The relevance of laboratory diagnosis of human cryptosporidiosis and other coccidia

Human infection by Cryptosporidium spp and other coccidia are due to opportunist non-host specific microorganisms. In HIV seropositive patients, the gastrointestinal symptoms accompanying such infections may be serious and prolonged and may include nausea, low-grade fever, abdominal cramps, anorexia and watery diarrhoea. We studied 188 stool samples from 111 patients (84 men and 27 women) with diarrhoea. A modified Ziehl-Nielsen technique for the detection of Cryptosporidium spp and Isospora belli was employed. The mean age of the patients was 31 years. Cryptosporidium spp was seen in 18% (n=20) of the patients, 90% (n=18) of whom were HIV seropositive. Isospora belli was recorded only from HIV seropositive patients (5.4% of all the patients studied and 6.5% of those who were HIV seropositive). These data confirm the good results obtained with this technique for the identification of Cryptosporidium spp and other coccidia and also reaffirm the clinical importance of correctly diagnosing the cause of diarrhoea, particularly in HIV seropositive patients.

The role of Public Health Laboratory in the problem os salmonellosis in São Paulo, Brazil

From 1950 to 1990 a total of 45,862 strains (31,517 isolates from human sources, and 14,345 of non-human origin) were identified at Instituto Adolfo Lutz. No prevalence of any serovars was seen during the period 1950-66 among human sources isolates. Important changing pattern was seen in 1968, when S. Typhimurim surprisingly increased becoming the prevalent serovar in the following decades. During the period of 1970-76, S. Typhimurium represented 77.7% of all serovars of human origin. Significant rise in S. Agona isolation as well as in the number of different serovars among human sources strains were seen in the late 70' and the 80's. More than one hundred different serovars were identified among non-human origin strains. Among serovars isolated from human sources, 74.9%, 15.5%, and 3.7% were recovered from stool, blood, and cerebrospinal fluid cultures, respectively. The outbreak of meningitis by S. Grumpensis in the 60's, emphasizes the concept that any Salmonella serovars can be a cause of epidemics, mainly of the nosocomial origin. This evaluation covering a long period shows the important role of the Public Health Laboratory in the surveillance of salmonellosis, one of the most frequent zoonosis in the world.

Concomitant rotavirus serotypes 1 and 4 infections in a diarrhoeic child from Belém, Brazil

Concomitant serotypes 1 and 4 infections were detected in a 15-month old female child with community-acquired diarrhoea which lasted 7 days and coursed with moderate dehydration. The evidence for dual rotavirus infection was offered by the following findings: a) enzyme-linked immunosorbent assay (ELISA) positive reactions to both 1 and 4 serotypes; and b) extra-migrating bands at electro-phoresis of RNA in polyacrylamide gel (PAGE). These results suggest that children living under poor sanitation conditions are heavily exposed to rotavirus infections; in addition, the co-circulation of different serotypes in the same setting sustains the current concept that a rotavirus vaccine should be rnultivalent, in order to protect children against the four epidemiologically important rotavirus G serotypes.

Prospective study of enteropathogens in two communities of Misiones, Argentina

Children under five years of age, from two communities of different socio-economic strata (97 from Zaiman and 55 from Las Dolores) were examined epidemiologically during 2 years, by means of quarterly visits of the working team, who carried out the collection of faecal samples. During the study, one or more enteropathogens were identified in 73.9% of samples in children from Zaiman and in 58.3% of the samples from Las Dolores, being associated to diarrhoea in 70.5% and to asymptomatic infections in 65.7%. The number of diarrheic episodes was higher in Zaiman (15.45%) than in Las Dolores (12.35%), being more frequent in the spring-summer seasons. In Zaiman, the bacterial enteropathogen proportion was relevantly higher (p< 0.005) in children with diarrhoea, whereas the presence of parasites was more frequent in asymptomatic children (p< 0.01). Rotavirus had an even distribution within diarrheic and asymptomatic children. In Las Dolores, no relevant differences were found in the detection of enteroparasites between diarrheic and asymptomatic children. Mixed infections were detected; enterotoxigenic Escherichia coli (ETEC)-rotavirus and ETEC-parasites being the most frequent ones. ETEC was involved in 85% of these infections. These data, together with the high enteropathogen carriage, suggest an elevated level of environmental contamination. The latter plays an important role in diarrheic diseases, and added to the most extreme poverty, it affects children's lives.

Salmonella-S. mansoni ASSOCIATION IN PATIENTS WITH ACQUIRED IMMUNODEFICIENCY SYNDROME

Two young men with Salmonella bacteraemia, active schistosomiasis and the acquired immunodeficiency syndrome are reported. The clinical presentation comprised nonspecific signs and symptoms, such as fatigue, malaise, weight loss, diarrhoea, prolonged fever, and hepatosplenomegaly. In one patient, liver biopsy showed poorly formed granulomata around Schistosoma mansoni eggs and hepatitis. Treatment of schistosomiasis alone induced consistent clinical improvement with eventual cure of both Salmonella and S. mansoni infections. Recognition of the Salmonella-S. mansoni association in patients with AIDS is important because treatment of schistosomiasis makes a difference, improving the prognosis of this otherwise, recurrent, potentially fatal bacteraemia.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Incidence of respiratory viruses in preterm infants submitted to mechanical ventilation

The objectives of this study were to determine the incidence of infection by respiratory viruses in preterm infants submitted to mechanical ventilation, and to evaluate the clinical, laboratory and radiological patterns of viral infections among hospitalized infants in the neonatal intensive care unit (NICU) with any kind of acute respiratory failure. Seventy-eight preterm infants were studied from November 2000 to September 2002. The newborns were classified into two groups: with viral infection (Group I) and without viral infection (Group II). Respiratory viruses were diagnosed in 23 preterm infants (29.5%); the most frequent was respiratory syncytial virus (RSV) (14.1%), followed by influenza A virus (10.2%). Rhinorrhea, wheezing, vomiting and diarrhea, pneumonia, atelectasis, and interstitial infiltrate were significantly more frequent in newborns with nosocomial viral infection. There was a correlation between nosocomial viral infection and low values of C-reactive protein. Two patients with mixed infection from Group I died during the hospital stay. In conclusion, RSV was the most frequent virus in these patients. It was observed that, although the majority of viral lower respiratory tract infections had a favorable course, some patients presented a serious and prolonged clinical manifestation, especially when there was concomitant bacterial or fungal infection.

Rotavirus serotypes and electropherotypes identified among hospitalised children in São Luís, Maranhão, Brazil

During June 1997-June 1999 rotavirus infection was screened in infants aged up to 2 years and hospitalised with acute diarrhoea in São Luís, Northeastern Brazil. Altogether, 128 stool samples were collected from diarrhoeic patients and additional 122 faecal specimens from age- and- temporal matched inpatients without diarrhoea were obtained; rotavirus positivity rates for these groups were 32.0% (41/128) and 9.8% (12/122), respectively (p < 0.001). Both electropherotyping and serotyping could be performed in 42 (79.2%) of the 53 rotavirus-positive stool samples. Long and short electropherotypes were detected at similar rates - 38.1% and 40.5% of specimens, respectively. Overall, a G serotype could be assigned for 35 (83.3%) of specimens, the majority of them (66.7%) bearing G1-serotype specificity. Taking both electropherotypes and serotypes together, G1 rotavirus strains displaying long and short RNA patterns accounted for 30.9% and 19.0% of tested specimens, respectively; all G2 strains had short electropherotype. Rotavirus gastroenteritis was detected year-round and, in 1998, the incidence rates tended to be higher during the second semester than in the first semester: 45.2% and 26.1% (p = 0.13), respectively. Rotavirus infections peaked at the second semester of life with frequencies of 30.1% and 13.5% for diarrhoeic children and controls, respectively. While the six rotavirus strains bearing G2-type specificity were circulating throughout the whole study period, G1 serotypes (n = 27) emerged as from June 1998 onwards, 20 (74.1%) of which clustering in 1998. These data underscore the importance of rotaviruses in the aetiology of severe infantile gastroenteritis in Northeastern Brazil and sustain the concept that a future vaccine should confer protection against more than one serotype.

Detection of Cryptosporidium sp. in non diarrheal faeces from children, in a day care center in the city of São Paulo, Brazil

The protozoan Cryptosporidium sp. has been frequently detected in faeces from children with persistent diarrhoea. This work achieved to investigate an outbreak of cryptosporidiosis, in a day care center, attending children of high socio-economic level, between 0 and six years old. The outbreak was detected through the network of public health, when stool samples, not diarrhoeic, were examined at the Parasitology Service of the Adolfo Lutz Institute. Among the 64 examined children, 13 (20.3%) showed oocysts of Cryptosporidium sp. in the faeces examined by Kinyoun technique: seven children one year old, three, two years old and three, three years old. Among the 23 examined adults, only a 22 years old woman, possibly having an immunocomprometiment, was positive. Clinical and epidemiological aspects were investigated by questionnaires, highlighting the occurrence of the outbreak in a very dry period.

Candiduria in a public hospital of São Paulo (1999-2004): characteristics of the yeast isolates

The study involved 100 yeast isolates, obtained from urine samples provided by a Public Pediatric Hospital of São Paulo, Brazil, from 1999 to 2004. The most frequent species was Candida albicans, followed by C. tropicalis, C. glabrata and C. parapsilosis. In regard to virulence, 97% of the isolates showed index 3 for proteinase and 63% index 2 for phospholipase. The most frequent killer biotypes were 511 and 888.

Aids-related progressive multifocal leukoencephalopathy: a retrospective study in a referral center in São Paulo, Brazil

Few data are available about progressive multifocal leukoencephalopathy (PML) in patients with acquired immunodeficiency syndrome (AIDS) from Brazil. The objectives of this study were to describe the main features of patients with PML and estimate its frequency among AIDS patients with central nervous system (CNS) opportunistic diseases admitted to the Instituto de Infectologia Emílio Ribas, São Paulo, Brazil, from April 2003 to April 2004. A retrospective and descriptive study was performed. Twelve (6%) cases of PML were identified among 219 patients with neurological diseases. The median age of patients with PML was 36 years and nine (75%) were men. Nine (75%) patients were not on antiretroviral therapy at admission. The most common clinical manifestations were: focal weakness (75%), speech disturbances (58%), visual disturbances (42%), cognitive dysfunction (42%), and impaired coordination (42%). The median CD4+ T-cell count was 45 cells/µL. Eight (67%) of 12 patients were laboratory-confirmed with PML and four (33%) were possible cases. Eleven (92%) presented classic PML and only one case had immune reconstitution inflammatory syndrome (IRIS)-related PML. In four (33%) patients, PML was the first AIDS-defining illness. During hospitalization, three patients (25%) died as a result of nosocomial pneumonia and nine (75%) were discharged to home. Cases of PML were only exceeded by cases of cerebral toxoplasmosis, cryptococcal meningoencephalitis, and CNS tuberculosis, the three more frequent neurologic opportunistic infections in Brazil. The results of this study suggest that PML is not an uncommon HIV-related neurologic disorder in a referral center in Brazil.

Immunization status of residents in pediatrics at the Federal University of São Paulo, Brazil

Vaccination of health care workers is an efficient way to reduce the risk of occupational infection and to prevent nosocomial transmission to vulnerable patients. Despite this, achieving high immunization rates among those professionals is a challenge. We assessed the immunization status of Residents in Pediatrics at the Federal University of São Paulo from June to December 2008. Their immunization records were checked and evaluated according to the Brazilian Immunization Schedule for health care workers. Considering all required vaccines, only 3.1% of the 64 Residents were up-to-date with their immunizations. Influenza was the vaccine with the lowest uptake (3.1%) and measles and rubella were diseases with the highest evidence of immunity (62.5% each). Only 37.5% of Residents had received three hepatitis B vaccine doses with a subsequent serology confirming seroconversion. Moreover, the vast majority of Residents in Pediatrics who were not up-to-date were unaware of the fact. Both medical schools and Pediatric Residence programs should not only offer information but also check vaccination records in an effort to keep their healthcare workers´ vaccinations up-to-date.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

SUMMARYIntroduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals.Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used.Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91.Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

POLYCLONAL OUTBREAK OF BLOODSTREAM INFECTIONS CAUSED BY Burkholderia cepacia COMPLEX IN HEMATOLOGY AND BONE MARROW TRANSPLANT OUTPATIENT UNITS

Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients.Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units.Findings: 14 patients had B. multivorans(one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred.Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.