antimicrobiana en estas tres poblaciones de manera a detectar en forma oportuna mecanismos de resistencia que pudieran afectar al ser humano a través de la cadena alimentaria.

Infection caused by Salmonella ssp. and Campylobacter spp. are the foodborne diseasesreported most frequently throughout the world, and chicken meat is considered one of themost important food vehicles for both. The objective was to present the first resultsobtained from the integrated antimicrobial surveillance of foodborne diseases of Salmonellaspp. and Campylobacter spp in three populations. In this descriptive cross - sectional ofconsecutive sampling, samples were collected from different sources of chicken meat and different populations for isolation, characterization and resistance profile. A prevalence of13% in food, 20% in clinical samples and 55% in cloacal feces was observed in the isolationof Campylobacter spp. with high prevalence of Campylobacter jejuni in all three populationsfollowed by 6% in food, 13% in clinical samples and 3% in birds cloacal feces of Salmonellaspp. with predominance in the isolation of the serotype Salmonella ser. Enteritidis in clinicalsample populations and birds cloacal feces. The resistance of Campylobacter spp. tociprofloxacin of 59-81%, stood out in the three populations under study, in contrast toSalmonella spp. A high resistance to nitrofurantoin of 73% was observed in poultry feces,55% in foods and 19.4% in humans. Resistance to tetracycline was found in foods (42%),5% in clinical samples and 9% in cloacal feces. A resistance of 72% was observed inanimals and 53% in clinical samples for nalidixic acid. It is important to strengthen theintegrated surveillance of antimicrobial resistance in these three populations in order totimely detect mechanisms of resistance that can affect the human being through the foodchain.

Detección molecular de Escherichia coli diarreogénica en pacientes pediátricos con síndrome diarreico agudo en Paraguay / Molecular detection of diarrheogenic Escherichia coli in pediatric patients with acute diarrheal syndrome in Paraguay

La Escherichia coli diarreogénica (ECD) se ha clasificado con base en criterios clínicos, epidemiológicos y moleculares en cinco grupos, cada uno con factores de virulencia específicos. El objetivo fue determinar la prevalencia de ECD en pacientes pediátricos con enfermedad diarreica aguda del Laboratorio Central de Salud Publica en el periodo 2012- 2015. Se procesaron muestras de heces con síndrome diarreico agudo, provenientes de pacientes pediátricos, en los cuales se buscó algún gen de virulencia ECD utilizando métodos convencionales de siembra y screening molecular, mediante PCR múltiple con cebadores diseñados específicamente para amplificar los genes de virulencia elt, est, eae, stx, ipaH y aggR. Del total de muestras analizadas, 13% (180/1379) de las muestras presentó algún factor de virulencia compatible con algún patotipo ECD con mayor predominio en niños de 1 a 3 años. La frecuencia de los distintos patotipos fue la siguiente: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC y 3 (2%) ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. El porcentaje de E. coli diarreogénicas detectado tiene similitud con lo reportado en otros países de la región, lo que nos indica que estos patógenos son parte importante de la etiología de la enfermedad diarreica aguda infecciosa en la población infantil en nuestro país. Se debe destacar que para el diagnóstico de las diferentes categorías ECD, es necesario disponer de un procedimiento diagnóstico específico dirigido a la detección de los factores de virulencia utilizando métodos moleculares o métodos inmunológicos.

Diarrheagenic Escherichia coli (DEC) has been classified based on clinical, epidemiological and molecular criteria in five groups, each with specific virulence factors. The objective was to determine the prevalence of DEC in pediatric patients with acute diarrheal disease of the Central Laboratory of Public Health in the 2012-2015 period. A total of 1447 fecal samples of acute diarrheal syndrome from pediatric patients were processed in which a DEC virulence gene was searched using conventional screening and molecular screening methods with multiple PCR primers specifically designed to amplify virulence genes, st, lt, eae, stx, ipaH and aggR. From the total of analyzed samples, 13% (180/1379) of the samples presented some virulence factor compatible with a DEC pathogen type with greater predominance in children from 1 to 3 years. The frequency of the different pathogen types was as follows: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC and 3 (2% ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. The percentage of DEC detected is similar to that reported in other countries of the region, which indicates that these pathogens are an important part of the etiology of acute infectious diarrheal disease in children in our country. It should be noted that for the diagnosis of different DEC categories, it is necessary to have a specific diagnostic procedure aimed at the detection of virulence factors using molecular methods or immunodiagnostic methods.

Development of a control region-based mtDNA SNaPshot™ selection tool, integrated into a mini amplicon sequencing method.

Mitochondrial DNA (mtDNA) analysis is regularly applied to forensic DNA samples with limited amounts of nuclear DNA (nDNA), such as hair shafts and bones. Generally, this mtDNA analysis involves examination of the hypervariable control region by Sanger sequencing of amplified products. When samples are severely degraded, small-sized amplicons can be applied and an earlier described mini-mtDNA method by Eichmann et al. [1] that accommodates ten mini amplicons in two multiplexes is found to be a very robust approach. However, in cases with large numbers of samples, like when searching for hairs with an mtDNA profile deviant from that of the victim, the method is time (and cost) consuming. Previously, Chemale et al. [2] described a SNaPshot™-based screening tool for a Brazilian population that uses standard-size amplicons for HVS-I and HVS-II. Here, we describe a similar tool adapted to the full control region and compatible with mini-mtDNA amplicons. Eighteen single nucleotide polymorphisms (SNPs) were selected based on their relative frequencies in a European population. They showed a high discriminatory power in a Dutch population (97.2%). The 18 SNPs are assessed in two SNaPshot™ multiplexes that pair to the two mini-mtDNA amplification multiplexes. Degenerate bases are included to limit allele dropout due to SNPs at primer binding site positions. Three SNPs provide haplogroup information. Reliability testing showed no differences with Sanger sequencing results. Since mini-mtSNaPshot screening uses only a small portion of the same PCR products used for Sanger sequencing, no additional DNA extract is consumed, which is forensically advantageous.

Evaluation of the predictive capacity of DNA variants associated with straight hair in Europeans.

DNA-based prediction of hair morphology, defined as straight, curly or wavy hair, could contribute to an improved description of an unknown offender and allow more accurate forensic reconstructions of physical appearance in the field of forensic DNA phenotyping. Differences in scalp hair morphology are significant at the worldwide scale and within Europe. The only genome-wide association study made to date revealed the Trichohyalin gene (TCHH) to be significantly associated with hair morphology in Europeans and reported weaker associations for WNT10A and FRAS1 genes. We conducted a study that centered on six SNPs located in these three genes with a sample of 528 individuals from Poland. The predictive capacity of the candidate DNA variants was evaluated using logistic regression; classification and regression trees; and neural networks, by applying a 10-fold cross validation procedure. Additionally, an independent test set of 142 males from six European populations was used to verify performance of the developed prediction models. Our study confirmed association of rs11803731 (TCHH), rs7349332 (WNT10A) and rs1268789 (FRAS1) SNPs with hair morphology. The combined genotype risk score for straight hair had an odds ratio of 2.7 and these predictors explained ∼ 8.2% of the total variance. The selected three SNPs were found to predict straight hair with a high sensitivity but low specificity when a 10-fold cross validation procedure was applied and the best results were obtained using the neural networks approach (AUC=0.688, sensitivity=91.2%, specificity=23.0%). Application of the neural networks model with 65% probability threshold on an additional test set gave high sensitivity (81.4%) and improved specificity (50.0%) with a total of 78.7% correct calls, but a high non-classification rate (66.9%). The combined TTGGGG SNP genotype for rs11803731, rs7349332, rs1268789 (European frequency=4.5%) of all six straight hair-associated alleles was identified as the best predictor, giving >80% probability of straight hair. Finally, association testing of 44 SNPs previously identified to be associated with male pattern baldness revealed a suggestive association with hair morphology for rs4679955 on 3q25.1. The study results reported provide the starting point for the development of a predictive test for hair morphology in Europeans. More studies are now needed to discover additional determinants of hair morphology to improve the predictive accuracy of this trait in forensic analysis.

Evaluation of DNA variants associated with androgenetic alopecia and their potential to predict male pattern baldness.

Androgenetic alopecia, known in men as male pattern baldness (MPB), is a very conspicuous condition that is particularly frequent among European men and thus contributes markedly to variation in physical appearance traits amongst Europeans. Recent studies have revealed multiple genes and polymorphisms to be associated with susceptibility to MPB. In this study, 50 candidate SNPs for androgenetic alopecia were analyzed in order to verify their potential to predict MPB. Significant associations were confirmed for 29 SNPs from chromosomes X, 1, 5, 7, 18 and 20. A simple 5-SNP prediction model and an extended 20-SNP model were developed based on a discovery panel of 305 males from various European populations fitting one of two distinct phenotype categories. The first category consisted of men below 50 years of age with significant baldness and the second; men aged 50 years or older lacking baldness. The simple model comprised the five best predictors: rs5919324 near AR, rs1998076 in the 20p11 region, rs929626 in EBF1, rs12565727 in TARDBP and rs756853 in HDAC9. The extended prediction model added 15 SNPs from five genomic regions that improved overall prevalence-adjusted predictive accuracy measured by area under the receiver characteristic operating curve (AUC). Both models were evaluated for predictive accuracy using a test set of 300 males reflecting the general European population. Applying a 65% probability threshold, high prediction sensitivity of 87.1% but low specificity of 42.4% was obtained in men aged <50 years. In men aged ≥50, prediction sensitivity was slightly lower at 67.7% while specificity reached 90%. Overall, the AUC=0.761 calculated for men at or above 50 years of age indicates these SNPs offer considerable potential for the application of genetic tests to predict MPB patterns, adding a highly informative predictive system to the emerging field of forensic analysis of externally visible characteristics.

Analysis of 36 Y-STR marker units including a concordance study among 2085 Dutch males.

The genotypes of 36 Y-chromosomal short tandem repeat (Y-STR) marker units were analysed in a Dutch population sample of 2085 males. Profiling results were compared for several partially overlapping kits, i.e. PowerPlex Y, Yfiler, PowerPlex Y23, and two in-house designed multiplexes with rapidly mutating Y-STRs. Nineteen Y-STR marker units, of which two are rapidly mutating, reside in at least two of these multiplexes, and for these markers concordance testing was performed. Two samples showed discordant genotyping results and the probable causative base change was revealed by Sanger sequencing. In addition, we encountered concordant, but aberrant genotyping results including one allele with low peak height and several null alleles. For 12 samples, this involved a null allele in two adjacent loci suggesting a large and recurrent deletion as the samples represent three distinct haplogroups. For each marker unit, the allele counts and frequencies are presented, as are the haplotype counts and haplotype diversities for several combinations of markers.