Profile of loiasis infection through clinical and laboratory diagnostics: the importance of biomarkers.

BACKGROUND: Detection of Loa loa microfilariae in peripheral blood is insensitive given only 30% of individuals are microfilaraemic while 70% are amicrofilaraemic with a variety of clinical signs. Biomarkers may improve the diagnosis of loiasis. METHODS: A total of 545 individuals exposed to L. loa were analysed using clinical data collected through a questionnaire (requesting information on eye worm, Calabar swelling, pruritis) and detection of microfilariae, immunoglobulin G4 (IgG4), DNA and antigens using microscopy, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR) and Western blot, respectively. RESULTS: The results revealed that the rates of detection of L. loa microfilariae in the blood, of DNA by qPCR, of IgG4 by ELISA and of antigen by Western blot were 4.7%, 5.5%, 15.60% and 10.09%, respectively. CONCLUSIONS: This study showed that clinical signs based on a questionnaire are highly subjective. Therefore it is imperative to use IgG4 and DNA biomarkers as well as antigens detected by Western blot to identify individuals infected with L. loa.

Production and immunological characterization of a recombinant subunit of a Loa loa polyprotein antigen.

Diagnosis of loiasis and analysis of the specific immune response are limited by a paucity of parasite material. To circumvent this problem, a Loa loa antigen has been expressed in a prokaryote vector (pTrcHis). Immunization of Balb/c mice with this soluble recombinant protein produced a strong antibody response, with antibodies recognizing 2 major bands of 38 and 20 kDa in a native crude extract of Loa loa adult worms and microfilariae on Western blots. The target molecule was located mainly in the hypodermis and cuticle of the adult worm. Analysis of human IgG subclasses against this antigen by enzyme-linked immunosorbent assay (ELISA) showed IgG1, IgG2 and IgG3 but not IgG4 reactivity. IgG2 against this recombinant antigen was 100% specific for loiasis when tested against samples from European donor individuals. The same IgG2 antibodies showed 91% specificity for loiasis by comparison with Wuchereria bancrofti, Onchocerca volvulus, Mansonnella perstans and other helminth infections. Furthermore, the IgG2 antibody level correlated with the density of Loa loa microfilariae (r=0.400; P=0.02). This recombinant 15r3 molecule and specific IgG2 assay may be useful for monitoring control programmes.

[Serological study on toxoplasmosis among pregnant women from Franceville, Gabon]. / Etude sérologique de la toxoplasmosechez les femmes enceintes de Franceville, Gabon.

The serological prevalence of Toxoplasma gondii was studied among 839 pregnant women in two hospitals from Franceville (Gabon), between May 2007 and December 2007. Specific T gondii IgG and IgM were measured by Enzyme Linked Fluorescent Assay (ELFA). Datation of the infection was carried out by avidity test. Fifty-six percent of women in this study were immunised compare to the 71% who were found as immunised in a previous study carried out fifteen years ago. 2.6% were found to be IgM positive. However, from the avidity test it was found that these infections occurred before pregnancy contact with cats and age increase this prevalence. The lack of information for pregnant women, the lack of continuous training for health personnel and lack of awareness about interpretation of laboratory diagnostic tests like avidity test in these hospitals reduce the level of counselling for women about T gondii.

Parasitological and immunological effects induced by immunization of Mandrillus sphinx against the human filarial Loa loa using infective stage larvae irradiated at 40 Krad.

Six mandrills were immunized with 150 Loa loa infective stage larvae (L3) irradiated with 40 Krad, and challenged with 100 L3, 60 days after initial vaccination. The parasitological outcome of this immunization was compared to results from six mandrills infected with normal L3. No clear association was seen between vaccination and microfilaremia until day 245 when a significant drop in the level of microfilaria occurred in vaccinated compared to infected animals (5 vs 10 mf/ml; p = 0.012). A one-year follow-up of the humoral immune response showed a strong adult, microfilariae (Mf) and L3 specific IgG response, with distinct profiles for each extract. In immunized animal a significant decrease in antibody level was systematically observed between days 90-145 for the anti-L3 and anti-adult IgG. However, in the same group anti-Mf antibody levels that peaked around 160-175 days post-challenge, were inversely correlated with the decrease in Mf density between day 200 and day 386. These results suggest that immunization with irradiated L3 using these specific conditions may affect the appearance of Mf.

Expression of filarial-specific IgG subclasses under different transmission intensities in a region endemic for loiasis.

Specific IgG subclasses were investigated in two villages (Okoumbi and Ndjokaye) in southeast Gabon with different Loa loa transmission intensities of approximately 9,000 and 1,300 infective larvae (L3) per person per year, respectively. IgG subclasses were measured by an enzyme-linked immunosorbent assay (ELISA) using extracts of L. loa L3, microfilariae (MF), or adult worms. Levels of L3-specific IgG3 were significantly higher in the village with low transmission (Ndjokaye) (P = 0.006). In contrast, MF-specific IgG2 was significantly higher in Okoumbi than in Ndjokaye (P = 0.0009). In the high-transmission village (Okoumbi), levels of both MF- and adult-specific IgG4 were significantly increased in MF carriers compared with amicrofilaremic subjects (P = 0.0015 and P = 0.003, respectively), while levels of L3- and MF-specific IgG1 were significantly higher in amicrofilaremic individuals compared with MF carriers (P = 0.04 and P = 0.03, respectively). Furthermore, among microfilaremic individuals, the level of the specific IgG1 subclass was much lower in Okoumbi than in Ndjokaye (P = 0.036). These results suggest that the expression of antigen-specific IgG3 and IgG2 is more likely to vary with transmission intensity, whereas antigen-specific IgG4 and IgG1 varies with adult worm and MF burden.

The relationship between parasitological status and humoral responses to Loa loa antigens in the Mandrillus sphinx model after immunization with irradiated L3 and infection with normal L3.

In order to identify antigens associated with protection and those associated with active infection, the humoral immune response of 6 Mandrillus sphinx immunized with 150 irradiated L3 and challenged with 100 normal L3 of Loa loa or 6 animals infected with 100 L3 were compared. The plasma of these animals was analysed by Western blot using adult, Mf and L3 antigens. Several antigens with molecular weights varying from 120 kDa to 13 kDa were recognized by the plasma of all animals. It was shown that early recognition of microfilarial antigens with molecular weights of 97, 68, 45 and 33 kDa correlated with the amicrofilaraemic state. A total of 83% of animals with circulating microfilariae had antibodies against the microfilariae 21 kDa antigen. Furthermore, the antibodies against the 21 kDa appeared 1 month before detection of microfilariae in the peripheral blood of 80% of these animals, and declined when animals became amicrofilaraemic. In contrast, when L3 antigen was used, a molecule with a relative molecular weight of 20 kDa was recognized by antibodies of the only animal which remained amicrofilaraemic for 1 year after immunization with irradiated L3. These results suggest that the microfilarial molecule of 21 kDa may be useful as a marker of Loa loa patent infection, whereas the 97, 68, 45 and 33 kDa molecules of microfilariae and the L3 molecule of 20 kDa may be associated with resistance against Loa loa.

IgG subclass recognition of Loa loa antigens and their correlation with clinical status in individuals from Gabon.

In endemic areas for Loa loa, a significant percentage of actively infected individuals have no circulating microfilariae, an observation which implies the existence of a stage-specific immune response. In an attempt to define the immunological basis of the amicrofilaraemic state, the reactivity of antigens from adult, microfilariae and infective larvae of L. loa was examined by Western blotting with individual serum samples from four clinically defined groups (high microfilaraemic, low microfilaraemic, amicrofilaraemic and endemic controls) using IgG subclass-specific reagents and IgE. In the adult parasite, a complex of antigens at 28-31 kDa was exclusively recognized by IgG1 from amicrofilaraemic individuals and, to a lesser extent, by IgG1 from endemic controls. However, this complex of antigens was recognized by IgG4 antibodies in serum samples from all individuals, including microfilaraemics. A microfilarial antigen of 21 kDa was recognized by IgG1 antibodies present in serum from amicrofilaraemic, endemic control and low microfilaraemic individuals. Persons with high levels of microfilariae did not recognise this antigen. In both the L3 and the microfilariae, a ladder antigen with increments of 15 kDa was the main target of IgG4 antibodies in amicrofilaraemic and microfilaraemic individuals. IgE antibodies recognized more antigens in the microfilarial stage than in the adult of L3. These results suggest that immunological differences between clinically defined groups are associated with the recognition of different antigens or epitopes.

High levels of parasite-specific IgG1 correlate with the amicrofilaremic state in Loa loa infection.

To investigate the mechanisms of protective immunity operating in Loa loa infection, 56 persons from a L. ioa-endemic village in southeast Gabon were examined over a 7-year period. The level of L. loa-specific IgG subclasses in defined parasitologic groups was compared by use of ELISA with either adult, microfilarial, or third-stage larval (L3) antigens of L. loa. With all antigen preparations, IgG1 levels were significantly higher in amicrofilaremic persons than in persons with high or low levels of microfilariae. Moreover, there was a significant negative correlation between IgG1 levels to L3 antigen and the density of microfilariae (Spearman's r(s) = -.701; P < .01). There was no correlation between density of microfilariae and levels of other IgG subclasses or of IgE. These data indicate that IgG1 may play a role in the effector mechanism(s) involved in resistance against L. loa and suggest that L3 antigens may be important in eliciting protective responses.

Markers of Loa loa infection in permanent residents of a loiasis endemic area of Gabon.

Different markers of infection were analyzed in 56 permanent residents of a Loa loa endemic village in Gabon. The population was divided into those with parasitological evidence of L. loa infection and those with no history of loiasis over the period of observation (c. 5 years). 26.7% of villagers had L. loa microfilariae, 33.9% had an ocular passage of an adult worm, and 17.8% had calabar oedema. Several other clinical symptoms were present in both groups of individuals, but none was considered to be pathognomonic for L. loa infection. Most of the villagers were polyparasitized, with Plasmodium falciparum and gastrointestinal parasites being particularly prevalent. Mansonella perstans was present in 80% of the villagers and was equally distributed between L. loa microfilaraemic and amicrofilaraemic individuals. Eosinophil levels were elevated in the whole population, and were not significantly different between the groups who were infected and non-infected with L. loa. Polyclonal immunoglobulin (Ig) E levels were high in both the Ambinda villagers and in Gambian serum from patients infected with M. perstans alone and there was no significant difference between the levels of L. loa specific IgG in the Ambinda villagers and the Gambian patients. However, the level of L. loa specific IgG4 was elevated in 75.6% of amicrofilaraemic individuals and could discriminate between most individuals infected with L. loa and those infected with M. perstans, suggesting that this is the best determinant of infection status in the absence of L. loa microfilariae.

Loa loa: structural diversity of a 15-kDa repetitive antigen.

A ladder antigen of Loa loa was identified on Western blots of all life cycle stages probed with loaisis sera. The smallest subunit has a relative M(r) of about 15 kDa and larger subunits represent size increments of 15.0 kDa. An 1800-bp genomic clone encoding this antigen was characterized further by restriction mapping. Southern blot analysis, and nucleotide sequencing. The antigen is encoded by multiple copies of a gene, linked in tandem repeats of 396 bp, each of which encodes 132 amino acids. These repeats have diverged sufficiently to produce distinct restriction enzyme sites and Southern blot hybridization patterns. The 1764-bp insert contains no introns and encodes 588 amino acids, representing one incomplete and four complete repeats. At the 3' end of three repeats, there are consensus proteolytic cleavage sites; one repeat has no cleavage site. Two perfect repeats show a 93.9% amino acid identity with one another; the rest of the repeats, despite being adjacent to one another, show only 31-42% identical amino acids. Putative asparagine N-linked glycosylation sites are expressed by only two of the repeats. Despite this structural diversity, each L. loa repeat showed homology to Ascaris suum allergen and the homologue protein described in Dirofilaria immitis and Brugia pahangi.

High levels of parasite-specific IgG4 in the absence of microfilaremia in Loa loa infection.

The specificity of IgG4 as marker of infection has been investigated. The study was based on two well defined clinical groups: amicrofilaremic individuals with verified ocular passage of adult worms of Loa loa, and microfilaremic patients. Both groups were compared to Africans not exposed to loiasis infection and to Europeans. The study revealed that there is no significant difference in the level of parasite-specific IgG4 between individuals with occult infection (i.e. amicrofilaraemic, but infected) and microfilaremic individuals, but there is a significant difference between L. loa infected individuals and Mansonella perstans exposed people. IgG4 of individuals exposed to L. loa infection recognised specific antigens in the molecular weight range 12-30 kDa. We conclude that the elevated level of filarial-specific IgG4 is therefore not dependent upon the presence of circulating microfilariae and that serology using homologous L. loa low molecular weight antigens, can facilitate specific diagnosis of occult loiasis in an endemic area with mixed filarial infections.

Cloning and characterization of a Loa loa-specific repetitive DNA.

A Loa loa EcoRI genomic library in lambda gt11 was screened with 32P-labeled L. loa DNA and 1 repetitive clone, LL20, was isolated. An 800-bp Rsa I fragment of LL20, which is L. loa specific, was subcloned into pUC19 and the recombinant plasmid was designated pRsa4. While the 3.8-kb Eco RI fragment of LL20 cross-hybridized to other filarial DNA under low stringency conditions, the 800-bp fragment of pRsa4 was L. loa specific under the same conditions. Further characterization of the insert of pRsa4 was therefore carried out. Its lower limit of detection is 800 pg of L. loa genomic DNA, it has a low copy number (50-100) and an interspersed distribution in the genome. As a probe it does not distinguish between simian and human L. loa DNA. The nucleotide sequence contains 69% A + T and 31% G + C and shows no notable internal repeats.

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.

Storage of filarial parasites in CsTFA and precipitation of filarial DNA using MTAB.

Immunological and biochemical studies on some parasite species are hampered by a limited availability of parasite material. Thomas Egwang Jean-Paul Akue and Margaret Pinder discuss a technique for preserving material from adult filarial worms during transport through endemic areas.

Age-related prevalence of antibody response against three different, defined Plasmodium falciparum antigens in children from the Haut-Ogooué province in Gabon.

The kinetics of the humoral response to defined Plasmodium falciparum antigens was studied in 543 children, 1 month to 15 years old, living in an area endemic for malaria. The antigens used for enzyme-linked immunosorbent assay were (i) the synthetic peptide (NANP)40 representing the immunodominant repeated region of the circumsporozoite protein, and (ii) the fusion peptide 31.1, representing the N-terminal portion of the 83 kDa polypeptide expressed at the surface of merozoites which is a processed product of the 190-200 kDa glycoprotein. In addition, glutaraldehyde-fixed infected red blood cells (RBC) were used to detect ring-infected erythrocyte surface antigen (RESA) and unfixed infected RBC to detect intra-erythrocytic asexual form (IEF) antigens by immunofluorescence. In the 1 to 2 months age group, 50%, 26% and 21% of the children had antibodies for IEF, (NANP)40 and 31.1 respectively, but none had anti-RESA antibodies. The proportions of positive subjects decreased until 3 to 6 months and then increased progressively for the 4 antigens, approaching, but not reaching, adult values by the age of 15 years. Antibodies against specific antigens were acquired concomitantly. Children born from (NANP)40-positive mothers showed enhanced anti-(NANP)40 IgG responses.