EXPANSION OF VISCERAL LEISHMANIASIS IN THE STATE OF RIO DE JANEIRO, BRAZIL: REPORT OF THE FIRST AUTOCHTHONOUS CASE IN THE MUNICIPALITY OF VOLTA REDONDA AND THE DIFFICULTY OF DIAGNOSIS

Visceral Leishmaniasis has been showing remarkable epidemiological changes in recent decades, with marked expansion and an emergence of cases in urban areas of the North, Southeast and Midwest regions of Brazil. The Kala-azar cases reported here, despite being very characteristic, presented a great difficulty of diagnosis, because the disease is not endemic in Volta Redonda. The child underwent two hospitalizations in different hospitals, but got the correct diagnosis only after 11 months of symptom onset. In this report we discuss the main differential diagnoses and call attention to the suspected symptoms of visceral leishmaniasis in patients with prolonged fever, hepatosplenomegaly and pancytopenia, even in areas not traditionally endemic for the disease.

DIFFERENTIATION BETWEEN Nocardia spp. AND Mycobacterium spp.: CRITICAL ASPECTS FOR BACTERIOLOGICAL DIAGNOSIS

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

SEROLOGICAL AND MOLECULAR SURVEY OF Leptospira spp. AMONG CART HORSES FROM AN ENDEMIC AREA OF HUMAN LEPTOSPIROSIS IN CURITIBA, SOUTHERN BRAZIL

Introduction: Cart horses are a re-emerging population employed to carry recyclable material in cities. Methods: Sixty-two horses were sampled in an endemic area of human leptospirosis. The microscopic agglutination test (MAT) and real-time polymerase chain reaction (qPCR) were performed. Results: A seropositivity of 75.8% with serovar Icterohaemorrhagiae in 80.8% of the horses was observed. Blood and urine were qPCR negative. MAT showed positive correlations with rainfall (p = 0.02) and flooding (p = 0.03). Conclusions: Although horses may be constantly exposed to Leptospira spp. in the environment mostly because of rainfall and flooding, no leptospiremia or leptospiruria were observed in this study.

SEROLOGICAL MARKERS OF VIRAL, SYPHILITIC AND TOXOPLASMIC INFECTION IN CHILDREN AND TEENAGERS WITH NEPHROTIC SYNDROME: CASE SERIES FROM MATO GROSSO STATE, BRAZIL

Some infections can be the cause of secondary nephrotic syndrome. The aim of this study was to describe the experience of a Renal Disease Reference Clinic from Central Brazil, in which serological markers of some infectious agents are systematically screened in children with nephrotic syndrome. Data were obtained from the assessment of medical files of all children under fifteen years of age, who matched nephrotic syndrome criteria. Subjects were tested for IgG and IgM antibodies against T. gondii and cytomegalovirus; antibodies against Herpes simplex, hepatitis C virus and HIV; and surface antigen (HBsAg) of hepatitis B virus. The VDRL test was also performed. 169 cases were studied. The median age on the first visit was 44 months and 103 (60.9%) patients were male. Anti-CMV IgG and IgM were found in 70.4% and 4.1%, respectively. IgG and IgM against Toxoplasma gondii were present in 32.5% and 5.3%, respectively. Two patients were positive for HBsAg, but none showed markers for HIV, hepatitis C, or Treponema pallidum. IgG and IgM against herpes simplex virus were performed on 54 patients, of which 48.1% and 22.2% were positive. IgM antibodies in some children with clinical signs of recent infection suggest that these diseases may play a role in the genesis of nephrotic syndrome.

DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca) IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

 This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

CANINE VISCERAL LEISHMANIASIS CASE INVESTIGATION IN THE JACARE REGION OF NITEROI, RIO DE JANEIRO, BRAZIL

SUMMARY American visceral leishmaniasis is a vector-borne zoonosis in expansion in Brazil. Dogs are the main urban reservoir. Departing from a case of canine visceral leishmaniasis (CVL) in Jacaré, Niterói, Rio de Janeiro State, an epidemiological canine and entomological study was performed to assess the extension of the disease at the location. Sample was collected around the case and the dogs identified by serological tests (rapid double platform immunochromatographic exams, immunoenzymatic assay/ELISA, indirect immunofluorescence/IFAT). The parasitological diagnosis was performed in animals positive in at least one of these tests. The entomological study was carried out by using light traps and manual collection. The associations between canine variables and outcome (ELISA and IFAT reagents) were assessed by the chi-square test and adjusted by multivariate logistic regression for those associations with p < 0.1 in the bivariate analysis. Seventeen cases of CVL were detected among 110 evaluated dogs (prevalence of 15.5%). Presence of ectoparasites (OR 6.5; 95% CI 1.1-37.4), animals with clinical signs (OR 9.5; 95% CI 1.2-76.6), and previous cases of CVL in the same house (OR 17.9; 95% CI 2.2-147.1) were associated with the outcome. Lutzomyia longipalpiswas not detected. Our results are indicative of an ongoing transmission in the area.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

COMPARISON OF PERMANENT STAINING METHODS FOR THE LABORATORY DIAGNOSIS OF TRICHOMONIASIS

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. The diagnosis is based on wet mount preparation and direct microscopy on fixed and stained clinical specimens. The aim of this study was to compare the performance of different fixing and staining techniques used in the detection of T. vaginalis in urine. The smears were fixed and submitted to different methods of permanent staining and then, the morphological aspects of the parasites were analyzed and compared. The Papanicolaou staining with ethanol as the fixative solution showed to be the best method of permanent staining. Our data suggest that staining techniques in association with wet mount examination of fresh specimens contribute to increase the sensitivity in the diagnosis of trichomoniasis.

SEROLOGICAL DETECTION OF HEPATITIS A VIRUS IN FREE-RANGING NEOTROPICAL PRIMATES (Sapajus spp., Alouatta caraya) FROM THE PARANÁ RIVER BASIN, BRAZIL

Nonhuman primates are considered as the natural hosts of Hepatitis A virus (HAV), as well as other pathogens, and can serve as natural sentinels to investigate epizootics and endemic diseases that are of public health importance. During this study, blood samples were collected from 112 Neotropical primates (NTPs) (Sapajus nigritus and S. cay, n = 75; Alouatta caraya, n = 37) trap-captured at the Paraná River basin, Brazil, located between the States of Paraná and Mato Grosso do Sul. Anti-HAV IgG antibodies were detected in 4.5% (5/112) of NTPs, specifically in 6.7% (5/75) of Sapajus spp. and 0% (0/37) of A. caraya. In addition, all samples were negative for the presence of IgM anti-HAV antibodies. These results suggest that free-ranging NTPs were exposed to HAV within the geographical regions evaluated.

LIVER BIOPSY: IMPORTANCE OF SPECIMEN SIZE IN THE DIAGNOSIS AND STAGING OF CHRONIC VIRAL HEPATITIS

Liver biopsy is the gold standard method for the grading and staging of chronic viral hepatitis, but optimal biopsy specimen size remains controversial. The aim of this study was to evaluate the quality of liver specimen (number of portal tracts) and to evaluate the impact of the number of portal tracts in the staging of chronic hepatitis. Material and Methods: 468 liver biopsies from consecutive patients with hepatitis C virus and hepatitis B virus infection from 2009 to 2010 were evaluated. Results: The length of fragment was less than 10 mm in 43 cases (9.3%), between 10 and 14 mm in 114 (24.3%), and ≥ 15 mm in 311 (64.4%); of these, in 39 (8.3%) cases were ≥ 20 mm. The mean representation of portal tracts was 17.6 ± 2.1 (5-40); in specimens ≥ 15 mm the mean portal tract was 13.5 ± 4.7 and in cases ≤ 15 mm was 11.4 ± 5.0 (p = 0.002). Cases with less than 11 portal tracts were associated with F3, and cases with 11 or more portal tracts with F2 (p = 0.001). Conclusion: this study demonstrated the good quality of liver biopsy and a relationship between the macroscopic size of the fragment and the number of portal tracts.

MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND

The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

OCULAR SYPHILIS IN A KIDNEY TRANSPLANT RECIPIENT

We present a case of ocular syphilis after a renal transplantation involving progressive vision loss without clinically identifiable ocular disease. Electroretinography showed signs of ischemia, especially in the internal retina. A serological test was positive for syphilis. Lumbar puncture revealed lymphocytic meningitis and a positive serologic test for syphilis in the cerebrospinal fluid. The patient was treated with penicillin, and had a quick vision improvement. In the case of transplant recipients, clinicians should always consider the diagnosis of ocular syphilis in cases with unexplained visual acuity decrement, as this condition may cause serious complications if not treated.

A parasitological and serological malária survey in Amazonas, 1971

Thick blood films and malaria indirect fluorescent antibody test (P. falciparum and P. vivax) were done in four different regions in Amazonas. There was a very low prevalence of parasites anã the antibody rates suggest a small amount of transmission and that P. vivax was the predominant parasite. The calculation of probability of being infected per year was about 8% in Tefé. Coari, Colonia Fernanão Costa and Labrea and 0.8% in Anori.