Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Rabies virus monitoring in bat populations in Rondônia state, Brazil

Abstract INTRODUCTION: The Jirau hydroelectric power plant built in Rondônia state has environmental impacts that could be relevant to rabies outbreaks. METHODS: Bat populations were monitored for rabies by fluorescent antibody testing and simplified fluorescent inhibition microtesting between 2010 and 2015. RESULTS: All 1,183 bats tested negative for rabies. The prevalence of rabies antibodies was 17.5% in 1,049 bats. CONCLUSIONS: The rabies antibody dosage was not reactive in samples collected before the environmental changes, and there was a progressive increase in subsequent collections that could indicate an increase in rabies virus circulation among bats and risk of a rabies outbreak.

A randomised double-blind clinical trial of two yellow fever vaccines prepared with substrains 17DD and 17D-213/77 in children nine-23 months old

This randomised, double-blind, multicentre study with children nine-23 months old evaluated the immunogenicity of yellow fever (YF) vaccines prepared with substrains 17DD and 17D-213/77. YF antibodies were tittered before and 30 or more days after vaccination. Seropositivity and seroconversion were analysed according to the maternal serological status and the collaborating centre. A total of 1,966 children were randomised in the municipalities of the states of Mato Grosso do Sul, Minas Gerais and São Paulo and blood samples were collected from 1,714 mothers. Seropositivity was observed in 78.6% of mothers and 8.9% of children before vaccination. After vaccination, seropositivity rates of 81.9% and 83.2%, seroconversion rates of 84.8% and 85.8% and rates of a four-fold increase over the pre-vaccination titre of 77.6% and 81.8% were observed in the 17D-213/77 and 17DD subgroups, respectively. There was no association with maternal immunity. Among children aged 12 months or older, the seroconversion rates of 69% were associated with concomitant vaccination against measles, mumps and rubella. The data were not conclusive regarding the interference of maternal immunity in the immune response to the YF vaccine, but they suggest interference from other vaccines. The failures in seroconversion after vaccination support the recommendation of a booster dose in children within 10 years of the first dose.

Serum survey for antibodies to coronavirus, herpesvirus, calicivirus, and parvovirus in domestics cats from Rio Grande do Sul, Brazil / Inquérito sorológico para anticorpos contra coronavírus, herpesvírus, calicivírus e parvovírus em gatos domésticos do Rio Grande do Sul, Brasil

A ocorrência da infecção por coronavírus felino (FCoV), herpesvírus felino tipo 1 (FHV-1), calicivírus felino (FCV) e parvovírus felino (FPV) foi investigada mediante a detecção de anticorpos no soro de 97 gatos domésticos de Pelotas, RS, pelo teste de soro-neutralização. Entre os animais estudados, 51 não eram vacinados, 11 haviam sido vacinados contra FHV-1, FCV e FPV com pelo menos uma dose, e 35 tinham histórico de vacinação desconhecido. Foram detectados anticorpos para o FCoV em 75,2% (73/97) dos gatos. Anticorpos contra o FHV-1 estavam presentes em 38,1% (37/97): 73% (8/11) dos gatos vacinados, 39,2% (20/51) dos não vacinados e 25,7% (9/35) dos gatos com histórico de vacinação desconhecido. Anticorpos para o FCV estavam presentes em 56,7% (55/97): 81,8% (9/11) dos gatos vacinados, 52,9% (27/51) dos não vacinados, e 54,3% (19/35) dos gatos com histórico de vacinação desconhecido. Para o FPV, havia anticorpos em 69,1% (67/97): 100% (11/11) dos vacinados, 66,6% (34/51) dos não vacinados e 62,8% (22/35) dos gatos com histórico de vacinação desconhecido. Os resultados sugerem alta exposição ao FCoV, FHV-1, FCV e FPV na população de gatos na área estudada.

Development of a western blot assay for detection of antibodies against HSV using purified HSV virions prepared by sucrose density gradient

Herpes simplex viruses [HSVs] have widespread and ubiquitous prevalence in the human population and they have received a great deal of attention due to the range of diseases, they caused as a result of an infection. It seems that the fast and reliable diagnostic methods are needed for detecting the herpes simplex virus type 1 [HSV1] antibodies especially in patients with HSV encephalitis, immunocompromised people, and neonatal infections. The aim of this study was designing a Western blotting method for HSV1 antibody detection, using the purified virus by sucrose density gradient centrifugation procedure. The most reliable method for HSV detection is virus neutralization test but it needs cell culture preparation, high expertise, as well as the high amounts of serum samples. Considering the difficulties of this method, we tried to run a new one for HSV antibody detection by propagating the viruses and then purify them by sucrose density gradient centrifugation method. The purified viruses used as antigens in Western blotting assay. Diluted sera [1:100, and 1:200 dilutions] used in Western blotting and two-fold dilutions of the sera applied in virus neutralization test. Five of twenty seven samples were negative in Western blotting and the same results obtained in virus neutralization test. Comparing with our gold standard, the sensitivity and specificity of the developed assay were both 100%. Our results show that the designed method is a reliable method for replacing the virus neutralization test in diagnostic laboratories. It can also, be used for confirming the ELISA results

Prevalência de anticorpos contra agentes virais e bacterianos em eqüídeos do município de Monte Negro, Rondônia, Amazônia ocidental brasileira / Seroprevalence of viral and bacterial agents in equids from Monte Negro municipality, State of Rondônia. brazilian western Amazon

Foram examinados 176 eqüídeos (15 muares e 161 eqüinos) do município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira, frente a agentes virais e bacterianos. A amostra correspondeu ao total de eqüídeos no município, considerando um nível de confiança de 99%, prevalência esperada de 50% e erro padrão de 10%. As infecções virais foram investigadas pelas provas de Imunodifusão em gel de Agar (Anemia Infecciosa Eqüina - AIE), Inibição da hemaglutinação (Influenza eqüina tipos 1 e 2 - IE-1 e 2) e Soroneutralização em cultura celular (Arterite Viral Eqüina - AVE, Herpesvírus Eqüino tipo 1 - HVE1, Estomatite Vesicular - EV e Encefalomielite Eqüina do Leste - EEE, do Oeste - WEE e Venezuela - VEE). Para o diagnóstico da leptospirose, foi utilizada a prova de Soroaglutinação Microscópica (SAM); para o diagnóstico da brucelose, o teste do Antígeno Acidificado Tamponado (AAT) foi utilizado como teste de triagem e as provas de Soroaglutinação Lenta em Tubos (SLT) e 2- mercaptoetanol como testes diagnósticos. Foram constatados 9,6% dos eqüídeos reativos para AIE, 22,7% para HVE1, 19,9% para IE- 1, 42,0% para IE-2, 21,0% para EEE, 11,3% para VEE, 3,4% para Brucella spp. e 91,4% para Leptospira spp. Os sorovares de leptospira mais freqüentes foram Bratislava (10,5%), Icterohaemorrhagiae (8,7%) e Autumnalis (8,7%) nos eqüinos e Patoc (26,6%) nos muares. Não foram encontrados animais com anticorpos contra AVE, EV e WEE.

Sera from 174 equidaes (15 mules and 161 equines) of Monte Negro municipality, Rondônia State were analyzed against viral and bacterial agents. The serum sample corresponded the total equid population in the municipality considering a confidence interval of 99%, expected prevalence of 50% and absolute desired of 10%. For the viral agents, sera were tested by the Agar Gel Immunodiffusion Test (Equine Infection Anemia - EIA), Inhibition Haemagglutination Test (Equine Influenza 1 and 2 - EI - 1 and 2), and Virusneutralizating Tests (Equine Viral Arteritis - EVA, Equine Herpesvirus 1 - EHV1, Vesicular Stomatitis - VS, Equine Encephalitis Eastern - EEE, Western - WEE and Venezuelan VEE). The diagnosis for brucellosis was made by Agglutination Tests and the Microscopic Agglutination Test was used for leptospirosis. The results showed positivity of 9.6% for EIA, 22.7% for HVE1, 19.9% for IE-1, 42.0% for IE-2, 21.0% for EEE, 11.3% for VEE, 3.4% for brucellosis, and 91.4% for leptospirosis. The most frequent serovars detected were Bratislava (10.5%), Icterohaemorrhagiae (8.7%), Autumnalis (8.7%) for equines and Patoc (26.6%) for mules. No one of the examined samples reacted to EVA, VS, or WEE.

Anticorpos contra lentivírus de pequenos ruminantes em caprinos e ovinos em abatedouros do estado de Pernambuco / Small ruminant lentivirus infection in goats and sheep from two abattoirs in Pernambuco State, Brazil

The prevalence of small ruminant lentivirus (SRL) infection was evaluated in goats and sheep in two counties of Pernambuco State, Brasil. Seriological examinations were performed from a total of 672 goats and 325 sheep, one year of age and older, at two abattoirs. Sera were analyzed by agar gel immunodiffusion using Maedi-Visna K-1514 antigens. There were 42 reactive samples (95 percent confidence interval 3.6 percent to 4.9 percent) in both slaughter houses. In São Lourenço da Mata county, 3.2 percent and 4.0 percent of goat and sheep sera were responsive, whereas in Paulista county, 5.1 percent of goat samples and 8.2 percent of sheep were reactive. Thus, the prevalence of small ruminant lentivirus was low in goats and sheep sampled from the region.

Levantamento soro-epidemiológico da infecção pelo vírus da Anemia Infecciosa Eqüina, da Influenza Eqüina-2 e do Herpesvírus Eqüino-1 em rebanhos do sul do Estado do Pará, Brasil / Seroepidemiological survey of infection of equine infectious anemia, equine influenza type 2 and equine herpesvirus type 1 in herds of south Pará State, Brazil

Os vírus da anemia infecciosa eqüina (EIAV), da influenza eqüina tipo 2 (EIV-2) e o herpesvírus eqüino tipo 1 (EHV-1) são agentes causadores de enfermidades que podem causar graves prejuízos econômicos. O objetivo deste presente estudo foi estimar a freqüência de anticorpos contra o EIAV, EIV-2 e o EHV-1 em rebanhos do sul do Estado do Pará, Brasil. Os anticorpos contra EIAV, EIV-2 e EHV- 1 foram detectados pelo teste de IDGA, pelo método de inibição da hemaglutinação e pela técnica de soroneutralização (TCID50 =100), respectivamente. Amostras de sangue de 672, 514 e de 506 equídeos saudáveis e sem histórico de vacinação contra nenhum dos três vírus foram testadas, respectivamente, para EIAV, EIV-2, EHV-1. A seguinte freqüência de soro reativos foi observada: 1,34% para o EIAV; 35,79% para o EIV-2; 45,45% para o EHV-1. Estes resultados indicam que estes agentes estão presentes no rebanho paraense e a adoção de medidas de manejo e profilaxia devem ser priorizadas, garantindo deste modo, a prosperidade da eqüideocultura brasileira.

Equine infectious anemia virus (EIA V), equine influenza virus type 2 (EIV-2) and equine herpesvirus type 1 (EHV-1) are the causal agents of diseases that may bring economical Iosses. The aim of this present study was to estimate the frequency of antibodies against EIAV, EIV-2 and EHV-1 in herds of south Pará State, Brazil. Antibodies against EIAV, EIV-2 and EHV-1 were detected by AGID, hemagglutination inhibition method and serum neutralization technique (TCID50 =100), respectively. Blood samples of 572, 514, and 506 healthy equine unvaccinated against any of the three viruses were tested, respectively, for EIAV, EIV-2 and EHV-1. The following frequencies of serum reactors animals were observed: EIAV,1,34%; EIV-2, 35,79%; EHV-1, 45,45%. These results show that the agents are present in herds from Pará herds and the adoption of measures of management and prophylaxis should be prioritized, ensuring, thereby, the prosperity of brazilian's breeding equine.

Soroprevalência da encefalomielite eqüina do leste e do oeste no Município de Uruará, PA, Brasil / Seroprevalence of holders farms for eastern equine encephalitis and western equine encephalomyelitis in Uruará Municipality, Pará State, Northen Brazil

O objetivo do presente foi estimar a soroprevalência de anticorpos contra o vírus da EEE e da WEE utilizando como unidades de análise os eqüídeos e as propriedades rurais do tipo familiar do município de Uruará, PA. Os anticorpos contra o vírus das EEE e da WEE foram pesquisados pela microtécnica de soroneutralização. As seguintes prevalências de animais sororeatores para os diferentes vírus foram observadas: EEE 27,37% (IC 15,33 - 39,21%), WEE 1,05% (IC 0,06 - 6,78%). Para as propriedades obtivemos as seguintes prevalências: EEE 53,12% (IC 35,03 - 70,49%), WEE 3,12% (IC 0,16-18,00%).

The aim of this study was to verify the prevalence of herds with EEEV and WEEV infected animals, in Uruará municipal district, Pará State-Brazil. In view of that, the serum neutralization test was utilized. The following prevalence of positive herds were observed: 17 positive herds for EEEV out of 32 herds, therefore, prevalence is 53.12% (IC 35.03 - 70.49%). One positive herd was found for WEEV out of the 32 studied herds, thus performing 3.12% (IC 0.16 - 18.00%) prevalence. The prevalence of serum reactors animals were observed: 27,37% (IC 15,33 - 39,21 %), WEE 1,05% (IC 0,06 - 6,78%).

Increase of genital HSV-1 and mixed HSV-1 and HSV-2 infection in Bangkok, Thailand.

From January 1998 to December 2004, 207 out of 1125 samples were HSV isolation positive and typed. Two groups of patients, Thai and foreigner, as well as site of infection, non-genital and genital area, were identified. The prevalence of non-genital HSV-1 infection from 27 samples of Thai patients was 81.84%. Out of 180 genital samples, 39.02% HSV-1 and 43.09% HSV-2 from 123 Thai patients and 36.84% HSV-1 and 49.12% HSV-2 of 57 foreigner patients were determined. Moreover mixed infection of HSV-1 and HSV-2 was found in both Thai and foreigner groups, 17.89% and 14.04%, respectively. The prevalence of genital HSV-1 infection in Thai patients chronologically increases from 1.6% to 56.91% from of 1985 to 2004. Increase of HSV-1 genital infection and mixed HSV-1 and HSV-2 infection in Thai people might probably be due to changing of sexual behavior in the AIDS era.

Prevalência de anticorpos antivírus da arterite dos eqüinos em cavalos criados no Estado de Säo Paulo / Prevalence of equine arteritis virus specific antibodies in horses of Säo Paulo State, Brazil

Utilizou-se a prova de soroneutralização em microplacas para detecção de anticorpos antivírus da arterite dos eqüinos em 659 amostras de soro sangüíneo de animais criados no Estado de São Paulo. A prevalência de anticorpos na população estudada foi igual a 18,2 por cento. A raça Mangalarga foi a que apresentou maior taxa de prevalência, 33,3 por cento. Animais na faixa etária de 6 a 24 meses de idade apresentaram a maior taxa de prevalência, 30,4 por cento, e as fêmeas apresentaram prevalência de 22, 9 por cento, mais alta do que nos machos

With the purpose of studying the prevalence of equine viral arteritis in horses raised in São Paulo State, Brazil, by the standard microtiter serum neutralization test, 659 serum samples were investigated. The prevalence of antibodies in the horse population was 18.2 per cent, which was significantly higher in Mangalarga horses (33.3 per cent) than in any other breed (Thoroughbred, Arab, Quarter Horse, mixed breeds and others). The distribuition of horses by age showed that horses between 6 to 24 months of age (30.4 per cent) had a higher prevalence (30.4 per cent) rate than others. The female horses prevalence rate of 22.9 per cent was significantly higher than in male horses.

Measles in Thai adults: three case reports and management of contact cases.

We report on three adults (one nurse and two medical students) diagnosed as having measles. All the patients presented with fever, cough, conjunctival injection and rash. They contracted measles from pediatric patients who had been treated at Chulalongkorn Hospital in the previous two weeks. Physical examination revealed Koplik's spots on the oral mucosa and typical maculopapular rash. The diagnosis was confirmed by viral isolation. Measles IgG antibodies were measured in 36 medical students who were in close contact with patient 2 and measles vaccines were given. Thirty-three specimens had positive measles IgG, two had equivocal results and one had negative result. The student with negative measles IgG eventually developed measles (patient 3). Except for patient 2 and 3, no further cases of measles were seen among the contacts.

Detection of human parvovirus B19 in a patient with hepatitis

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3 percent) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis

Cerebelite aguda causada por vírus Epstein-Barr: relato de caso / Acute cerebellitis caused by Epstein-Barr virus: case report

A cerebelite aguda pode ocorrer em associação a infecção pelo vírus da varicela-zoster, enterovirus, caxumba, micoplasma e outros agentes infecciosos. A cerebelite aguda é uma complicação rara da infecção pelo vírus Epstein-Barr (EBV). Relatamos o caso de uma mulher de 21 anos com história de 12 dias de evolução com náuseas, vômitos, ataxia de marcha e membros, tremor cefálico e de membros, opsoclono, mioclonias e rash cutâneo. Sorologia para EBV foi positiva. A infecção pelo EBV, com complicações neurológicas, pode não se apresentar com os sinais e sintomas clássicos da mononucleose infeciosa.

Anti-VP1 and anti-VP2 antibodies detected by immunofluorescence assays in patients with acute human parvovirus B19 infection

Acute human parvovirus B19 infection is followed by an antibody response to the structural proteins of the viral capsid (VP1 and VP2). We used 80 sera collected from 58 erythema infectiosum and 6 transient aplastic crisis patients to test IgM and IgG antibodies against these two proteins in an immunofluorescence assay (IFA) using Sf9 cells infected with recombinant baculovirus expressing either VP1 or VP2 antigen. Although less sensitive than IgM capture enzyme immunoassay using native antigen (MACEIA), we could detect anti-VP1 or anti-VP2 IgM antibodies by IFA in 49 patients with acute infection (76.6 percent). Detection of IgG anti-VP1 and anti-VP2 by IFA, however, was as sensitive as IgG detection by indirect enzyme immunoassay. By applying IgG avidity IFA to sera of the 15 IgM IFA negative patients we were able to confirm acute infection in further 12 cases by IFA. Overall, acute infection was confirmed by IFA in 61 (95.3 percent) of the 64 patients

Three year seroepidemiological study of varicella-zoster virus in São Paulo, Brazil

A serosurvey of varicella has been carried out in children attending the public school network of São Paulo city, Brazil, from 1992 to 1994. This study was performed in order to establish the age related prevalence of antibodies against varicella-zoster virus (VZV) and its age specific transmission dynamics pattern in these children. Among 2500 schools in the city of São Paulo public network, 304 were randomly selected; 7 children of a given age (ranging from 1 to 15 years) were randomly selected in each school, and blood samples were obtained by fingerprick into filter paper. Blood eluates were analyzed for the presence of antibodies to VZV by ELISA. Proportion of seropositivity were calculated for each age group. Samples consisted of 1768 individuals in 1992, 1758 in 1993, and 1817 in 1994, resulting in 5343 eluates. A high proportion of seropositive children from 1 to 3 years of age was observed, ascending until 10 years of age and reaching a plateau around 90 percent afterwards. VZV transmission in this community was similar along the three years of the study. In children attending public schools in the city of São Paulo, contact with VZV occurs in early childhood. If immunization against VZV is considered it should be introduced as soon as possible.

Human T-lymphotropic retrovirus type-1 in Thailand.

Seroprevalence of antibodies to human T-lymphotropic virus type-1 (HTLV-1) was surveyed among the Thai population by the particle agglutination test and Western blotting test. None of a total of 727 individuals from seven ethnic groups were positive for the specific antibody to HTLV-1. Among hospital based 3,427 subjects in Southern Thailand, one patient with a brain tumor showed positivity in the Western blotting test, however, HTLV-1 proviral genome was not identified by PCR. The present data suggest that HTLV-1 is not endemic in the Thai population and that HTLV-1 is not a major public health problem in Thailand because HTLV-1 rarely causes its associated diseases.

Seroprevalencia de infeccion por el virus encefalitis San Luis en la Provincia de Formosa / Seroprevalence of infection by St. Louis encephalitis virus in the Province of Formosa

El objetivo del presente trabajo fue conocer la prevalencia de anticuerpos para el virus encefalitis San Luis (ESL) en sueros humanos del Laboratorio de Vigilancia Epidemiológica del departamento Formosa (provincia de Formosa, Argentina), extraídos en los años 1995 y 1997, se emplearon las pruebas de inhibición de la hemaglutinación (IH) y de neutralización (NT). El tamizaje realizado mediante la prueba de NT mostró una prevalencia del 21 por ciento (60/284) y 32 por ciento (50/157) en las muestras obtenidas en los años 1995 y 1997 respectivamente. El 14 por ciento de los sueros del año 1995 presentaron títulos de anticuerpos neutralizantes (NT) bajos (1/20 y 1/40) mientras que en el año 1997 el 19 por ciento de los sueros tenían títulos de anticuerpos NT iguales o mayores a 1/80. En la titulación simultánea de anticuerpos por NT e IH se observaron sueros con títulos bajos de anticuerpos NT (1/20-1/40) y negativos en IH, mientras que otros poseían títulos altos por ambas pruebas. Esta relación entre los títulos de anticuerpos indica la presencia de infecciones pasadas y recientes y la circulación continua de este virus. Por otra parte la prevalencia de anticuerpos NT en la población estudiada se incrementó significativamente en 2 años (p < 0.0075), lo que confirma la endemicidad de este agente en la zona y muestra la necesidad de realizar estudios de las enfermedades febriles de etiología viral no confirmadas, a fin de conocer su importancia como patógeno humano en nuestro país.