Frequência do Papilomavírus Humano na placenta, no colostro e no sangue do cordão umbilical

OBJETIVO: Determinar a frequência do Papilomavírus Humano (HPV) na placenta, no colostro e no sangue do cordão umbilical de parturientes e seus neonatos atendidos no Ambulatório de Ginecologia e Obstetrícia do Hospital Universitário de Rio Grande (RS), Brasil. MÉTODOS: Foram coletadas biópsias de 150 placentas do lado materno, 150 do lado fetal, 138 amostras do sangue do cordão umbilical e 118 amostras de colostro. As biópsias de placenta foram coletadas da porção central e periférica. O DNA foi extraído segundo protocolo do fabricante e conforme referência encontrada na literatura. O HPV foi detectado pela técnica da reação em cadeia da polimerase aninhada (PCR-Nested) com os primers MY09/11 e GP5/GP6. A genotipagem foi por sequenciamento direto. As participantes responderam a um questionário autoaplicado com dados demográficos e clínicos, a fim de caracterizar a amostra. RESULTADOS: O HPV foi detectado em 4% (6/150) do lado materno das placentas, 3,3% (5/150) do lado fetal; 2,2% (3/138) no sangue do cordão e 0,8% (1/118) no colostro. A taxa de transmissão vertical foi de 50%. O genótipo de baixo risco oncogênico encontrado foi o HPV-6 (60%) e de alto risco, os HPV-16 e HPV-18 (20% cada). CONCLUSÕES: Esses resultados sugerem que o HPV pode infectar a placenta, o colostro e o sangue do cordão umbilical.

Detecção de Toxoplasma gondii no sêmen de ovinos naturalmente infectados

O objetivo deste trabalho foi estudar a eliminação de Toxoplasma gondii no sêmen de carneiros naturalmente infectados. Foram utilizados 65 reprodutores submetidos inicialmente à pesquisa de anticorpos anti-T. gondii por meio da técnica de imunofluorescência indireta (IFI). Os carneiros sorologicamente positivos foram submetidos à colheita de sêmen para detecção do DNA de T. gondii. Na sorologia observaram-se 6/65 (9,2%) carneiros positivos, enquanto no PCR nested de sêmen 4/6 (66,6%) carneiros foram positivos. Conclui-se que a detecção, por meio da técnica da PCR nested, da forma proliferativa de T. gondii no sêmen de carneiros naturalmente infectados, reforça a necessidade de se pesquisar sobre a possibilidade da transmissão horizontal do parasito via sêmen na espécie ovina.

Freqüência de fungos dermatófitos em (Felis catus) infectados e não infectados pelo vírus da imunodeficiência felina

Pós-graduação em Medicina Veterinária - FMVZ

Determinação da infecção em Anopheles por diagnóstico molecular

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo epidemiológico e genético dos vírus da imunodeficiência felina identificados no Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Epidemiologia da leishmaniose tegumentar no município de Juruti, Pará

A leishmaniose tegumentar (LT) encontra-se em expansão no Estado do Pará, Brasil. Juruti é um dos 143 municípios desse Estado e atualmente cenário de grandes transformações ambientais devido à mineração de bauxita, o que poderá influenciar o padrão de transmissão. Objetivo: Este estudo buscou elucidar aspectos epidemiológicos relevantes para o controle da LT em Juruti. Materiais e Métodos: A frequência de LT e o perfil dos pacientes no hospital municipal "Francisco Barros" foram determinados de janeiro a dezembro/2007. Espécies de flebotomíneos silvestres existentes no entorno de uma área de prospecção da bauxita foram também descritas, durante levantamento entomológico em janeiro/2008 (armadilha Shannon/18h às 20h/2 noites). Em 21 indivíduos, portadores de lesão cutânea suspeita de LT, biópsias de pele foram realizadas entre fevereiro e junho de 2007. Neste grupo procedeu-se ao diagnóstico parasitológico (esfregaço corado e cultura), molecular e teste intradérmico de Montenegro. Utilizaram-se sondas de DNA ribossomal (PCR-SSUrDNA) gênero específicas (S4, S12; S17, S18) e de G6PD, para distinguir o subgênero Viannia (ISVC, ISVA: ISVC, ISVG) e a espécie L. (V.) braziliensis (ISVC, ISVA; ISVC, ISVB). Resultados: No ano de 2007 foram confirmados 42 casos novos de LT, com média mensal inferior a quatro (3,5 ± 0,8), maior frequência em julho (11) e menor em junho e novembro (0). A maioria dos pacientes foi de homens (41/42, 98%) com menos de 20 anos (<10 anos: 30%; 10-20: 57%; 20-40: 12%). A maioria também residia em localidades rurais (33/42, 79%), incluindo áreas impactadas pela mineração (19/42, 45%), e exercia atividades de risco (28/42, 67%). Doze eram funcionários de empresas (29%). A análise molecular das 21 amostras identificou 12 resultados positivos para o gênero Leishmania (57%), sendo 11 (52%) parasitologicamente confirmados. A PCRG6PD identificou 75% das amostras como sendo L. (V.) braziliensis. As demais (3/12, 25%) não hibridizaram com os oligonucleotídeos da PCR-G6PD e, por isso, os produtos da reação de nested-PCR SSUrDNA foram clonados e sequenciados, confirmando que se tratavam de Leishmania (Viannia) sp. Apenas 9/12 (75%) casos confirmados pelos métodos parasitológico e/ou olecular tiveram reações de hipersensibilidade tardia em resposta ao antígeno de Montenegro, cujos diâmetros variaram de 7 a 40mm (16,3 ± 3,2). Capturaram-se 105 flebotomíneos de 13 espécies nas seguintes frequências: 1- Lutzomyia (Ps.) geniculata (23, 22%), 2- Lutzomyia (Ps.) paraensis (21, 20%), 3- Lutzomyia (Ps.) complexa (18, 17%), 4-Lutzomyia (Ps.) davisi (10, 10%), 5- Lutzomyia (N.) flaviscutellata (13, 13%) e outras oito espécies (20, 18%). Discussão: Espécies de Leishmania do subgênero Viannia, sobretudo L. (V.) braziliensis predominam em Juruti, o que é compatível com o extenso diâmetro das reações cutâneas observadas ao antígeno de Montenegro e com os relatos comuns de persistência e recidiva, apesar do tratamento específico. Entre os flebotomíneos antropófilos destacam-se L. (Ps.) complexa (17%) e L. (Ps.) flaviscutellata (13%) por serem vetores de L. (V.) braziliensis e L. (L.) amazonensis respectivamente, associadas às formas severas da LT humana. Conclusão: Medidas de controle em Juruti devem priorizar a redução da morbidade, diagnóstico precoce, busca ativa de LT humana, vigilância entomológica e de microambientes no entorno da área de impacto de mineração.

Epidemiologia molecular do Vírus da imunodeficiência humana 1 (HIV-1) em mulheres (mães e grávidas) dos Estados do Acre e Tocantins, Brasil

A transmissão vertical é a principal fonte de infecção pelo HIV em crianças, e pode ocorrer antes, durante e depois do nascimento, dessa forma sendo um grande problema de saúde pública mundial. O presente trabalho teve como objetivo descrever a epidemiologia molecular e o perfil de susceptibilidade/resistência das cepas de HIV-1 identificadas em mulheres nos estados do Acre e do Tocantins. Coletou-se amostra de sangue de 36 mulheres, sendo 9 grávidas e 16 mães de Palmas (Tocantins) e 1 grávida e 10 mães do Rio Branco (Acre) entre abril de 2007 a outubro de 2008. Realizou-se a técnica molecular Reação em Cadeia mediada pela Polimerase (PCR) Nested, para a amplificação de duas regiões genômicas (pro e tr) do DNA proviral, seguido de sequenciamento nucleotídico e análise filogenética. No Acre, tanto em relação ao segmento da protease quanto da transcriptase reversa, todas as amostras foram do subtipo B. Em Tocantins, quanto ao segmento da protease, todas as amostras pertenceram ao subtipo B, já em relação ao segmento da transcriptase reversa 87,5% foram do subtipo B e 12,5% pertencente ao subtipo F. No estado do Acre, todas as cepas analisadas foram suscetíveis aos inibidores de protease (IP) e apenas uma grávida de Tocantins (4,7%) apresentou cepa com resistência aos IP utilizados atualmente. Além disso, verificou-se uma baixa prevalência de cepas com mutações de resistência aos inibidores de transcriptase reversa nucleosídicos (ITRN) e não nucleosídicos (ITRNN), sendo que a resistência as três classes desses ARV foi observada em apenas uma amostra proveniente do estado do Tocantins. Dessa forma, a maioria das cepas de HIV isoladas mostrou susceptibilidade aos ARV utilizados, indicando que há baixa circulação de cepas do HIV resistentes a estes medicamentos nesses estados.

Ocorrência do subtipo B do vírus da imunodeficiência felina em gatos domésticos da região sul do estado do Rio Grande do Sul, Brasil

In Brazil there are few studies on the occurrence of the feline immunodeficiency virus (FIV) infection and its subtypes, which are essential for the development of vaccines and new diagnostic tests. The present study investigated the occurrence of the FIV infection between 2010 and 2011 in domestic cats submitted to medical attendance in the city of Pelotas and nearby area. Total blood samples of seventy cats, suspected (28) or not (42) of infection by FIV were analyzed by nested PCR in order to perform a diagnosis. The results pointed to a FIV infection frequency of 15.7% (11/70) and the analysis of the risk factors related to infection (sex, age and clinical condition) evidenced a greater occurrence in cats up to 10 years of age with chronic and recurrent infections. Eight samples found positive by nested PCR were submitted to DNA sequencing indicating that only the subtype B was detected in the studied region.

Diferentes métodos de diagnóstico molecular na diferenciação das espécies de Sarcocystis spp

The Sarcocystis genus includes obligatory two-host life cycle protozoan parasites. It is the most numerous of the six genera of the Sarcocystidae family. The infection caused by parasites of this genus is a zoonotic and cosmopolitan disease known as sarcosistosis or sarcosporidiosis. The sarcositosis though frequently asymptomatic in its definitive hosts can be fatal in its intermediate hosts. The usual diagnoses of sarcosistosis takes place through a histological demonstration of schizonts in blood vessels and organs, and the presence of cysts in muscle tissue by necropsy or biopsy, this second method still more common and based on morphological features of the sarcocyst. However, these methods can be inadequate to a precise identification of the infector species once that, besides the genus being of numerous species, these often present similar morphological features. Another factor that makes the diagnostic more difficult is the non specificity of some Sacocystis species to their hosts. Consequently, molecular diagnostic methods have been used in order to identify the infector species and the parasite specific biological cycles, demonstrating also new species and coevolutive aspects between parasite and host. Among the most employed molecular techniques the Polimerase Chain Reaction (PCR), the nested-PCR and the Restriction Fragment Length Polymorphism (RFLP) stands out

Comparação entre métodos diagnósticos da tuberculose em bovinos abatidos em matadouros-frigoríficos do Estado de São Paulo

Pós-graduação em Medicina Veterinária - FCAV

Detection of human T-cell lymphotropic virus type 1 in plasma samples

Human T-cell lymphotropic virus type 1 (HTLV-1) is-an RNA virus responsible for diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATL). Cell-to-cell contact and Tax-induced clonal expansion of infected cells are the main modes of virus replication, making virus detection during the viremic stage difficult. Consequently, the proviral load is the current virologic marker for disease monitoring, but the mechanisms of progression have not been established yet. Thus, this study investigated the presence of virus in plasma from asymptomatic HTLV-1 carriers and from HAM/TSP patients. Real-time PCR was performed on DNA from 150 plasma samples; 12(8%) had detectable DNA amplification, including 6(4%) asymptomatic HTLV-1 carriers and 14(26%) HAM/TSP patients (p < 0.005). Of the 33 samples submitted for nested PCR, six (18%, p = 0.02) were positive for HTLV-1 RNA in the plasma. Additionally, 26 plasma samples were treated with DNAse enzyme to eliminate any DNA contamination before RNA extraction. Two of them (8%) showed amplification for HTLV-1 (p = 0.5). Therefore, this study described for the first time the detection of free HTLV-1 RNA in plasma from HTLV-1-infected subjects, regardless of their clinical status. Thus, HTLV-1 viral replication does occur in plasma, and other transmission pathways for HTLV-1 should be investigated further. (C) 2011 Elsevier B.V. All rights reserved.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (â^ž), NLR (0.017), and Ef (99%).

Human Herpesvirus Infections Of The Central Nervous System: Laboratory Diagnosis Based On Dna Detection By Nested Pcr In Plasma And Cerebrospinal Fluid Samples.

Infections of the central nervous systems (CNS) present a diagnostic problem for which an accurate laboratory diagnosis is essential. Invasive practices, such as cerebral biopsy, have been replaced by obtaining a polymerase chain reaction (PCR) diagnosis using cerebral spinal fluid (CSF) as a reference method. Tests on DNA extracted from plasma are noninvasive, thus avoiding all of the collateral effects and patient risks associated with CSF collection. This study aimed to determine whether plasma can replace CSF in nested PCR analysis for the detection of CNS human herpesvirus (HHV) diseases by analysing the proportion of patients whose CSF nested PCR results were positive for CNS HHV who also had the same organism identified by plasma nested PCR. In this study, CSF DNA was used as the gold standard, and nested PCR was performed on both types of samples. Fifty-two patients with symptoms of nervous system infection were submitted to CSF and blood collection. For the eight HHV, one positive DNA result-in plasma and/or CSF nested PCR-was considered an active HHV infection, whereas the occurrence of two or more HHVs in the same sample was considered a coinfection. HHV infections were positively detected in 27/52 (51.9%) of the CSF and in 32/52 (61.5%) of the plasma, difference not significant, thus nested PCR can be performed on plasma instead of CSF. In conclusion, this findings suggest that plasma as a useful material for the diagnosis of cases where there is any difficulty to perform a CSF puncture.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle. (C) 2010 Elsevier B.V. All rights reserved.