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.

Lethality of triatomines (Hemiptera: Reduviiae), vectors of Chagas' disease, feeding on blood baits containing synthetic insecticides, under laboratory conditions

A laboratory study was conducted to test the toxicity of synthetic insecticides added to defibrinated sheep blood kept at room temperature and offered as food to the following triatomine species: Triatoma infestans, Panstrongylus megistus, Triatoma vitticeps, Triatoma pseudomaculata, Triatoma brasiliensis and Rhodnius prolixus. The insecticides used, at a concentration of 1g/l, were: HCH, DDT, Malathion and Trichlorfon, and the lethalithy observed at the end of a 7-day period varied according to the active principle of each. HCH was the most effective by the oral route, killing 100% of the insects, except P. megistus (95.7%) and T. pseudomaculata (94.1%). Trichlorfon killed the insects at rates ranging from 71.8% (T. vitticeps) to 98% (R. prolixus). Malathion was slightly less efficient, killing the insects at rates from 56.8% (T. vitticeps) to 97% (T.brasiliensis). DDT was the least effective, with a killing rate of 10% (T. vitticeps) to 75% (T.brasiliensis). Since the tests were performed at room temperature, we suggest that baits of this type should be tried for the control of triatomines in the field.

Lethal effect of a bait for Rhodnius prolixus (Hemiptera: Reduviidae), the vector of Chagas' disease, containing hexachlorocyclohexane (HCH), under laboratory conditions

The lethal effect of a bait containing an aqueous hexachlorocyclohexane (HCH) suspension at the concentration of 1g/l and maintained at room temperature was studied in the laboratory over a period of 12 weeks. The suspension was placed in a latex bag hanging inside a 1000-ml beaker tightly covered with nylon netting, and left there with no changes for 85 days. Sixteen groups of R. prolixas bugs, consisting on average of 30 specimens each, were successively exposed to the bait and observed at different intervals for one week each. The mortality rate was 100% for all groups, except for the 16th, whose mortality rate was 96.7%. As the groups succeeded one another, mortality started to occur more rapidly and was more marked at the 6- and 24-h intervals. Later tests respectively started at 6:00 a.m. and 6:00 p.m. showed that diurnal and nocturnal periodicity in the offer of food had no effect on mortality. First- and 2nd- instar nymphs and adults male were more sensitive and 5th- instar nymphs were more resistant to the active principle of the bait.

Laboratory acquired infection by the virus SP H 114202 (Arenavirus: Arenaviridae): clinical and laboratory findings

Here in is described the clinical and laboratorial findings of a laboratory-acquired infection caused by the virus SP H 114202 (Arenavirus, family Arenaviridae) a recently discovered agent responsible for a viral hemorrhagic fever. The patient was sick for 13 days. The disease had an abrupt onset characterized by high fever (39ºC.), headache, chills and myalgias for 8 days. In addition, on the 3rd day, the patient developed nauseas and vomiting, and in the 10th, epigastralgia, diarrheia and gengivorrhagia. Leucopenia was seen within the 1 st week of onset, with counts as low as 2,500 white cells per mm³. Counts performed after the 23th day of the onset were within normal limits. With the exception of moderate lymphocitosis, no changes were observed in differential counts. An increase in the liter of antibodies by complement fixation, neutralization and ELISA (IgM) was detected. Suckling mice and baby hamsters were inoculated intracerebrally with 0.02 ml of blood samples collected in the 2nd and 7th days of disease. Attempts to isolate the virus were also made in Vero cells. No virus was isolated. This virus was isolated before in a single occasion in São Paulo State, in 1990, from the blood of a patient with hemorrhagic fever with a fatal outcome. The manipulation of the virus under study, must be done carefully, since the transmission can occur through aerosols.

Severe scorpion envenomation in Brazil: clinical, laboratory and anatomopathological aspects

Scorpion stings in Brazil are important not only because of their incidence but also for their potential ability to induce severe, and often fatal, clinical situations, especially among children. In this report we present the clinical and laboratory data of 4 patients victims of scorpion stings by T. serrulatus, who developed heart failure and pulmonary edema, with 3 of them dying within 24 hours of the sting. Anatomopathologic study of these patients revealed diffuse areas of myocardiocytolysis in addition to pulmonary edema. The surviving child presented enzymatic, electrocardiographic and echocardiographic changes compatible with severe cardiac involvement, which were reversed within 5 days. These findings reinforce the need for continuous monitoring of patients with severe scorpion envenoming during the hours immediately following the sting.

Cell and humoral immunity in endemic pemphigus foliaceus

A study was conducted on 16 patients with pemphigus foliaceus, ten of them with the localized form (group G1) and six with the disseminated form (group G2). These patients were submitted to full blood counts, quantitation of mononuclear cell subpopulations by monoclonal antibodies, study of blastic lymphocyte transformation, and quantitation of circulating antibodies by the indirect immunofluorescence test, in order to correlate their clinical signs and symptoms and laboratory data with their immunological profile, and to determine the relationship between circulating autoantibody titers and lesion intensity and course of lesions under treatment. Leucocytosis was observed especially in group G2. All patients showed decreased relative CD3+ and CD4+ values and a tendency to decreased relative values of the CD8+ subpopulation. Blastic lymphocyte transformation indices in the presence of phytohemagglutinin were higher in patients (group G1+G2) than in controls. The indirect immunofluorescence test was positive in 100% of G2 patients and in 80% of G1 patients. The median value for the titers was higher in group G2 than in group G1. Analysis of the results as a whole permits us to conclude that cell immunity was preserved and that there was a relationship between antibody titers detected by the direct immunofluorescence test and extent of skin lesions.

Evaluation of nutritional status in patients with endemic pemphigus foliaceus

Sixteen patients with endemic pemphigus foliaceus were submitted to nutritional evaluation. Ten had the localized form of the disease (Group G1) and six the disseminated form (Group G2). The patients were submitted to anthropometric measurements (weight, height, Quetelét index, tricipital skin fold, subscapular skin fold, arm circumference, arm muscle circumference, arm area, arm muscle area, and arm adipose area) and to laboratory evaluation by protein electrophoresis. Arm circumference, arm area and arm muscle area showed lower values in G2 than in G1. Weight and arm muscle circumference tended to the lower in G2 than in G1. Protein electrophoresis showed decreased albumin levels in both groups, with lower values in G2. Overall analysis of the results permits us to conclude that patients with endemic pemphigus foliaceus present signs and symptoms of protein, but not calorie, malnutrition and that this malnutrition is more marked in patients with disseminated pemphigus foliaceus.

The relevance of laboratory diagnosis of human cryptosporidiosis and other coccidia

Human infection by Cryptosporidium spp and other coccidia are due to opportunist non-host specific microorganisms. In HIV seropositive patients, the gastrointestinal symptoms accompanying such infections may be serious and prolonged and may include nausea, low-grade fever, abdominal cramps, anorexia and watery diarrhoea. We studied 188 stool samples from 111 patients (84 men and 27 women) with diarrhoea. A modified Ziehl-Nielsen technique for the detection of Cryptosporidium spp and Isospora belli was employed. The mean age of the patients was 31 years. Cryptosporidium spp was seen in 18% (n=20) of the patients, 90% (n=18) of whom were HIV seropositive. Isospora belli was recorded only from HIV seropositive patients (5.4% of all the patients studied and 6.5% of those who were HIV seropositive). These data confirm the good results obtained with this technique for the identification of Cryptosporidium spp and other coccidia and also reaffirm the clinical importance of correctly diagnosing the cause of diarrhoea, particularly in HIV seropositive patients.

The role of Public Health Laboratory in the problem os salmonellosis in São Paulo, Brazil

From 1950 to 1990 a total of 45,862 strains (31,517 isolates from human sources, and 14,345 of non-human origin) were identified at Instituto Adolfo Lutz. No prevalence of any serovars was seen during the period 1950-66 among human sources isolates. Important changing pattern was seen in 1968, when S. Typhimurim surprisingly increased becoming the prevalent serovar in the following decades. During the period of 1970-76, S. Typhimurium represented 77.7% of all serovars of human origin. Significant rise in S. Agona isolation as well as in the number of different serovars among human sources strains were seen in the late 70' and the 80's. More than one hundred different serovars were identified among non-human origin strains. Among serovars isolated from human sources, 74.9%, 15.5%, and 3.7% were recovered from stool, blood, and cerebrospinal fluid cultures, respectively. The outbreak of meningitis by S. Grumpensis in the 60's, emphasizes the concept that any Salmonella serovars can be a cause of epidemics, mainly of the nosocomial origin. This evaluation covering a long period shows the important role of the Public Health Laboratory in the surveillance of salmonellosis, one of the most frequent zoonosis in the world.

Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

COLONIZATION AND LABORATORY MAINTENANCE OFAnopheles albimanus WIEDEMANN IN VENEZUELA

Anopheles albimanus is one of the main vectors of malaria in Central America and the Caribbean, based on its importance, there are previous reports of the successful colonization of this species in Latin America countries. Mosquitoes were collected in the Aragua State of Venezuela colonized in the laboratory, using a simple and efficient maintenance method. Based on life table calculations under well established laboratory conditions, the Survival Rate Probability was constant and always close to 1 in immature stages, the Reproductive Net Rate (Ro) was 3.83, the generation time (Tc) was 24.5 days and the Intrinsic Growth Rate (rm) was 0.0558. This is the first report of the colonization of A. albimanus in Venezuela.