A serosurvey for hantavirus infection in wild rodents from the states of Rio de Janeiro and Pernambuco, Brazil.

Sera from 269 rodents obtained during the routine surveillance operations in plague areas of Rio de Janeiro and Pernambuco states, Brazil were tested by ELISA for specific IgG antibodies against a recombinant nucleocapsid (N) protein of Araraquara hantavirus. ELISA-positive sera were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) for amplification of the virus genome and later sequencing for identification of the viral variant. The samples from the state of Pernambuco were antibody negative, and although four from Rio de Janeiro were ELISA-positive, they failed to yield viral cDNA by RT-PCR. This is the first report of the presence of antibodies to a hantavirus among rodents from Rio de Janeiro and suggests the possibility of human cases of hantavirus pulmonary syndrome (HPS) in that state, although no case has yet been reported.

The Brazilian flaviviruses.

Ten flaviviruses occur in Brazil: Bussuquara, Cacipacoré, dengue 1, 2 and 4, Iguape, Ilhéus, Rocio, Saint Louis encephalitis and yellow fever. Aspects of sylvatic maintenance cycles and human diseases caused by these viruses are analyzed. Large dengue outbreaks are occurring in Brazil and there is a risk of yellow fever urbanization.

Phylogenetic analysis of Brazilian Flavivirus using nucleotide sequences of parts of NS5 gene and 3' non-coding regions.

Viruses of the genus Flavivirus, which are arboviruses, of the Flaviviridae family, are amongst the most important agents of infectious disease in Brazil, causing human infections with a high morbility and mortality. In this work, the phylogeny of 14 virus amplicon sequences that were obtained by RT-PCR with universal primers for mosquito-borne Flavivirus were studied. The amplicons included a region of the Flavivirus genome of 129 nucleotides at the 3' terminus of the NS5 gene and the 145 initial nucleotides of the 3' non-coding region (NS5-3'NCR). Based on phylogenetic trees, most Brazilian Flaviviruses were grouped into two main branches, including a yellow-fever branch and a second main branch divided into a dengue branch that in its turn is subdivided into serotype 1, 2 and 4 branches, and another (Japanese Encephalitis Virus Complex) branch including SLE and Ilhéus. Rocio and Cacipacoré viruses were included in the Japanese Encephalitis Virus Complex branch in one of the two phylogenetic trees. Iguape virus appears in phylogenetic trees as a separate distant branch.

Congenital cytomegalovirus infection in preterm and full-term newborn infants from a population with a high seroprevalence rate.

BACKGROUND: Cytomegalovirus (CMV) is the most frequent cause of congenital infections in humans. Prematurity occurs in as many as 34% of infants with symptomatic congenital CMV infection. OBJECTIVE: To determine the clinical presentation and frequency of congenital CMV infection among preterm infants and full-term infants from a population with a high seroprevalence rate. DESIGN/METHODS: A total of 289 preterm infants (median gestational age, 34 weeks; median birth weight, 1,757 g) and 163 term infants (median gestational age, 39 weeks; median birth weight, 3,150 g) sequentially born were included in the study. Serum IgG antibodies to CMV were measured in all mothers. One urine sample was collected within the first 7 days of age from all newborns. Virus isolation in urine samples was performed by tissue culture, and viral DNA was detected by a multiplex PCR. CMV infection was diagnosed in infants with virus excretion detected by both methods on at least two occasions within the first 3 weeks of life. RESULTS: Maternal CMV seropositivity rate was 95.7%. Congenital CMV infection was detected in 6 of 289 (2.1%) (95% confidence interval, 0.84 to 4.68) preterm infants and in 3 of 163 term infants (1.8%) (95% confidence interval, 0.48 to 5.74) (P > 0.05). Four of 6 preterm infants with congenital CMV infection were symptomatic, but none of the term infants was symptomatic (P = 0.16). CONCLUSION: The frequency of congenital CMV infection in preterm newborn infants from mothers with a high seropositive rate was similar to that found in term infants. No significant difference was found between the proportion of symptomatic infants among preterm and term infants. Our finding of symptomatic congenital CMV infection underscores the need of further evaluation of correlates of congenital symptomatic infection in highly immune populations.

Identification of Brazilian flaviviruses by a simplified reverse transcription-polymerase chain reaction method using Flavivirus universal primers.

We report a simplified reverse transcription-polymerase chain reaction (RT-PCR) method for identification of Brazilian flaviviruses based on the patterns of electrophoretic separation of the amplicons. The RT-PCR was done on the culture fluids of Aedes albopictus C6/36 cells infected with Brazilian flaviviruses, without previous extraction of viral RNA, using Flavivirus universal primers that anneal to highly conserved sequences within the nonstructural protein 5 and 3'- non translated region of the virus genome. Genomes of 13 Brazilian Flavivirus isolates were amplified. It was not possible to amplify the genome of Bussuquara virus. Analysis of the RT-PCR products gave reproducible results and three distinct amplicon patterns were observed. Cacipacoré (800-850 basepairs [bp]) and yellow fever viruses (600 bp) yielded a single amplicon; dengue virus types 1 and 2 (650 and 550 bp), dengue virus type 4 (550 and 450 bp), Iguape (650-600 bp and 750-700 bp), St. Louis encephalitis (700 and 650-600 bp), and Rocio viruses (600 and 500-550 bp) yielded two amplicons; and Ilheus virus yielded five amplicons, two larger than 1,000 bp, one 650-700 bp, one 550-600 bp, and one 450-500 bp. The analysis of amplicon DNA sequences of six viruses showed homology with the 3'- nontranslated region of Flavivirus genome. The use of the Flavivirus universal primers in this simple RT-PCR technique is suitable as a screening test for the genus Flavivirus, with the exception of Bussuquara virus, in Brazilian isolates in tissue culture fluid.

Isolation of a newly recognized Bunyamwera serogroup virus from a febrile human in Panama.

A virus, strain 86MSP18, was isolated from the acute phase serum of a U.S. soldier with a febrile illness. He was stationed at Fort Sherman in the Republic of Panama when the onset of his illness occurred. A rise in neutralizing antibody to the viral isolate was observed between the patient's acute and convalescent-phase serum samples. Virus strain 86MSP18 has been shown by plaque reduction neutralization to be closely related to but distinct from Cache Valley virus and known subtypes. It appears to be a newly recognized subtype of Cache Valley virus and is believed to be the second isolation of a Cache Valley virus subtype from a human with a febrile illness. The name "Fort Sherman" virus for strain 86MSP18 is proposed.

An enzyme immunoassay for dengue antibody using infected cultured mosquito cells as antigen.

A simple enzyme immunoassay (EIA) for dengue virus antibody detection used infected tissue culture cells as antigen. C6/36 Aedes albopictus cells infected with dengue virus, and uninfected control cells were fixed with 3.3% formalin. This technique eliminated microplate coating and laborious antigen preparation, and it facilitated rapid screening of large numbers of sera. Formalin also inactivated dengue virus infectivity. Microplates prepared by this technique could be stored at -20 or -70 degrees C for at least two months. Human sera were adsorbed with C6/36 cells prior to testing in the EIA in order to reduce nonspecific binding to C6/36 cells.

Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique.

A rapid test for the diagnosis of congenital CMV infection is still needed. This study evaluated the usefulness of dried blood and urine samples collected on filter paper for detecting cytomegalovirus (CMV) by the polymerase chain reaction (PCR) assay compared with the use of liquid urine. Samples were obtained from 332 infants aged 1-7 days. Liquid urine samples were collected into bags, cultured in human fibroblasts, and processed using a multiplex PCR technique. Dried urine samples were obtained by placing a piece of filter paper in contact with the infant's genitals. The heels of neonates were punctured and capillary blood was blotted onto filter paper and dried. Dried blood and urine specimens were analyzed by multiplex PCR and nested-PCR assays. A diagnosis of congenital CMV infection was established by isolating the virus, and by detecting viral DNA in the liquid urine. Of the 332 liquid urine samples collected from 332 neonates, seven (2.1%) were positive for CMV and 325 were negative, by both cell culture and PCR assay. In dried samples, CMV DNA was detectable only with a nested PCR assay. Compared with known CMV infection status, 5/7 (71.4%) neonates were positive for congenital CMV infection using dried blood samples. All 325 uninfected neonates were negative. In the dried urine samples, 4/4 CMV-infected infants gave positive tests, and all 262 uninfected infants were negative. Although further improvements in sample collection and/or processing are still needed, PCR testing on dried urine or blood collected on filter paper is a promising approach in the diagnosis of neonatal CMV infection.

Culture conditions affect cytotoxin production by Serratia marcescens.

Cytotoxins have been implicated in the pathogenesis of bacterial infections. In this study, the influence of different culture conditions was evaluated on cytotoxin production of Serratia marcescens. Parameters such as culture media, incubation temperature, starting pH of culture medium, aeration, anaerobiosis, carbon sources, iron concentration in he culture media, and release of cell-bond toxin by polymyxin B were investigated. The data suggest that this cytotoxin is predominantly extracellular and is not induced by iron limitation. Aerobic culture with shaking resulted in higher cytotoxicity than static aerobic or anaerobic culture. Bacteria grown in glucose, sucrose or galactose were more cytotoxic than those grown in inositol or maltose. The culture conditions that were identified as optimal for cytotoxin production by Serratia marcescens were incubation temperature ranging from 30 to 37 degrees C, in medium adjusted pH 8.5, with shaking. This work will contribute to further studies on the identification of this cytotoxic activity.

Enzyme immunoassay for the detection of dengue IgG and IgM antibodies using infected mosquito cells as antigen.

The detection of immunoglobulin (Ig)G and IgM antibodies to dengue 1 virus was studied by a simple enzyme immunoassay, in which infected cultured cells infected with dengue virus were used as antigen (EIA-ICC). Detection of anti-dengue 1 IgG by EIA-ICC was correlated with haemagglutination assays. EIA-ICC anti-dengue 1 IgM detection was less sensitive than IgM capture enzyme-linked immunosorbent assay. IgG and IgM responses in dengue 1 infection were studied by EIA-ICC, using sera collected at different intervals after onset of illness: IgM and IgG appeared on the 4th day of disease; the highest IgM mean titres were detected on the 7th day and IgM was not detected in sera obtained after the 60th day; the highest mean titres of anti-dengue 1 IgG were seen in sera obtained between 22 and 30 d after onset of illness. EIA-ICCs for 6 flaviviruses and 1 alphavirus were conducted with sera from patients infected with dengue 1, and primary and secondary infections of other flaviviruses. The results showed that anti-dengue 1 IgG detection was sensitive, and the antibodies were cross-reactive among the flaviviruses. Anti-dengue 1 IgM detected in dengue 1 patients was mostly type specific. The pattern of secondary dengue infection, i.e. the presence of IgG and a low titre or absence of IgM antibodies, was observed in the sera of 6 patients obtained in the first week after onset of illness. EIA-ICC is useful for dengue diagnosis, surveillance and sero-epidemiological studies.

Identification of Simbu, California and Bunyamwera serogroup bunyaviruses by nested RT-PCR.

We describe a reverse transcription-polymerase chain reaction (RT-PCR) with primers that anneal to the 5' and 3' ends and amplify the Bunyavirus S RNA segments. The RT-PCR was done on the fluids of C6/36 cells infected with each of 21 bunyaviruses. The bunyaviruses studied, with the exception of Catu virus, produced amplicons having 700 to 1300 base pairs and probably contained the whole S RNA segment sequence. A nested PCR performed with these amplicons distinguished California and most Bunyamwera serogroup viruses from other bunyaviruses by use of BBC specific internal primers for the S RNA segment, and distinguished Simbu serogroup viruses from others by use of BS specific internal primers. The nested-PCR amplicons of Guaroa, Maguari, California encephalitis, Bunyamwera, and Oropouche viruses were sequenced. The sequences were aligned with previously known sequences of the S RNA segment of the same viruses, showing a high degree of homology and thus confirming the specific origin of these amplicons. The nested RT-PCR is suitable as a specific screening for most California and Bunyamwera serogroup and Simbu serogroup viruses depending on the use of BBC or BS internal primers. Oropouche virus is an important public health problem in Brazil and the nested PCR with BS primers could be used for the detection of this virus in tissue culture and mouse brain isolates as well as in clinical samples.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR.

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2%) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2% of the patients with CMV infection were symptomatic.

Donated organs as a source of cytomegalovirus (CMV) in renal transplant patients.

Two patients receiving the same cadaver kidney graft were investigated prospectively for cytomegalovirus (CMV) infection using the polymerase chain reaction (PCR) and serologic tests (ELISA and IFI). The data indicate that a strain of CMV was probably transmitted from the same donor to both kidney recipients including one who was seropositive for CMV.

Detection of cytomegalovirus infections by PCR in renal transplant patients.

Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants. To evaluate the incidence and the clinical importance of CMV infection in renal transplants in Brazil, 37 patients submitted to renal allograft transplants were tested periodically for the presence of cytomegalovirus DNA in urine using the polymerase chain reaction (PCR), and for the presence of IgM and IgG antibodies against CMV by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). The PCR-amplified products were detected by gel electrophoresis and confirmed by dot-blot hybridization with oligonucleotide probes. Thirty-two of the 37 patients (86.4%) were positive by at least one of the three methods. In six patients, PCR was the only test which detected the probable CMV infection. Ten patients had a positive result by PCR before transplantation. In general, the diagnosis was achieved earlier by PCR than by serologic tests. Active infection occurred more frequently during the first four months after transplantation. Sixteen of the 32 patients (50%) with active CMV infection presented clinical symptoms consistent with CMV infection. Five patients without evidence of active CMV infection by the three tests had only minor clinical manifestations during follow-up. Our results indicate that PCR is a highly sensitive procedure for the early detection of CMV infection and that CMV infection in renal transplant patients is a frequent problem in Brazil.

[Prospective study with infants whose mothers had dengue during pregnancy]. / Estudo prospectivo com lactentes cujas mães tiveram dengue durante a gravidez.

Dengue congenital disease was not confirmed in 10 children whose mothers had the infection during pregnancy. The fetal sera presented anti-dengue IgG antibodies which progressively declined, and disappeared after 8 months. IgM antibodies to dengue were not observed in the sera. Other normal data suggesting the healthy state of the children included: absence of malformations, pregnancy time, Apgar index, weight, and placenta aspect.

Infective endocarditis (IE) first diagnosed at autopsy: analysis of 31 cases in Ribeirão Preto, Brazil.

Thirty one infective endocarditis (IE) fatal cases whose diagnosis was first obtained at autopsy were studied. The clinical data of these patients (Group 1) showed significant differences compared to other 141 IE cases (Group 2). The average age of 53 years in Group 1 patients was 18 years higher than that of Group 2. The Group 1 patients had a low frequency of IE predisposing heart disease. Both patient groups presented fever (about 87%), but a significant low frequency of cardiac murmur (25.8%) was observed in Group 1 patients and echocardiography tests were performed in only 16.1%, suggesting that IE diagnosis was not suspected. Likewise, although most Group 1 patients appeared with severe acute illness, they did not present the classic IE clinical presentation. Blood cultures were performed in only 64.5% of the Group 1 patients. However, bacteria were isolated in 70% of these blood cultures and Staphylococcus aureus was isolated in 71.4%. The bacteria attacked mitral and aortic valves. Complications such as embolizations and cardiac failure occurred in almost half of the cases and they also presented with infections of the lungs, urinary tract, and central nervous system. Medical procedures were performed in practically all fatal cases whose diagnosis was first obtained at autopsy. Sepsis occurred in about half of the patients and it was followed by shock in more than 25%. This form of IE must be suspected in mature and in old febrile hospitalized patients having infection predisposing diseases, embolization, and suffering medical procedures.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method.

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.

[Laboratory diagnosis and symptoms of dengue, during an outbreak in the Ribeirão Preto region, SP, Brazil]. / Estudo sobre diagnóstico laboratorial e sintomas do dengue, durante epidemia ocorrida na região de Ribeirão Preto, SP, Brasil.

A dengue type 1 outbreak started in the Ribeirao Preto Region, North of Sao Paulo State, Brazil, in November of 1990. About 3500 dengue cases were confirmed by blood tests until February of 1991. The Virus Research Unit of The Faculty of Medicine of Ribeirao Preto-Sao Paulo State University, studied 502 dengue suspect cases. The serologic diagnosis of dengue type 1 was confirmed by haemagglutination inhibition test (HAI) in 19% of the cases. Diagnosis was done later by using an enzyme immuno assay on infected cultured cells (EIA-ICC) which discriminated IgG and IgM dengue, antibodies. EIA-ICC was less sensitive (89%) but more effective than HAI. EIA-ICC is a simple technique. It dispenses a second serum sample for diagnosis and it can be completed in about 5 hours. Dengue virus was isolated from the blood of 21 patients by inoculation in culture of mosquito C6/36 cells. The isolated virus were identified by indirect immunofluorescent test, by using an antisera pool to the flavivirus family and dengue type specific monoclonal antibodies. The dengue most frequent symptoms in 71 patients were observed: fever (90%), myalgias (57%) and arthralgias (41%).

Hantavirus pulmonary syndrome (HPS) in Guariba, SP, Brazil. Report of 2 cases.

Human infections caused by a hantavirus were reported in different regions of the State of São Paulo (SP), Brazil during the first six months of 1998. Two cases of fatal pulmonary syndrome occurred in May of 1998 in the City of Guariba, located in the Northeastern Region of SP. Both patients worked in a corn storage barn infested by rodents. These patients, after 2 or 3 days of non-specific febrile illness, developed a severe interstitial pneumonia spreading widely in both lungs, causing respiratory failure and death. At autopsy both patients showed lung interstitial edema with immunoblast-like mononuclear cell infiltrates, consistent with a viral etiology. Hantavirus infection was diagnosed by ELISA in both cases and by RT-PCR in one of the patients. Aspects of the clinical presentation, physiopathology and differential diagnosis of Hantavirus Pulmonary Syndrome are discussed.

[Yellow fever in the Ribeirão Preto region at the turn of the 19th century: its scientific importance and economic repercussions]. / A febre amarela na região de Ribeirão Preto durante a virada do século XIX: importancia científica e repercussões econômicas.

Yellow fever in the Region of Ribeirao Preto at the turn of XIX century: scientific importance and economic repercussion. This historical review describes the bad situation of public health in Brazil during the XIX Century caused by multiple yellow fever outbreaks. The knowledge regarding to yellow fever at that time is also described. A short history is presented of the development of the Region of Ribeirao Preto, located in the Northeast of Sao Paulo State, Brazil, emphasising the actuation of immigrants and pioneer coffee farmers like Luiz Pereira Barreto. Yellow fever outbreaks occurred in the City of Sao Simao in 1896, 1898, and 1902 are described as well as an outbreak in the City of Ribeirao Preto occurring in 1903. It is shown that yellow fever outbreaks were stopped in the 2 cities by Emilio Ribas who led the fight against the transmitting mosquito Aedes aegypti. Emilio Ribas, helped by Adolpho, Lutz and Luiz Pereira Barreto, promoted scientific experiments in order to confirm the vectorial transmission of yellow fever and to annul the supposed importance of other kinds of contagion. The yellow fever outbreaks caused damage to the development of Sao Simao and influenced the transference of the economic pole of the region to the City of Ribeirao Preto. The vector control work done during yellow fever outbreak and the scientific experiments on the transmission of yellow fever were important for the development of medical science and fpublic health in Brazil.