DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

Anopheles (Kerteszia) cruzii (DIPTERA: CULICIDAE) IN PERIDOMICILIARY AREA DURING ASYMPTOMATIC MALARIA TRANSMISSION IN THE ATLANTIC FOREST: MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES INDICATES HUMANS AS PRIMARY INTERMEDIATE HOSTS

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

IDENTIFICATION OF Leishmania infantum IN PUERTO IGUAZÚ, MISIONES, ARGENTINA

 The emergence of zoonotic visceral leishmaniasis (ZVL) in Latin America is a growing public health problem. The urbanization of ZVL has been observed in different countries around the world, and there are a growing number of reports drawing attention to the emergence of this infection in new locations, as well as its increase in previously established areas of endemicity. In the city of Posadas, Misiones province, Northeastern Argentina, the transmission of ZVL associated with canines and Lutzomyia longipalpis was first reported in 2006. In the city of Puerto Iguazú, also in Misiones province, the first human case of ZVL was reported in February 2014. From 209 surveyed dogs, 15 (7.17%) were identified as positive by serological and/or parasitological methods. Amplification was observed in 14 samples and in all cases the species implicated was Leishmania infantum. To the authors’ knowledge, this is the first molecular characterization of L. infantum from dogs in this area.

Leishmania mexicana in Proechimys iheringi denigratus Moojen (Rodentia, Echimyidae) in a region endemic for American cutaneous leishmaniasis

Three isolates of Leishmania were recovered from five of 27 specimens of the rodent Proechimys iheringi denigratus Moojen captured near Três Braços in the Atlantic Forest region of Bahia, Brazil. Two of these isolates were recovered from hamsters inoculated with a pooled triturate of liver, spleen and skin tissue from apparently healthy P. i. denigratus. The third isolate was recovered from a triturate of only skin tissue from another. Metastasis was observed in the inoculated hamsters, the parasites grew abundantly in artificial media and a typical suprapylarial pattern of infection in Lutzomyia longipalpis was produced indicating that the parasites belong to the Leishmania mexicana complex. All isolates reacted with Leishmania mexicana mexicana and Leishmania mexicana amazonensis monoclonal antibodies. The isoenzyme analysis differentiated these isolates from standard isolates of L. m. mexicana, L. m. amazonensis, L. m. aristedesi, L. m. pifanoi, L. m. garnhami and L. m. ssp.(Goiás-W. Barbosa). These isolates seem to be a subspecies of L. mexicana very closely related to L. m. amazonensis from which they differ by decreased electrophoretic mobility of GPI, PEP and ALAT. This is the first record of the isolation of a parasite of thegenus Leishmania in a rodent captured in the State of Bahia.

The FML (Fucose Mannose Ligand) of Leishmania donovani: a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovanii infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0 % in antibody response (p<0.001) and reduction of 85.5 % parasite liver burden (p<0.001) was detected in animals immunized with FML saponin, unrespectivety of the immunization route.

Tegumentary leishmaniasis in Northern Argentina: distribution of infection and disease, in three municipalities of Salta, 1990-1992

This work describes the epidemiological pattern of tegumentary leishmaniasis in an area north of Salta, Argentina. The prevalence and incidence were estimated by means of a cross-sectional study and two follow-up studies during two consecutive years. The Montenegro Skin Test (MST) was administered to 7336 subjects at baseline. The prevalence and incidence between 1990 and 1992 of infection (MST reactive) was 38‰ persons and 4.5‰ persons/year respectively. The prevalence and incidence of tegumentary leishmaniasis (presence of clinical signs) was 1.8‰ and 0.8‰ persons/year, respectively. A physical examination performed on 264 patients with MST reactive during three years revealed that 130 cases (49.2%) had some evident sign of infection (scar and/or lesion), with a clinical presentation compatible with leishmaniasis. Our study demonstrated that after the epidemic outbreak of 1985 the transmission in the study area returned to endemic levels in 1992, and also demonstrated the presence of the asymptomatic infection in the area.

Multiple lymphoid nodules in bone marrow biopsy in immunocompetent patient with cytomegalovirus infection: an immunohistochemical analysis

In Brazil, a high prevalence of cytomegalovirus (CMV) infection has been documented. In immunocompetent adults CMV infection is usually asymptomatic and therefore morphologic and immunophenotypic bone marrow changes have rarely been described. The authors report the case of a previously healthy patient who developed fever of undetermined origin. The diagnosis of acute CMV infection was based on serological testing. A computed tomographic scan showed mediastinal lymphadenopathy. A bone marrow biopsy revealed a hypercellular haematopoiesis with eosinophilia and large mixed T- and B-cell lymphoid aggregates. In spite of bcl-2 positivity, their reactive nature was demonstrated. Polymerase chain reaction (PCR) and immunohistochemistry were unable to detect CMV-DNA in paraffin-embedded bone marrow sections. Much like in other systemic disorders, the lymphoid nodules in this case seemed to be caused by immunological mechanisms, possibly due to cytokines released in response to the systemic infectious process.

Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin® and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.

Histological evaluation of the lesion induced by inoculation of Leishmania mexicana in the cheek pouch of the hamster

We have studied the role of the immune response in the morphology of the leishmaniotic granuloma induced in the cheek pouch of hamsters, an immunologically privileged site, after inoculation of 3 x 10(5) Leishmania mexicana. Animals were histologically and immunologically evaluated until 120 days after inoculation. Independent of the time of sacrifice, the animals were always non-reactors to the footpad test (FPT). At histology, the introduction of L. mexicana in the cheek pouch leads to an abscess that evolves to a granulomatous reaction rich in amastigote forms, and later it leads to resolution, even in the absence of immune response detectable by FPT. Our results demonstrate that the development of immune response is not preponderant for the control of infection induced by L. mexicana inoculated subcutaneously in the cheek pouch of the hamster. It also suggests that the macrophages present in the leishmaniotic granuloma are capable of eliminating this parasite, even in the absence of immune response evaluated by FPT.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30µg of each protein at 15-day intervals combined with 250µg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14% were demonstrated for each purified protein.

Ocular lesions in HTLV-1 infected patients from Salvador, State of Bahia: the city with the highest prevalence of this infection in Brazil

In order to determine the prevalence of ocular lesions in HTLV-1 infected patients in Salvador Bahia, a transversal study was conducted on 140 HTLV-1 infected patients (90 asymptomatic and 50 tropical spastic paraparesis/HTLV-1-associated myelopathy) between June 2004 and November 2005. The ophthalmological examination included visual acuity measurement, ocular motility, biomicroscopy of the anterior and posterior chambers, intraocular pressure and evaluation of lachrymal secretion. Observation verified 4 (2.8%) out of 140 patients with uveitis (two patients had intermediate uveitis and two had pan-uveitis) and 39 (36.4%) out of 107 patients with keratoconjunctivitis sicca. The prevalence of Keratoconjunctivitis sicca was significantly higher among the TSP/HAM patients (OR age adjusted=3.64; 95%CI 1.59-8.32). Uveitis and corneal opacities were also important findings, indicating the strong need for periodic ophthalmological follow-up in all HTLV-1 subjects.

Human papillomavirus genotypes in asymptomatic young women from public schools in Rio de Janeiro, Brazil

INTRODUCTION: The aim of this work was to survey HPV information from a random population of young women from Rio de Janeiro, Brazil. METHODS: This cross-sectional study included cervical samples from 241 female students. To determine human papillomavirus status, polymerase chain reaction amplification was performed. HPV typing was determined by restriction fragment length polymorphism analysis. Demographic data, life style, sexual and gynecological history were obtained through use of a structured questionnaire. RESULTS: The average age of the women was 19.6 years-old (SD=3.4 years). HPV prevalence was 27.4%. Nineteen different HPV genotypes were detected, including 13 high risk types. HPV 16 was the most prevalent type (6.2%), followed by 31 (4.1 %) and 66 (3.7%). Most of the oncogenic types belonged to the A9 species (28/48). The frequency of women infected by at least one oncogenic type was significantly higher than those only infected by low risk types (18.7% versus 7.5%). Cervical changes were detected in 12.5% of the sample and were significantly linked to infection with HPV types of the A9 species. Demographic variables, sexual initiation, or number of sexual partners were not associated with HPV prevalence, variety of HPV genotypes or oncogenic types. CONCLUSIONS: The relative frequency of HPV genotypes other than vaccine types in young females should be taken into account when evaluating vaccination strategies. Due to the high prevalence of HPV infection among the population studied, implementation of sex education in schools, promotion of condom use and an organized screening program to prevent cervical cancer must be encouraged for this age group.

Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania) amazonensis in the state of Mato Grosso do Sul, mid-western Brazil

INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae) and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania) amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus) as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3%) and Bi flaviscutellata (41.4%), present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania) amazonensis. CONCLUSIONS: Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.

Characterization of Leishmania infantum species in dogs from the urban area of Cuiabá, State of Mato Grosso, Brazil

INTRODUCTION: Visceral leishmaniasis presents urban behavior in some Brazilian cities, with domestic dogs as the main infection source. In Cuiabá, MT, canine visceral leishmaniasis was diagnosed and characterized as recommended by the Ministry of Health. METHODS: Biological samples from suspected canine carriers were analyzed by the isoenzyme electrophoresis technique. The 6PGDH enzyme and reference strain IOC/L0566 (MHOM/BR/1975/M2903) of Leishmania (Leishmania) infantum was used as one of the controls. RESULTS: Electrophoresis analysis revealed that the canine isolates belonged to the species L. (L.) infantum. CONCLUSIONS: The authors emphasize the importance of species characterization, particularly in areas of mixed infection like Cuiabá.

Slow clinical improvement after treatment initiation in Leishmania/HIV coinfected patients

INTRODUCTION: In Brazil there is a large area of overlap of visceral leishmaniasis (VL) and HIV infection, which favored a increased incidence of coinfection Leishmania/HIV. METHODS: This study evaluated 65 consecutive patients with VL and their clinical response to treatment in two health care settings in Belo Horizonte, Brazil. RESULTS: At baseline, the clinical picture was similar between both groups, although diarrhea and peripheral lymphadenomegaly were more frequent in HIV-infected subjects. HIV-positive patients had lower median blood lymphocyte counts (686/mm³ versus 948/mm³p = 0.004) and lower values of alanine aminotransferase (ALT) (48IU/L versus 75.6IU/L p = 0.016) than HIV-negative patients. HIV-positive status (hazard ratio = 0.423, p = 0.023) and anemia (HR = 0.205, p = 0.002) were independent negative predictors of complete clinical response following antileishmanial treatment initiation. CONCLUSIONS: This study reinforces that all patients with VL should be tested for HIV infection, regardless of their clinical picture. This practice would allow early recognition of coinfection with initiation of antiretroviral therapy and, possibly, reduction in treatment failure.