Patient-based dengue virus surveillance in Aedes aegypti from Recife, Brazil.

BACKGROUND & OBJECTIVES: Dengue is currently one of the most important arthropod-borne diseases and may be caused by four different dengue virus serotypes (DENV-1 to DENV-4), transmitted mainly by Aedes aegypti (Diptera: Culicidae) mosquitoes. With the lack of a dengue vaccine, vector control strategies constitute a crucial mode to prevent or reduce disease transmission. In this context, DENV detection in natural Ae. aegypti populations may serve as a potential additional tool for early prediction systems of dengue outbreaks, leading to an intensification of vector control measures, aimed at reducing disease transmission. In Brazil, this type of surveillance has been performed sporadically by a few groups and has not been incorporated as a routine activity in control programs. This study aimed at detecting DENV in natural Ae. aegypti from Recife, Pernambuco, to check the circulating serotypes and the occurrence of transovarial transmission in local mosquito populations. METHODS: From January 2005 to June 2006, mosquitoes (adults and eggs) were collected in houses where people with clinical suspicion of dengue infection lived at. RNA was extracted from pooled mosquitoes and RT-PCR was performed in these samples for detection of the four DENV serotypes. RESULTS & CONCLUSION: Out of 83 pools of adult mosquitoes collected in the field, nine were positive for DENV: five for DENV-1, two for DENV-2 and two for DENV-3. From 139 pools of adult mosquitoes reared from collected eggs, there were 17 positive pools: three for DENV-1, 10 for DENV-2, and four for DENV-3. These results are discussed in the paper in regard to the local dengue epidemiological data. The conclusions clearly point to the informative power and sensitivity of DENV entomological surveillance and to the importance of including mosquito immature forms in this strategy.

Resistance to the organophosphate temephos: mechanisms, evolution and reversion in an Aedes aegypti laboratory strain from Brazil.

Insecticide resistance is one of the main problems in vector control programs. Because insects have developed resistance to all classes of available chemical insecticides, a proper surveillance and management of resistance in areas where these compounds are being utilized is crucial for the success of control programs. Since the mechanisms and molecular bases of resistance are various, they must be characterized to allow efficient monitoring strategies. Here we report the establishment of an Aedes aegypti strain resistant to temephos, named RecR, selected under laboratory conditions. The parental A. aegypti population was obtained from eggs collected in an area where temephos had been used for 8 years, and presented a baseline resistance ratio (RR) of 7. After 17 generations under selective pressure, the RR has increased to 180. Biochemical assays indicate that metabolic mechanisms are involved on temephos resistance in the selected strain. These experiments showed that, compared to the susceptible colony Rockefeller, RecR present higher activity of glutathione S-transferases (GSTs), alpha- and beta-esterases, and, to a lesser degree, mixed function oxidases (MFO). At the 14th or 17th generations, there was no cross resistance of these insects to deltamethrin, cypermethrin and malathion, while a low resistance level (RR=3) was observed for pyriproxyfen, a juvenile hormone analogue. Experiments on resistance reversal, performed through three different field simulated schemes using the resistant strain, showed that temephos susceptibility can be recovered. The establishment of an A. aegypti colony resistant to temephos is extremely valuable for a deeper understanding of resistance mechanisms and thus for further improvements in control strategies against this vector. With the urgent need on improving methodologies to monitor resistance, molecular studies such as microarrays, and resistant colonies such as RecR will certainly hasten such studies.

Phagocytosis of latex beads and bacteria by hemocytes of the triatomine bug Rhodnius prolixus (Hemiptera: Reduvidae).

Insect circulating hemocytes are primarily responsible for the immune defense against parasites and pathogens. Here, we have analyzed phagocytosis of both biotic (bacteria) and abiotic (latex) particles by circulating hemocytes of 5th-instar nymphs of the triatomine bug Rhodnius prolixus. The following hemocyte types were identified: prohemocytes, plasmatocytes, granulocytes, oenocytoids and adipohemocytes. There was a considerable change in the relative percentage of plasmatocytes and prohemocytes in the hemolymph after challenge with both latex beads and bacteria. Granulocytes and oenocytoids also change their relative percentage in response to latex bead and Staphylococcus aureus, respectively. No significant change was observed in adipohemocytes at any time or treatment. Our data demonstrated that plasmatocytes were the only cell type involved in phagocytosis of foreign particles. As in mammal cells, phagocytosis by both zipper and trigger mechanisms were observed for the uptake of latex beads and bacteria. Neither melanization nor micro-aggregation was observed towards latex particles or Escherichia coli. On the other hand, R. prolixus produced a strong melanization reaction against S. aureus, thus showing that differences exist in the responses to E. coli and to S. aureus. Ultrastructural changes observed in plasmatocytes, adipohemocytes and oenocytoids suggest that these hemocyte types are directly involved in the immune defense of R. prolixus against foreign particles.

Genetic differentiation of Aedes aegypti (Diptera: Culicidae), the major dengue vector in Brazil.

In 2000, Brazil reported 180,137 cases of dengue, approximately 80% of the total in the Americas. However, little is known about gene flow among the vector populations in Brazil. Random amplified polymorphic DNA (RAPD) was used to study the genetic structure of Aedes aegypti in 15 populations from five states, with a range extending 2,800 km. An analysis of 47 polymorphic RAPD loci estimated gene flow at the macro- (different states) and micro- (different cities) geographical levels. Genetic polymorphism was high (H(S) = 0.274), and high levels of genetic differentiation existed both between different states (G(ST) = 0.317) and between cities or neighborhoods in each state (G(ST) = 0.085-0.265). These values are higher than those described for any other populations of A. aegypti.

Screening of exogenous enzymes for ruminant diets: relationship between biochemical characteristics and in vitro ruminal degradation.

With the objective of developing a rational approach for the selection of feed enzymes for ruminants, 22 commercial enzyme products were examined in terms of protein concentration, enzymic activities on model substrates, and hydrolytic capacity, the latter determined from the release of reducing sugars from alfalfa hay and corn silage. An in vitro ruminal degradation assessment was carried out using the same substrates, untreated or treated with the 22 enzyme products at 1.5 microL/g forage DM. Stepwise regressions were then performed to establish relationships between these factors. Protein concentration and enzymic activities explained at least 84% (P < 0.01) of the variation in the release of reducing sugars from alfalfa and corn silage. Alfalfa DM degradation after incubation with ruminal fluid for 18 h was positively related to xylanase activity (R2 = 0.29, P < 0.01), but the same activity was negatively related to DM degradation of corn silage (R2 = 0.19, P < 0.05). Protease activity explained a further 10% of the alfalfa DM degradation (P < 0.10). Following sequential steps involving the determination of rate and extent of DM and fiber degradation, the best candidates for alfalfa and corn silage were selected. Enzyme products effective with alfalfa hay seemed to exert part of their effect during the pretreatment period, whereas enzymes effective with corn silage worked exclusively after ruminal fluid was added. This finding suggests that different modes of action of exogenous enzymes are attacking different substrates and may partly explain enzyme-feed specificity. In alfalfa, it seems that effective enzymes work by removing structural barriers that retard the microbial colonization of digestible fractions, increasing the rate of degradation. In corn silage, effective enzymes seem to interact with ruminal enzymes to degrade the forage more rapidly, which is consistent with previous findings of synergism between exogenous and ruminal enzymes.

Genetic diversity in Brazilian populations of Aedes albopictus.

Random amplified polymorphic DNA (RAPD) analysis technique was undertaken in Aedes albopictus populations from three states in Brazil, Rio de Janeiro (RJ), Minas Gerais (MG) and Pernambuco (PE), to estimate the level of genetic variability and levels of genetic exchange between populations. Allele and genotype frequencies were measured on 47 RAPD loci. Average observed heterozigosity (Ho) ranged from 0.282 in MG to 0.355 in Casa Forte (PE) population. Genetic distances estimates indicated that RJ and MG were more genetically similar than populations from PE. Genetic variation observed in local Brazilian populations was attributed to genetic drift associated with restricted gene flow in recently established populations.

Genetic diversity in Brazilian populations of Aedes albopictus

Random amplified polymorphic DNA (RAPD) analysis technique was undertaken in Aedes albopictus populations from three states in Brazil, Rio de Janeiro (RJ), Minas Gerais (MG) and Pernambuco (PE), to estimate the level of genetic variability and levels of genetic exchange between populations. Allele and genotype frequencies were measured on 47 RAPD loci. Average observed heterozigosity (Ho) ranged from 0.282 in MG to 0.355 in Casa Forte (PE) population. Genetic distances estimates indicated that RJ and MG were more genetically similar than populations from PE. Genetic variation observed in local Brazilian populations was attributed to genetic drift associated with restricted gene flow in recently established populations

Effect of a fibrolytic enzyme preparation from Trichoderma longibrachiatum on the rumen microbial population of dairy cows.

The effects of supplementing a dairy cow diet with incremental levels of a fibrolytic enzyme preparation (preparation B) from Trichoderma longibrachiatum on the rumen microbial population were investigated. Two cows fitted with rumen cannulae were each fed a diet containing barley-based concentrate (52%), maize silage (29%), and chopped alfalfa hay (19%), supplemented with 0, 1, 2, 5, or 10 L of preparation B per tonne of dry matter (DM). Preparation B stimulated numbers of total viable bacteria in a quadratic manner (P < 0.05), to approximately 230, 330, 390, and 250% at 1, 2, 5, and 10 L x t(-1) DM, respectively. Preparation B increased the numbers of cellobiose-utilizing (P < 0.01), xylanolytic (P < 0.05), and amylolytic bacteria (P < 0.05), but had no effect (P > 0.05) on numbers of cellulolytic bacteria. However, when bacterial numbers enumerated on each substrate were expressed as a proportion of total viable bacterial numbers, only cellobiose utilizers were stimulated, and this stimulation was limited to the 1 L x t(-1) DM level of preparation B (P < 0.05). The results of this study demonstrate that the inclusion of an exogenous fibrolytic enzyme preparation in dairy cow diets increased the numbers of rumen bacteria that utilize hemicelluloses and secondary products of cellulose digestion.

Efficacy of Bacillus sphaericus in control of the filariasis vector Culex quinquefasciatus in an urban area of Olinda, Brazil.

The efficacy of Bacillus sphaericus 2362 against Culex quinquefasciatus was tested in 1991-94 in a major Brazilian endemic zone for bancroftian filariasis. Continuous selection pressure against the mosquito population was sustained for 18 months through treatment of 2500 potential breeding sites occurring within a 5.7-km2 urban area in the Metropolitan Region of Recife. The impact of this control intervention was evaluated by comparing entomological indices with those from an untreated area. Application of the larvicide kept the Cx. quinquefasciatus population density significantly lower when compared to the untreated area, despite some operational difficulties. Adult densities remained lower for at least 5 months after spraying ceased. Pre-trial microfilaria rates from the untreated and operational area were 13.1% and 7.2%, respectively. A 60% reduction in human exposure to infective bites was recorded as a consequence of this vector population control.

[Views on the municipalization of lymphatic filariasis control in greater metropolitan Recife]. / Perspectivas da municipalização do controle da filariose linfática na região metropolitana do Recife.

Lymphatic filariasis is caused by the nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti. The disease occurs in developing countries and is more frequent in urban areas. An estimated 4 billion people live in at-risk areas. In Brazil the endemic is caused by W. bancrofti and was first documented in 1878. It was first detected in Recife in 1952. Currently, Recife and Belém are the only cities in Brazil where the endemic is considered a public health problem. The objectives of this study are to discuss the epidemiology and control of lymphatic filariasis and review its control since it was reported by Rachou in 1952. We analyze the "campanhista" or campaign-oriented model employed by the Sucam/FNS institutional program, as well as several proposed innovative methods. We present available strategies for control of filariasis through primary health care services, decentralization to the local level (or "municipalization"), and community-based health programs.

Diagnosis of Wuchereria bancrofti infection by the polymerase chain reaction using urine and day blood samples from amicrofilaraemic patients.

A sensitive and specific polymerase chain reaction (PCR) based on a highly repeated deoxyribonucleic acid (DNA) sequence (188 bp; SspI repeat) was tested for the detection of Wuchereria bancrofti DNA in blood and urine samples collected during the day from individuals in Coque, Recife, Brazil, an endemic area for W. bancrofti. All microfilaraemic individuals were also positive by PCR, irrespective of the samples used. The PCR system was capable of detecting W. bancrofti DNA in amicrofilaraemic individuals: c. 93% were positive by PCR when day blood samples were used and 59.7% when urine samples collected at 07:00 were used. Thus, nocturnally periodic W. bancrofti infection can be detected in blood samples collected during the day, which is convenient for large-scale screening. In addition, non-invasive urine collection provided suitable samples for PCR, which is clearly advantageous for preliminary mass diagnosis.

Absence of lysozyme (muramidase) in the intestinal Paneth cells of newborn infants with necrotising enterocolitis.

AIM: To determine immunocytochemically whether preterm and newborn infants with necrotising enterocolitis (NEC) show differences in numbers of lysozyme positive Paneth cells compared with normal controls, and to relate the findings to the possibility that lysozyme deficiency may facilitate the bacterial infections thought to be associated with this condition. METHODS: Tissues from 10 infants with NEC and from 11 matched controls were sectioned and stained immunocytochemically for lysozyme. Differences in the numbers of Paneth cells and degree of lysozyme positivity in the tissues were assessed. RESULTS: Tissues from NEC patients showed no, or very few, lysozyme positive Paneth cells, whereas controls showed strong positive staining. CONCLUSIONS: A deficiency or developmental defect in Paneth cells, resulting in an absence of lysozyme, may render the intestine more susceptible to bacterial infection, allowing organisms to adhere and translocate across the mucosa. Such enhancement of infection may contribute to the pathogenesis of NEC.

The LYMFASIM simulation program for modeling lymphatic filariasis and its control.

The LYMFASIM modeling framework for the transmission and control of the tropical parasitic disease lymphatic filariasis is described and its use in the context of an endemic community in north-eastern Brazil is illustrated. Lymphatic filariasis is a disease with a complex natural history with many unknowns. This complicates decision making with respect to control strategies. With LYMFASIM, a variety of hypotheses can be tested about the life history of the parasite Wuchereria bancrofti, its transmission from man to man through mosquitoes, the role of the immune system in regulating parasite numbers, the development of disease symptoms, and the effects of control measures (drug treatment or mosquito control). The implications of alternative assumptions and uncertainty about the quantification of parameters for the effectiveness of control strategies can be investigated. Thanks to the use of stochastic microsimulation, LYMFASIM is highly flexible and can be adapted and extended as new knowledge emerges.

Intra-abdominal sepsis: an immunocytochemical study of the small intestine mucosa.

AIM: To investigate immunocytochemical changes in intestinal tissues from patients with intra-abdominal sepsis, and to relate the changes to the possibility of enhanced bacterial adhesion and translocation. METHODS: Tissues from 17 patients suffering from intra-abdominal sepsis and from controls were sectioned and stained immunocytochemically for IgA, IgM, secretory component, J chain, and HLA-DR. Differences in the distribution and characteristics of positively staining cells between the patient groups were assessed. RESULTS: Patients with intra-abdominal sepsis had noticeable reductions in numbers of IgA and IgM plasma cells, reduced J chain staining, and had little immunoglobulin on the surfaces of enterocytes. In contrast, HLA-DR positive cells were increased in the sepsis compared with the control group. The plasma cells present showed cytological changes suggestive of apoptosis. CONCLUSIONS: Stress associated with sepsis and its immediate causes might result in increased plasma glucocorticoid levels that bring about apoptosis of mucosal plasma cells (or their precursors). The consequent reduction in expression of IgA and IgM may favour bacterial adhesion to the enterocytes and facilitate bacterial translocation into the tissues.

Bancroftian filariasis in two urban areas of Recife, Brasil: the role of individual risk factors.

Bancroftian filariasis is spreading in towns of endemic areas in Recife, northeastern Brazil, where it is a major public health problem. This paper deals with the prevalence of microfilaraemia and filarial disease in two urban areas of Recife, studying their association with individual characteristics and variables related to the exposure to the vectors. The parasitologic survey was performed through a "door-to-door" census and microfilaraemia was examined by the thick-drop technique using 45 microliters of peripheral blood collected between 20:00 and 24:00 o'clock. 2,863 individuals aged between 5 and 65 years were interviewed and submitted to clinical examination. Males aged between 15 and 44 years old presented the greatest risk of being microfilaraemic. Microfilaraemia was also significantly associated with no use of bednet to sleep. The risk of being microfilaraemic was greater among those who had lived in the studied areas for more than 5 years. The overall disease prevalence was 6.3%. Males presented the greatest risk of developing acute disease. The risk of developing chronic manifestations was also greater among males and increased with age. We found no association between time of residence, bednet use, microfilaraemia and acute and chronic disease. We may conclude that in endemic areas there are subgroups of individuals who has a higher risk of being microfilariae carriers due to different behaviours in relation to vector contact.

Abnormal lymphatic function in presymptomatic bancroftian filariasis.

Despite the common association of filarial infection with elephantiasis, the great majority of those infected are in fact clinically asymptomatic microfilariae carriers. The assumption has been that infection but not disease exists in these presymptomatic persons. In an area Brazil where Wuchereria bancrofti is endemic, flow studies done with dynamic radionuclide lymphoscintigraphy were used to compare 30 limbs from asymptomatic microfilaremic subjects with 16 control limbs. Geometric mean values for T1/2 (19.8 vs. 37.7 min; P < .001), appearance time (7.9 vs. 27.9 min; P < .001), percent uptake at the region of interest (0.67% vs. 0.14%; P < .001), and peak activity (62.6 vs. 2.6 cps; P < .001) each indicated an enhanced pattern of rapid, increased lymph flow in asymptomatic microfilaremic subjects. The abnormal lymphatic function in these subjects indicates that current passive intervention strategies may need to change if the debilitating sequelae of this parasitic infection are to be avoided.

Lymphoscintigraphic assessment of the effect of diethylcarbamazine treatment on lymphatic damage in human bancroftian filariasis.

Despite many millions of doses administered over the past 40 years, basic and crucial issues regarding the use, mode of action, and effectiveness of diethylcarbamazine (DEC) in many clinical situations remain unresolved. To directly investigate whether the well-known microfilaricidal and macrofilaricidal actions of DEC actually result in subsequent improvement in existing damage to lymphatic vessels or lymph nodes, 29 study subjects in Recife, Brazil were stratified into three groups according to the severity of clinical manifestations of lymphatic insufficiency. After baseline radionuclide lymphoscintigraphy was performed, subjects were treated with two courses of DEC separated by at least a six-month interval and then rescanned one year after the baseline scan. A side-by-side comparison of images obtained at baseline with those obtained at follow-up in 13 asymptomatic microfilaremic individuals, six individuals with filarial fever, and in 10 individuals with chronic pathology demonstrated essentially unchanged lymphatic morphology in all but one individual whose disease actually progressed in the face of therapy. We conclude that two 12-day treatment courses of DEC did not have a demonstrable direct or indirect effect on existing structural damage to the lymphatic system even in those individuals with preclinical disease.

A monoclonal antibody against blood forms of Trypanosoma cruzi lyses the parasite in vitro and inhibits host cell invasion.

Monoclonal antibodies (mAbs) of the IgM isotypes were produced from mice immunized with blood forms of Trypansoma cruzi Y strain. Characterization of the epitope recognized by one of the mAbs, 164C11, as well as the effects of this mAb on complement-mediated lysis and host cell invasion are reported. Immunocytochemical analysis showed that the mAb was reactive with various strains of T. cruzi (Y, WSL, and Colombiana) as well as other trypanosomatids. The mAb 164C11 demonstrated a high complement-mediated lytic activity against bloodstream trypomastigotes, being more effective than chronic mouse serum. A protein with an apparent molecular weight of 72 kDa was detected by this mAb on all developmental stages of T. cruzi. Studies using periodate and endoglycosidase treatments suggested that the epitope is not a carbohydrate and seems to be located on the parasite membrane. In addition, preliminary results are presented, suggesting that the 72-kDa protein is involved in adhesion/or internalization of bloodstream trypomastigotes.

Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife.

Integrated control measures against Culex quinquefasciastus have been implemented in a pilot urban area in Recife, Brazil. About 3,000 breeding sites found within the operational area were responsible for very high mosquito densities recorded during the pretrial period. Physical control measures have been applied to cess pits before starting a series of 37 treatments of the other sites with Bacillus sphaericus strain 2362, over 27 months. In spite of the difficulties due to environmental conditions, very significant reductions in preimaginal population of C. quinquefasciatus were achieved and, as a consequence, low adult mosquito densities were maintained for a relatively long period of time. Entomological and environmental data gathered in this pilot project can contribute to design an integrated mosquito control program in Recife city.