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Artículo sustentado el 30 de Enero 2014 para la obtención del título profesional Médico Cirujano en la Escuela de Medicina, Facultad de Ciencias de la Salud. Universidad Peruana de Ciencias Aplicadas - UPC. / Artículo publicado el 27 de Abril de 2014 en la Revista Transactions of the Royal Society of Tropical Medicine & Hygiene (Oxford University Press). / Background: Gastroenteritis by rotavirus is responsible for approximately 810 annual deaths/year in children under 5 years in Peru and emerging rotavirus genotypes have led to concerns regarding cross-protection by the vaccines available. Moreover, there are no reports on the molecular-epidemiology of rotavirus diarrhea in Peru Methodology: A total of 131 stool samples were obtained from children under 5 years old hospitalized from January 2010 to December 2012 in the Hospital Regional de Cajamarca, Peru. ELISA and RT-PCR techniques were performed for rotavirus detection. G and P typing of rotavirus-positive samples were obtained by semi-nested multiplex RT-PCR and sequencing was performed to confirm the PCR results. Results: Of the 117 samples available, 18.80% (22/117) tested positive for rotavirus by ELISA and 35.90% (42/117) by RT-PCR. Among the G-genotype identified, G9 in 35.71% (15/42) and G12 in 33.33% (14/42) were the most prevalent. With the most common combination being G12/P6 in 23.81% (10/42). Conclusions: A high prevalence of the G12/P6 genotype was detected. It is know that this genotype is not covered by the current vaccines available. More in depth studies are needed to know the current rotavirus genotypes presents in Peru.

Els calicivirus humans (HuCV) són una causa important de brots de gastroenteritis (GEA) en adults, el paper però d'aquests virus com a causa de casos esporàdics de GEA en nens és desconegutObjectius1. Estudiar la prevalença i les característiques clíniques del les infeccions per HuCV en nens hospitalitzats als USA2. Determinar la diversitat genètica dels HuCV causants d'aquesta patologia3. Determinar el cost mèdic derivat d'aquestes infeccionsMetodesEs va realitzar un estudi prospectiu a tres hospitals pediàtrics dels USA, a Cincinnati, OH; Oakland, CA; i Norfolk, VA. Nens de 14 dies fins a 5 anys d'edat ingressats per un quadre de diarrea, vòmits o febre sense causa de menys de 7 dies d'evolució van ser inclosos. Es van recollir dades clíniques i demogràfiques, així com una mostra de femta. La femta es va analitzar per la presencia de rotavirus (RV) per EIA i per HuCV per RT-PCR utilitzant els cebadors 289/290 i 289hi/290hijk, dirigits a la regió de la polimerasa i que són capaços de detectar NLV i SLV. Totes les mostres positives per RT-PCR van ser confirmades per sequenciació utilitzant SequiTherm EXCEL II long-Read DNA Sequencing kit-ALF en un seqüenciador automàtic. Els arbres filogenètics es van obternir utilitzant l'algoritme de Maximum likelihood. Es va calcular el cost del periode pre, hospitalització i posthospitalització a partir del nombre de visites a serveis mèdics, les medicacions prescrites, les exploracions complementàries solicitades i el cost de la hospitalització. ResultatsEs van obtenir 1844 mostres. Es van detectar RV en 27% de les mostres i HuCV en un 8%. Dels 155 HuCV, 25 eren SLVs i 130 NLVs. L'edat dels pacients infectats per RV era superior a la dels infectats per HuCV (mitjana 13 mesos vs 8 mesos). Els RV van mostrar un predomini estacional durant l'hivern, mentre que els HuCV van ser més freqüents a finals d'hivern-principis de primavera. Es va detectar co-infecció amb RV en 12% dels pacients infectats per HuCV. El símptoma més freqüent en les infeccions per HuCV van ser els vòmits (79% dels pacients). La febre va ser el signe més freqüent en nens de menys de 3 mesos d'edat. La gravetat dels quadres produits per SLV i NLV van ser similars. La mitjana de cost per pacient va ser de $3,574 (interval $820-$116,088).L'analisi filogenètic de les soques aïllades mostrà 3 soques pertanyent als SLV amb menys d'un 70% d'identitat amb les soques de referència i 7 NLV que no pertanyien a cap dels dos genotips, mostrant menys d'un 65% identitat a nivell nucleòtid amb les soques de referènciaConclusions Els HuCVs són una causa important de GEA en nens als USA, afectant pacients de menor edat que els RV i mostrant un patró estacional diferent.Els HuCV causant de GEA són geneticament diversos. No es van poder demostrar diferències en la presentació clínica ni en les característiques epidemiològiques de les infeccions pels diferents géneres.El cost economic i social d'aquestes infeccions és important / Human calicivirus (HuCV) are an important cause of gastroenteritis (AGE) outbreaks in adults but the role of this virus as a cause of sporadic gastroenteritis in children is not known.Objectives1. To asses the prevalence and clinical characteristics of HuCV infection among children resulting in hospitalization in the USA.2. To determine the genetic diversity of the HuCV causing this severe illness3. To determine the direct medical cost and burden cause by these infections MethodsPatients were enrolled in a longitudinal prospective maneer at three pediatric hospitals in Cincinnati, OH; Oakland, CA; and Norfolk, VA. Children 14 days to 5 years of age admitted because of diarrhea, vomiting or fever of less than 7 days duration at admission were enrolled. Clinical and demografic information were collected, as well a stool sample. Stools were tested for rotavirus (RV) by EIA and stored at -70ºC until being tested for HuCV. Samples were tested for HuCV by RT-PCR using primers 289/290 and 289hi/290hijk targeting the polimerase region and able to detect NLV and SLV. All the positives by RT-PCR were confirmed by sequencing using SequiTherm EXCEL II long-Read DNA Sequencing kit-ALF on an automated sequencer. Phylogenetic trees were obtained using Maximum likelihood algoritm. The cost of pre, hospitalization and posthospitalization period was calculated based on the cost of visits to a health care provider, tests ordered, medications prescribed and cost of hospitalization. Results1844 samples were available for testing. The detection rates for RV and HuCV were 27% and 8% respectively. Of 155 samples positive for HuCV, 25 were SLVs and 130 NLVs. Patients infected by RV were older than those infected by HuCV (median 13 months vs 8 months). RV had winter seasonality while HuCV was more frequent during late winter/early spring. Co-infection with RV occurred in 12% of the HuCV -positive patients. The most common symptom in HuCV infected patients was vomiting (79%). SLV was associated with more vomiting (86%) compared to NLV (79%). Fever was a frequent symptom, specially in children less than 3 months of age. Severity for SLV and NLV were similar. The median cost for calicivirus infected patients was $3,574 (range $820-$116,088).Phylogenetic analysis of the isolated strains showed 3 SLV samples that were less than 75% identical to the reference strains. Seven of the NLV strains did not fall in any ot the two genotypes showing less than 65% identity at nucleotide level to any of the reference strains.Conclusions HuCVs are a relevant cause of severe AGE in children in USA. Seasonality and epidemiology for RV was different than for HuCV.HuCV causing AGE in children are genetically diverse. No differences regarding clinical presentation and demographic characteristics could be proved for the different genera or clusters.HuCV infection has an important economical and social cost.

Background: Gastroenteritis by rotavirus is responsible for approximately 810 annual deaths/year in children under 5 years in Peru and emerging rotavirus genotypes have led to concerns regarding cross-protection by the vaccines available. Moreover, there are no reports on the molecular-epidemiology of rotavirus diarrhea in Peru Methodology: A total of 131 stool samples were obtained from children under 5 years old hospitalized from January 2010 to December 2012 in the Hospital Regional de Cajamarca, Peru. ELISA and RT-PCR techniques were performed for rotavirus detection. G and P typing of rotavirus-positive samples were obtained by semi-nested multiplex RT-PCR and sequencing was performed to confirm the PCR results. Results: Of the 117 samples available, 18.80% (22/117) tested positive for rotavirus by ELISA and 35.90% (42/117) by RT-PCR. Among the G-genotype identified, G9 in 35.71% (15/42) and G12 in 33.33% (14/42) were the most prevalent. With the most common combination being G12/P6 in 23.81% (10/42). Conclusions: A high prevalence of the G12/P6 genotype was detected. It is know that this genotype is not covered by the current vaccines available. More in depth studies are needed to know the current rotavirus genotypes presents in Peru. / Tesis

La gastroenteritis ocasionada por rotavirus se encuentra entre las principales causas de morbilidad y mortalidad infantil. En el Perú rotavirus representa el 30% de las muertes por diarrea y 4% del total de defunciones, afectando principalmente a niños menores de 5 años. Desde el año 2009 el Perú implementó la vacuna contra rotavirus en su programa de vacunación universal, sin embargo, debido a la aparición de genotipos emergentes, es importante monitorear la diversidad del virus para evaluar los posibles efectos sobre la eficacia de la vacuna. Pese a esto, existen escasos reportes de la epidemiología de rotavirus en nuestro país. El objetivo del presente estudio es determinar la presencia y los genotipos G y P de Rotavirus en muestras provenientes de niños con gastroenteritis aguda atendidos en un hospital de Lima entre Octubre del 2013 y Octubre del 2014. Como metodología se utilizó el RT-PCR en tiempo real (qRT- PCR) para la detección del rotavirus y el RT PCR convencional para la genotipificación de las muestras positivas, asimismo se secuenciaron algunas muestras para determinar los genotipos finales. De las 448 muestras analizadas en el estudio, 45 (10%) tuvieron resultado positivo para rotavirus. Entre los genotipos identificados, G12 (40.9%), G2 (25%) y P[8] (77.3%) fueron los más frecuentes y la combinación G/P más dominante fue G12P[8] (54.5%). Los resultados evidenciaron una alta prevalencia de G12P[8]entre los casos positivos, genotipo no reportado previamente en el Perú. Se recomienda realizar nuevos estudios para evaluar las variaciones en la diversidad de rotavirus circulantes en el Perú y los posibles efectos ante la presión selectiva de la vacuna.Rotavirus gastroenteritis is one of the leading causes of morbidity and mortality in children. In Peru rotavirus represents 30% of deaths due to diarrhea and is responsible of 4% of the total deaths, affecting mostly children under 5 years. Peru implemented the rotavirus vaccine in its universal vaccination program since 2009. Due to the appearance of emerging genotypes, it is important to evaluate the diversity of the virus in order to determine possible effects on vaccine efficacy. However there are few reports on the molecular epidemiology of rotavirus in our country. The aim of this study was to determine the presence and Rotavirus G and P genotypes in samples from children with acute gastroenteritis attended in a hospital in Lima between October 2013 and October 2014. The method usedfor the detection of rotavirus was real time RT-PCR (qPCR) and conventional RT-PCR to determine the genotype of positive samples. Additionally, sequencing was used to confirm the final genotype of some samples. Of the 448 samples analyzed, 45 (10%) were positive for rotavirus.G12 (40.9%), G2 (25%) and P[8] (77.3%) were the most frequent genotypes and the most prevalent G/P combination was G12P[8] (54.5%). The results showed a high prevalence of G12P[8] among the positive cases. G12P[8] has not been previously reported in Peru. We recommend more in deep studies to identify the diversity of circulating genotypes in Peru.

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Propósito: Evaluar el valor del Vesikari Scoring System (VSS) como herramienta diagnóstica en la predicción de patógenos en niños con gastroenteritis aguda. Métodos: En un estudio retrospectivo, se analizó 247 historias clínicas y registros laboratoriales del Hospital de Emergencias Pediátricas (Lima, Perú), utilizando el diagnóstico de CIE-10 A00-A009 (enfermedades intestinales infecciosas). Se dividió según los patógenos detectados: no específico, viral y bacteriana. Se detectaron las bacterianas por coprocultivo y las virales por inmunoanálisis. Los casos no específicos tenían ambos resultados negativos o no hubo pruebas laboratoriales. Se ha calculado la sensibilidad, especificidad, valores predictivos negativos (VPN) y positivos (VPP), índice de probabilidad (LR) y el área bajo la curva (ROC). Resultado: El área bajo la curva ROC del VSS no fue significativo para determinar la etiología bacteriana ni viral. En el punto de corte de 9 en VSS, se analizó VPP (18.29%) y VPN (89.47%). Los leucocitos en heces tuvieron un área bajo la curva ROC de 0.8831(IC95% 0,79-0,96 y p=0.04) para etiología bacteriana, con una sensibilidad de 80.95%, especificidad de 88.24% y LR- de 0.22. Los leucocitos en heces con un punto de corte de ≥20, tuvo una precisión aceptable para diagnosticar gastroenteritis bacteriana. La prueba de moco en heces tuvo una sensibilidad de 83.33%, especificidad de 82.35% y VPP de 95.89%. Conclusión: El VSS no es una herramienta adecuada para determinar etiología de gastroenteritis viral y bacteriana. Sin embargo, se ha determinado que los leucocitos en heces con un punto de corte de ≥20 leucocitos por campo y moco en heces permite identificar la gastroenteritis aguda bacteriana. / Purpose: To evaluate the Vesikari Scoring System (VSS) as a valuable diagnostic tool for predicting pathogens in children with acute gastroenteritis. Methods: In this retrospective study were analyzed 247 clinical and laboratory records of the Pediatric Emergency Hospital (Lima, Peru), with the diagnosis of ICD-10 A00-A009 (Intestinal Infectious diseases). According to the pathogens detected were divided into groups: non-specific, viral, and bacterial. Bacteria were detected by stool culture, viral pathogens by immune analysis. The non-specific group had negative on both tests, or they did not perform. We calculated sensitivity, specificity, negative and positive predictive values, negative and positive likelihood ratios, and receiver operating characteristic curves.  Results: The area under the ROC curve of VSS was not significant for viral and bacterial etiology. The cut-off point of 9 in VSS was calculated by analyzing PPV (18,29%) and NPV (89,47%). The fecal leukocyte had a ROC of 0.8831 (IC95% 0.79-0.96 and p=0.04) for bacterial group, with a sensibility of 80,95%, specificity of 88,24% and LR- of 0,22. At a cut-off point of ≥20 fecal leukocytes was an acceptable diagnostic accuracy for bacterial gastroenteritis. The stool mucus for the bacterial group had a sensibility of 83,33%, a specificity of 82,35%, and a PPV of 95,89%. Conclusion: VSS is not an adequate diagnostic tool to differentiate viral from bacterial etiology. Nevertheless, we determine that a test of fecal leukocytes, with a cut-off point of ≥20 leukocytes per field and mucus in stools, allow us to identify the bacterial etiology of gastroenteritis. / Tesis