Pathogenesis of lymphatic disease in bancroftian filariasis: a clinical perspective.

The pathogenesis of lymphatic filariasis has been a matter of debate for many decades. Here, Gerusa Dreyer and colleagues propose a dynamic model of bancroftian filariasis, integrating clinical, parasitological, surgical, therapeutic, ultrasonographic and histopathological data. This model has profound implications for filariasis control programs and the management of the individual patient.

Evaluation of PCR-based methods for the diagnosis of infection in bancroftian filariasis.

The value of the polymerase chain reaction (PCR) in the diagnosis of Wuchereria bancrofti infection was evaluated in comparison to microscopical examination of night blood smears, Nuclepore filtration, serology and ultrasonography. No correlation was found between PCR-based deoxyribonucleic acid (DNA) probing and serology. We did not find any evidence of free filarial DNA in either blood plasma or chylocoele fluid. We conclude that the 2 PCR-based techniques evaluated are not more sensitive than Nuclepore filtration for detection of W. bancrofti microfilaraemia, need at least 1 intact microfilaria in the volume of blood used for DNA extraction, and were much inferior to ultrasonography for detection of amicrofilaraemic adult worm carriers.

Extralymphatic disease due to bancroftian filariasis.

Infection with Wuchereria bancrofti, Brugia malayi, or B. timori not only affects the structure and function of lymphatic vessels but is also associated with extralymphatic pathology and disease. Because it is now possible to detect living adult worms by ultrasonography, much emphasis is placed on lymphatic pathology. However, the finding of renal damage in asymptomatic microfilaremic carriers has led to increased recognition of the importance of extralymphatic clinical manifestation in bancroftian filariasis. The authors present a number of clinical syndromes that may be manifestations of extralymphatic filarial disease and discuss possible mechanisms that cause these conditions. The main purpose of this paper is to raise the awareness of students and physicians of the prevalence and the importance of extralymphatic disease in bancroftian filariasis so that it is diagnosed and treated properly and also to alert for the need of additional research in this area.

Extralymphatic disease due to bancroftian filariasis

Infection with Wuchereria bancrofti, Brugia malayi, or B. timori not only affects the structure and function of lymphatic vessels but is also associated with extralymphatic pathology and disease. Because it is now possible to detect living adult worms by ultrasonography, much emphasis is placed on lymphatic pathology. However, the finding of renal damage in asymptomatic microfilaremic carriers has led to increased recognition of the importance of extralymphatic clinical manifestation in bancroftian filariasis. The authors present a number of clinical syndromes that may be manifestations of extralymphatic filarial disease and discuss possible mechanisms that cause these conditions. The main purpose of this paper is to raise the awareness of students and physicians of the prevalence and the importance of extralymphatic disease in bancroftian filariasis so that it is diagnosed and treated properly and also to alert for the need of additional research in this area.

[Studies on recombinant chitinase and SXP-1 antigens as antimicrofilarial vaccines].

AIM: To determine whether immunization with recombinant filarial chitinase or a fragment containing the epitope recognized by McAbMF1 and SXP-1 could protect jirds against microfilaremia resulting from infection with B. malayi. METHODS: Test jirds were immunized with the following recombinant parasite antigens: filarial chitinase, the c-terminal fragments F7R2 or F8R2 of r-chitinase, filarial SXP-1, myosin or maltose binding protein (MBP). Employing immunochemical techniqe (SDS-PAGE, Western Blotting) and serology (ELISA) measured antifilarial antibodies level. RESULTS: Immunization of jirds with recombinant chitinase induced partial protection against microfilaremia resulting from subsequent infection with B. malayi, but did not reduce adult worm burdens. Vaccination was much less effective when administered during the prepatent stage of infection and was ineffective when given to microfilaremic jirds. Immunization of jirds with SXP-1, an antigen present in multiple worm stages also reduced microfilaremia and, in some experiments, adult worm burdens. CONCLUSION: The recombinant chitinase, fragments F7R2 and F8R2 and SXP-1 could provide partial protection against microfilaremia in jirds.

Acute attacks in the extremities of persons living in an area endemic for bancroftian filariasis: differentiation of two syndromes.

The natural history of lymphatic disease in human filariasis remains unclear, but recurrent episodes of acute lymphangitis are believed to constitute a major risk factor for the development of chronic lymphoedema and elephantiasis. Prospective analysis of 600 patients referred to the filariasis clinic of the Centro de Pesquisas Aggeu Magalhães/FIOCRUZ in Recife, Brazil, indicated that 2 distinct acute syndromes accompanied by lymphangitis occur in residents of this filariasis-endemic area. One syndrome, which we call acute filarial lymphangitis (AFL), is caused by the death of adult worms. It is relatively uncommon in untreated persons, usually is asymptomatic or has a mild clinical course, and rarely causes residual lymphoedema. The second syndrome, of acute dermatolymphangioadenitis (ADLA), is not caused by filarial worms per se, but probably results from secondary bacterial infections. ADLA is a common cause of chronic lymphoedema and elephantiasis in Recife as well as in other areas of Brazil where lymphatic filariasis is not present. The syndromes of AFL and ADLA can be readily distinguished from each other by simple clinical criteria.

Abnormalities of the leg lymphatics are not specific for bancroftian filariasis.

Studies using conventional angiography or non-invasive scintigraphy have revealed widespread abnormalities in the lymphatics of the legs of patients with bancroftian filariasis, regardless of whether clinical lymphoedema is present. To determine if the observed changes were specific for filarial infections, we imaged the lymphatics of both legs in native residents of an area in Brazil where filariasis is not endemic. Study participants were matched by age, socioeconomic status and physical activities to patients with filariasis in Recife, evaluated in parallel. Based on textbook criteria, only one of 15 study participants had a completely normal lymphoscintigram. Modest to severe pathology of the leg lymphatics was observed in the remaining 14 residents of the non-endemic area and in 49 of 50 patients with bancroftian filariasis. These results indicated that factors other than filarial worms are a common cause of subclinical pathology of the leg lymphatics in north-eastern Brazil, and that the latter is not specific for bancroftian filariasis.

Evaluation of recombinant chitinase and SXP1 antigens as antimicrofilarial vaccines.

Prior studies indicate that a microfilarial stage-specific chitinase is a possible candidate antigen for a transmission-blocking vaccine against Brugian filariasis. The antigen is a functional enzyme that progressively appears as microfilariae mature and become able to infect and develop in a susceptible mosquito vector. It is recognized by a monoclonal antibody that reduces microfilaremia in infected animals and by a subset of sera from infected persons who remain amicrofilaremic. Immunization of jirds with recombinant chitinase induced partial protection against microfilaremia resulting from subsequent infection with Brugia malayi, but did not reduce adult worm burdens. Vaccination was much less effective when administered during the prepatent stage of infection and was ineffective when given to microfilaremic jirds. The protective epitope appears to be located close to the carboxy terminus of the chitinase molecule. Immunization of jirds with SXP1, an antigen present in multiple worm stages, also reduced microfilaremia and, in some experiments, adult worm burdens, but hyperimmunization with a recombinant filarial myosin was not protective. These observations indicate that the relative timing of immunization and infection is an important factor in the efficacy of antimicrofilarial vaccines.

B-cell outgrowth and ligand-specific production of IL-10 correlate with Th2 dominance in certain parasitic diseases.

In many parasitic infections, dominant T helper cell (Th) type-2 CD4+ T cell responses exacerbate the disease. We have previously demonstrated that lacto-N-fucopentaose-III (LNFPIII), a sugar found on soluble egg antigens (SEA) of Schistosoma mansoni, stimulates splenic B cells from parasite-infected mice to proliferate and produce IL-10, a cytokine that promotes the generation of Th2 immune responses. In the present study, we extend our observations on ligand-specific activation of IL-10 producing B cells to leishmaniasis and lymphatic filariasis. We report here that infection with Leishmania major increases the splenic B220+ B cell subset in BALB/c mice, but not BALB/c. xid (lacking B-1 cells and carrying defective B-2 cells). In addition, these B cells secrete large amounts of IL-10 in vitro in response to stimulation with soluble leishmanial extract (LSE), LNFPIII, or SO4-Lewis(x). We also observed that injection of LSE increased the level of peritoneal exudate (PeC) B-1 cells (CD5+B220+) in BALB/c mice, but not C57BL/6, as compared to buffer-injected controls. Further, LSE elicited PeC B cells secreted IL-10 in response to LSE as well as to the sugars tested. A similar differential secretion of IL-10 by splenic B cells from BALB/c and BALB/c.xid was seen after S. mansoni infection. Likewise, injection of soluble microfilarial extract (MFX) resulted in an increase in percentage of PeC B-1 cells in BALB/c mice, but not C57BL/6, and these cells secreted IL-10 in response to stimulation with MFX or phosphorylcholine (PC). Collectively, these results suggest a correlation between expansion of ligand-specific IL-10 producing B and B-1 cells with dominance of Th2-type T cells in mice with the susceptible phenotype for these diseases.

Studies on the periodicity and intravascular distribution of Wuchereria bancrofti microfilariae in paired samples of capillary and venous blood from Recife, Brazil.

We examined the periodicity and intravascular distribution of Wuchereria bancrofti microfilariae (mf) and determined the effect of these parasite properties on the accuracy of blood filming and filtration methods for diagnosis of bancroftian filariasis in the endemic area of Recife, Brazil. Microfilariae in both venous and capillary blood exhibited a nocturnal periodicity pattern with a relatively high amplitude. Overall, capillary blood contained approximately 1.25 times the number of mf present at the same time in the same volume of venous blood. However, the ratio of mf present in capillary and venous blood varied over a 24-hour period, so that the fewest mf were present in the capillary bed of the skin at the time when biting activity of the local Culex vector is the lowest. Twenty or 60 microliters blood films did not reliably detect carriers with fewer than 100 or 60 mf/ml venous blood, respectively, and were thus inadequate for the identification of low density mf carriers. In contrast, all carriers with > 1 mf/20 or 60 microliters blood smear at night could be identified during daytime hours by filtration of 1 micromilligram venous blood.

Histological evidence for adulticidal effect of low doses of diethylcarbamazine in bancroftian filariasis.

The ability of diethylcarbamazine (DEC) to kill adult Wuchereria bancrofti worms was evaluated by examining lymphatic nodules formed after treatment with 4 different treatment schedules of 193 males living in the endemic area of Greater Recife, Brazil. Lymphatic nodules appeared in the spermatic cord or upper extremities in 43 of 138 microfilaraemic individuals, in 3 of 30 amicrofilaraemic patients with filarial disease manifestations, and in 1 of 25 asymptomatic amicrofilaraemic residents of the endemic area treated with DEC. Fourteen of these nodules were surgically removed 10-150 d after the start of treatment. Regardless of the DEC dosage and schedule used, all nodules contained damaged and degenerating adult worms. An exuberant granulomatous process with large numbers of eosinophils and progressive fibrosis gradually developed around the dead parasites. The mechanism(s) by which DEC killed adult W. bancrofti could not be determined.

Lymphatic pathology in Wuchereria bancrofti microfilaraemic infections.

To determine the extent of lymphatic disease in persons infected with Wuchereria bancrofti who were microfilaraemic, we examined the superficial lymphatics of the legs by scintigraphy. In 4 endemic control subjects and in 10 of 14 clinically asymptomatic microfilaraemic individuals, lymphoscintigraphy revealed one major channel of lymphatic drainage in each leg. However, while trunk lymphatics were bilaterally symmetrical in the control, marked differences in the calibre of lymphatic vessels were observed in the microfilaraemic persons. Non-discrete lymphatics and a diffuse symmetrical distribution of collateral vessels in both legs were observed in all of 5 amicrofilaraemic patients with grade 2 lymphoedema. A similar diffuse drainage pattern was also seen in 3 previously microfilaraemic persons who had remained amicrofilaraemic and asymptomatic following treatment with diethylcarbamazine citrate (DEC). Thus, clearance of microfilaraemia by DEC therapy did not appear to reverse the type of lymphatic pathology observed in microfilaraemic subjects. The lymphoscintigraphy patterns did not correlate with serum levels of antibodies to 3 recombinant filarial antigens. Virtually all the asymptomatic microfilaraemic individuals infected with W. bancrofti examined had subclinical lymphatic disease detected by the non-invasive imaging technique of lymphoscintigraphy.

Pathogenesis of onchocercal dermatitis: possible role of parasite proteases and autoantibodies to extracellular matrix proteins.

We examined the immunogenicity of various connective tissue proteins in patients with chronic onchocercal dermatitis and the effect of filarial proteases on this host-parasite interaction. Sera from patients with onchocerciasis reacted strongly with cuticular collagens from filarial parasites and with mammalian laminin. Some sera also contained antibodies to elastin and collagen type IV, but none reacted with collagen types I-III or fibronectin. This pattern of reactivity was characteristic for onchocerciasis: sera from patients with mansonellosis reacted strongly with collagen type IV but only weakly with laminin. Reactivity with mammalian laminin or collagen could not be absorbed with cuticular proteins from filarial worms and vice versa. Digestion fragments of laminin treated with filarial proteases retain antigenic determinants recognized by sera from patients with onchocerciasis. In contrast, proteases from Onchocerca volvulus adults and microfilariae drastically decreased the reactivity of the same sera with collagen type IV. These results indicate that filarial proteases may contribute to the pathogenesis of chronic onchocercal dermatitis, directly, by enzymatically destroying connective tissue of the skin, and indirectly, by triggering autoimmune responses to self-determinants on connective tissue proteins that are normally hidden within the supramolecular structure of the extracellular matrix complex.

Diagnosis of active Paragonimus westermani infections with a monoclonal antibody-based antigen detection assay.

We evaluated the performance of Dot-enzyme-linked immunosorbent assays designed to detect species- and stage-specific antigens of Paragonimus westermani in sera with monoclonal antibodies as serodiagnostic tests for active paragonimiasis. Sera from all donors with parasitologically confirmed infections with P. westermani contained adult worm antigens, as did a high proportion of sera from persons suspected to be infected with this parasite. A smaller proportion of these sera also contained metacercarial stage-specific antigens. Sera from donors with other helminth infections, with confirmed pulmonary tuberculosis, or from healthy Chinese donors were nonreactive in the assay. Treatment of experimentally infected animals with praziquantel triggered a marked but transient increase in serum levels of adult P. westermani antigens, which then gradually disappeared within the next two months. The results of our studies indicate that the antigen-detection assay we have developed is a highly specific and sensitive diagnostic test for active infections with P. westermani.

[Efficacy of ivermectin for control of microfilaremia recurring after treatment with diethylcarbamazine: immunological observation].

We compared the effect of a single dose of ivermectin (100 micrograms/kg) with that of a standard course of diethylcarbamazine (DEC) (6 mg/kg) on several parameters of the host's antifilarial immune response in 60 patients with bancroftian filariasis enrolled in a double-blind drug trial. All participants had measurable serum levels of antifilarial antibodies and parasite antigens. Drug-induced clearance of microfilaremia was associated with a temporary increase in HC11 antigenemia and a decrease in serum levels of antibodies to soluble filarial antigens. Antigenemia progressively declined in patients who remained a microfilaremic after treatment, but declined and then rose in persons with recurrent microfilaremia. Treatment triggered a sustained increase in serum levels of IL-1, IL-6, TNF alpha and IFN gamma in all patients. Although Ivermectin and DEC are believed to exert their antiparasite activity via different mechanisms, the same pattern of serological changes was observed in patients treated with either drug.

Filarial parasites contain a ras homolog of the TC4/ran/Spil family.

We have isolated and characterized a gene encoding a novel GTP-binding protein of the GTPase superfamily in the filarial parasites Brugia malayi and Onchocerca volvulus. The deduced amino acid sequence of the cloned molecule has approximately 30% overall homology to ras proteins and approximately 90% homology to the 'ras-like' nuclear proteins TC4, ran and Spil. Rabbit antisera to bacterially expressed filarial protein detect a 24-22 kDa doublet in extracts of adult B. malayi and mature microfilariae, which is absent from immature microfilariae. Increased expression of the native parasite protein occurs when worms are cultured in the presence of epidermal growth factor.

A cloned antigen for serological diagnosis of Wuchereria bancrofti microfilaremia with daytime blood samples.

By differentially screening an adult Brugia malayi cDNA library with sera from microfilaremic and amicrofilaremic donors infected with Wuchereria bancrofti, we have identified a novel parasite antigen denoted SXP-1. Recombinant SXP-1 filarial antigen is preferentially recognized by sera from microfilaremic persons with bancroftian filariasis and from skin snip-positive patients with onchocerciasis. Antibodies to SXP-1 are restricted to the IgG4 subclass and gradually decline after treatment with diethylcarbamazine. These findings indicate that it may be possible to replace microscopic examination of night blood films with a serological test designed to detect antibodies to a mix of SXP-1 and other suitable antigens for the diagnosis of microfilaremia due to bancroftian filariasis.