The Experience of Testing for Coronavirus Disease (COVID-19) at a Single Diagnostic Center in Paraguay before the Introduction of Vaccination.

Soon after the declaration of the COVID-19 pandemic, the Institute for Health Sciences Research (IICS) of the National University of Asunción, Paraguay became a testing laboratory (COVID-Lab) for SARS-CoV-2. The COVID-Lab testing performance was assessed from 1 April 2020 to 12 May 2021. The effect of the pandemic on the IICS and how the COVID-Lab contributed to the academic and research activities of the institute were also assessed. IICS researchers and staff adjusted their work schedules to support the COVID-Lab. Of the 13,082 nasopharyngeal/oropharyngeal swabs processed, 2704 (20.7%) tested positive for SARS-CoV-2 by RT-PCR. Of the individuals testing positive, 55.4% were female and 48.3% were aged 21-40 years. Challenges faced by the COVID-Lab were unstable reagent access and insufficient staff; shifting obligations regarding research, academic instruction, and grantsmanship; and the continuous demands from the public for information on COVID-19. The IICS provided essential testing and reported on the progress of the pandemic. IICS researchers gained better laboratory equipment and expertise in molecular SARS-CoV-2 testing but struggled to manage their conflicting educational and additional research obligations during the pandemic, which affected their productivity. Therefore, policies protecting the time and resources of the faculty and staff engaged in pandemic-related work or research are necessary components of healthcare emergency preparedness.

SARS-CoV-2 Variants in Paraguay: Detection and Surveillance with an Economical and Scalable Molecular Protocol.

SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.

An exploration of the political, social, economic and cultural factors affecting how different global regions initially reacted to the COVID-19 pandemic.

Responses to the early (February-July 2020) COVID-19 pandemic varied widely, globally. Reasons for this are multiple but likely relate to the healthcare and financial resources then available, and the degree of trust in, and economic support provided by, national governments. Cultural factors also affected how different populations reacted to the various pandemic restrictions, like masking, social distancing and self-isolation or self-quarantine. The degree of compliance with these measures depended on how much individuals valued their needs and liberties over those of their society. Thus, several themes may be relevant when comparing pandemic responses across different regions. East and Southeast Asian populations tended to be more collectivist and self-sacrificing, responding quickly to early signs of the pandemic and readily complied with most restrictions to control its spread. Australasian, Eastern European, Scandinavian, some Middle Eastern, African and South American countries also responded promptly by imposing restrictions of varying severity, due to concerns for their wider society, including for some, the fragility of their healthcare systems. Western European and North American countries, with well-resourced healthcare systems, initially reacted more slowly, partly in an effort to maintain their economies but also to delay imposing pandemic restrictions that limited the personal freedoms of their citizens.

Chagas disease: national survey of seroprevalence in children under five years of age conducted in 2008

BACKGROUND Since the early 1990s, programs to control Chagas disease in South America have focused on eradicating domiciliary Triatoma infestans, the main vector. Seroprevalence studies of the chagasic infection are included as part of the vector control programs; they are essential to assess the impact of vector control measures and to monitor the prevention of vector transmission. OBJECTIVE To assess the interruption of domiciliary vector transmission of Chagas disease by T. infestans in Paraguay by evaluating the current state of transmission in rural areas. METHODS A survey of seroprevalence of Chagas disease was carried out in a representative sample group of Paraguayans aged one to five years living in rural areas of Paraguay in 2008. Blood samples collected on filter paper from 12,776 children were tested using an enzyme-linked immunosorbent assay. Children whose serology was positive or undetermined (n = 41) were recalled to donate a whole blood sample for retesting. Their homes were inspected for current triatomine infestation. Blood samples from their respective mothers were also collected and tested to check possible transmission of the disease by a congenital route. FINDINGS A seroprevalence rate of 0.24% for Trypanosoma cruzi infection was detected in children under five years of age among the country’s rural population. Our findings indicate that T. cruzi was transmitted to these children vertically. The total number of infected children, aged one to five years living in these departments, was estimated at 1,691 cases with an annual incidence of congenital transmission of 338 cases per year. MAIN CONCLUSION We determined the impact of vector control in the transmission of T. cruzi, following uninterrupted vector control measures employed since 1999 in contiguous T. infestans-endemic areas of Paraguay, and this allowed us to estimate the degree of risk of congenital transmission in the country.

Chagas disease: national survey of seroprevalence in children under five years of age conducted in 2008.

BACKGROUND: Since the early 1990s, programs to control Chagas disease in South America have focused on eradicating domiciliary Triatoma infestans, the main vector. Seroprevalence studies of the chagasic infection are included as part of the vector control programs; they are essential to assess the impact of vector control measures and to monitor the prevention of vector transmission. OBJECTIVE: To assess the interruption of domiciliary vector transmission of Chagas disease by T. infestans in Paraguay by evaluating the current state of transmission in rural areas. METHODS: A survey of seroprevalence of Chagas disease was carried out in a representative sample group of Paraguayans aged one to five years living in rural areas of Paraguay in 2008. Blood samples collected on filter paper from 12,776 children were tested using an enzyme-linked immunosorbent assay. Children whose serology was positive or undetermined (n = 41) were recalled to donate a whole blood sample for retesting. Their homes were inspected for current triatomine infestation. Blood samples from their respective mothers were also collected and tested to check possible transmission of the disease by a congenital route. FINDINGS: A seroprevalence rate of 0.24% for Trypanosoma cruzi infection was detected in children under five years of age among the country's rural population. Our findings indicate that T. cruzi was transmitted to these children vertically. The total number of infected children, aged one to five years living in these departments, was estimated at 1,691 cases with an annual incidence of congenital transmission of 338 cases per year. MAIN CONCLUSION: We determined the impact of vector control in the transmission of T. cruzi, following uninterrupted vector control measures employed since 1999 in contiguous T. infestans-endemic areas of Paraguay, and this allowed us to estimate the degree of risk of congenital transmission in the country.

Coinfección leishmania infantum, VIH e histoplasma capsulatum: manifestaciones dermatológicas / Leishmania infantum, HIV and hystoplasma capsulatum coinfection: dermatologic manifestations

La coinfección Leishmania/VIH es frecuente y por lo general son casos de leishmaniosis visceral (LV), sin embargo, Leishmania infantum, el agente etiológico, también puede causar formas cutáneas en pacientes VIH-positivos. Los parásitos llegan a la piel por difusión, en una nueva infección o reactivación de una infección latente. Existen técnicas moleculares que confirman el diagnóstico y caracterizan a la especie. En estos pacientes también se presenta como infección oportunista la histoplasmosis, reconocida como marcador de SIDA y causada por Histoplasma capsulatum. En este trabajo describimos un caso de coinfección SIDA, histoplasmosis y LV asociado a lesiones cutáneas. Paciente de sexo masculino, adulto, residente en J.A. Saldívar, VIH positivo (2010), sin tratamiento antirretroviral. En el 2012 es internado en el Instituto de Medicina Tropical (IMT) con fiebre, anemia, hepato-esplenomegalia, frotis de médula ósea y rk39 negativos para Leishmania, recibe tratamiento con Anfotericina B. En el 2013 presenta síndrome febril prolongado y en médula ósea se encuentran amastigotes de Leishmania sp. En el 2014 presenta úlceras en el rostro en las que se observan es porosmicóticos de H. capsulatum y amastigotes de Leishmania sp. Se confirma L. infantum por técnicas de biología molecular. En el caso estudiado concluimos que la presentación dérmica es causada por L. infantum, caracterizada por técnicas moleculares, eH. capsulatum que se presenta en concomitancia debido al estado inmunológico. Cabe resaltar la importancia del diagnóstico diferencial para aplicar el tratamiento correcto y además la manifestación clínica que normalmente no es asociada a esta especie de Leishmania.

Leishmania/HIV coinfection is frequent and generally they are visceral leishmaniasis cases(VL). However, Leishmania infantum, the etiological agent, could also cause cutaneousforms in HIV-positive patients. The parasites reach the skin through diffusion, a newinfection or a reactivation of a latent infection. There are molecular techniques that confirmthe diagnosis and characterize Leishmania species. These patients also present otheropportunistic infections like histoplasmosis, well known as an AIDS marker and caused byHistoplasma capsulatum. In this work, we describe one HIV/histoplasmosis/VL coinfectioncase associated to cutaneous lesions. The patient was a male, adult, coming from the city ofJ.A. Saldívar, HIV positive (since 2010), without antiretroviral treatment. He was hospitalized in 2012 at the Instituto de Medicina Tropical (IMT) with fever, anemia,hepatosplenomegaly, bone marrow smear and rK39 both negative for Leishmania. He wastreated with Amphotericin B. In 2013, he presented a prolonged febrile syndrome andLeishmania amastigotes were observed in the bone marrow. In 2014, the patient presentedulcerations on the face where H. capsulatum fungal spores and Leishmania amastigoteswere observed. L. infantum was confirmed by molecular methods. In the case under studywe can conclude that the dermal manifestation was caused by L. infantuma ccording to thecharacterization by molecular techniques, and simultaneously H. capsulatum was presentdue to his immunological status. It should be emphasized the importance of the differentialdiagnosis in order to apply the right treatment and also the uncommon clinicalmanifestation that is not associated to this Leishmania species.

Triatoma sordida: indicadores de adaptación y transmisión de trypanosoma cruzi en intradomicilio del Chaco Paraguayo / Triatoma sordida: indicators of adaptive capacity and trypanosoma cruzi intradomicile transmission in the Paraguayan Chaco

El gran Chaco es una eco-región que incluye Argentina, Bolivia y Paraguay; donde la trasmisión vectorial de la enfermedad de Chagas por Triatoma infestans (vector principal), constituye hasta la fecha un problema de salud pública. El Chaco paraguayo ocupa el 25% de esta región, caracterizada por una baja densidad poblacional y localidades dispersas. El objetivo de este estudio fue determinar el rol potencial de la especie Triatoma sordida (vector secundario) en el ciclo doméstico de transmisión de Trypanosoma cruzi. Se aplicaron técnicas moleculares asociadas a indicadores entomológicos y epidemiológicosa 436 ejemplares de T. sordida capturados en 147 viviendas del Chaco Paraguayo. Se detectó infestación y colonización en el intradomicilio y peridomicilio por T. sórdida en 12 (8.2%) y 79 (53.7%) viviendas de las 147 evaluadas, respectivamente. Al menos un ejemplar infectado con T. cruzi fue detectado por PCR en las 12 viviendas con colonización intradomiciliar y en dos de ellas por caracterización molecular se detectó en ninfas el genotipo TC2. Adultos y ninfas en el peridomicilio de 4 viviendas dieron positivo para el genotipo TC1. Se estima un elevado riesgo de transmisión de T. cruzi intradomiciliar del 87%. Estos resultados evidencian capacidad adaptativa de esta especie en el domicilio, y un incremento de su potencial vectorial para transmitir la enfermedad de Chagas en el Chaco Paraguayo.

The Gran Chaco is an eco-region that includes Argentina, Bolivia and Paraguay; where the vectorial transmission of Chagas disease by Triatoma infestans (main vector) is a public health problem up to date. The Paraguayan Chaco occupies 25% of this region, characterized by low population density and dispersed locations. The aim of this study wasto determine the potential role of the specie Triatoma sordida (secondary vector) in the domestic cycle of transmission of Trypanosoma cruzi. Molecular techniques associated with entomological and epidemiological indicators were applied in 436 specimens of T. sordida captured in 147 dwellings of the Paraguayan Chaco. Infestation rate and colonization by T. Sordida was detected in the intradomicile and peridomiciliary in 12 (8.2%) and 79 (53.7%)of the 147 dwellings, respectively. In 12 houses were colonization was observed, at leastone specimen was PCR-T. cruzi positive, and in two of them genotype TC2 of T. cruzi were detected innymphs by molecular characterization. Adults and nymphs in the peridomiciliary of 4 houses were positive for genotype TC1.A high risk of indoor transmission of T. cruzi isestimated in 87%. These results demonstrate adaptive capacity of this specie in the intradomicile, and increased vector potential to transmit Chagas disease in the Paraguayan Chaco.

Estandarización de la técnica PCR-RFLP del gen mitocondrial cyt b como herramienta para la identificación de fuentes de alimentación de insectos hematófagos / Standardization of a PCR-RFLP technique based on RESUMEN mitochondrial cyt b gen as a tool to identify feeding sources of hematophagous insects

La identificación de la fuente de alimentación de insectos hematófagos puede proporcionar información sobre la capacidad vectorial, patrones de alimentación en condiciones naturales y proveer indirectamente datos sobre probables reservorios de enfermedades. Varias técnicas de identificación son empleadas, entre ellas las más utilizadas son las basadas en reacciones antígeno-anticuerpo. Actualmente, se han desarrollado ensayos moleculares, algunos de ellos permiten detectar e identificar solo sangre humana. Otros como la reacción en cadena de la polimerasa (PCR) del gen mitocondrial citocromo b (cyt b), que ha mostrado alto grado de sensibilidad y especificidad, permite detectar e identificar otras especies de vertebrados. El objetivo del trabajo fue estandarizar la técnica PCR-RFLP del gen mitocondrial citocromo b (cyt b) para determinar la fuente de alimentación sanguínea de insectos. Inicialmente se realizó un análisis bioinformático para la búsqueda y alineamiento de secuencias del gen cyt b de los potenciales huéspedes, con secuencias que están disponible en el GenBank. Se utilizaron 10 muestras de sangre de potenciales huéspedes vertebrados (humano, perro, gallina y roedor) y para la reacción de PCR se empleó un par de cebadores universales que amplifican una región del gen cyt b, seguido de cortes con dos enzimas de restricción (RFLP) (Hae III y Mwo I), generando patrones de electroforesis específicos para los diferentes vertebrados. Se logró la estandarización de la técnica de PCR del gen cyt b que fue capaz de detectar ADN de al menos 1 μL de sangre observándose el producto de amplificación de 358 pb. El análisis de los patrones de bandas obtenidos con el corte de las enzimas mostró los tamaños de fragmentos esperados para humano, gallina, perro y roedor. Estos resultados muestran la utilidad de la técnica PCR-RFLP del gen cyt b que, con un simple par de cebadores seguido del corte con dos enzimas de restricción...

Identification of feeding sources of hematophagous insects can provide informationabout the vectorial capacity,feeding patternsin natural conditionsand indirectlyprovidedataon possible disease reservoirs preferences.Several identificationtechniquesare used,most of them based on antigen-antibody reactions.Recently molecular assayshave been developedand, some ofthese assayscandetect andidentificateonlyhuman blood. Otherassays,likethepolymerase chain reaction (PCR)basedonmitochondrialcytochromebgene,have shown high sensitivity and specificityallowingdetectionand identificationofothervertebrate species. The aim of this studywas tostandardizea PCR-RFLP basedonmitochondrialcytochrome bgene(cyt b) inorder to determineblood mealfrom insects.Initially bioinformatic analysiswasperformed forsearching andalignmentofcyt bsequencesofpotential hosts availableattheGenBankdatabase.Blood samplesfrom potentialvertebrate hostswere usedand PCR was performed using specificprimersthat amplify aregionofcyt bgene. Theproducts were digestedwithrestriction enzymes (RFLP),generating specificelectrophoresis patterns forseveral vertebrates. The PCR technique forcyt bgene wasstandardized allowing the detection of at least 1μLof blood.The 359 bp band wascorrectly amplified and the profiles obtained after the enzyme digestion withHaeIIIandMwoI were the expected for human, chicken, dog and rodents. These resultsshowed the usefulness of the PCR-RFLP ofcyt bgenethat,with a single pair of primersfollowed digestionusing two restriction enzymes,allowed the differentiationof thevertebrate species of our interestthrough the patterns obtained without sequencinghavingalso the advantage of detecting small volumesof blood sample.

Aplicación de la PCR para la detección de género y complejos de Leishmania en diferentes tipos de muestras biológicas / Application of PCR for detection of Leishmania genus and complexes in different types of biological samples

La leishmaniosis es una enfermedad producida por diferentes especies del protozoario Leishmania agrupados en complejos, es transmitida por flebótomos presentando una variedad de síntomas clínicos. La técnica reacción en cadena de la polimerasa (PCR) permite amplificar regiones blanco específicas del género Leishmania y de los complejos Leishmania donovani y Leishmania braziliensis, directamente en muestras biológicas sin cultivos in vitro previo. En este estudio se realizó una evaluación descriptiva, durante el periodo 2001 a 2006, analizando 169 biopsias (piel y mucosas) y 31 aspirados de médula ósea en pacientes con sospecha clínica de leishmaniosis, además se evaluaron 44 muestras de bazo de caninos. Las muestras procedían de distintas zonas endémicas del país. Se detectó género Leishmania en el 71% de las biopsias (piel y mucosas), en 68% en los aspirados de médula ósea y en 82% de los bazos de caninos. Se identificó el complejo L. braziliensis en los pacientes con leishmaniosis cutánea y mucosa y el complejo L. donovani en pacientes con la forma visceral. Estos resultados han demostrado la utilidad de la técnica de PCR para la detección y caracterización de los parásitos de Leishmania en distintas muestras biológicas.

Reglamento del comité de ética en investigación del Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción (IICS-UNA) / Regulation of the committee of ethics in Investigation of the Institute of Investigations in Sciences of the Health of the National University of Asunción (IICS-UNA)

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Complejo Leishmania braziliensis: descripción de un caso pediátrico y revisión de la literatura / Leishmania braziliensis complex: description of a pediatric case and review of the literature

La leishmaniasis es una enfermedad infecciosa, no contagiosa, de evolución crónica, causada por un protozoario del género Leishmania y transmitida al hombre a través de la picadura del flebótomo hembra infectado, vector de la enfermedad. Presentamos el caso de un lactante menor con leishmaniasis cutánea localizada, que tuvo buena respuesta al tratamiento con antimoniato de N-metilglucamina.

Leishmaniasis is a chronic, infectious but not contagious disease caused by a protozoan of the genus Leishmania that is transmitted to humans through the bite of infected female sand fly, which is the vector of the disease. We present the case of an infant with localized cutaneous leishmaniasis who had a good response to the treatment with N-methylglucamine antimoniate.

Trypanosoma cruzi lineages detected in congenitally infected infants and Triatoma infestans from the same disease-endemic region under entomologic surveillance in Paraguay.

Trypanosoma cruzi II is associated with Chagas disease in the southern part of South America. We analyzed T. cruzi variants in field-collected triatomines and congenitally infected infants living in the same disease-endemic region in Paraguay. Results of polymerase chain reactions for T. cruzi kinetoplast DNA and satellite DNA were positive in 83 triatomine feces samples and 58 infant blood samples. However, lineages were detected in 33 and 38 samples, respectively. Trypanosoma cruzi genotypes were determined in 56 (97%) blood samples after hybridization by using specific probes. The Tc I genotype was not detected. The prevalent sublineage was Tc IId in triatomines (27 of 33) and infant blood (36 of 58) as assessed by amplification of the 24Salpha ribosomal RNA and the mini-exon region genes. The Tc IIc genotype was detected in 20 infant blood samples and in 1 triatomine. This study shows T. cruzi II is the predominant lineage circulating in triatomines and humans in endemic areas of eastern region of Paraguay.