St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

Difficulties in the diagnosis of HTLV-2 infection in HIV/AIDS patients from Brazil: comparative performances of serologic and molecular assays, and detection of HTLV-2b subtype

The current diagnosis of human T-lymphotropic virus type-2 (HTLV-2) infection is based on the search of specific antibodies; nevertheless, several studies conducted in Brazil pointed deficiencies of the commercially available kits in detecting HTLV-2, mostly in HIV/AIDS patients. This study searched for the presence of HTLV-1 and -2 in 758 HIV/AIDS patients from Londrina, Paraná, Brazil. Serum samples were screened for HTLV-1/2 antibodies using two EIA kits (Vironostika and Murex), and confirmed by WB (HTLV Blot 2.4, Genelabs). The results obtained by EIA disclosed 49 (6.5%) reactive sera: 43 positive by both EIA kits, and six with discordant results. WB confirmed HTLV-1 infection in seven samples (0.9%) and HTLV-2 in 21 sera (2.8%). Negative and indeterminate results were detected in four (0.5%) and 16 (2.1%) sera, respectively. Blood from 47 out of 49 HTLV seroreactive patients were collected and analyzed for the presence of env, LTR and tax genomic segments of HTLVs by PCR. PCR confirmed six cases of HTLV-1 and 37 cases of HTLV-2 infection (14 out of 16 that were found to be WB indeterminate). Restriction analysis of the env PCR products of HTLV-2 disclosed 36 isolates of HTLV-2a/c subtype, and one of HTLV-2b subtype. These results emphasize the need of improving serologic tests for detecting truly HTLV-2 infected patients from Brazil, and confirm the presence of HTLV-2b subtype in the South of this country.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

Circulation of human papillomavirus (HPV) genotypes in women from Córdoba, Argentina, with squamous intraepithelial lesions

Human papillomavirus (HPV) can induce a wide spectrum of squamous intraepithelial lesions (SIL) of varying severity. The aim of the present study was to establish the frequency of HPV infection and identify the genotypes circulating in women from Córdoba, Argentina, in relation to age and cytology. A total of 186 women, between 18 and 65 years old, with antecedents of SIL, underwent a pelvic examination and had cervical cells collected for cytology and HPV DNA detection. Ninety-six samples (51.6%) were positive for HPV detection, and sixty-three (65.6%) of them showed the presence of at least one HR-HPV. Low- and high-grade SIL showed significant association in patients younger than 35 years of age. We found 18 different genotypes, with a greater presence of HR-HPV. Genotypes 16 and 6 were the most frequent. Seven (7.3%) multiple infections, 85.7% of which had at least one HR-HPV, were detected. The detection of a large number of different HPV genotypes is a warning sign. It is thus necessary to strengthen the monitoring of the circulation of high-risk genotypes, currently less prevalent in intraepithelial lesions, as a control measure for the possible impact of the implementation of vaccines against genotypes 16 and 18.

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.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Yellow fever epizootics in non-human primates, São Paulo state, Brazil, 2008-2009

Since 2000, the expansion of Sylvatic Yellow Fever (YF) has been observed in the southeast of Brazil, being detected in areas considered silent for decades. Epizootics in non-human primates (NHPs) are considered sentinel events for the detection of human cases. It is important to report epizootic events that could have impact on the conservation status of susceptible species. We describe the epizootics in NHPs, notified in state of São Paulo, Brazil, between September 2008 to August 2009. Ninety-one epizootic events, involving 147 animals, were reported in 36 counties. Samples were obtained from 65 animals (44.2%). Most of the epizootics (46.6%) were reported between March and April, the same period during which human cases of YF occurred in the state. Biological samples were collected from animals found dead and were sent to Instituto Adolfo Lutz, in São Paulo. Two samples, collected in two counties without an indication for YF vaccination, were positive for the virus. Another 48 animals were associated with YF by clinical-epidemiological linkage with laboratory confirmed cases. Because the disease in human and NHPs occurred in the same period, the detection of the virus in NHPs did not work as sentinel, but aided in the delineation of new areas of risk.

LACK OF ASSOCIATION BETWEEN HERPESVIRUS DETECTION IN SALIVA AND GINGIVITIS IN HIV‑INFECTED CHILDREN

The aims of this study were to compare the detection of human herpesviruses (HHVs) in the saliva of HIV-infected and healthy control children, and to evaluate associations between viral infection and gingivitis and immunodeficiency. Saliva samples were collected from 48 HIV-infected and 48 healthy control children. Clinical and laboratory data were collected during dental visits and from medical records. A trained dentist determined gingival indices and extension of gingivitis. Saliva samples were tested for herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) by nested polymerase chain reaction assays. Thirty-five HIV-infected and 16 control children had gingivitis. Seventeen (35.4%) HIV-infected children and 13 (27%) control children were positive for HHVs. CMV was the most commonly detected HHV in both groups (HIV-infected, 25%; control, 12.5%), followed by HSV-1 (6.2% in both groups) and HSV-2 (HIV-infected, 4.2%; control, 8.3%). The presence of HHVs in saliva was not associated with the presence of gingivitis in HIV-1-infected children (p = 0.104) or healthy control children (p = 0.251), or with immunosuppression in HIV-infected individuals (p = 0.447). Gingivitis was correlated with HIV infection (p = 0.0001). These results suggest that asymptomatic salivary detection of HHVs is common in HIV-infected and healthy children, and that it is not associated with gingivitis.

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.

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Reemergence of yellow fever: detection of transmission in the State of São Paulo, Brazil, 2008

INTRODUCTION: Following yellow fever virus (YFV) isolation in monkeys from the São José do Rio Preto region and two fatal human autochthonous cases from the Ribeirão Preto region, State of São Paulo, Brazil, two expeditions for entomological research and eco-epidemiological evaluation were conducted. METHODS: A total of 577 samples from humans, 108 from monkeys and 3,049 mosquitoes were analyzed by one or more methods: virus isolation, ELISA-IgM, RT-PCR, histopathology and immunohistochemical. RESULTS: Of the 577 human samples, 531 were tested by ELISA-IgM, with 3 positives, and 235 were inoculated into mice and 199 in cell culture, resulting in one virus isolation. One sample was positive by histopathology and immunohistochemical. Using RT-PCR, 25 samples were processed with 4 positive reactions. A total of 108 specimens of monkeys were examined, 108 were inoculated into mice and 45 in cell culture. Four virus strains were isolated from Alouattacaraya. A total of 931 mosquitoes were captured in Sao Jose do Rio Preto and 2,118 in Ribeirão Preto and separated into batches. A single isolation of YFV was derived from a batch of 9 mosquitoes Psorophoraferox, collected in Urupês, Ribeirão Preto region. A serological survey was conducted with 128 samples from the municipalities of São Carlos, Rincão and Ribeirão Preto and 10 samples from contacts of patients from Ribeirão Preto. All samples were negative by ELISA-IgM for YFV. CONCLUSIONS: The results confirm the circulation of yellow fever, even though sporadic, in the Sao Paulo State and reinforce the importance of vaccination against yellow fever in areas considered at risk.

Detection of Dientamoeba fragilis in patients with HIV/AIDS by using a simplified iron hematoxylin technique

INTRODUCTION: Studies strongly indicate Dientamoeba fragilis as one of the causes of diarrhea in human immunodeficiency virus (HIV) patients. METHODS: The objective of the present study was to evaluate the prevalence of D. fragilis associated with the causes of diarrhea in 82 HIV/ AIDS patients hospitalized at the Instituto de Infectologia Emílio Ribas from September 2006 to November 2008. RESULTS: In total, 105 samples were collected from 82 patients. Unprotected sex was the most frequent cause of HIV infection (46.3%), followed by the use of injectable or non-injectable drugs (14.6%). Patients presented with viral loads of 49-750,000 copies/ mL (average: 73,849 ± 124,850 copies/mL) and CD4 counts ranging of 2-1,306 cells/mm³ (average: 159 ± 250 cells/mm³). On an average, the odds of obtaining a positive result by using the other techniques (Hoffman, Pons and Janer or Lutz; Ritchie) were 2.7 times higher than the chance of obtaining a positive result by using the simplified iron hematoxylin method. Significant differences were found between the methods (p = 0.003). CONCLUSIONS: The other techniques can detect a significantly greater amount of parasites than the simplified iron hematoxylin method, especially with respect to Isospora belli, Cryptosporidium sp., Schistosoma mansoni, and Strongyloides stercoralis, which were not detected using hematoxylin. Endolimax nana and D. fragilis were detected more frequently on using hematoxylin, and the only parasite not found by the other methods was D. fragilis.

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

INTRODUCTION: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. METHODS: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5′-3′-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. RESULTS: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95%I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. CONCLUSIONS: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA) assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP) presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62). In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively), whereas ELISA using egg antigens (ELISA-SEA) detected samples after 140 days (p=0.03). CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis

Visceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ, under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8 and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could be looked upon as an interesting option for the treatment of VL.