Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses.

The filarial nematode Mansonella perstans is endemic throughout Africa, northern South America and the Caribbean. Interestingly, M. perstans-infected individuals present no distinct clinical picture associated with certain pathology. Due to its relatively silent nature, research on this tropical disease has been neglected, especially M. perstans-driven immune responses. A hindrance in obtaining data on M. perstans-specific responses has been the inability to obtain adult worms since their habitats in serous cavities are difficult to access. Thus, in this study, for the first time, we used Mansonella perstans worm antigen extract as stimulant to obtain filarial-specific recall and immunoglobulin responses from M. perstans microfilaremic individuals (Mp MF+) from Cameroon. Moreover, systemic immune profiles in sera and immune cell composition in peripheral blood from Mp MF+ and amicrofilaremic individuals (Mp MF-) were obtained. Our data reveal that Mp MF+ individuals showed significantly reduced cytokine (IL-4, IL-6 and IL-12p70) and chemokine levels (IL-8 and RANTES), but significantly higher MIP-1ß as well as increased M. perstans-specific IgG4 levels compared to Mp MF- individuals. In contrast, upon re-stimulation with worm antigen extract, IFN-γ, IL-13, IL-10 and IL-17A secretion was enhanced in cell cultures from Mp MF+ individuals when compared to those from cultures of healthy European individuals. Moreover, analysis of immune cell composition in peripheral blood from Mp MF+ individuals revealed increased type 2 helper T (Th2), natural killer (NK), regulatory B and T cell (Breg and Treg) subsets but decreased type 1 regulatory T (Tr1) cells. In summary, this study deciphers for the first time, M. perstans-specific immune responses using worm antigen extract and shows that patent M. perstans infections have distinct Th2, Breg and Treg subsets accompanied with reduced systemic innate and adaptive immune responses and dominant filarial-specific IgG4 levels.

Immune response in Mansonella ozzardi infection modulated by IL-6/IL-10 axis in Amazon region of Brazil.

Mansonellosis is an endemic disease in the South and Central America. In Brazil, one of the etiological agents is Mansonella ozzardi. This filarial infection is yet poorly understood, with a controversial morbity, presenting since a oligosymptoms, malaria-like signs or without complaint in humans. The knowledge of the human immune response to microfilariae infection is limited mainly by different evolutionary cycles of the parasite in the host. In addition, the prevalence of this filarial parasite infection is high in several regions of Amazonas State. A cross-sectional study was conducted in an endemic area for microfilariae of M. ozzardi (MF) infection in the Amazonas State, Brazil. Proinflammatory and regulatory cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-gamma, and IL-17A) were measured in cryopreserved serum using the Cytometric Bead Array techniques (CBA) in 54 patients diagnosed with M. ozzardi infection and 55 individuals without the infection were included in the study (Controls). The IL-4, IL-6 and IL-10 level increased in infected patients with MF infection, while IL-17A increased in control only. When we compared controls to patients with high or low parasite load, the increased level of IL-6 and IL-10 were maintained. IL-6 contributes to the proinflammatory activity and IL-10 modulates Th1, Th2 and Th17 immune response. Furthermore, IL-4 was detected as a marker in the MF infection and MF patients with low parasite load, indicating the action of the Th2 cell response. The complex network of cytokines acting during M. ozzardi infection depends on a fine balance to determine a host protective effect or filarial persistence. Therefore, these results suggest that the immune response in MF infection is modulated by IL-6/IL-10 axis.

Imported Infections with Mansonella perstans Nematodes, Italy.

We report 74 patients in Italy infected with Mansonella perstans nematodes, a poorly described filarial parasite. M. perstans nematodes should be included in the differential diagnosis for patients with eosinophilia from disease-endemic countries. Serologic analysis is useful for screening, and testing for microfilaremia in peripheral blood should be performed for parasite-positive patients.

Influence of Mansonella perstans microfilaraemia on total IgE levels in Gabonese patients co-infected with Loa loa.

Mansonella (M.) perstans filariasis is widely found in Africa, including Gabon where Loa loa is also endemic. This study reports the total IgE titres according to different bioclinical forms of single or co-infection with L. loa and M. perstans in 138 patients and 20 healthy controls. The median parasite density was significantly higher in cases of loiasis. IgE titres were higher in patients with microscopic dual-infection and in the group of patients with occult loiasis plus M. perstans microfilaraemia (8425 [5292-20,679]KUI/L and 6304 [1045-10,326]KUI/L, respectively), compared to individuals with either microfilaraemic Loa loa (3368 [1414-7074]KUI/L) or Mansonella (4370 [1478-7334]KUI/L) single infections (p<0.01). IgE levels were positively correlated with M. perstans microfilaraemia (rho=0.27; p<0.01). Compared to single infections, dual M. perstans-L. loa infection induces very high total IgE titres. Studies correlating IgE titres and clinical symptoms are needed to confirm the involvement of this immunoglobulin in the pathological processes during filariasis.

At homeostasis filarial infections have expanded adaptive T regulatory but not classical Th2 cells.

Despite the well-documented immune suppression associated with human helminth infections, studies characterizing the immune response at the single-cell level are scanty. We used multiparameter flow cytometry to characterize the type of effector (Th1, Th2, and Th17) and regulatory (natural T regulatory cells [nTregs] and adaptive Treg cells [aTreg/type 1 regulatory cells (Tr1s)]) CD4(+) and CD8(+) T cells in filaria-infected (Fil(+)) and -uninfected (Fil(-)) individuals at homeostasis (in the absence of stimulation). Frequencies of CD4(+) lymphocytes spontaneously producing IL-4, IL-10, and IL-17A were significantly higher in Fil(+), as were those of IL-10(+)/IL-4(+) double-producing CD4(+) cells. Interestingly, frequencies of Th17 and aTreg/Tr1s but not classical Th1 or Th2 cells were significantly increased in Fil(+) compared to Fil(-) individuals. Although the frequency of nTreg was increased in Fil(+), IL-10 was overwhelmingly produced by CD4(+)CD25(-) cells. Moreover, the concentration of IL-10 produced spontaneously in vitro strongly correlated with the integrated geometric mean fluorescence intensity of IL-10-producing aTreg/Tr1s in Fil(+). Together, these data show that at steady state, IL-10-producing aTreg/Tr1 as well as nTreg and effector Th17 CD4(+) cells are expanded in vivo in human filarial infections. Moreover, we have established baseline ex vivo frequencies of effector and Tregs at homeostasis at a population level.

Rapid, novel, specific, high-throughput assay for diagnosis of Loa loa infection.

The ability to diagnose Loa loa infection readily and accurately remains a demanding task. Among the available diagnostic methods, many are impractical for point-of-care field testing. To investigate whether luciferase immunoprecipitation systems (LIPS) can be used for rapid and specific diagnosis of L. loa infection, a LIPS assay was developed based on immunoglobulin G (IgG) and IgG4 subclass antibodies to a recombinant L. loa SXP-1 (designated LlSXP-1) antigen and tested with sera from healthy controls or patients with proven infection with L. loa, Mansonella perstans, Onchocerca volvulus, Strongyloides stercoralis, or Wuchereria bancrofti. A LIPS test measuring IgG antibody against LlSXP-1 readily differentiated L. loa-infected from uninfected patients and demonstrated markedly improved sensitivity and specificity compared with an LlSXP-1 IgG4-based enzyme-linked immunosorbent assay (67% sensitivity and 99% specificity). No significant immunoreactivity was observed with S. stercoralis-infected sera, but a small number of patients infected with O. volvulus, M. perstans, or W. bancrofti showed positive immunoreactivity. Measuring anti-IgG4-specific antibodies to LlSXP-1 showed a significant correlation (r approximately 0.85; P < 0.00001) with the anti-IgG results but showed no advantage over measuring the total IgG response alone. In contrast, a rapid LIPS format (called QLIPS) in which the tests are performed in less than 15 minutes under nonequilibrium conditions significantly improved the specificity for cross-reactive O. volvulus patient sera (100% sensitivity and 100% specificity). These results suggest that LIPS (and the even more rapid test QLIPS) represents a major advance in the ability to diagnose L. loa infection and may have future applications for point-of-care diagnostics.

Mansonella perstans causing symptomatic hypereosinophilia in a missionary family.

Mansonella perstans is rarely pathogenic. The rare reports of symptomatic cases, however, include severe complications. Three cases of symptomatic hypereosinophilia with multi-organ involvement are described in a missionary family returning from tropical Africa. Pathogenicity may be related to the induction of hypereosinophilia rather than direct host-parasite interactions.

Immunodiagnostic studies on Onchocerca volvulus and Mansonella perstans infections using a recombinant 33 kDa O. volvulus protein (Ov33).

A total detergent-soluble extract of adult female Onchocerca volvulus (OvAg) and a recombinant O. volvulus protein (Ov33) linked to glutathione-S-transferase (GST) were compared with regard to their serodiagnostic suitability for differentiating between O. volvulus and Mansonella perstans infections in a region endemic for both filarial worms in western Uganda. Using OvAg in an enzyme-linked immunosorbent assay (ELISA), 98.8% sensitivity was obtained examining 84 O. volvulus microfilariae (mf) carriers living in the hyperendemic area. However, 5 of 18 (28%) sera from M. perstans mf carriers without O. volvulus mf, from another area hypoendemic for O. volvulus, cross-reacted with OvAg. Using the recombinant antigen Ov33-GST in an ELISA and Western blot assay, sensitivity for O. volvulus remained high (97.2% and 98.8% respectively) while none of 90 sera from M. perstans mf carriers reacted positively. Both antigens were used to examine a batch of sera from 260 persons living in the onchocerciasis hyperendemic area who did not have mf in their skin snips (9.5% of 2728 persons examined); 116 of these sera (44.6%) were positive in the OvAg ELISA, compared to 85 (32.7%) and 69 (26.5%) which were positive in Ov33-GST ELISA and Ov33-GST Western blot, respectively. Reaction with GST alone was minimal. The recombinant antigen Ov33 efficiently differentiates between O. volvulus and M. perstans infections, and is sensitive when used to detect patent and prepatent or low-level O. volvulus infections.

Mansonella ozzardi: the course of patency in experimentally-infected patas monkeys.

Twenty-five of 30 patas monkeys (Erythrocebus patas) inoculated with varying numbers (35 to 135) of third-stage larvae of Mansonella ozzardi developed patent infections in an average of 163 days. There was no correlation between the size of the inoculum and the length of the prepatent period. Ten of the monkeys were monitored thereafter by regular blood examination for extended periods of time in order to characterize the onset, course and duration of patency. Typically, with the onset of patency, microfilaremias increased steadily, peaking at about 20 weeks and then decreased slowly stabilizing at low levels for up to 48 weeks. Thereafter microfilariae disappeared from the blood and occasionally reappeared in scanty numbers. Laparotomies and followup studies indicated that the spleen was involved in the suppression of peripheral microfilaremia as had been observed earlier in patas monkeys infected with Loa loa. In ten monkeys splenectomized after the initial "wave" of microfilaremia, it was observed that (a) 30% of the animals remained amicrofilaremic, (b) another 30% reestablished patent infections but microfilaremias were lower than presplenectomy levels, and (c) in the remaining 40%, levels of microfilaremia equaled or exceeded pre-splenectomy levels. Patent infections persisted for up to 212 weeks. One monkey splenectomized prior to inoculation with 87 larvae developed a patent infection with microfilaremia which persisted for 156 weeks. Three monkeys with low and high levels of microfilaremia bled at four hour intervals over a 28 hour period showed no evidence of periodicity in the microfilaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific detection of human antibodies to Onchocerca volvulus.

Specific diagnosis of antibodies to Onchocerca was achieved through (1) the construction of direct and indirect ELISA systems, and (2) restricting ELISA assays to the IgG4 class. The direct ELISA was based on the isolation of a surface derived, low molecular weight surface antigen preparation containing two main antigens (M. wt. 16.2 and 12.8 kDA) as defined by Western blot analysis. The direct ELISA system detected antibodies in children of six years old, and may therefore be applicable to detecting reinvasion in OCP areas of Onchocerca volvulus control. The indirect ELISA system was a competitive binding ELISA-based assay using a monoclonal antibody recognising two Onchocerca components (M. wts. 15.6 and 25.9) on a Western blot. The direct and indirect ELISA systems were similarly specific and sensitive when evaluated in a preliminary survey. The direct ELISA system yielded a specificity and sensitivity of: 100% and 100% respectively, using Mexican endemic and Mexican intestinal nematode infection sera as positive and negative controls respectively: 91% and 96% respectively, using Venezuelan endemic and Venezuelan Mansonella ozzardi infection sera as positive and negative controls, respectively: 87% and 93% respectively, using African endemic and Papuan (New Guinea) Wuchereria bancrofti infection sera as positive and negative controls respectively: 93% and 93% respectively, using African endemic and Indian W. bancrofti infection sera as positive and negative controls respectively. Similar specificity and sensitivity levels were obtained when the same comparisons were made using the indirect (inhibition) ELISA assay. These values may be contrasted with the currently used PBS extract of O. volvulus which yielded specificities of less than 10% in all the above comparisons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell adherence to microfilariae of Onchocerca volvulus: a comparative study.

The conditions were examined for in vitro antibody-mediated adherence of granulocytes to microfilariae of Onchocera volvulus and Dirofilaria immitis. Reactivity in human sera from patients in endemic foci in Sudan was specific for O. volvulus and no reactions were observed with heterologous Onchocerca species or with Mansonella perstans. Microfilariae from skin, nodules or adult female worms were satisfactory targets for cell adherence, and the cells involved were almost exclusively eosinophils. The reaction was inhibited by indomethacin but not by nordihydroguaiaretic acid, an inhibitor of leukotriene production. Agents that slowed or stopped microfilarial motility (e.g. nifedipine, lidocaine, chloroquine) inhibited the reaction, probably by reducing target/cell contact. Ivermectin did not enhance the reaction, and in the absence of cells exerted only slight effects on the movement of microfilariae at higher concentrations (greater than 10 micrograms/ml). Antibody activity was labile, and did not persist well through freeze-thaw cycles. Some differences between homologous and heterologous mixtures (microfilariae/cells/serum) were seen but they could not be resolved satisfactorily. There were no apparent geographical differences between microfilariae from different foci in Sudan. In the D. immitis system neutrophils were the dominant cell type adhering to microfilariae, and the activity was stable to storage and freeze-thaw. No enhancement was detectable with diethylcarbamazine. Antibody activity was absorbable with microfilarial antigens and was reduced by agents that inhibited microfilarial motility. In dogs, adherence-mediating antibody was seen only in amicrofilaraemic animals with occult infection, and in only a minority of these sera. In humans the relationship to clinical findings was less clear, but patients with punctate keratitis were the most likely to have positive serum and were the most reactive in the assay. This system may therefore offer some insights into disease mechanisms in vivo, and its molecular mechanisms deserve further characterization.

Lack of evidence for transplacental transfer of microfilarial antigens in filariasis due to Loa loa and Mansonella perstans.

The transplacental transfer of microfilarial antigens could induce prenatal tolerance, or conversely resistance, which would influence the development of filariasis after birth. The passage across the placenta of immunogens constitutive of a crude microfilarial Loa loa extract has been investigated by dosing specific IgG, IgM and IgE antibodies in 92 maternal and corresponding cord blood samples, IgG levels were shown to be identical in about half of the mother-cord paired samples while in the other half the cord serum had a lower titre. Specific IgM or IgE could not be evidenced in any of the cord blood samples, even from those mothers who where harbouring L. loa microfilariae and/or specific antibodies. It is concluded that prenatal sensitization to the immunogenic preparation used is unlikely to have occurred.

Mansonella ozzardi in Haiti. IV. Evaluation of antibody reactivity to heterologous antigens.

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.