Immunomodulation of allergic response in children and adolescents: What we can learn from lymphatic filarial infection.

BACKGROUND: Although it is well known that allergic diseases involve a strong Th2 immune response, with production of high levels of specific IgE allergen, knowledge on the association between filarial infection and allergies, among paediatric patients is scarce. OBJECTIVE: To evaluate the allergic response patterns in cases of filarial infection by comparing peripheral eosinophils, total IgE levels, immediate hypersensitivity and cytokine levels in children and adolescents in Brazil. METHODS: This was an exploratory study with three groups: (I) with filarial infection and without allergic diseases; (II) without filarial infection and with allergic diseases; and (III) without filarial infection and without allergic diseases. The prick test and specific IgE tests for aeroallergens were performed using five antigens. Peripheral eosinophils and total IgE were also evaluated. IL-4 and IL-5 were determined using whole-blood culture stimulated by three antigens. RESULTS: Eosinophilia and elevated levels of total IgE (≥400IU/dl) were observed in all groups. The prick test was positive in 56.6% of the cases. Group I presented hypersensitive responses similar to the allergic disease groups. In the whole-blood culture stimulated by Dermatophagoides pteronyssinus, average IL-4 production did not differ significantly among the groups, but IL5 production resulting from stimulation was greater in the allergic disease groups (p<0.05). CONCLUSIONS: The allergic response pattern in group with filarial infection was similar to that of the groups with and without allergic diseases, but the response to IL-5 in the culture stimulated by D. pteronyssinus was an exclusive characteristic of the allergic group.

Screening and evaluation of lymphatic filariasis in immigrants from endemic countries residing in a focus where it is considered eliminated in the Southern Region of Brazil: A risk of reemergence?

Lymphatic filariasis (LF) has been targeted by the World Health Organization for elimination by the year 2020. However, migration of infected individuals from areas where LF is endemic to areas considered non-endemic or foci for the control and elimination may jeopardize the achievement of this goal. The aim of the present study was to evaluate the occurrence of filarial infection by way of circulating filarial antigen (CFA) circulation using the point of care AD12-immunochromatography card (POC-ICT) among immigrants from Haiti residing in Chapecó, Santa Catarina, between May and October 2015. Of the 420 subjects examined, 77.4% were male, aged 19-54 years. Ten (2.38%) were POC-ICT positive. Of this total, one was not found. Two individuals were negative for Og4C3-ELISA and DNA/Wb-PCR in all biological samples, but positive for the anti-filarial antibody Bm14 and only one showed microfilaremia (1mf/mL). These findings point to the importance of the Brazilian surveillance action to reduce the possibility of reintroduction of LF in Chapecó, Santa Catarina, by infected immigrants, and to guarantee the success of the National LF Elimination Plan.

Measuring changes in transmission of neglected tropical diseases, malaria, and enteric pathogens from quantitative antibody levels.

BACKGROUND: Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. METHODS/PRINCIPAL FINDINGS: We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman's rho = 0.75). In both high- and low transmission settings, mean antibody curves revealed changes in population mean antibody levels that were masked by seroprevalence measures because changes took place above or below the seropositivity cutoff. CONCLUSIONS/SIGNIFICANCE: Age-dependent antibody curves and summary means provided a robust and sensitive measure of changes in transmission, with greatest sensitivity among young children. The method generalizes to pathogens that can be measured in high-throughput, multiplex serological assays, and scales to surveillance activities that require high spatiotemporal resolution. Our results suggest quantitative antibody levels will be particularly useful to measure differences in exposure for pathogens that elicit a transient antibody response or for monitoring populations with very high- or very low transmission, when seroprevalence is less informative. The approach represents a new opportunity to conduct integrated serological surveillance for neglected tropical diseases, malaria, and other infectious diseases with well-defined antigen targets.

Longitudinal monitoring of the development of antifilarial antibodies and acquisition of Wuchereria bancrofti in a highly endemic area of Haiti.

Antifilarial antibody testing has been established as a sensitive and specific method of diagnosing lymphatic filariasis. However, the development of serological responses to specific filarial antigens and their relationship to acquisition of infection is poorly understood. In order to evaluate whether the development of antigen specific antifilarial antibodies precedes microfilaremia and antigenemia, we compared the antibody responses of serum samples collected between 1990 and 1999 from a cohort of 142 Haitian children followed longitudinally. Antigen status was determined using the Og4C3 ELISA and the presence of microfilaremia was detected using microscopy. Antibody responses to Wb123, a Wuchereria bancrofti L3 antigen, were measured using a Luciferase Immunoprecipitation System (LIPS) assay. Antibody responses to Bm14 and Bm33, Brugia malayi antigens and to a major surface protein (WSP) from Wolbachia were analyzed using a multiplex bead assay. Over follow-up, 80 (56%) of the children became antigen-positive and 30 (21%) developed microfilaremia. Detectable antibody responses to Bm14, Bm33, Wb123, and WSP developed in 95%, 100%, 92%, and 29% of children, respectively. With the exception of WSP, the development of antibody responses generally preceded detection of filarial antigen. Our results show that antifilarial antibody responses can serve as an important epidemiological indicator in a sentinel population of young children and thus, may be valuable as tool for surveillance in the context of lymphatic filariasis elimination programs.

[Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae]. / Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti.

A study of protein characterization and recognition of the antigenic profile was accomplished in extracts of infective larvae (L3) from Wuchereria bancrofti. Two proteins of relative molecular weight of 49 and 55 kDa were recognized as antigenic in all extracts by the tested sera. The secretory/excretory antigen presented the largest number of recognized bands (105, 100, 76, 55, 49, 39 and 32 kDa) followed by the somatic antigen (100, 76, 55 and 49 kDa) when incubated with pools of sera from healthy individuals resident in endemic areas (normal endemics). Human sera and parasitized blood used to infect mosquitoes in order to obtain L3, were collected from microfilaraemic individuals living in a filariasis endemic area. From 792 persons screened with the thick smear technique, 87 (11%) were positive. No statistical significance was observed between genders. The group between 11 and 19 years of age presented higher percentage of infection (36.8%).

Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti / Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae

A caracterização protéica dos extratos de larvas infectantes (L3) de Wuchereria bancrofti foi realizada por eletroforese em gel de poliacrilamida, em presença de dodecil sulfato de sódio (SDS-PAGE) e o reconhecimento antigênico das proteínas por Western-blot. O maior número de bandas protéicas reconhecidas foi evidenciado nos extratos AgSE (105, 100, 76, 55, 49, 39 e 32 kDa) e AgS (100, 76, 55, e 49 kDa) na presença de soros de indivíduos endêmicos normais. As bandas de 49 e 55 kDa foram reconhecidas pelos soros dos microfilarêmicos, endêmicos normais (residentes de área endêmica livres de infecção filarial) e portadores da forma crônica da doença. As larvas infectantes foram obtidas pela dissecção de mosquitos Culex quinquefasciatus infectados com sangue microfilarêmico de voluntários portadores de microfilaremia, residentes do Município de Olinda-PE. Nos 792 indivíduos investigados pela técnica da gota espessa mensurada (60æl de sangue) 87 foram positivos (11 por cento). A diferenca da positividade entre homens e mulheres não foi significativa e a faixa etária de 11 a 19 anos foi a de maior prevalência.

Characterization of antibody responses to Wolbachia surface protein in humans with lymphatic filariasis.

Symbiotic Wolbachia organisms of filarial nematodes have received much attention as possible chemotherapy targets and disease-causing organisms. In order to further investigate the association between anti-Wolbachia immune responses and chronic filarial disease in humans, antibody responses to Wolbachia surface protein (WSP) were assayed in serum samples collected from 232 individuals living in Leogane, Haiti, an area where Wuchereria bancrofti infection is endemic, and from 67 North Americans with no history of lymphatic filariasis. As opposed to antifilarial antibody responses, which were largely influenced by the patient's infection status, the prevalence and levels of anti-WSP immunoglobulin G (IgG) antibodies among individuals with lymphedema or hydrocele were significantly greater than those in gender- and infection-matched individuals without disease. In at least one case, the anti-WSP IgG response was coincident with the onset of lymphedema development, and among anti-WSP-positive women with lymphedema, anti-WSP IgG levels were negatively correlated with the duration of lymphedema. The presence of anti-WSP IgG was also associated with the severity of inguinal adenopathy among men with hydrocele. In addition to the presence of anti-WSP antibodies among Haitians, 15 of 67 (22%) serum samples collected from individuals from North America, where filariasis is not endemic, were also positive for anti-WSP antibodies. In comparison to those from Haitians, anti-WSP antibodies from North Americans primarily recognized a distinct region of WSP located within the highly conserved second transmembrane domain. The results of this study demonstrate that anti-WSP antibody responses are associated with the presence of chronic filarial morbidity and not filarial infection status in humans and suggest that WSP should be further studied as a potential trigger for the development of filarial disease.

Influence of infection with non-filarial helminths on the specificity of serological assays for antifilarial immunoglobulin G4.

Serological assays based on the detection of immunoglobulin (Ig) G4 antibodies to crude filarial extracts are widely used for epidemiological and diagnostic purposes. We tested 195 samples collected in 1998 from an area of Brazil where filariasis is not endemic and 13 (6.7%) had levels of antifilarial IgG4 antibodies that were defined as positive. Both Strongyloides infection and the presence of Strongyloides antibody responses were associated with higher antifilarial antibody responses. None of the specimens had a positive response to the Brugia malayi recombinant antigen (Bm14). These data suggest that serodiagnostic assays based on the use of crude filarial antigens should be interpreted with caution because of the potential for cross-reactivity with Strongyloides.

Reactivity to bacterial, fungal, and parasite antigens in patients with lymphedema and elephantiasis.

Both secondary infections and antifilarial immunity are thought to play roles in the development and progression of lymphedema. To investigate this issue, immune responses to a panel of bacterial, fungal, and parasite antigens were examined for women with lymphedema and elephantiasis (n = 28) and for women with no clinical evidence of lymphatic dysfunction who were either microfilaremic (Mf+, n = 23) or microfilaria- and filarial antigen-negative (Ag-, n = 24). The prevalence and intensity of delayed-type hypersensitivity (DTH) responses was similar for most recall antigens; for individual antigens, lymphedema patients were significantly more likely to be reactive only to Proteus. Lymphedema patients with a history of three or more attacks of adenolymphangitis in the last 18 months showed increased DTH reactivity to Trichophyton. Proliferative responses to fungal and bacterial antigens were similar for all three groups; however, antigen-negative women, independent of disease status, mounted greater responses to filarial antigen. In contrast, lymphedema patients had higher levels of antifilarial specific IgG1, IgG2, and IgG3 and higher IgG responses to streptolysin O than either Ag- or Mf+ women. In persons with lymphatic filariasis, immune reactivity is influenced by disease status as well as infection status.

The pathogenesis of filarial lymphedema: is it the worm or is it the host?

Our understanding of the pathogenesis of filarial lymphedema, although evolving, is still limited. Recurrent bacterial infections play a major role in the progression of lymphedema to elephantiasis, but the host and parasite factors that trigger disease development are not known. Field studies in Haiti show that lymphedema and host responses to parasite antigens cluster in families, consistent with the hypothesis that host genes influence lymphedema susceptibility. The recent recognition that filarial parasites harbor the endosymbiotic bacteria, Wolbachia, also raises questions about the potential contribution of the inflammatory response to Wolbachia antigens to lymphedema development. In this review, we discuss potential risk factors for lymphedema and try to integrate these in a model of pathogenesis.

Immunocytochemical localization of antigens recognized by asymptomatic microfilaremic patient's antisera in microfilariae of Wuchereria bancrofti.

Ultrathin sections of microfilariae of Wuchereria bancrofti embedded in hydrophilic resin were incubated with sera from patients, using antisera from asymptomatic microfilaremic patients with different microfilarial densities [1-100 microfilariae (mf)/ml, 101-500 mf/ml, > 1,000 mf/ml]. All groups studied showed reactivity against relevant epitopes in all tissues of microfilariae of W. bancrofti, instead of being localized in a specific nematode region, although the number of colloidal per square micron was inversely proportional to the microfilaremia. Such results confirm data obtained by other authors and indicate a possible role for the humoral response in the mechanism for the destruction of circulating microfilariae.

[Tropical filarial pulmonary eosinophilia and its differential diagnosis]. / Eosinofilia pulmonar tropical filariótica e o seu diagnóstico diferencial.

The authors present a comprehensive review of Tropical Pulmonary Eosinophilia (TPE) of filarial etiology and describe its differential diagnosis with similar syndromes. Epidemiological, clinical, diagnostic, therapeutic and phisiopathological aspects are considered, with an emphasis on new advances in our knowledge of lymphatic filariasis and their implication for improved understanding of TPE and similar syndromes. A TPE-like syndrome, which is caused by intestinal helminth infections, occurs in filariasis-endemic and non-endemic areas alike. The authors suggest guidelines for interpreting epidemiological, clinical, laboratory, radiologic (including ultrasonographic) and therapeutical data and properly diagnosing TPE syndromes. This guidelines also should be useful for physicians in areas where filariasis is not endemic but to which patients from endemic area (e.g., Greater Recife-PE, Maceió-AL and Belém-PA) frequent migrate.

Filariasis and erisipela in Santo Domingo.

This study examined acute-convalescent changes in diagnostic anti-streptococcal antibodies by the anti-streptolysin O (ASO) and anti-DNAase B (ADAB) tests among patients (n 28) with lymphedema and recurrent erisipela of the lower limb, comparing them with endemic normal control residents (n=25). The study was based in Villa Francisca, an urban focus of Bancroftian filariasis in eastern Santo Domingo, capital of the Dominican Republic. The acute signs and symptoms of erisipela were consistent with a diagnosis of bacterial cellulitis. The ASO test was especially successful at demonstrating a rise in mean titer during convalescence, whereas the ADAB produced about the same frequency of significant increases (0.2 log titer) as did the ASO. When subjects were scored as responders if mounting a minimal titer increase by either test, patients were found more frequently positive than were controls (chi2=5.3, P=0.02). About half (54%) of all patients mounted at least a minimal antibody increase. Filaria-specific IgG4 antibodies were absent from all sera of 20 residents of a nonendemic Dominican mountain town but appeared in about two-thirds of the sampled residents of the endemic barrio. Notably however, levels did not change between the acute phase and convalescence. These findings are consistent with the hypothesis that recurrent streptococcal invasion of the lymphatics may be a significant factor triggering or amplifying lymphedema and elephantiasis in patients with chronic filariasis.

The presence or absence of active infection, not clinical status, is most closely associated with cytokine responses in lymphatic filariasis.

Twenty-eight Brazilians from an area in which Wuchereria bancrofti is endemic were classified as asymptomatic microfilaremic or having clinical filariasis with active infection or without current active infection. Total accumulation of antigen-specific interleukin (IL)-4 and IL-5 in 48 h peripheral blood mononuclear cell supernatants was not significantly different between groups. However, when cytokine kinetics were examined, responses segregated according to infection status. Sustained production of IL-4 and IL-5 beyond the first 24 h of stimulation and production of interferon-gamma were seen only in the group with clinical filariasis without active infection. CD8 T cells were the major source of IL-5 production in this group, while CD8 production of IL-5 was undetectable in any subject with active infection (asymptomatic microfilaremic or with clinical filariasis and active infection). These findings indicate that active infection, rather than clinical status, is most closely associated with cytokine patterns in lymphatic filariasis.

Immunocytochemical localization of surface and intracellular antigens recognized by human sera in microfilariae of Wuchereria bancrofti.

Sera from patients with various clinical pictures of lymphatic filariasis, including tropical pulmonary eosinophilia (TPE), were used for the localization of surface and intracellular antigens in microfilariae of Wuchereria bancrofti embedded in Lowicryl K4M. Very few or no antigenic sites were located on the outer face of the sheath. The most inner layer, as well as the space between the cuticle and the sheath, was intensely labeled. Sera from TPE patients intensely labeled the cuticle and the cytoplasm of muscle cells.

Analysis of isotype-specific antifilarial antibody levels in a Haitian pediatric population.

Previous studies of antifilarial antibodies in a pediatric population residing in an area with endemic Wuchereria bancrofti filariasis have demonstrated age related shifts in antifilarial immunity. To further characterize humoral responses in Haitian children, serum samples from 129 patients (3 months-15 years of age) were analyzed by ELISA for isotype-specific antifilarial antibody responses. Age-stratified analysis of geometric mean antibody titers showed significant increases in antibody titers of all isotypes with age in the amicrofilaremic population. Antifilarial IgG1, 2, and 3 levels were higher in amicrofilaremic children than in microfilaremic children, significantly so for IgG2 and IgG3. In contrast, IgG4 antibody levels were higher in microfilaremic subjects than in amicrofilaremic subjects. A multivariate, unconditional, logistic regression model was developed from these data to predict infection status. The model correctly classified 91.6% of the amicrofilaremic subjects, but only 55.6% of the microfilaremic subjects.

Heightened anti-filarial immune responsiveness in a Haitian pediatric population.

The immunological consequences of exposure to filarial infection were examined by cross-sectional serological studies. Serum samples from 121 pediatric patients (18 months-15 years of age) were analyzed in parallel with a panel of sera from adults residing in the same area of Haiti. Parasite antigen specific IgG and IgE levels were determined by ELISA. IgG levels in children were significantly elevated in humoral immunoreactivity to Brugia pahangi extracts compared to adults. In addition, anti-filarial IgG levels in amicrofilaremic children were significantly greater than in microfilaremic children. In contrast, IgG levels in adults were equivalent independent of microfilaremic status. Anti-filarial IgE levels in sera from both children and adults were low in comparison to that of a subject with tropical pulmonary eosinophilia and were unrelated to clinical status. No correlations were found between humoral responses and age, sex, or degree of parasitemia. Sera from amicrofilaremic children and, to a lesser extent, adults recognize more antigens, particularly those of high molecular weight (greater than 55 kDa), than sera from microfilaremic patients.

Manifestaçoës clínicas da filariose linfática bancroftiana / Clinical manifestations of lymphatic bancroftian filariasis

Os autores inicialmente detêm-se, de forma sucinta, nos aspectos básicos da filariose bancroftiana, envolvendo principalmente a biologia do parasita e os aspectos imunológicos e patogênicos. Em seguida, na base de suas experiências pessoais e dos dados da literatura, sugerem uma classificaçäo geral das formas clínicas da infecçäo, cujo amplo espectro distribuem em seis grupos: 1§) indivíduos endêmicos "normais" (negativos endêmicos); 2§) indivíduos microfilarêmicos assintomáticos; 3§) pacientes com manifestaçöes agudas (ex. linfangite, orquiepididimite); 4§) pacientes com manifestaçöes crônicas (ex. edema, elefantíase, hidrocele, quilúria); 5§) pacientes com eosinofilia pulmonar tropical (EPT); 6§) pacientes com formas frustas ou controversas. Em apítulos sucessivos, os autores procuram descrever, de forma sumária, em cada grupo de pacientes, os aspectos pertinentes a clínica e ao diagnóstico, assinalando em cada caso as prováveis respostas imunes do hospedeiro. Finalizando, os autores tecem algumas consideraçöes sobre o tratamento da infecçäo

[Clinical manifestations of lymphatic bancroftian filariasis]. / Manifestações clínicas da filariose linfática bancroftiana.

A review of clinical manifestations of bancroftian filariasis, based on the literature and in the authors' experience, suggests a general classification in to six groups: 1) Normal endemics; 2) Individuals bearing microfilaremia; 3) Acute manifestations; 4) Chronic manifestations; 5) Tropical Pulmonary Eosinophilia; 6) Controversial forms. An overview on diagnostic procedures and the immunologic relationship between host and parasite was made in each group. Final considerations about treatment was also considered.

Bancroftian filariasis in Haiti: preliminary characterization of the immunological responsiveness of microfilaremic individuals.

Patent infections with the lymphatic filariae, Wuchereria bancrofti and Brugia malayi, are associated with suppressed in vitro cellular responsiveness to filarial antigens. In studies of bancroftian filariasis in Haiti, a significant number of microfilaremic individuals can be characterized as "responders" to filarial antigens. Cells from 37/74 untreated microfilaremic subjects responded to B. pahangi antigen (stimulation ratio greater than 2) as detected by in vitro blastogenesis. A comparison of responders to nonresponders revealed a significant difference in mean B. pahangi reactivity (15,822 vs. 4,538 cpm, P less than 0.001), but no significant differences with respect to age, microfilaremia, PPD or PHA reactivity, or B. pahangi-specific antibody levels. Subtle differences may exist between these groups with respect to recognition of specific antigens on Western blots.