Avaliação epidemiológica de doenças negligenciadas em escolares: filariose linfática e parasitoses intestinais / Epidemiological assessment of neglected diseases in children: lymphatic filariasis and soil-transmitted helminthiasis

OBJETIVO: Descrever a prevalência de infecção filarial e de parasitoses intestinais em escolares numa área endêmica de filariose e refletir sobre a opção terapêutica utilizada no Brasil no tratamento coletivo para filariose. MÉTODOS: Estudo transversal envolvendo 508 alunos na faixa etária de 5-18 anos cadastrados em escolas públicas do município de Olinda-PE. Realizou-se a investigação da parasitose intestinal em três amostras de fezes, analisadas pelo método de Hoffmann, Pons e Janer. A investigação filarial foi feita com teste antigênico pela técnica de imunocromatográfica rápida (ICT) e pesquisa de microfilárias, utilizando filtração em membrana de policarbonato. Para análise de dados utilizou-se a estatística descritiva através do programa EpiInfo versão 7. RESULTADOS: A prevalência de filariose por ICT foi de 13,8% e por microfilaremia de 1,2%, enquanto a de parasitoses intestinais foi 64,2%. A concomitância do diagnóstico filarial e de parasitoses intestinais foi de 9,4% e, 31,5% eram isentos de ambas as parasitoses. Entre os indivíduos com parasitoses intestinais, 55% eram monoparasitados e 45% poliparasitados. A presença de geohelmintos ocorreu em 72,5% dos parasitados. No grupo com infecção filarial a ocorrência de geohelmintíase foi de 54,5%. CONCLUSÕES: O diagnóstico simultâneo de infecção filarial e parasitose intestinal, bem como a elevada frequência de geohelmintos justificam uma reavaliação da estratégia terapêutica do tratamento coletivo no programa de filariose no Brasil.

OBJECTIVE: To report the prevalence of lymphatic filariasis and intestinal parasitic infections in school-aged children living in a filariasis endemic area and discuss about the therapeutic regimen adopted in Brazil for the large-scale treatment of filariasis. METHODS: A cross-sectional study including 508 students aged 5-18 years old, enrolled in public schools within the city of Olinda, Pernambuco. The presence of intestinal parasites was analyzed using the Hoffman, Pons and Janer method on 3 stool samples. The diagnosis of filarial infection was performed using the rapid immunochromatographic technique (ICT) for the antigen, and the polycarbonate membrane filtration for the presence of microfilariae. Descriptive statistics of the data was performed using EpiInfo version 7. RESULTS: The prevalence of filariasis was 13.8% by ICT and 1.2% by microfilaraemia, while intestinal parasites were detected in 64.2% of cases. Concurrent diagnosis of filariasis and intestinal parasites was 9.4%, while 31.5% of students were parasite-free. Among individuals with intestinal parasites, 55% had one parasite and 45% had more than one parasite. Geohelminths occurred in 72.5% of the parasited individuals. In the group with filarial infection the prevalence of soil-transmitted helminthiasis was 54.5%. CONCLUSIONS: The simultaneous diagnosis of filariasis and intestinal parasites as well as the high frequency of geohelminths justify the need to reevaluate the treatment strategy used in the Brazilian filariasis large-scale treatment program.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

Prevalence of bancroftian filariasis on the Thai-Myanmar border.

To achieve the goal of eliminating lymphatic filariasis by the year 2020, close monitoring systems and effective control strategies need to be implemented and the real disease burden needs to be assessed. Bancroftian filariasis is endemic at the Thai-Myanmar border. However, there are only limited data on the prevalence of this disease in Thailand available. We employed microscopic examination, together with ELISA kits to detect W. bancrofti-specific Og4C3 circulating antigen and specific anti-filarial IgG4 antibodies to determine the burden of bancroftian filariasis in an endemic area at the Thai-Myanmar border in Umphang District, Tak province, Thailand. A total of 433 Thai-Karen blood samples were analyzed. The microfilarial rate determined by microscope was 6% and the W. bancrofti-specific Og4C3 antigenemia rate was 22%, while the specific anti-filarial IgG4 antibody rate was 54%. There were statistically significant higher levels of W. bancrofti-specific Og4C3 antigen in the microfilaremic-antigenemic group than in the amicrofilaremic-antigenemic group (unpaired Student's t-test; p < 0.001), similar to the specific anti-filarial IgG4 antibody results (unpaired Student's t-test; p < 0.001). A statistically significant correlation of moderate degree between the presence of W. bancrofti-specific Og4C3 antigen and of specific anti-filarial IgG4 antibody was found in the amicrofilaremic group (r = 0.474, p < 0.001), but not in the microfilaremic group (r = 0.291, p > 0.05). Our study revealed a very high prevalence of bancroftian filariasis in this endemic area and thus emphasized the importance of using highly sensitive and specific diagnostic tools to evaluate the true prevalence of the disease.

Transmission of filarial infection through blood transfusion.

Microfilariae can be transmitted by blood transfusion and they may be circulated in the recipient's blood but they do not develop into adult worms. Mortality associated with transfusion associated filarial infection is not documented but it may give rise to morbidity in transfusion recipients in terms of allergic reaction. The present study was carried out to investigate the association of post transfusion reactions and filarial infections in an endemic area. About 11,752 transfusion recipients were followed up and in 15 months period, 47 (0.4%) post transfusion reactions (PTR) were reported. Routine investigations for post transfusion reaction were carried out in all 47 patients and their respective blood donor. Moreover, blood culture, microfilaria detection by concentration technique, filarial antibody and antigen detection (both by ELISA) were done in all subjects. Out of 47 patients showing post transfusion reaction, 29 (61.7%) patients developed allergic reaction. Eighteen (38.3%) patients having allergic reaction did not have previous history of blood transfusion and 14 (29.8%) of them received transfusion from blood donors who was either positive for microfilaria, filarial antigen or antibody. Microfilaremia was demonstrated in 4 (8.5%) patients and 5 (10.6%) blood donors. Microfilaria was concurrently present in 2 patients and their respective donors. Filarial antibody was detected in 27 (56.5%) patients and 26 (55.3%) blood donors but microfilaria was detected in 3 (6.4%) and 4 (8.5%) subjects, respectively. Antigen detection test correlated with microfileraemic state of subjects. The result shows that transfusion associated filarial infection may be a probable cause for transfusion-associated morbidity in endemic areas. In 14 (29.8%) patients having allergic reactions, the probable cause was transfusion-associated filarial infection. Filarial antigen detection test was found to be more useful in detecting infections. Blood donors with active history of filarial infection should be deferred from donating blood. Filarial antigen detection test may be employed as screening test for blood donors, if possible.

A preliminary analysis of Wuchereria bancrofti microfilarial antigens for potential use in diagnosis.

Several antigens from the microfilarial stage of Wuchereria bancrofti have been identified using immunoblots of microfilarial antigens and screening with immune sera and tropical pulmonary eosinophilia (TPE) sera. This analysis revealed an array of antigens with apparent molecular weights of 14kDa, 35kDa, 42kDa, 63kDa, 88kDa, 97kDa and 200kDa. Among these only the 14kDa and 42kDa antigens were consistently recognized by most of the immune sera. A 132kDa antigen was recognized only by TPE sera. Analysis of rabbit immune sera revealed that the 42kDa antigen was shared by two developmental stages of W. bancrofti, namely L3 and mF. This antigen could become a potential vaccine candidate. The 14kDa antigen seems specific for the microfilarial stage and therefore could be a diagnostic marker for active infection. The 132kDa antigen could aid in the diagnosis of TPE.

Comparative study of Dot-immunogold silver staining and Dot-ELISA for the detection of serum antibodies against Wuchereria bancrofti.

Dot-immunogold silver staining (Dot-IGSS) and Dot-ELISA, using the soluble antigen of Brugia malayi, were employed to detect anti-Wuchereria bancrofti antibodies in 50 cases of Wuchereria bancrofti microfilaremia. The positive rates were 100% and 90% in Dot-IGSS and Dot-ELISA respectively. The average titer in the 45 positive cases was 1:184 (1:10-1:2560) for Dot-IGSS and 1:150 (1:10-1:2560) for Dot-ELISA, with 30 cases showing the same titer in both tests, 13 cases showing higher titer in Dot-IGSS than in Dot-ELISA and 2 cases in the former showing lower titers than in the latter. There was a linear relationship between the titers of antibodies detected by Dot-IGSS and by Dot-ELISA (r = 0.8443). Dot-IGSS, similar to Dot-ELISA, is easy to carry out and the result is easy to read. It is seen that Dot-IGSS is highly sensitive and specific and is practicable for immunodiagnosis and surveillance of filariasis.

Detection of circulating antigens and parasite specific antibodies in filariasis.

In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.

Detection of filarial antigen using antibodies raised against Wuchereria bancrofti microfilarial SDS soluble antigen.

Polyclonal antibodies raised in mouse ascitic fluid against Wuchereria bancrofti microfilarial antigens (Wb Mf SDS S Ag) were studied for their diagnostic use in bancroftian filariasis using a dip stick, enzyme-linked immunosorbent assay. In sandwich ELISA, 100% of microfilaremic sera (30 out of 30) 53% of acute filarial sera (7/13), 40% of subacute filarial sera (6 out of 15), 13% of chronic filarial sera (2/15) and 20% of endemic area normal sera (3/15) showed the presence of filarial antigen. Determination of filarial antigen titer in microfilaremic sera showed an apparent positive correlation between microfilarial density and antigen titer. The antibody raised against Wb Mf SDS S Ag was found to be cross reactive with phosphorylcholine epitopes. The filarial antigen detected by anti Wb Mf SDS S Ag antibodies in sandwich ELISA is possibly associated with the active stage (microfilaremia) of infection.

Evaluation of RNA rich fraction of Litmosoides carinii in induction of protection in infected rats.

The RNA rich fraction of adult L. carinii worms was evaluated in evoking a protective response in infected rats. The RNA immunization was seen to be effective in limiting the microfilaraemia in peripheral blood as well as the adult worm burden. The antibodies to both RNA antigen and adult worm antigen were high in this group of animals at the peak of infection. The RNA immunization was seen to evoke hyperresponsiveness in lymphocytes to mitogens like adult worm antigen, PHA and Con A.

Fractionation and characterization of urinary filarial antigen in bancroftian filariasis.

Urine samples from microfilaraemic patients were concentrated and fractionated by gel chromatography on Ultrogel AcA 44. Four protein fractions labelled as UFA C1, UFA C2, UFA C3 and UFA C4 were tested for filarial antigenicity by sandwich ELISA. UFA C1 and UFA C2 showed antigenic activity. On further analysis by SDS-PAGE, UFA C1 and UFA C2 showed antigenic components with MW ranging from 10.4 K to 123 K. UFA C1-1 and UFA C2-2 showed high antigen titre in ELISA. Urinary albumin was observed as a major component in UFA C2. Absorption of albumin from UFA C2 enhanced its antigenic activity considerably. As little as 0.01 pg antigenic protein per test was found to be sufficient for the detection of filarial antibody in ELISA. Biochemical characterization indicated a glycoprotein nature of UFA C2.

Mantenimiento in vitro de microfilarias / In vitro maintenance of microfilariae

Con el propósito de hacer estudios inmunológicos de los productos metabólicos de microfilarias, se realiza el mantenimiento in vitro de las mismas. Se trabaja con 3 especies de filarias humanas: Wuchereria bancrofti, Dipetalonema perstans, Loa loa y con una filaria canina, Dirofilaria immitis. Se utilizan en los primeros 20 estudios, 2 medios de cultivo, 2 pH y 2 temperaturas diferentes. De esta primera experiencia se escogen las mejores condiciones para realizar la segunda fase del trabajo. El tiempo de sobrevivencia establecido, teniendo en cuenta la no utilización de suplementos en el medio, está en correspondencia con el objetivo del trabajo

Brugia malayi: serum dependent cell-mediated reactions to microfilariae.

Sheathed and exsheathed microfilariae of Brugia malayi are killed by normal rat cells in the presence of immune serum in vitro. Immune serum heated at 56 degrees C for 1 hour lost this activity which was largely restored by the addition of fresh normal rat serum. EDTA but not EGTA abolished this activity indicating the operation of complement by alternate pathway. Fresh normal rat serum alone promoted cellular adherence without exerting cytotoxicity to the microfilariae. The activity in the immune serum could be removed with Staphylococcus aureus cells containing Protein A or anti-IgG antiserum. The activity could also be absorbed to and eluted from Protein A--sepharose CL-4B suggesting the involvement of IgG. Neutrophils and macrophages participate in the antibody dependent cell-mediated cytotoxicity phenomenon. Eosinophils while adhering to the microfilariae exert cytotoxicity only to the exsheathed parasites.

Studies on filariasis in Singapore: immunodiagnosis using indirect immunofluorescence.

Filarial antibodies were detected by the indirect fluorescent antibody (IFA) technique using sonicated microfilariae of Brugia malayi as antigen. Of the 324 sera of patients with clinical symptoms suggestive of filarial infection, 90 (28%) had detectable antibodies with titres ranging from 1 : 4 to 1 : 4096. Forty-six percent of patients with eosinophilic lung were positive with titres ranging from 1 : 4 to 1 : 1024. Highest rates of positives were seen in Indians (48%) with lower rates in Malays (36%) and Chinese (15%).