A ten-year follow-up of human leptospirosis in Uruguay: an unresolved health problem

Leptospira spp. are delicate bacteria that cannot be studied by usual microbiological methods. They cause leptospirosis, a zoonotic disease transmitted to humans through infected urine of wild or domestic animals. We studied the incidence of this disease in the Uruguayan population, its epidemiologic and clinical features, and compared diagnostic techniques. After examining 6,778 suspect cases, we estimated that about 15 infections/100,000 inhabitants occurred yearly, affecting mainly young male rural workers. Awareness about leptospirosis has grown among health professionals, and its lethality has consequently decreased. Bovine infections were probably the principal source of human disease. Rainfall volumes and floods were major factors of varying incidence. Most patients had fever, asthenia, myalgias or cephalalgia, with at least one additional abnormal clinical feature. 30-40% of confirmed cases presented abdominal signs and symptoms, conjunctival suffusion and altered renal or urinary function. Jaundice was more frequent in patients aged > 40 years. Clinical infections followed an acute pattern and their usual outcome was complete recovery. Laboratory diagnosis was based on indirect micro-agglutination standard technique (MAT). Second serum samples were difficult to obtain, often impairing completion of diagnosis. Immunofluorescence was useful as a screening test and for early detection of probable infections.

PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10 -3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS

Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.

Detection of HTLV-IIa in blood donors in an urban area of the Amazon Region of Brazil (Belém, PA)

The human lymphotropic viruses type I (HTLV-I) and type II (HTLV-II) are members of a group of mammalian retroviruses with similar biological properties, and blood transfusion is an important route of transmission. HTLV-I is endemic in a number of different geographical areas and is associated with several clinical disorders. HTLV-II is endemic in several Indian groups of the Americas and intravenous drug abusers in North and South America, Europe and Southeast Asia. During the year of 1995, all blood donors tested positive to HTLV-I/II in the State Blood Bank (HEMOPA), were directed to a physician and to the Virus Laboratory at the Universidade Federal do Pará for counselling and laboratory diagnosis confirmation. Thirty-five sera were tested by an enzyme immune assay, and a Western blot that discriminates HTLV-I and HTLV-II infection. Two HTLV-II positive samples were submitted to PCR analysis of pX and env genomic region, and confirmed to be of subtype IIa. This is the first detection in Belém of the presence of HTLV-IIa infection among blood donors. This result emphasizes that HTLV-II is also present in urban areas of the Amazon region of Brazil and highlights the need to include screening tests that are capable to detect antibodies for both types of HTLV.

Molecular detection of Treponema pallidum sp. pallidum in blood samples of VDRL-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern Brazil

INTRODUCTION: Although control measures of maternal and congenital syphilis are available in Brazil, difficulties exist within the healthcare network in providing a laboratory diagnosis of the infection during the prenatal period. The objective of this study was to confirm the presence of Treponema pallidum by PCR in women with positive VDRL serology and lethal pregnancy outcomes, i.e., abortion, stillbirth and neonatal death. METHODS: A retrospective study was conducted on VDRLseroreactive women with lethal pregnancy outcomes admitted to the Fundação Santa Casa de Misericórdia do Pará (FSCM-PA) between January and July 2004. Serum samples and DNA from whole blood were obtained at the time of screening by the VDRL test. These samples were analyzed by IgG ELISA, IgM FTA-Abs and simple PCR (polA). RESULTS: During the study period, 0.7% (36/4,912) of women with lethal pregnancy outcomes presented a positive VDRL test. The polAgene was amplified in 72.7% (24/33) of these women, with 55.6% (20/36) and 94.4% (34/36) presenting IgM and IgG antibodies against T. pallidum, respectively. Comparison of these results showed a significant difference, with agreement between the PCR and IgM FTA-Abs results, suggesting that maternal syphilis was an active infection. No basic cause of death of the conceptus was reported in 97.2% (35/36) of cases. Among women who were submitted to the VDRL test during the prenatal period, only four of the nine seroreactive patients underwent treatment. CONCLUSIONS: The high frequency of syphilis in the group studied indicates the fragility of the service of infection diagnosis, treatment and monitoring, compromising epidemiological control.

Interpretation of the presence of IgM and IgG antibodies in a rapid test for dengue: analysis of dengue antibody prevalence in Fortaleza City in the 20th year of the epidemic

INTRODUCTION: The diagnosis of dengue and the differentiation between primary and secondary infections are important for monitoring the spread of the epidemic and identifying the risk of severe forms of the disease. The detection of immunoglobulin (Ig)M and IgG antibodies is the main technique for the laboratory diagnosis of dengue. The present study assessed the application of a rapid test for dengue concerning detection of new cases, reinfection recognition, and estimation of the epidemic attack rate. METHODS: This was a retrospective, cross-sectional, descriptive study on dengue using the Fortaleza Health Municipal Department database. The results from 1,530 tested samples, from 2005-2006, were compared with data from epidemiological studies of dengue outbreaks in 1996, 2003, and 2010. RESULTS: The rapid test confirmed 52% recent infections in the tested patients with clinical suspicion of dengue: 40% detected using IgM and 12% of new cases using IgG in the non-reactive IgM results. The positive IgM plus negative IgG (IgM+ plus IgG-) results showed that 38% of those patients had a recent primary dengue infection, while the positive IgG plus either positive or negative IgM (IgG+ plus IgM+/-) results indicated that 62% had dengue for at least a second time (recent secondary infections). This proportion of reinfections permitted us to estimate the attack rate as >62% of the population sample. CONCLUSIONS: The rapid test for dengue has enhanced our ability to detect new infections and to characterize them into primary and secondary infections, permitting the estimation of the minimal attack rate for a population during an outbreak.

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

Frequency of amoebiasis and other intestinal parasitoses in a settlement in Ilhéus City, State of Bahia, Brazil

Introduction: This study evaluated the frequency of intestinal parasites, emphasizing the identification and differentiation of Entamoeba spp. Methods: Multiplex polymerase chain reaction (PCR), coproantigen tests and morphometric analysis were performed for Entamoeba spp. differentiation. Results: The overall frequency of intestinal parasites was 65%. Entamoeba histolytica was detected by the coproantigen test, and the PCR showed that Entamoeba dispar predominated in the population. In contrast, morphometric analysis was important for identifying Entamoeba hartmanni. Conclusions: It is possible to identify the causative agent of amoebiasis and to differentiate this agent from other species by combining techniques.

Toxoplasmosis: an examination of knowledge among health professionals and pregnant women in a municipality of the State of Paraná

Introduction The aim of this study was to investigate the knowledge of toxoplasmosis among professionals and pregnant women in the public health services in Paraná, Brazil. Methods A cross-sectional observational and transversal study of 80 health professionals (44 nurses and 36 physicians) and 330 pregnant women [111 immunoglobulin M (IgM)- and IgG-non-reactive and 219 IgG-reactive] was conducted in 2010. An epidemiological data questionnaire was administered to the professionals and to the pregnant women, and a questionnaire about the clinical aspects and laboratory diagnosis of toxoplasmosis was administered to the professionals. Results The participants frequently provided correct responses about prophylactic measures. Regarding the clinical and laboratory aspects, the physicians provided more correct responses and discussed toxoplasmosis with the pregnant women. The professionals had difficulty interpreting the avidity test results, and the physicians stated that they referred pregnant women with high-risk pregnancies to a county reference center. Of the professionals, 53 (91.4%) reported that they instructed women during prenatal care, but only 54 (48.6%) at-risk pregnant women and 99 (45.2%) women who were not at risk reported receiving information about preventive measures. The physicians provided verbal instructions to 120 (78.4%) women, although instructional materials were available in the county. The pregnant women generally lacked knowledge about preventive measures for congenital toxoplasmosis, but the at-risk pregnant women tended to respond correctly. Conclusions This study provides data to direct public health policies regarding the importance of updating the knowledge of primary care professionals. Mechanisms should be developed to increase public knowledge because prophylactic strategies are important for preventing congenital toxoplasmosis.

Percutaneous closure of ductus arteriosus with the amplatzer prosthesis. The Brazilian experience

OBJECTIVE - To report the results of percutaneous occlusion of persistent ductus arteriosus with the Amplatzer prosthesis in 2 Brazilian cardiological centers. METHODS - From May 1998 to July 2000, 33 patients with clinical and laboratory diagnosis of persistent ductus arteriosus underwent attempts at percutaneous implantation of the Amplatzer prosthesis. The median age was 36 months (from 6 months to 38 years), and the median weight was 14kg (from 6 to 92kg). Sixteen patients (48.5%) were under 2 years of age at the time of the procedure. All patients were followed up with periodical clinical and echocardiographic evaluations to assess the presence and degree of residual shunt and possible complications, such as pseudocoarctation of the aorta and left pulmonary artery stenosis. RESULTS - The minimum diameter of the arterial ducts ranged from 2.5 to 7.0mm (mean of 4.0±1.0, median of 3.9). The rate of success for implantation of the prosthesis was 100%. Femoral pulse was lost in 1 patient. The echocardiogram revealed total closure prior to hospital discharge in 30 patients, and in the follow-up visit 3 months later in the 3 remaining patients. The mean follow-up duration was 6.4±3.4 months. All patients were clinically well, asymptomatic, and did not need medication. No patient had narrowing of the left pulmonary artery or of the aorta. No early or late embolic events occurred, nor did infectious endarteritis. A new hospital admission was not required for any patient. CONCLUSION - The Amplatzer prosthesis for persistent ductus arteriosus is safe and highly effective for occlusion of ductus arteriosus of varied diameters, including large ones in small symptomatic infants.

Virological study of a dengue type 1 epidemic at Rio de Janeiro

A dengue outbreak started in March, 1986 in Rio de Janeiro and spread very rapidly to other parts of the country. The great majority of cases presented classical dengue fever but there was one fatal case, confirmed by virus isolation. Dengue type 1 strains were isolated from patients and vectors (Aedes aegypti) in the area by cultivation in A. albopictus C6/36 cell line. The cytopathic effect (CPE) was studied by electron microscopy. An IgM capture test (MAC-ELISA) was applied with clear and reproducible results for diagnosis and evaluation of virus circulation; IgM antibodies appeared soon after start of clinical disease, and persisted for about 90 days in most patients. The test was type-specific in about 50% of the patients but high levels of heterologous response for type 3 were observed. An overall isolation rate of 46,8% (813 virus strains out of 1734 specimens) was recorded. The IgM test increased the number of confirmed cases to 58,2% (1479 out of 2451 suspected cases). The importance of laboratory diagnosis in all regions where the vectors are present is emphasized.

Morphometric Analysis of McCoy Cells Inoculated with Cerebrospinal Fluid from Patients with Rabies

To demonstrate the potential of McCoy cells for the isolation of rabies virus from the cerebrospinal (CSF) fluid of a patient with a diagnosis of rabies, McCoy cells were inoculated with CSF from a patient with a clinical diagnosis of rabies and investigated in terms of morphometric aspect using the JAVA analysis system for the quantification of the increased size of infected cells compared to noninfected cells. The cells were also examined in terms of specific staining for the diagnosis of rabies by the method of Sellers for the observation of intracytoplasmic inclusions and by specific immunofluorescence staining for rabies virus. Infected cells showed changes in cell permeability and morphologic modifications which differed significantly compared to normal cells (P<0.001) when analyzed by the Mann-Whitney and Kruskal-Wallis tests. Intense activity of the endoplasmic reticulum was also observed, as indicated by the presence of intracytoplasmic inclusions visualized by specific staining. The present study demonstrated the isolation of rabies virus from the CSF of a patient with rabies, showing that McCoy cells can be used for the laboratory diagnosis of patients suspected to have rabies.

Dengue situation in Brazil by year 2000

Dengue virus types 1 and 2 have been isolated in Brazil by the Department of Virology, Instituto Oswaldo Cruz, in 1986 and 1990 respectively, after many decades of absence. A successful continental Aedes aegypti control program in the Americas, has been able to eradicate the vector in most countries in the 60's, but the program could not be sustained along the years. Dengue viruses were reintroduced in the American region and the infection became endemic in Brazil, like in most Central and SouthAmerican countries and in the Caribbean region, due to the weaning of the vector control programs in these countries. High demographic densities and poor housing conditions in large urban communities, made the ideal conditions for vector spreading. All four dengue types are circulating in the continent and there is a high risk of the introduction in the country of the other two dengue types in Brazil, with the development of large epidemics. After the Cuban episode in 1981, when by the first time a large epidemic of dengue hemorrhagic fever and dengue shock syndrome have been described in the Americas, both clinical presentations are observed, specially in the countries like Brazil, with circulation of more than one dengue virus type. A tetravalent potent vaccine seems to be the only possible way to control the disease in the future, besides rapid clinical and laboratory diagnosis, in order to offer supportive treatment to the more severe clinical infections.

Diphtheria remains a threat to health in the developing world: an overview

Changes in the epidemiology of diphtheria are occurring worldwide. A large proportion of adults in many industrialized and developing countries are now susceptible to diphtheria. Vaccine-induced immunity wanes over time unless periodic booster is given or exposure to toxigenic Corynebacterium diphtheriae occurs. Immunity gap in adults coupled with large numbers of susceptible children creates the potential for new extensive epidemics. Epidemic emergencies may not be long in coming in countries experiencing rapid industrialization or undergoing sociopolitical instability where many of the factors thought to be important in producing epidemic such as mass population movements and difficult hygienic and economic conditions are present. The continuous circulation of toxigenic C. diphtheriae emphasizes the need to be aware of epidemiological features, clinical signs, and symptoms of diphtheria in vaccine era so that cases can be promptly diagnosed and treated, and further public health measures can be taken to contain this serious disease. This overview focused on worldwide data obtained from diphtheria with particular emphasis to main factors leading to recent epidemics, new clinical forms of C. diphtheriae infections, expression of virulence factors, other than toxin production, control strategies, and laboratory diagnosis procedures.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.