Comparação entre métodos de estocagem de DNA extraído de amostras de sangue, sêmen e pêlos e entre técnicas de extração / Comparison between storage methods of DNA extracted from blood, semen and hair and between the techniques of extraction

DNA samples of six bovines obtained from three tissues (blood, semen and hair) were extracted using two different techniques. After the extraction procedures the samples were divided in six fractions. Three were stored at -20º C and three at 4º C. Every three months one sample of each tissue/extraction procedure was analyzed in spectrophotometer, to determine the quantity of the DNA and the extract was amplified using the primer RM 29. No differences in the DNA quantity or in the level of protein contamination among the three periods of analyses were observed. All the DNA extracted by quick extraction technique showed good amplification patterns during the nine months, meaning that this technique can be used in laboratory routine instead of the permanent extraction technique. The extract obtained from blood, using the permanent extraction technique, showed the higher quantity of DNA with the smaller index of protein contamination. The high quantity of protein contamination found in the semen samples preserved in egg yolk demanded modifications in both extraction techniques. After that the results were positive, showing good amplification patterns.

Teste de DNA para verificaçäo de parentesco em cäes: avaliaçäo do método näo automatizado com o auxílio do primer CMR S / DNA test for parentage verification in dogs: evaluation of the non-automatized method with assistance of the primer CMR S

To evaluate the precision of the DNA tests using the non-automatized technique for individual identification and parentage tests, 105 Rottweiler dogs were studied using the primer CMR S. The sample was composed of 39 animals belonging to 11 complete families and their progenies, and 66 non related individuals until the second generation, derived from kennels located in the states of Minas Gerais and Säo Paulo. The CMR S primer was used for the Polimerase Chain Reaction (PCR). The results showed the inefficiency of the technique, even when analyzed through the automated gel analysis system. Also showed the impossibility of its commercial use due to the fact of does not permit the storage of data for subsequent use

Microssatélites BM2113, ILSTS005, ILSTS008, ETH131 e RM88 em testes de verificaçäo de parentesco para bovinos da raça Gir / Microsatellites BM2113, ILSTS005, ILSTS008, ETH131 and RM88 for parentage tests in bovines of Gir breed

Foram utilizados 46 animais da raça Gir, registrados na Associaçäo Brasileira de Criadores de Zebu, provenientes de cinco fazendas situadas no Estado de Minas Gerais, com o objetivo de avaliar a eficiência dos microssatélites BM2113, ILSTS005, ILSTS008, ETH131 e RM88 em testes de verificaçäo de parentesco. Os locos BM2113, ILSTS005, ETH131 e RM88 mostraram-se eficientes, apresentando valores de PE2 (probabilidade de exclusäo quando os dois progenitores säo genotipados) entre 0,62 e 0,69 e PIC2 (conteúdo de informaçäo polimórfica quando os dois progenitores säo genotipados) entre 0,78 e 0,83. O mesmo näo ocorreu para o loco ILSTS008, o qual apresentou baixos valores de PE2 (0,24) e PIC2 (0,41)

Isolation of yellow fever virus from nulliparous Haemagogus (Haemagogus) janthinomys in eastern Amazonia.

In 1998, an epizootic of yellow fever (YF) killed many howler monkeys (Alouatta spp.) in eastern Amazonia near the city of Altamira. An infection level with YF virus of approximately 3.6% was determined from analysis of 456 females of Haemagogus janthinomys Dyar, the main enzootic YF vector in South America. One month later, a second study of 164 females captured in the same place led to infection levels of 0.8% for parous and 2.9% for nulliparous females. These results lead to the conclusion that vertical transmission, one of the key elements in the epidemiology of YF, occurs in South America as it does in Africa.

Epidemic of jungle yellow fever in Brazil, 2000: implications of climatic alterations in disease spread.

Seventy-seven human cases of sylvatic yellow fever were reported in Brazil during the period January-June 2000. The first cases were reported 1 week after New Year's day and originated at Chapada dos Veadeiros, a tourist canyon site in Goiás state, near Brasília, the Brazilian capital. The laboratory procedures used for diagnoses included serology with an IgM capture assay and plaque reduction neutralization test, virus isolation in suckling mice and C6/36 cells, and immunohistochemistry. All cases were diagnosed by at least two different laboratory procedures, with the exception of the first three fatal cases, which were diagnosed on the basis of clinical and epidemiological information. The cases were reported in eight Brazilian states as follows: Goiás with 64.9% (50 cases); Amazonas (1); Bahia (10); Distrito Federal (1); Mato Grosso (4); Minas Gerais (2); Pará (1); São Paulo (2); and Tocantins (6). Patient ages were within the following ranges: 13-74 years old (mean 34.3), 64 (84.4%) were male, especially agricultural workers (n = 30), but tourists (n = 11), carpenters (n = 4), fishermen (n = 4), students (n = 3), truck drivers (n = 3), and other people (n = 22) were also sickened. The case fatality rate was 50.6% (39/77). In Bahia state, a serologic survey that was carried out has suggested a symptomatic/asymptomatic coefficient of 1:4. Field studies developed in Distrito Federal, Goiás, and São Paulo states showed that Haemagogus janthinomys was the mosquito species associated with the transmission. A single strain was also obtained from Aedes scapularis in Bahia. Epizootic occurrence (monkey mortality) was observed in 49 municipalities mainly in Goiás state, where 40 municipalities made reports, 21 of which also diagnosed human cases. Data obtained by the National Institute of Meteorology in Brazil showed an increase in temperature and rain in December 1999 and the first 3 months of 2000 in Goiás and surrounding states, which perhaps has contributed to the intense and widespread transmission of the yellow fever virus. The relatively small number of cases probably reflects the extensive use of yellow fever 17D-vaccine during the last 3 years, in which about 45 million doses were used. During the last months of 1999, 16 and 11 yellow fever cases were reported in Tocantins and Goiás states, respectively. It is noteworthy that the last reported autochthonous cases of sylvatic yellow fever in São Paulo and Bahia, both states outside the endemic/enzootic area, had occurred in 1953 and 1948, respectively.

Yellow fever in Pará State, Amazon region of Brazil, 1998-1999: entomologic and epidemiologic findings.

Yellow fever (YF) is frequently associated with high severity and death rates in the Amazon region of Brazil. During the rainy seasons of 1998 and 1999, 23 (eight deaths) and 34 (eight deaths) human cases of YF were reported, respectively, in different geographic areas of Pará State; most cases were on Marajó Island. Patients were 1 to 46 years of age. Epidemiologic and ecological studies were conducted in Afuá and Breves on Marajó Island; captured insects yielded isolates of 4 and 11 YF strains, respectively, from Haemagogus janthinomys pooled mosquitoes. The cases on Marajó Island in 1999 resulted from lack of vaccination near the focus of the disease and intense migration, which brought many nonimmune people to areas where infected vectors were present. We hypothesize that YF virus remains in an area after an outbreak by vertical transmission among Haemagogus mosquitoes.

Inadequate management of natural ecosystem in the Brazilian Amazon region results in the emergence and reemergence of arboviruses.

A total of 187 different species of arboviruses and other viruses in vertebrates were identified at the Evandro Chagas Institute (IEC) from 1954 to 1998, among more than 10,000 arbovirus strains isolated from humans, hematophagous insects, and wild and sentinel vertebrates. Despite intensive studies in the Brazilian Amazon region, especially in Pará State, very little is known about most of these viruses, except for information on date, time, source, and method of isolation, as well as their capacity to infect laboratory animals. This paper reviews ecological and epidemiological data and analyzes the impact of vector and host population changes on various viruses as a result of profound changes in the natural environment. Deforestation, mining, dam and highway construction, human colonization, and urbanization were the main manmade environmental changes associated with the emergence and/or reemergence of relevant arboviruses, including some known pathogens for humans.

Phenotypic and molecular analyses of yellow fever 17DD vaccine viruses associated with serious adverse events in Brazil.

The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.

Dengue epidemic in Belém, Pará, Brazil, 1996-97.

We describe clinical and epidemiologic findings during the first epidemic of dengue fever in Belém, Pará State, Brazil, in 1996-97. Of 40,237 serum samples, 17,440 (43%) were positive for dengue by virus isolation or serologic testing. No hemorrhagic cases or deaths were reported. Mycobacterium tuberculosis

[A seroepidemiological survey on the island of São Luis during a dengue epidemic in Maranhão]. / Inquérito soro-epidemiológico na ilha de São Luis durante epidemia de dengue no Maranhão.

The island of São Luis in the State of Maranhão, constituted by the municipalities of São Luis-SL (835,428 inhabitants), São José de Ribamar-SJR (60,633 inhabitants) and Paço do Lumiar-PL (80,274 inhabitants), has been suffering dengue (DEN) fever epidemics since 1995, caused by DEN-1. In 1996, from August through October, an aleatory sero-epidemiologic survey was carried out in order to estimate the incidence of DEN infection and to analyze other clinical and epidemiological parameters. A questionnaire was applied and serum samples were simultaneously obtained. Serum samples were tested by hemagglutination inhibition (HI). Results were analyzed using Lotus 123, Epi-info 6.0, Excel 5.0 and STATA softwares. A total of 1,217 serum samples were obtained (101 of PL, 100 of SJR and 1017 of SL). The rate of DEN was 55.4% in PL, 28% in SJR and 41.4% in SL, suggesting the occurrence of 401,933 infections. No difference was seen between males and females, but infection occurred more in the upper social class than in poor people (p < 0.003), and was more frequent in adults than in children (p < 0.0004). In SL, the incidence was stratified into seven sanitary districts (SD), and prevalence was found to range from 26.1% in SD4 to 56.8% in SD1 (p < 0.0001). Symptoms were more frequently reported by people whose HI was positive: they included fever, headache, chills, dizziness, retrobulbar pains, muscle and joint pains, nausea, anorexia and skin rash. In spite of the high incidence of infection, no hemorrhagic cases were reported.

Involvement of the central nervous system in dengue fever: three serologically confirmed cases from Fortaleza Ceará, Brazil.

Three cases of dengue fever involving the central nervous system (CNS) are reported. All occurred in 1994 during a dengue (DEN) epidemic caused by serotypes DEN-1 and DEN-2. The first case examined was a 17-year-old girl who complained of fever, nuchal rigidity and genital bleeding. Three blood samples were positive by anti-dengue IgM ELISA and showed hemagglutination-inhibition (HI) test titers > or = 1,280. The second case concerned a 86-year-old women with fever, muscle and joint pains, altered consciousness, syncope, nuchal rigidity and meningismus. Her blood sample showed an HI titer of 1:320 for flaviviruses, and an IgM ELISA positive for dengue. The third case was a 67-year-old women with fever, abnormal behaviour, seizures, tremor of extremities, thrombocytopenia, increased hematocrit and leukopenia. The patient suffered a typical case of dengue hemorrhagic fever with ensuing shock and a fatal outcome. A single blood sample showed HI antibodies of > or = 1,280 and an IgM ELISA positive for dengue. No virus could be isolated from any patient by inoculation of blood into C6/36 cells and suckling mice. No other agent of disease was encountered in the patient.

[Dengue epidemic in Fortaleza, Ceará: randomized seroepidemiologic survey]. / Epidemia de dengue em Fortaleza, Ceará: inquérito soro-epidemiológico aleatório.

OBJECTIVE: A seroepidemiological random survey was carried out in Fortaleza city, State of Ceará, Brazil, following an epidemic of dengue virus type 2 (DEN 2), with the purpose of evaluating the frequency of clinical manifestations (signs and symptoms) and the prevalence of dengue infection. METHOD: A questionnaire calling for information on address, sex, age, clinical, epidemiological and economic status was applied to the population, followed by venupuncture collection of 5-10 ml of blood for testing by hemagglutination-inhibition (HI). The sample was calculated to obtain a prevalence of 20% with relative risk of 10% and confidence interval of 95%. All information obtained was analyzed by computer using Epi Info 5.0, Lotus 123, Excel 5.0, and Stata software. RESULTS AND CONCLUSIONS: A total of 1,341 serum samples were obtained from nine Health Districts (SD) and tested by hemagglutination inhibition. Of these, 589 (44%) were positive and 752 (56%) negative. Of the positive results, 93 primary responses (PR) (7%) to DEN-2 and 496 secondary responses (SR) (37%) were observed. The global prevalence in the SD ranged from 21% to 71%. There were 41% (243/589) asymptomatic infections and 59% (346/589) symptomatic infections. Data analysis showed no difference in frequency by sex, age, on schooling, although a highly statistically significant difference was found as between the different social classes, the infection most commonly observed being among people of better social status. The stratification of positive cases showed greater prevalence of AI (p < 0.001) and SI (p < 0.0001) in both sexes, among people with SR rather than PR. The most prevalent symptoms were fever, headache, muscle pains, rash, dizziness, and joint pains. Moreover, itching, retro-bulbar pain, rash, and gingival bleeding, showed statistically significant differences. On the other hand, dizziness and joint pains were more associated in the patients with SR than PR, and statistically significant differences were also observed.

Evidências sorológicas da circulação do arbovírus Rocio (Flaviviridae) na Bahia / Serological evidence of the circulation of the Rocio arbovirus (Flaviviridae) in Bahia

Oito casos com anticorpos anti-Rocio são descritos, de quatro cidades do Estado da Bahia, sendo seis portadores de anticorpos IgG (IH e TN) e dois IgM (ELISA e TN). Os autores comentam sobre a circulação deste arbovírus no Estado, e as possibilidades de reações cruzadas com outros vírus antigenicamente relacionados.

Eight antibody anti-Rocio cases, from four distinct cities in the state of Bahia, are described; six of them being carriers of the antibody IgG (HI and NT) and two IgM (ELISA and NT). The authors comment on the circulation of these arboviruses in the state of Bahia and on the possibility of cross reactions with other antigenically related viruses.

An epidemic of sylvatic yellow fever in the southeast region of Maranhao State, Brazil, 1993-1994: epidemiologic and entomologic findings.

Yellow fever virus transmission was very active in Maranhao State in Brazil in 1993 and 1994. An investigation was carried out to evaluate the magnitude of the epidemic. In 1993, a total of 932 people was examined for yellow fever from Maranhao: 70 were positive serologically, histopathologically, and/or by virus isolation, and another four cases were diagnosed clinically and epidemiologically. In Mirador (17,565 inhabitants), the incidence was 3.5 per 1,000 people (case fatality rate [number of deaths/number of cases diagnosed] = 16.4%), while in a rural yellow fever risk area (14,659 inhabitants), the incidence was 4.2 and the case-fatality rate was 16.1% (10 of 62). A total of 45.2% (28 of 62) asymptomatic infections were registered. In 1994, 49 serum samples were obtained and 16 cases were confirmed (two by virus isolation, two by seroconversion, and 12 by serology). No fatal cases were reported. In 1993, 936 potential yellow fever vectors were captured in Mirador and a single strain was isolated from a pool of Haemagogus janthinomys (infection rate = 0.16%). In 1994, 16 strains were isolated from 1,318 Hg. janthinomys (infection rate = 1.34%) and one Sabethes chloropterus (infection rate = 1.67%). Our results suggest that this was the most extensive outbreak of yellow fever in the last 20 years in Brazil. It is also clear that the lack of vaccination was the principal reason for the epidemic, which occurred between April and June, during the rainy season, a period in which the mosquito population in the forest increases.

[Serological evidence of the circulation of the Rocio arbovirus (Flaviviridae) in Bahia]. / Evidências sorológicas da circulação do arbovírus Rocio (Flaviviridae) na Bahia.

Eight antibody anti-Rocio cases, from four distinct cities in the state of Bahia, are described; six of them being carriers of the antibody IgG (HI and NT) and two IgM (ELISA and NT). The authors comment on the circulation of these arboviruses in the state of Bahia and on the possibility of cross reactions with other antigenically related viruses.

[Outbreak of oropouche virus fever in Serra Pelada, municipality of Curionópolis, Pará, 1994]. / Epidemia de febre do Oropouche em Serra Pelada, município de Curionópolis, Pará, 1994.

In the final of November 1994, an outbreak of a febrile disease was observed in the Serra Pelada gold mine (5 degrees 35'S: 49 degrees 30'W) in the Southeast region of Pará State. Twenty samples were collected and sent to the laboratory of Arbovirus of Instituto Evandro Chagas. The tests showed that the disease was caused by Oropouche virus (Bunyaviridae, Bunyavirus, Simbu serological group). Between 8-22 December 296 serum samples were taken (54 from febrile patients, 16 paired samples and 242 from contacts and convalescent patients) of the 73 familiar groups. From febrile patients, ten Oropouche virus strains were obtained. From paired serum, six seroconversions were obtained and 242 other Oropouche infections were diagnosed by HI and MAC ELISA. The clinical-picture of febrile disease accompanied by severe bedache, chills, myalgia, photophobia retrobulbar pain and malaise was observed. Involvement of central nervous system was not observed. Based on the serological data, we estimated that in the outbreak of Serra Pelada around 5,000 cases occurred corresponding to a prevalence of 83%.

[Dengue and its vectors in Brazil]. / La dengue et ses vecteurs au Brésil.

History of dengue in Brazil is covered from the first citations in the XIXth century to the great outbreaks of the last ten years. DEN-1 and DEN-4 viruses have been isolated for the first time in 1982 during an epidemic in Boa Vista, Roraima State. In 1986-1987, epidemics of dengue type 1 covered an extended area from Rio de Janeiro/Sào Paulo States to the North East States of Brazil. During 1990-1991, dengue type 2 epidemics have been notified in the South East (Rio de Janeiro/São Paulo) and in some States of the interior of the country (Mato Grosso do Sul, Tocantins). DEN-1 virus was also circulating the same year in São Paulo and Minas Gerais States. Recently (1994), an important outbreak has been studied in Ceará State, where DEN-2 and DEN-1 viruses have been isolated. In Rio de Janeiro and Ceara (1990 and 1994, respectively), it is probably the succession of infections by DEN-1 and DEN-2 viruses which has caused many DHF/DSS cases. The urban vector has always been the mosquito Aedes aegypti, from which 4, 7 and 16 strains of DEN-4, DEN-1 and DEN-2 have been isolated, respectively. In Brazil, transovarial transmission of dengue viruses by this species has not yet been shown to occur in nature.

[Laboratory-acquired human infection with SP H 114202 virus (Arenavirus: Arenaviridae family): clinical and laboratory aspects]. / Infecção humana adquirida em laboratório causada pelo virus SP H 114202 (Arenavirus: família Arenaviridae): aspectos clínicos e laboratoriais.

Here in is described the clinical and laboratorial findings of a laboratory-acquired infection caused by the virus SP H 114202 (Arenavirus, family Arenaviridae) a recently discovered agent responsible for a viral hemorrhagic fever. The patient was sick for 13 days. The disease had an abrupt onset characterized by high fever (39 degree C.), headache, chills and myalgias for 8 days. In addition, on the 3rd day, the patient developed nausea and vomiting, and in the 10th, epigastralgia, diarrhea and gengivorrhagia. Leucopenia was seen within the 1st week of onset, with counts as low as 2,500 white cells per mm3. Counts performed after the 23rd day of the onset were within normal limits. With the exception of moderate lymphocytosis, no changes were observed in differential counts. An increase in the titer of antibodies by complement fixation, neutralization and ELISA (IgM) was detected. Suckling mice and baby hamsters were inoculated intracerebrally with 0.02 ml of blood samples collected in the 2nd and 7th days of disease. Attempts to isolate the virus were also made in Vero cells. No virus was isolated. This virus was isolated before in a single occasion in São Paulo State, in 1990, from the blood of a patient with hemorrhagic fever with a fatal outcome. The manipulation of the virus under study, must be done carefully, since the transmission can occur through aerosols.

[Outbreak of classical fever of dengue caused by serotype 2 in Araguaiana, Tocantins, Brazil]. / Epidemia de febre clássica de dengue causada pelo sorotipo 2 em Araguaina, Tocantins, Brasil.

We report the first outbreak of dengue fever caused by dengue 2 (DEN 2) in Araguaina, Tocantins State. Four hundred people of 74 families, living at S. João, Araguaina Sul and Neblina districts were questioned and then bled, in order to obtain sera to test for anti-dengue antibodies. If a person was sick, a small quantity of blood was collected for virus isolation. The main clinical picture of disease was characterized by fever, headache, myalgias, arthralgias and skin rash. Were obtained 1,105 (56 females and 45 males of Culex quinquefasciatus and 567 females and 437 males of Aedes aegypti) mosquitoes from larvae collected in Araguaina. The females of Aedes aegypti obtained from larvae were allowed to feed on 8 febrile patients. The diagnosis of infection was made by both virus isolation into Aedes albopictus (C6/36) cells, and serology, by Hemagglutination-inhibition (HI) and IgM capture ELISA (MAC ELISA). No virus was isolated from mosquitoes. Although five strains of DEN 2 were obtained from humans, and another 111 infections were diagnosed serologically (IgM positive). The positivity rate of the samples was 27.75% (111 of 400), while that of the families was 66.2% (45 of 72), where at least one member of the each family was infected. It was also detected 26.1% of asymptomatic infections. All age groups were affected. Therefore, the infection was more frequent in females (33.5%) than males (23.8%).(ABSTRACT TRUNCATED AT 250 WORDS)