Monitoramento da infecção filarial por Wuchereria bancrofti através da cinética de anticorpos com o antígeno recombinante Bm14, em áreas endêmicas da RMR-PE submetidas ao tratamento coletivo para filariose / Filarial infection monitoring by Wuchereria bancrofti through kinetic antibodies with the recombinant antigen Bm14, in endemic areas of the RMR-PE subject to collective treatment for filariasis

A Filariose Linfática (FL) no Brasil é causada pela espécie Wuchereria bancrofti e consiste em um problema de saúde pública. O principal foco ativo de transmissão atualmente no país é a Região Metropolitana do Recife - PE, que desde 2003 iniciou o Programa de Controle/Eliminação da FL, tendo como estratégia principal o Tratamento Coletivo (TC) com Dietilcarbamanzina (DEC). Este trabalho, aprovado pelo Comitê de Ética em Pesquisa do Centro de Pesquisas Aggeu Magalhães, analisou o TC nessas áreas, acompanhando 30 moradores, no período de 2003 a 2009. Para essa análise além das ferramentas tradicionais da pesquisa filarial - Filtração (MF/mL de sangue) e Antígeno Circulante Filarial (Og4C3) - também foi utilizada a pesquisa de anticorpos através de um antígeno recombinante (Bm14). Essa nova metodologia desenvolvida é recomendada para ser empregada como uma forma de avaliar o progresso dos programas de controle e eliminação da FL nas áreas sob intervenção. Os resultados obtidos indicam redução na positividade para a FL pelas três metodologias: o Bm14 reduziu de 90 por cento para 80,00 por cento, o Og4C3 de 100 por cento para 60,00 por cento e a microfilaremia (MF) de 100 por cento para 0 por cento. A análise da densidade de MF/mL de sangue e a positividade para o Bm14 revelou que o grupo com maior densidade de MF/mL no sangue (= 57 MF/mL) apresentou maior percentual de redução na positividade para o anticorpo do que o grupo de menor densidade ( 57 MF/mL) em 2009. A taxa de anticorpos-positivos apresentou um percentual de redução de 11,11 por cento no último ano. A diminuição nas taxas de positividade apresentadas pelo Bm14 e o padrão de decaimento observado na análise das Densidades Óticas média e mediana do anticorpo durante os seis anos da pesquisa indicam que o monitoramento dos anticorpos com o antígeno recombinante Bm14 foi capaz de reconhecer indivíduos infectados e também de identificar redução dos níveis de anticorpos produzidos por eles após exposição aos parasitos filariais. Sugerindo que o TC com DEC teria surtido efeito na eliminação dos vermes adultos e conseqüente desaparecimento das microfilárias da circulação sanguínea.

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.

Antibody responses of Wuchereria bancrofti patients to recombinant Brugia pahangi superoxide dismutase.

Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis.

Depressed specific and nonspecific immune responses in secondary Brugia pahangi infection in jirds.

The immune responsiveness to specific antigens or mitogens was examined in jirds after primary and secondary infections with Brugia pahangi. When spleen cells were obtained from secondarily infected jirds, their proliferative responses to mitogens such as Con A or LPS, or to specific antigens prepared from infective larvae or adult worms were significantly lower than those of spleen cells obtained from primarily infected jirds. The proliferative responses of the peripheral blood mononuclear cells obtained from animals undergoing primary and secondary infections also showed a similar tendency. The depressed proliferative responses of the secondary infected spleen cells to Con A or LPS was partially restored by removing adherent/phagocytic cells from the original cell populations. After deletion of the adherent cells, however, antigen-specific proliferative responses were not altered and remained at low level. These results suggest that at least two different mechanisms of depression, namely adherent cell-mediated antigen-nonspecific suppression and unresponsiveness of lymphocytes to filarial antigens, are induced in jirds in the secondary infection.

Isolation and concentration of microfilariae from peripheral blood of wuchereria bancrofti infected patients by density gradient centrifugation

A Ficoll-Hypaque gradient centrifugation technique was used for isolation and concentration of microfilaria from peripheral blood of 30 subjects with clinically and parasitologically diagnosed Wuchereria bancrofti infections. 86% of the microfilaria were found in the Ficoll-Hypaque layer. None were detected in the plasma, leucocyte layer or lower erythrocyte layer. 14% of microfilaria were identified on the top part of the erythrocyte layer. A 35-fold concentration and 88% quantitative recovery of parasites was achieved by conventional centrifugation of microfilaria-rich Ficoll-Hypaque layer. Following the centrifugation procedures, living motile microfilaria were separated. These results indicated that Ficoll- Hypaque centrifugation technique could be an effective method for the detection of low levels of microfilaremia, and for obtaining relatively pure suspensions of living microfilaria for metabolic studies, production of antigen-rich excretory-secretory products and antigen analysis

Course of antibody production by the DIG-ELISA method in neonatally infected and juvenile infected rats after primary infection with Breinlia booliati (Filarioidea: Onchocercidae).

The course of antibody production in Wistar neonatal and juvenile rats after primary infection with Breinlia booliati was studied by the DIG-ELISA technique using filter papers impregnated with capillary blood drawn from the infected rat tails at 7, 14, 28, 60 and 90 days post infection. Sera of neonatally infected rats did not react with adult worm antigen until day 7 and the titers of antibody remained at very low levels for the next 7 days. There was little tendency to eliminate the filarial larvae during this time. The antibody levels then rose rapidly throughout the next fortnight and increased to a maximum at day 60 after which the titer leveled out at a constant high value until early patency at day 90. On the other hand, antibodies could be detected in sera of juvenile infected rats as early as day 7 and the levels of antibody rose markedly to a maximum at day 28. During the period from day 60 to day 90 at early patency, the antibodies declined gradually to lower levels. The humoral immune responses of 42 neonatally infected rats and 53 juvenile infected rats of 3 strains (Lewis, Wistar and Sprague Dawley) were tested against soluble B. booliati antigens from both female (1:50) and male (1:10) worm extracts by the DIG-ELISA method. Antibodies were detected in sera from all the microfilaremic and amicrofilaremic rats belonging to neonatally and juvenile infected groups. Sera of clean neonatal rats did not give a positive reaction zone.

Differential recognition of Brugia malayi antigens by bancroftian filariasis sera.

Individuals residing in an area endemic to Wuchereria bancrofti infection were broadly categorised as endemic normals (EN), microfilaraemics (mf + ve) and elephantoids i.e., chronic lymphatic filariasis (EL). The immune status of these three groups was examined in terms of (i) specific antibody levels; (ii) ability to induce antibody dependent cellular cytotoxicity (ADCC) to microfilariae; and (iii) ability to recognise different microfilarial antigens by immunoblotting. All three groups of endemic residents were indistinguishable in their antibody levels as measured by ELISA with B. malayi microfilarial antigen. Many endemic normal sera and most elephantoid sera exerted strong cytotoxicity against W. bancrofti microfilariae whereas none of the mf + ve sera had any such activity. Immunoblotting studies revealed that a protein with mol. wt of 79 KDa was the only one among the proteins of B. malayi microfilarial extracts that was consistently recognised by sera from all endemic residents. Endemic normal sera and elephantoid sera, which exerted maximum cytotoxicity, together specifically recognised three proteins with molecular weights 25, 58 and 68 KDa and these three proteins could be among the candidate antigens that induce resistance to filarial infection.

Relationship of C3 & C4 levels with filarial immune complexes in bancroftian filariasis.

C3 and C4 levels were determined by radial immuno-diffusion technique and filarial circulating immune-complexes by anti-C3 ELISA in microfilaraemic individuals and patients with clinical filariasis. Decreased levels of C3 and C4 were observed in both groups of filarial patients. Low levels of complement components were associated with low levels of circulating immune complexes.

Antigenic analysis of excretory-secretory products of Wuchereria bancrofti and Brugia malayi infective larval forms by SDS-PAGE.

Excretory-secretory (ES) products of W. bancrofti and the closely related B. malayi infective larval forms were analysed for their antigenic activity by SDS-PAGE followed by Western blotting as well as by gel elution-sandwich ELISA using filarial serum immunoglobulin-G (FSIgG) as a capture antibody. In W. bancrofti infective larval ES products, the protein molecules of 66, 46, 35, 33, 30 and 14 kDa molecular wt. showed antigenic activity by immuno blotting technique. In sandwich ELISA technique eventhough all SDS-PAGE fractions except ESA 6 (55-47 kDa) showed antigenic positivity, the fractions ESA 8 (37-31 kDa) and ESA 9 (31-25 kDa) showed high reciprocal antigen titre of 262144 and 32768 respectively. In B. malayi infective larval ES products, the protein molecules of 109, 102, 97 and 77 kDa molecular wt. showed reactivity with FSIgG by blotting technique, where as in sandwich ELISA except ESA 7 (47-37kDa), all fractions showed antigenic positivity. However, these fractions failed to show high antigen titre similar to W. bancrofti ES products with FSIgG.

Antifilarial and anti PPD IgM antibodies in cord blood.

Tuberculosis and Filariasis are commonly encountered in South India, more so in the low socio-economic group. This study was undertaken to find out the fetal immune response to maternal filariasis and tuberculosis. The presence of anti-filarial and anti-PPD IgG and IgM antibodies in the mother and cord blood at the time of delivery was identified by ELISA. Six out of 29 cases showed IgM against Brugia malayi antigen in the cord blood. In both instances, 6 out of 29 for PPD and 7 out of 29 for B. malayi, the maternal blood too showed the presence of IgM to these two antigens. Comparison with anti-Ascaris lumbricoides IgM antibodies in mother and cord blood in these (6 out of 29 against PPD and 7 out of 29 against B. malayi) cases did not show a similar distribution of IgM antibodies in mother and cord blood, indicating that there is a fetal response to maternal filariasis and tuberculosis.