Diagnostic value of IgG isotype responses against Brugia malayi antifilarial antibodies in the clinical spectrum of brugian filariasis

To study the diagnostic significance of antifilarial IgG subclasses in the clinical spectrum of brugian filariasis, IgG1, IgG3 and IgG4 antifilarial antibodies were determined in an exposed population comprising 74 asymptomatic amicrofilaraemics, 30 microfilaraemics, 20 lymphangitis and 16 elephantiasis patients resident in Narathiwart province, an area endemic for Brugia malayi lymphatic filariasis in southern Thailand. The dominant isotype of antifilarial antibody was IgG4. A significantly higher percentage of individuals were positive for IgG1 in the microfilaraemic and lymphangitis groups compared with the elephantiasis and endemic normal patients, while a significantly higher positive rate of IgG3 was found in those with lymphangitis. The possible role of these isotypes for diagnostic purposes and the pattern of antibody response in various clinically manifesting groups are discussed.     

IgG antibody subclasses in human filariasis. Differential subclass recognition of parasite antigens correlates with different clinical manifestations of infection

The four subclasses of IgG are distinct in structure, function, and degree of participation in the antibody response to complex antigens. Looking for differential responsiveness of potential pathogenetic significance, we have analyzed both quantitatively and qualitatively the filaria-specific IgG subclass responses of 20 patients with lymphatic filariasis presenting either with chronic lymphatic obstructive pathology and elephantiasis (CP) or with asymptomatic microfilaremia (MF). Subclass-specific monoclonal antibodies were used in an enzyme-linked immunosorbent assay to study IgG filarial antibodies quantitatively and in immunoblot analyses to determine qualitatively the subclass antibody specificities. Quantitatively, the most significant differences among patient groups were in levels of IgG4, which were more than 17 times higher in MF patients (geometric mean, 64.7 micrograms/ml) than in those with CP (mean, 3.7 micrograms/ml). When qualitative analyses were done on the same sera, major differences were noted, particularly in the recognition profiles of the IgG1, IgG3, and IgG4 responses. IgG1 and IgG3 responses to antigens were seen especially to antigens with m.w. greater than 68,000 in all patients with elephantiasis, whereas MF patients showed most of their reactivity to antigens less than 68,000. For IgG4, the MF patients showed prominent recognition of antigens throughout the entire range of m.w., whereas those with CP had very little IgG4 recognition of antigens of any m.w. Interestingly, this relationship was essentially reversed in the IgG3 antibody responses (especially to antigens greater than 68,000) and, to a lesser extent, the IgG1 responses. These findings demonstrate correlations of potential cause/effect significance between IgG4 antibody responsiveness and the immunomodulated asymptomatic MF form of clinical filariasis and between IgG3/IgG1 antibody responsiveness and the clinical presentation of CP.     

The geographical distribution of lymphatic filariasis infection in Malawi

Background

Papua New Guinea is the only endemic country in the Western Pacific Region that has not yet introduced a countrywide programme to eliminate lymphatic filariasis. However, on Misima Island in Milne Bay Province, government and private sectors have collaborated to implement a pilot elimination programme. Although interim evaluation indicated that the programme has been parasitologically successful, an appreciation that sustainable health gains depend on understanding and accommodating local beliefs prompted this qualitative study.

Methods

We investigated Misima community members knowledge and attitudes about lymphatic filariasis and the elimination programme. A combination of focus groups and key informant interviews were used to explore participants perceptions of health; knowledge of the aetiology and symptoms of filariasis, elephantiasis and hydrocele; attitudes towards the disease and mass drug distribution; and the social structure and decision-making protocols within the villages.

Results

Focus group discussions proved inferior to key informant interviews for gathering rich data. Study participants did not consider lymphatic filariasis ("pom") a major health problem but were generally positive about mass drug administration campaigns. A variety of conditions were frequently and incorrectly attributed to filariasis. Participants expressed the belief that individuals infected with filariasis always had visible manifestations of disease. A common misconception was that taking drugs during campaigns provided long-term immunity against disease. The role of mosquito vectors in transmission was not generally appreciated and certain clinical presentations, particularly hydrocele, were associated with supernatural forces. Multiple adverse events were associated with mass drug administration campaigns and most study participants mentioned community members who did not participate in campaigns.

Conclusion

Important issues requiring educational intervention and elimination activity modification in the Misima region were identified during this study. Research outcomes should assist Papua New Guinea in developing and implementing a national elimination strategy and inform discussions regarding the appropriateness of current elimination strategies.     

Rapid assessment procedures to detect hidden endemic foci in areas not subjected to mass drug administration in Sri Lanka

For the declaration of elimination of lymphatic filariasis, reliable epidemiological data in all parts of a country are required. In Sri Lanka, due to social disturbance, there are 3 provinces whose endemicity has been declared unknown. Further, a recent report revealed an endemic pocket, which is on the border with the district that was not covered by the national elimination program. These facts indicate the necessity of more extensive studies to discover hidden endemic foci. To facilitate such studies, we evaluated 2 methods of Rapid Assessment Procedure (RAP) in Hambantota district, where the filariasis endemicity was low: (1) indirect questioning by mailing a questionnaire to each local leader (IndQ), asking about the presence of clinical cases, and (2) focus group discussion (FGD) by villagers. The information given by people was validated with clinical examination by doctors (CE) and IgG4 ELISA using urine samples. In the results: there was a strong positive correlation between CE and ELISA rates. The hydrocele rates obtained by FGD or IndQ were associated significantly with CE rates. The rates by FGD or Cluster-IndQ (‘modified’ IndQ) were also associated significantly with ELISA rates. The IndQ was most cost-effective. Based on these findings, we have concluded that screening by IndQ and confirmation by the ELISA would be an effective and practical way in Sri Lanka to locate endemic foci in hitherto unsurveyed districts.     

A survey of Brugia malayi infection on the Heugsan Islands,Korea

Lymphatic filariasis due to Brugia malayi infection was endemic in several areas of South Korea. The infection was controlled, or disappeared, in most areas, with the exception of the remote southwestern islands of Jeonranam-do, including the Heugsan Islands. To discover its current situation, a small-scale survey was performed on the Heugsan Islands in September 2000. A total of 378 people, 151 male and 227 female, living in 8 villages (6 on Daeheugsan-do, 1 on Daejang-do, and 1 on Yeongsan-do) were subjected to a night blood survey for microfilaremia, and physical examination for elephantiasis on the extremities. There were 6 (1.6%) microfilaria positive cases, all in females aged 57-72 years, and from only two villages of the Daeheugsan-do area. There were 4 patients with lower leg elephantiasis, but they showed no microfilaremia. The results show that a low-grade endemicity of filariasis remains on the Daeheugsan-do.     

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.     

Onchocerca volvulus and Wuchereria bancrofti: fluorescent antibody staining of frozen homologous sections for diagnosis

Antibody responses to two filarial diseases of man, onchocerciasis and bancroftian filariasis, were evaluated with the indirect fluorescent antibody technique (IFAT), using antigens derived from the appropriate etiologic agent. Antigenic preparations consisted of frozen cut sections of the adult Onchocerca volvulus and stage III larvae of Wuchereria bancrofti fixed to glass slides. Little difference between the preparations was demonstrated in tests for the diagnosis of onchocerciasis. Of 105 sera from individuals with biopsy-proven infections, 102 (97%) reacted with homologous O. volvulus antigen, and 19 of 22 (86%) with W. bancrofti antigen. In bancroftian filariasis, however, the homologously derived antigen was superior for diagnosis, and the highest seropositive rates occurred in acute, symptomatic infections. All such sera (8) reacted with homologous antigen. In contrast, only 75% (6) reacted with onchocercal antigen. Of those with chronic disease, characterized by long-standing elephantiasis or lymphedema without microfilaremia, 79% (22) were reactors to homologous antigen and 32% (9) to heterologous. The lowest seropositive rates occurred where microfilaremia was unaccompanied by local or systemic symptoms: 38% (3) were positive to homologous antigen and none to onchocercal antigen.Of sera from seven individuals apparently free of bancroftian filariasis, but living in a hyperendemic area, five reacted with bancroftian antigen and four with onchocercal antigen. These reactions could be attributed to occult infections, but more likely resulted from repeated exposure to nondeveloping infective larvae.Cross-reactions in nonfilarial infections were rare with either antigen, and no positive reactions occurred in sera from healthy controls.     

An analysis of in vitro B cell immune responsiveness in human lymphatic filariasis

The immunoregulatory mechanisms involved in B cell function in patients with varying clinical manifestations of bancroftian filariasis were examined by studying the ability of peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from patients with elephantiasis, asymptomatic microfilaremia (MF), and acute tropical pulmonary eosinophilia (TPE) to produce polyclonal and parasite-specific antibody in vitro, both spontaneously and in response to a mitogen (PWM) and to parasite antigen. When the spontaneous or mitogen-driven polyclonal responses were examined, all groups produced significant amounts of IgM and IgG; those with TPE produced extremely high levels. However, when in vitro parasite antigen-specific responses were examined, those with MF were unable to produce filaria-specific antibody either spontaneously or in response to PWM or parasite antigen; in contrast, patients with chronic lymphatic obstruction or TPE produced large quantities. Removal of neither adherent cells nor T8+ T cells affected the parasite-specific B cell anergy seen in those with MF. This absent or severely diminished capacity to produce antibody on parasite antigenic stimulation in patients with MF is likely responsible for the low levels of parasite-specific antibody seen in this most common clinical manifestation of bancroftian filariasis. Its inability to be reversed by the removal of "suppressor elements" suggests a state of B cell unresponsiveness to the parasite.     

Identification of Brugia malayi immunogens by an immunoproteomics approach

Filariasis remains a health problem in tropical countries. Identification of immunogens from its causative organism would lead to development of a better diagnostic test, as well as vaccine discovery to effectively prevent this disease. We applied immunoproteomics to define potential immunogens of adult Brugia malayi that were recognized by IgM, IgG1 and IgG4 in sera of patients with four distinct clinical spectra of filariasis, including endemic asymptomatic, lymphangitis, elephantiasis and microfilaremia (n=5/group). Sera of healthy individuals (n=5) from non-endemic area served as the negative control. Brugian proteins were resolved by 2-DE and subjected to 2-D Western blot analysis probed with these sera. A total of 30 immunoreactive proteins recognized by IgM, IgG1 and IgG4 in sera from all four filarial groups were identified by Q-TOF MS and MS/MS analyses. Interestingly, only three immunogens were recognized by IgM in lymphangitis, elephantiasis and microfilaremia, but not in endemic asymptomatic group. IgG1 recognized 20 immunogens in endemic asymptomatic, lymphangitis and microfilaremia (mostly in endemic asymptomatic group), but not in elephantiasis, whereas IgG4 recognized 28 immunogens in all four filarial groups (mostly in microfilaremia). This large data set is an important resource for further development of a new diagnostic test and/or vaccine for filariasis.     

Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis

BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

上海市人群Echo30病毒性脑膜炎隐性感染调查

为了了解上海市不同年龄组人群的Echo30病毒隐性感染情况及IgG抗体阳性率分布。采集上海市412份不同年龄组人群血清,用间接酶联免疫吸附试验(ELISA)检测血清中的Echo30IgG抗体。发现受检普通人群血清中Echo30IgG抗体阳性率为25.8%。其中1岁以下婴幼儿中未见抗体阳性者,15岁以下儿童抗体阳性率较低(10%～16.7%),15岁以上人群抗体阳性率水平明显升高(45.0%～46.7%)。孕妇与普通人群抗体阳性率无显著性差异。研究结果提示上海市人群中存在隐性感染者,人群通过自然感染获得免疫保护,15岁以下儿童为Echo30感染及发病的高危人群,母体传递给婴幼儿的抗体水平较低,不能为婴幼儿提供先天性免疫保护。     

IgE responses in human filariasis. IV. Parallel antigen recognition by IgE and IgG4 subclass antibodies

Immediate hypersensitivity responses are highly modulated in filariasis, and with few exceptions, the majority of infected individuals do not develop allergic manifestations. One possible mechanism for this modulated responsiveness could involve the high levels of IgG "blocking antibodies" shown to be present in filariasis and other chronic helminth infections. When immunoblot analyses were done to analyze the immunoglobulin (Ig) E and IgG antibody responses of patients simultaneously, remarkable similarity in the patterns of antigen binding was observed. In this study, the four IgG subclasses were analyzed in a similar manner in relation to IgE. The results clearly demonstrate that IgG4 was primarily responsible for this "parallel" recognition that was seen previously between IgG and IgE antibodies. These results lend additional support to the possibility that IgG4 may play an important role in modulating IgE-mediated allergic responses in vivo.     

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.     

Spatial clustering of filarial transmission before and after a Mass Drug Administration in a setting of low infection prevalence

BACKGROUND: In the global program for the elimination of lymphatic filariasis (LF) longitudinal assessment of the prevalence of microfilaremia and antigenemia is recommended to monitor the effect of mass treatment on transmission. Additional monitoring tools such as entomologic and antibody methods may be useful in identifying residual foci of infection. In this study, we characterized serologic markers of infection and exposure spatially both before and after mass treatment, in an area of initial low Wuchereria bancrofti infection prevalence. METHODS: Consenting persons in the sentinel community were tested for circulating microfilaria and antigen (by immunochromatographic test) before and after the 1st annual mass drug administration of diethylcarbamazine and albendazole. A cohort of 161 persons provided serum specimens both years that were tested for antifilarial IgG (1 and 4) antibody. Every house was mapped using a differential Global Positioning System; this information was linked to the serologic data. W. bancrofti infection in the mosquito vector was assessed with year-round collection. Multiple linear regression was used to investigate the influence of antigen-positive persons on the antifilarial antibody responses of antigen-negative neighbors. RESULTS: After mass treatment, decreases were observed in the sentinel site in the overall prevalence of antigen (10.4% to 6.3%) and microfilaremia (0.9 to 0.4%). Of the persons in the cohort that provided serum specimens both years, 79% received treatment. Antigen prevalence decreased from 15.0% to 8.7%. Among 126 persons who received treatment, antigen and antifilarial IgG1 prevalence decreased significantly (p = 0.002 and 0.001, respectively). Among 34 persons who did not receive treatment, antifilarial IgG1 prevalence increased significantly (p = 0.003). Average antifilarial IgG1 levels decreased in households with high treatment coverage and increased in households that refused treatment. Each 10-meter increase in distance from the residence of a person who was antigen-positive in 2000 was associated a 4.68 unit decrease in antifilarial IgG1 level in 2001, controlling for other factors (p = 0.04). DISCUSSION: Antifilarial antibody assays can be used as a measure of filarial exposure. Our results suggest that micro-scale spatial heterogeneity exists in LF exposure and infection. Treatment appeared to be associated with reduced exposure at the sub-community level, suggesting the need to achieve high and homogeneous coverage. Public health messages should note the benefits of having one's neighbors receive treatment with antifilarial drugs.     

Filarial-specific IgG4 response correlates with active Wuchereria bancrofti infection

The filarial-specific humoral immune response of adult residents of two areas of Papua New Guinea, differing in transmission of Wuchereria bancrofti infection was compared. The majority of residents of the village of Bonahoi, in an area where transmission of filariasis had been interrupted by a 20-year insecticide spray program to control malaria, showed no parasitologic signs of active W. bancrofti infection and were negative for both circulating phosphorylcholine Ag and peripheral blood microfilariae. In contrast, adult residents of the village of Nanaha were in an area exposed to infection, and were phosphorylcholine-Ag- and microfilariae-positive. The antibody response of these two groups to both adult worm excretory/secretory (ES) Ag and somatic antigen extract was examined to determine which components of the filarial-specific immune response were dependent on active infection. Identification of these immune responses may point to immunologic methods to evaluate control programs for lymphatic filariasis. Adults from Bonahoi were found to have significant immune responses to [35S] methionine-labeled ES Ag by immunoprecipitation and to adult somatic antigen extracts by ELISA and by immunoblotting. This result is consistent with the fact that these individuals were previously exposed to and/or infected with W. bancrofti. Similarly, residents of the endemic village had detectable immune responses to these Ag irrespective of if they were microfilaremic. The most striking immunologic difference observed between the two groups was that residents of Bonahoi had a dramatically reduced filarial-specific IgG4 antibody response to both adult somatic Ag and adult ES Ag. These data suggest that longitudinal measurement of filarial-specific IgG4 levels may be a useful seroepidemiologic indicator of changes in W. bancrofti infection status.     

Seroprevalence to the circumsporozoite protein peptide antigen of Plasmodium vivax in Korean children

Recently, malaria re-emerged in the Republic of Korea (ROK), but there have been only a few reports on malaria seroprevalence. For the epidemiological study in children, a total of 1,176 serum samples were obtained from children and adolescent inhabitants from the three different regions, Pajoo, the Guro district in the western part of Seoul (Guro), and Ansan, from June to September 2002, when the transmission rate was high. Anti-circumsporozoite protein (CSP) antibody levels were assessed in samples using an indirect enzyme-linked immunosorbent assay (ELISA) method. Among the three regions, the highest IgG seroreactivity against the CSP antigen of Plasmodium vivax was found in the children from Pajoo (8.0%), followed by the children from Guro (3.2%), and those in Ansan (0.02%) had the lowest seroreactivity. In Pajoo, the profile of antibody response showed the highest in age 9, but decreased with age towards 12 years old. We found significant correlation between the seroprevalence and annual incidence in the investigated areas, suggesting that the area-related patterns reflected the differences of inoculation rates in children.     

Age of appearance of IgG, IgM, and IgE antibodies specific for Loa loa in Gabonese children

IgG, IgM, and IgE antibodies against the filaria Loa loa were measured in umbilical cord blood and in blood from young Gabonese children by an ELISA technique using a homologous metabolic antigen. For children in eight consecutive age groups and adults the percentage of the population positive for each of the antibody classes was determined. The number of children with maternal IgG decreased until one year of age when new synthesis began to become apparent. IgM antibodies were detected only after six months, probably indicating an early infancy as opposed to a fetal infection. The percentage of individuals positive for IgM or IgE reached a peak between two and three years old, followed by a slight decline. Over half of the individuals over one year of age had IgM antibody against L. loa, indicating long-term synthesis of this class of immunoglobulin in many people. In the first two years of life, IgE antibodies were usually accompanied by L. loa-specific IgM. This specific IgE did not appear to trigger the synthesis of nonspecific IgE. By the age of two, 95% of the population had some antibodies against L. loa and by five the percentage of individuals positive for each antibody class had reached adult levels.     

Lymphatic filariasis in children: clinical features, infection burdens and future prospects for elimination

Lymphatic filariasis (LF), a common parasitic infection in tropical countries, causes lymphoedema of limbs, hydrocele and acute attacks of dermato-lymphangio-adenitis. Recent advances in diagnosis have helped to recognize that LF infection is often acquired in childhood. Newly available diagnostic techniques like sensitive antigen and antibody assays, Doppler ultrasonography and lymphoscintigraphy have helped to understand the subclinical pathology caused by this infection, which was hitherto generally believed to be irreversible. Recent studies indicate that drugs used in the mass drug administration (MDA) programme under GPELF are capable of reversing the sub-clinical lymphatic damage in children and provide benefits other than interruption of transmission. Albendazole and ivermectin used in MDA are effective against soil-transmitted helminthic infections common in children in LF endemic areas. Thus MDA had other 'beyond LF' benefits in treated children including increased appetite, weight gain, greater learning ability and concentration, better school attendance and prevention of anaemia. MDA should no longer be viewed as a measure for interrupting transmission alone. Recent findings of reversibility of early lymphatic pathology in treated children indicate that both MDA and 'foot-hygiene' measures are effective strategies in preventing and managing morbidity. Programme managers should effectively utilize this information to strengthen their advocacy efforts to achieve high and sustainable coverage in MDA.     

T-cell activation and the balance of antibody isotypes in human lymphatic filariasis

Human filarial infection presents a spectrum of clinical states with two major poles: asymptomatic microfilaraemia and amicrofilaraemic chronic disease. Microfilaremia is associated with a Th1-type tolerance, and maximal IgG4 antibodies, while elephantiasis patients react across a broad range of immune parameters. In this review, Rick Maizels and his colleagues discuss recent advances in the immunology of human filariasis and present a summary of their latest studies in an endemic area of Indonesia.