Malária no município de Humaitá, estado do Amazonas: XXI. Prevalência da deficiência de glicose-6-fosfato desidrogenase (G6PD) Em amostra da população e em doentes com malaria causada pelo Plasmodium falciparum

Os autores estudaram a prevalência da deficiência de glicose-6-fosfato desidrogenase (G6PD), pelo método de BREWER et alii, em 141 indivíduos da população do município de Humaitá, Estado do Amazonas. Destes, 128 eram amazônides, 67 dos quais nunca tiveram malária, enquanto que 61 tinham tido ou estavam tendo a doença; os 13 restantes que estavam com malária não eram arnazônides. Os resultados revelaram que 7 arnazônides (4,96%), eram deficientes. Destes, 5 eram do sexo feminino e 2 do masculino. Em todos os indivíduos do sexo feminino o teste foi positivo com comportamento do tipo heterozigoto. Dos indivíduos deficientes, 4 nunca tinham tido malária; dos outros 3, 2 apresentavam a reação de hemaglutinação positiva com título 1/16 e o terceiro estava tendo malária causada pelo Plasmodium falciparum pela primeira vez. Este doente apresentou forma benigna de malária evoluindo para cura clínica e parasitológica no 3.° dia de tratamento com a clindamicina. Nenhum dos 13 doentes não arnazônides apresentava deficiência de G6PD. Dessa forma, não houve diferença na prevalência da deficiência de G6PD em arnazônides que nunca tiveram malária e em arnazônides que tinham tido ou estavam tendo a doença. Portanto, os indivíduos com deficiência de G6PD estão sujeitos a infecções por Plasmodium falciparum na mesma proporção que os não deficientes. Por outro lado, o aumento da prevalência da deficiência de G6PD, na amostra estudada, poderia estar relacionado com a pressão seletiva exercida pela malária em população submetida à homozigose.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05) for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s.), respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent) binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01) for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01) before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1 female) were also studied. The G6PD and glutathione reductase were partially activated, the change being more intense in males. On the basis of race and of the laboratory characteristics observed, it is possible to suggest that the G6PD deficiency of these individuals is of the African type and that the female is heterozygous for this deficiency. Analysis of the results as a whole permitted us to conclude that the methods proposed here were efficient for evaluating the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The latter is dependent on the pentose pathway, which generates NADPH, and on riboflavin, a FAD precursor vitamin.

Ecological aspects of American cutaneous leishmaniasis: 6. Man-biting sandfly fauna of a patch of residual forest in the middle and north-eastern region of the São Paulo State, Brazil

This report shows the phlebotomine vectorial role in the endemic cutaneous leishmaniasis situated in the middle and north-eastern region of the São Paulo, Brazil. Analysis of information encloses a sandfly man-biting fauna of four different patches of residual forest. So, using the human-bait and Shannon trap for a period of one year we have caught 16,869 sandflies. The predominance of Lutzomyia intermedia (85,6%) was clear. Moreover, the low density of Lutzomyia whitmani and Lutzomyia pessoai and their epidemiological implications at present and in the past are discussed. Information about the daily activity of some species is given and we emphasize for the first time, the diurnal activity of Lutzomyia firmatoi in the São Paulo State.

Avaliação sorológica de imunização contra o sarampo com duas doses administradas aos 6 e 11 meses de idade: estudo prospectivo

A eficácia sorológica de um esquema de vacinação contra o sarampo empregando duas doses da vacina BIKEN CAM 70, sendo a primeira dose administrada aos 6 meses de idade e a segunda aos 11 meses de idade foi avaliada através de um estudo prospectivo. A amostra de sangue foi colhida entre 6 e 12 meses (média de 8,0 ± 1,7 meses) após a segunda dose da vacina, tendo-se empregado para pesquisa de anticorpos específicos a reação de imunofluorescência indireta (RIFI) e a técnica imunoenzimática ELISA. Anticorpos para o sarampo na amostra de sangue pós-vacinal foram detectados em 88,5% (85/96) das crianças quando foi empregada a RIFI e em 96,8% (93/96) quando se empregou a técnica imunoenzimática ELISA. Nenhuma das crianças apresentou, durante o período do estudo, quadro clínico compatível com sarampo. Em regiões em que uma proporção significativa de casos ocorrem antes dos 9 meses de idade, o esquema de vacinação de 2 doses, a primeira aos 6 e a segunda aos 11 meses de idade, pode representar alternativa válida para o controle do sarampo.

Estudio comparativo de la patogenicidad y la antigenicidad de 6 cepas de Paracoccidioides brasiliensis

Fueron estudiadas en forma comparativa 6 cepas de P. brasiliensis con el propósito de determinar su patogenicidad para la rata y su antigenicidad. Las mismas fueron aisladas de : 1) biopsia de cuello uterino en 1989 (U), 2) biopsia de mucosa bucal en 1988 (V), 3) aspiración ósea en 1991 (63265), 4) testículo de cobayo 1984(C24), 5) punción-aspiración ganglionaren 1986 (G) y 6) cepa proveniente de la Escola Paulista de Medicina (339). Se prepararon antigenos citoplasmáticos liofilizados de cada una de ellas, en la concentración final de 100 mg/ml y se realizaron pruebas de inmunodifusión frente a 6 sueros patrones positivos de ratas. En este ensayo todos los antígenos presentaron dos ó tres bandas de precipitación. Para estudiar el poder patógeno se inocularon, en total, 120 ratas Wistar, de ambos sexos de 200 g de peso, por via intracardíaca con suspensiones de la fase levaduriforme del P. brasiliensis, en concentraciones de 3x10(7) y 5x10(7) células/ml de cada cepa. Los animales que no murieron espontáneamente fueron sacrificados a los 14,28,42, 56 y 70 dias post-infección y se evaluaron los siguientes parámetros: A) exámenes macro y microscópicos de pulmones, hígado, bazo y riñones; B) cultivos de un pulmón y C) prueba de inmunodifusión con antígeno homólogo. Se consideró además, el porcentaje de muertes espontáneas por cada cepa. Los resultados de estos estudios fueron los siguientes:No se observó relación entre la patogenicidad y la antigenicidad. La cepa más virulenta correspondió a un aislamiento reciente a partir de una forma juvenil grave y la más antigénica fue una cepa, morfológicamente atípica, que no provocó lesiones macroscópicas ni microscópicas en los órganos de las ratas.

Association of human herpesvirus 6 infection with exanthem subitum in Belem, Brazil

Recent human herpesvirus 6 (HHV-6) infection was detected in cases of exanthem subitum (ES) involving four children, aged 10 to 24 months, between April and August 1994, in Belém, Brazil. By using the indirect immunofluorescence antibody assay (IFA), significant increases (at least eight times) in antibody concentrations were noted from the acute to the convalescent serum samples, with titers ranging from <1:10/1:80 to <1:10/1:640 (patients 3 and 2, respectively). All children had high fever (over 39ºC) for three days, followed by generalized, maculo-papular skin rash. A physical examination of the children also revealed concomitant, cervical lymph node swelling and tonsillar pharyngitis in two of them.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Outbreaks of human-herpes virus 6 (HHV-6) infection in day-care centers in Belém, Pará, Brazil

A total of 730 children aged less than 7 years, attending 8 day-care centers (DCCs) in Belém, Brazil were followed-up from January to December 1997 to investigate the occurrence of human-herpes virus 6 (HHV-6) infection in these institutional settings. Between October and December 1997 there have been outbreaks of a febrile- and -exanthematous disease, affecting at least 15-20% of children in each of the DCCs. Both serum- and- plasma samples were obtained from 401 (55%) of the 730 participating children for the detection of HHV-6 antibodies by enzyme-linked immunosorbent assay (ELISA), and viral DNA amplification through the nested-PCR. Recent HHV-6 infection was diagnosed in 63.8% (256/401) of them, as defined by the presence of both IgM and IgG-specific antibodies (IgM+/IgG+); of these, 114 (44.5%) were symptomatic and 142 (55.5%) had no symptoms (p = 0.03). A subgroup of 123 (30.7%) children were found to be IgM-/IgG+, whereas the remaining 22 (5.5%) children had neither IgM nor IgG HHV-6- antibodies (IgM-/IgG-). Of the 118 children reacting strongly IgM-positive ( > or = 30 PANBIO units), 26 (22.0%) were found to harbour the HHV-6 DNA, as demonstrated by nested-PCR. Taken the ELISA-IgM- and- nested PCR-positive results together, HHV-6 infection was shown to have occurred in 5 of the 8 DCCs under follow-up. Serological evidence of recent infections by Epstein-Barr virus (EBV) and parvovirus B19 were identified in 2.0% (8/401) and 1.5% (6/401) of the children, respectively. Our data provide strong evidence that HHV-6 is a common cause of outbreaks of febrile/exanthematous diseases among children attending DCCs in the Belém area.

Evidence of active herpesvirus 6 (variant-A) infection in patients with lymphadenopathy in Belém, Pará, Brazil

A total of 323 patients with lymphadenopathy were selected in Belém, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates are found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across < 5 through 41-50-year age-groups for patients whose serum samples were IgM+, with rates ranging from 7.7% (female subjects aged < 5 years) to 80.0% (male, 11-20 years). Among patients whose serological status was IgG+, HHV-6 DNA was detected in < 5, 6-10, 21-30 and > 50 age-groups at rates that ranged from 15.4% (male, < 5 years of age) to 100.0% (female aged 11-20 years). Swelling cervical lymph nodes were the most common sign, accounting for 9 (32.0%) cases in each gender group. Among patients (n = 28) with active infection by HHV-6A variant, duration of symptoms lasted 1-5 days in 35.7% of subjects, whereas in 64.3% of them the disease lasted 6-20 days. Our data suggest that it is worth seeking for HHV-6 infection whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level.Bifidobacterium animalis subsp. lactisprovided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probioticB. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection.

Experiências terapêuticas com a alfa-6-deoxi-5-oxitetraciclina (doxiclina): um nôvo antibiótico de largo espectro

A Doxiciclina, um nôvo antibiótico derivado da oxitetraciclina, foi administrada a 54 pacientes, 16 com diversas entidades infecciosas, 18 portadores de Staphylococcus aureus e 20 portadores de Streptococcus beta haemolyticus. O medicamento, que foi empregado em dose única diária, por via oral, durante períodos variáveis de acôrdo com a entidade clínica e com a evolução do caso, mostrou-se de excelente tolerância e de grande eficiência, particularmente nas infecções por Staphylococcus aureus e Streptococcus beta haemolyticus que representam a maior incidência desta casuística.

Observações sôbre a terapêutica da ascaridiose com o hidroclorato e com o ciclamato de 2,3,5,6 – Tetrahidro - 6 fenil - imidazo (2,1-b) tiazol

Os autores estudam a ação do ciclamato e do cloridrato de 2, 3, 5, 6, - Tetrahidro-6-fenil-imidazo (2,1-b) tiazol no tratamento da ascaridiose. Foram tratados ao todo 84 indivíduos sendo 48 com o cloridrato e 36 com o ciclamato. O índice de cura com o primeiro foi de 83,9% e com o segundo de 76,4%. Nenhuma reação colateral foi observada com o emprêgo do cloridrato sendo que com o ciclamato 53,6 dos pacientes apresentaram sintomas discretos de intolerância. Os índices de cura assemelham-se aos observados com a piperazina que é a medicação clássica para terapêutica da ascaridiose.

Infecções bacterianas tratadas com Ro 6-2580

Nova combinação medicamentosa composta de sulfametoxazól e trimetoprim ("Bactrim"), com efeito bactericida sobre germes Gram negativos e Gram positivos, foi ensaiada no tratamento de 15 casos de salmonelose e 9 casos de infecções bacterianas. Os resultados foram favoráveis, tanto nas salmoneloses, como em infecções por estafilococos, shigelas, pneumococos e coliformes. A remissão do estado tóxico nos casos de febre tifóide foi observada dentro de 48 horas, e o tempo médio para desaparecimento da febre e demais sintomas, nessa infecção, foi de 3 dias. Foram constatados efeitos secundários em alguns pacientes, tais como hipertermia medicamentosa, farmacodermia, vômitos, linfomonocitose e eosinofilia, possivelmente relacionados a posologia excessiva, e todos remissíveis. Os autores concluem pela utilidade do novo medicamento na terapêutica de salmoneloses, estafilococcias e outras infecções.