Microfilaremic Individuals Research Articles

Species-specific sequence in the repeat 3 region of the gene encoding a putative Loa loa allergen: a diagnostic tool for occult loiasis.

A polymerase chain reaction (PCR)-based method to detect Loa loa DNA in the blood lysate of infected individuals is described. A set of primers was designed to amplify the repeat 3 sequence (15r3) of the gene encoding a putative L. loa allergen. The qualitative PCR was carried out using blood lysates from subjects from an L. loaendemic area of Gabon where loiasis exists sympatrically with Mansonella perstans, and from individuals from a loiasis-free area in Togo infected concomitantly with M. perstans and Onchocerca volvulus. No specific amplification was observed after ethidium bromide staining of a gel containing M. perstans and O. volvulus control samples. In contrast, a 396-basepair (bp) DNA was detected in all L. loa microfilaremic individuals and in seven of the 20 L. loa amicrofilaremic subjects diagnosed by leukoconcentration. Qualitative Southern blots carried out at high stringency (65 degrees C) using 15r3 oligonucleotide probe revealed hybridization only with L. loa samples (5 of 5 microfilaremic individuals and 15 of 20 amicrofilaremic individuals), confirming the results obtained with ethidium bromide staining of PCR products. We conclude that this 396-bp sequence could be used as a species-specific diagnostic tool for occult loiasis in an endemic area with concurrent filarial infections.

Th1-like antifilarial immune responses predominate in antigen-negative persons.

To characterize immune responses associated with the putatively immune state in bancroftian filariasis (that is, both microfilaria and antigen free), humoral and cellular responses were compared among antigen- and microfilaria-negative, antigen-positive and microfilaria-negative, and microfilaria-positive individuals. Antifilarial isotype levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cell responses were measured by proliferation, by bioassay for interleukin-2 (IL-2) and IL-10, and by reverse transcription-PCR for IL-4, IL-5, and gamma interferon. The absence of circulating filarial antigen was associated with Th1-like responses, including significantly higher proliferative (P < 0.001) and IL-2 (P = 0.008) responses and a higher prevalence of gamma interferon (0.02 < P < 0.1) responses. Significantly elevated antifilarial immunoglobulin G4 (IgG4) levels (P = 0.0035) were associated with antigenemia, whereas microfilaremia was associated with significantly decreased antifilarial IgG2 levels (P = 0.0014). IL-4 mRNA levels were not significantly different among the three groups; however, there was a subpopulation of microfilaremic individuals who did not make detectable levels of IL-4 mRNA and who produced low antifilarial IgG4 levels compared with those of individuals who had detectable levels of IL-4 mRNA. IL-5 mRNA levels also were not significantly different among groups; however, more microfilaremic individuals produced IL-5 mRNA in response to adult filarial antigens, and total parasite-specific IL-4 and IL-5 mRNA levels were significantly correlated (P = 0.05). Although longitudinal data are not currently available, the elevated Th1-like responses in antigen- and microfilaria-negative individuals are consistent with the hypothesis that these responses contribute to protection in putatively immune individuals.

Immunoregulation in human lymphatic filariasis: the role of interleukin 10.

In humans with lymphatic filariasis microfilaremia is associated with a parasite antigen-specific hyporesponsiveness when assessed by cell proliferation and secretion of interleukin-2 and interferon-gamma. Hyporesponsiveness in these individuals is not only parasite antigen-specific but appears to be limited to Th1-type responses. Th2 mediated responses such as IL-5 secretion and IgE antibody production to parasite antigens are generally strong and usually no different than those seen in immunologically more reactive amicrofilaremic individuals with chronic lymphatic pathology. The mechanisms by which Th1 responses are inhibited have not yet been elucidated, but some studies suggest that down-regulatory cytokines such as IL-10 may be involved in this process. Mononuclear cells from microfilaremic individuals have been found to secrete greater quantities of IL-10 spontaneously and in response to parasite antigens. In this review, mechanisms by which IL-10 may be induced by the parasite and the mode by which IL-10 may regulate parasite antigen-specific Th1 responses in these individuals are discussed.

Longitudinal Survey of Loa loa Filariasis in Southern Cameroon: Long-Term Stability and Factors Influencing Individual Microfilarial Status

A longitudinal, one-year survey of Loa loa infection was carried out in an endemic area of southern Cameroon. Parasitologic samplings (calibrated thick blood smears) were performed every two months to study the evolution of loiasis infection at both the population and the individual level. The mean number of measurements by subject was 3.8 (range 1-6). At the population level, prevalence of infection and microfilarial load were found to be very stable over time. This observation is consistent with the existence of an important reserve of parasitic material available for vectors and the maintenance of high levels of transmission. At the individual level, both the microfilarial status (microfilaremic/nonmicrofilaremic) and the level of parasitemia showed a remarkable stability over time. Age was the relevant factor that influenced the individual microfilarial status in the whole population. When only microfilaremic individuals were taken into account, age did not influence the level of microfilaremia, suggesting that loiasis could be considered as a noncumulative disease. The stability of individual microfilarial status and the pattern of infection variations observed with age support the view that genetic factors might be involved in host defense mechanisms against loiasis infection.

Lymphoscintigraphic Assessment of the Effect of Diethylcarbamazine Treatment on Lymphatic Damage in Human Bancroftian Filariasis

Despite many millions of doses administered over the past 40 years, basic and crucial issues regarding the use, mode of action, and effectiveness of diethylcarbamazine (DEC) in many clinical situations remain unresolved. To directly investigate whether the well-known microfilaricidal and macrofilaricidal actions of DEC actually result in subsequent improvement in existing damage to lymphatic vessels or lymph nodes, 29 study subjects in Recife, Brazil were stratified into three groups according to the severity of clinical manifestations of lymphatic insufficiency. After baseline radionuclide lymphoscintigraphy was performed, subjects were treated with two courses of DEC separated by at least a six-month interval and then rescanned one year after the baseline scan. A side-by-side comparison of images obtained at baseline with those obtained at follow-up in 13 asymptomatic microfilaremic individuals, six individuals with filarial fever, and in 10 individuals with chronic pathology demonstrated essentially unchanged lymphatic morphology in all but one individual whose disease actually progressed in the face of therapy. We conclude that two 12-day treatment courses of DEC did not have a demonstrable direct or indirect effect on existing structural damage to the lymphatic system even in those individuals with preclinical disease.

Age-specific prevalence of antigenemia in a Wuchereria bancrofti-exposed population.

Antigen detection assays serve as a useful adjunct to blood examinations for studies of filariasis, in terms of the diagnostic and epidemiologic information provided. We examined the utility of the Og4C3 antigen detection enzyme-linked immunosorbent assay for field studies and analyzed the distribution of Wuchereria bancrofti antigenemia in a Haitian population. Using serum samples collected following venipuncture, antigenemia levels were correlated with microfilaremia (P < 0.001). The microfilariae had a pronounced nocturnal periodicity while the sensitivity of the antigen assay was the same whether serum samples were collected during the day or at night. To determine whether the Og4C3 assay could be used in conjunction with fingerprick blood examinations, nocturnal blood surveys were conducted. Of 419 persons surveyed, 207 (49.4%) were antigen-positive with the Og4C3 assay. Serum specimens from all 121 microfilaremic individuals were antigen positive (100% sensitivity). The age prevalence of antigenemia increased from 24.5% for 1-5-year-old children to 70% for persons greater than 50 years of age. These results demonstrate that the Og4C3 assay is a sensitive tool for the detection of infection and raise questions about the expression of protective immunity in populations exposed to infection.

Primary structure of and immunoglobulin E response to the repeat subunit of gp15/400 from human lymphatic filarial parasites.

We have isolated and sequenced clones encoding the repeated subunit of the surface-associated glycoprotein gp15/400 from the two nematode species predominantly responsible for lymphatic filariasis in humans: Brugia malayi and Wuchereria bancrofti. The amino acid sequence of the 15-kDa subunit, derived from the nucleotide sequence of the gene fragment from B. malayi, is identical to that previously reported for B. pahangi, whereas the derived W. bancrofti protein sequence differs in only 7 of 132 residues. The identity of the protein in the two Brugia species allowed us to use a recombinant from B. pahangi to examine the serological response of adult Indonesian subjects infected with B. malayi. The polymerase chain reaction-amplified subunit was expressed in Escherichia coli via the pDS56/RBS11 plasmid and purified by nickel-chelating chromatography. A significant proportion of individuals produced antigen-specific immunoglobulin E (IgE). This was most pronounced in the individuals with elephantiasis, with 14 of 15 showing elevated titers and a mean of 3.2 ng of specific IgE ml-1. Only 2 of 15 microfilaremic individuals possessed elevated titers of specific IgE, with a mean of 0.045 ng ml-1 for the group as a whole. Asymptomatic amicrofilaremic residents showed approximately equal numbers of responders (defined as having a value in the radioimmunoassay greater than two standard deviations above controls) and nonresponders, with a group mean of 1.2 ng of antigen-specific IgE ml-1.

Characterization of a myosin heavy chain gene from Brugia malayi

We have previously shown that an antigen recognized by antibodies in sera of several microfilaremic individuals from a Wuchereria bancrofti endemic area bears strong homology to an invertebrate muscle protein. We have cloned and sequenced the entire gene containing this antigen encoding fragment and present data that confirms that the antigen is myosin heavy chain (MHC). This gene, which we have named Bmmyo-1 extends over 11 kb and has the potential to encode a protein of 1957 amino acids. The coding sequence is interrupted by 14 introns, most of which are larger than those in the myosin gene of the free-living nematode, Caenorhabditis elegans. The protein encoded by this gene bears greatest homology (75.1% identity) to the C. elegans myosin isoform MHC-B, encoded by the unc-54 gene. MHC-B is the major body wall myosin in C. elegans.

Differential recognition of Loa loa antigens by sera of human subjects from a loiasis endemic zone.

Somatic antigens of Loa loa adult worms with molecular weights of 15-180 kDa were identified by Western blot analysis using sera from 3 categories of parasitologically and clinically defined subjects from a loiasis endemic zone. Sera of occult, amicrofilaremic (OL), and 'resistant' individuals with no clinical signs of infection (R) reacted with an antigen of 160 kDa; sera of highly microfilaremic individuals (ML) did not. ML sera strongly reacted with an antigen of 18 kDa which was recognized only weakly or not at all by OL and R sera. At higher dilutions, OL sera only reacted with antigens at 23 and 160 kDa and ML sera reacted with antigens at 18 and 23 kDa, whereas R sera reacted with antigens at 23, 42, 54, 70, 100, and 160 kDa. These data suggested that R sera contained a higher concentration of antibodies which reacted with denatured, nitrocellulose-bound antigens. The IgG4 isotype predominated for all groups of sera, while IgG3 antibody responses were observed only with R sera. IgG1 antibodies were seen in all groups but reacted with fewer antigens than IgG4 antibodies, and no IgG2 antibody responses were detected. Sera against Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, and Dirofilaria immitis cross-reacted with somatic antigens greater than 70 kDa, whereas none reacted with Loa loa antigens less than 23 kDa.

Protective immunity in bancroftian filariasis. Selective recognition of a 43-kD larval stage antigen by infection-free individuals in an endemic area.

There is little information about naturally occurring protective immunity in individuals living in areas endemic for lymphatic filariasis, though an immunologically hyperresponsive, uninfected group of "endemic normal" individuals that may be immune has been previously recognized. To analyze the nature of the hyperresponsiveness and its potential relation to a state of protective immunity in such individuals, strict clinical, parasitological, and serological criteria were applied to select seven "infection-free" endemic normal individuals (ENs) from a population of 459 persons resident in an area heavily endemic for bancroftian filariasis. Immunoblot analysis was used to compare the qualitative antigen recognition patterns of these endemic normal individuals to those of a group of 12 clearly infected microfilaremic individuals (MFs) from the same endemic area. Though immunoblot analysis using microfilarial and adult stage filarial antigens revealed no distinct differences in antigen recognition patterns between the two groups, when responses to infective larval stage antigens were assessed, 7/7 (100%) of the ENs were found to recognize a 43-kD antigen that was recognized by only 1/12 (8%) of the MFs. These findings are consistent with the concept that recognition of unique larval antigens may induce protective immunity to human filarial parasites and they identify a candidate immunogen for further functional assessment.

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.

Differential recognition of a protective filarial antigen by antibodies from humans with bancroftian filariasis.

The objectives of this study were to identify filarial antigens which induce enhanced clearance of circulating microfilariae and to establish if human antibody reactivity with these molecules correlates with the apparent parasite burdens of residents of an endemic area of Bancroftian filariasis. Mice immunized with an extract of Brugia malayi microfilariae develop IgG antibodies to four major filarial antigens with an apparent molecular weight (Mr) of approximately 112,000, 60,000, 45,000, and 25,000. Animals immunized with gel slices containing the approximately 25,000-Mr antigen are resistant to intravenous challenge with live microfilariae (78-98% reduction in parasitemia vs. controls, P less than 0.01). A group of 22 amicrofilaremic humans had a significantly higher (P less than 0.025) mean antibody titer to the Mr 25,000-Mr antigen (1: 424) than 16 microfilaremic individuals (1:95). There were no significant differences between the two groups in antibody titers to filarial antigens of Mr approximately 112,000, 60,000, and 45,000 Mr. These data suggest that a high degree of reactivity to the 25,000-Mr antigen in humans with lymphatic filariasis correlates with a parasitologic status that is least conducive to transmission of infection.

Mansonella Ozzardi in Haiti

Sera from individuals in an area of Haiti endemic for Mansonella ozzardi were analyzed for reactivity to antigens of Brugia pahangi, Dirofilaria immitis, Mansonella llewellyni or Ascaris lumbricoides using either an enzyme-linked immunosorbent assay or an indirect immunofluorescent antibody test. IgM and IgG reactivity to all antigens was observed with sera from both microfilaremic and amicrofilaremic individuals when compared to reactivity of sera from individuals from nonendemic areas. Antibody reactivity to B. pahangi was greater than that to other antigens. IgG reactivity of sera from endemic patients to filarial antigens was consistently greater than that of IgM. Antibody reactivity was not correlated with age or microfilarial density.

IgE responses in human filariasis. III. Specificities of IgE and IgG antibodies compared by immunoblot analysis.

IgE and IgG immune responses were qualitatively characterized in three well-defined groups of patients with different clinical manifestations of lymphatic filariasis caused by infection with Wuchereria bancrofti; viz. tropical pulmonary eosinophilia (TPE), chronic lymphatic obstruction with elephantiasis (CP), and asymptomatic microfilaremia (MF). A complex filarial antigen preparation extracted from adult filarial parasites was separated on SDS-polyacrylamide gels, and was electrophoretically transferred to nitrocellulose paper before being incubated with individual sera and being probed with radiolabeled anti-IgE or protein A. Three clinical groups of patients showed distinct patterns of antigen recognition by both IgE and IgG antibodies, with TPE patients showing the most diverse patterns and microfilaremic individuals the most restricted responses for both antibody isotypes. "Dual recognition" of antigens by IgE and IgG antibodies seemed to be the rule rather than the exception for each individual's immune response to the parasite antigens, but the relative magnitudes of IgG and IgE responses differed among the three groups. The ratio of IgG to IgE antibody was generally greater in patients with MF and CP than in those with TPE. These findings of the comparative specificities and relative abundance of IgE and IgG antibodies in infected patients may have fundamental importance in explaining the clinical expression (especially allergic reactivity) and disease pathogenesis of human filarial infections.

A monoclonal antibody-based immunoradiometric assay for detection of circulating antigen in Bancroftian filariasis.

A monoclonal antibody designated Gib 13 has been used in an immunoradiometric assay (IRMA) to detect circulating antigen in the sera of Wuchereria bancrofti-infected subjects from an endemic area of Papua New Guinea. A clear association between the presence of patent infection and the Gib 13 target epitope in serum was established because 93% of microfilaremic individuals were antigen-positive. Moreover, there was a significant correlation between levels of serum antigen and blood microfilarial counts. Detection of circulating antigen in amicrofilaremic subjects with acute symptoms of lymphatic filariasis, and 53% of asymptomatic amicrofilaremic subjects, but not in nonendemic controls, suggests that the Gib 13 IRMA will also be of value in the diagnosis of occult filariasis. However, as in all IRMA based on detection of potentially immunogenic molecules in man, antibodies can be expected to be the major contributor to reduced sensitivity of the assay.

Parasitological, serological, and clinical studies of Wuchereria bancrofti in Limbe, Haiti.

A survey for Wuchereria bancrofti in Limbe, Haiti (est. pop. = 10,500) revealed that 17% (231/1,450) had a patent infection. Nearly half of those surveyed harbored fewer than 10 microfilariae (mf) per 20 mm3 of finger-prick blood; the median mf density for females and males was 12.4 and 9.5, respectively. Parasitemias occurred as early as age 4. Antibody titers greater than or equal to 1:20 against adult D. viteae antigen were observed in 38% of microfilaremic individuals and in 29% of amicrofilaremic individuals. Peak antibody responsiveness (40%) was observed between 5 and 9 years of age. In all age groups there was no correlation between mf density and antibody titer. Among the mf carriers, 5.6% had no clinical symptoms. Lymphangitis was a common feature with 14.3% having lymphedema, 8.2% with edema of the lower extremities, and 1.3% reporting episodes of chyluria. Genital involvement among women was rare, but in males 5.4% had genital swelling and 4.5% had hydroceles. Culex pipiens quinquefasciatus (Say) was observed to support the complete development of W. bancrofti in Limbe.