Influence of infection on malaria-specific antibody dynamics in a cohort exposed to intense malaria transmission in northern Uganda.

The role of submicroscopic infections in modulating malaria antibody responses is poorly understood and requires longitudinal studies. A cohort of 249 children ≤5 years of age, 126 children between 6 and 10 years and 134 adults ≥20 years was recruited in an area of intense malaria transmission in Apac, Uganda and treated with artemether/lumefantrine at enrolment. Parasite carriage was determined at enrolment and after 6 and 16 weeks using microscopy and PCR. Antibody prevalence and titres to circumsporozoite protein, apical membrane antigen-1 (AMA-1), merozoite surface protein-1 (MSP-119 ), merozoite surface protein-2 (MSP-2) and Anopheles gambiae salivary gland protein 6 (gSG6) were determined by ELISA. Plasmodium falciparum infections were detected in 38·1% (194/509) of the individuals by microscopy and in 57·1% (284/493) of the individuals by PCR at enrolment. Antibody prevalence and titre against AMA-1, MSP-119 , MSP-2 and gSG6 were related to concurrent (sub-)microscopic parasitaemia. Responses were stable in children who were continuously infected with malaria parasites but declined in children who were never parasitaemic during the study or were not re-infected after treatment. These findings indicate that continued malaria infections are required to maintain antibody titres in an area of intense malaria transmission.

Population-based validation of dihydrofolate reductase gene mutations for the prediction of sulfadoxine-pyrimethamine resistance in Uganda.

Mutations in the dihydrofolate reductase gene (dhfr) of Plasmodium falciparum have been proposed as molecular markers for the surveillance of sulfadoxine-pyrimethamine (SP)-resistant malaria, but such proposals have not been validated. At 7 Ugandan sites in 1999, we determined the population-based prevalence of infections with mutations and the mutant allele frequency of dhfr codons 108, 51, and 59 using a random sample of infected individuals aged 1-45 years. Sulfadoxine-pyrimethamine treatment failure was independently estimated by in vivo tests in 327 children aged 6-59 months with clinical malaria. The prevalence of infections with the single point mutations and the dhfr codons 108 and 51 mutant allele frequency were not correlated to SP treatment failure. However, the dhfr codon 59 mutant allele frequency was positively correlated to SP treatment failure (r = 0.72, P = 0.06). The ratio of the infections with the mutant to wild genotype (M/W) and that of the mutant to wild allele (MA/WA) had the same values. Both dhfr codon 59 M/W and MA/WA ratio were significantly and positively correlated to SP treatment failure (r = 0.73, P = 0.05). Moreover, the prevalence of infections with only 2 mutations (Asn-108 plus Ile-51) was significantly and inversely correlated to the prevalence of infections with 3 mutations (Asn-108 plus Ile-51 plus Arg-59) (r = 0.92, P = 0.004), suggesting the stepwise accumulation of the dhfr mutations is Asn-108 Ile-51 Arg-59 and further supporting the idea of using the dhfr codon 59 M/W ratio as a molecular index for the prediction of SP treatment failure. Atthe population level, the dhfr codon 59 M/W ratio is a simple and stable index for the estimation of SP treatment failure.

The Lys-76-Thr mutation in PfCRT and chloroquine resistance in Plasmodium falciparum isolates from Uganda.

Recent molecular studies of chloroquine (CQ) resistance of Plasmodium falciparum have demonstrated an association between a mutation in the PfCRT gene and CQ resistance. We identified wild type and mutant alleles of the PfCRT codon 76 in baseline pre-CQ treatment P. falciparum isolates collected during 1999 and investigated their relationship to CQ efficacy in 3 different sites with different levels of CQ parasite resistance in Uganda. Of 32 isolates from Mulago Hospital, all were mutant (100%), while of 45 isolates from Tororo, 5 (11%) were mixed wild type and mutant and 40 (89%) were mutants only. Of 41 isolates from Apac, 13 (32%) were mixed wild type and mutant whereas 28 (68%) were mutants only. The finding of 100% prevalence of the Thr-76 mutant allele in all isolates at the 3 sites was remarkable. We found no association between the presence of Thr-76 mutation and treatment outcome at all the sites. However, the prevalence of the wild-type Lys-76 allele was higher in Apac, an area with lower CQ parasite resistance, compared to Tororo and Mulago which have relatively higher CQ parasite resistance. The Thr-76 allele as a marker of CQ resistance is probably useful in regions where the allele frequency has not yet plateaued.

Natural human immunoglobulin G subclass responses to Plasmodium falciparum serine repeat antigen in Uganda.

Serum samples from Ugandan residents of a malaria-hyperendemic region were tested by enzyme-linked immunosorbent assay for reactivity against recombinant constructs of the 47 (SE47')- and 50 (SE50A)-kDa fragments of Plasmodium falciparum serine repeat antigen (SERA). Immunoglobulin (Ig) G3 and IgG1 were the predominant subclass responses to SE47' and SE50A, respectively. The geometric mean optical density (OD) for IgG3 anti-SE47' was significantly lower in children < 15 years compared with adults > or = 15 years (P < 0.0001). By contrast, the geometric mean IgG1 anti-SE50A was slightly higher in children compared with adults (P < 0.01). The proportion of high responders (ODs > 0.5) to SE47' was significantly lower in children compared with adults (P < 0.001), whereas the proportion of high responders to SE50A was comparable in children and adults (P = 0.07). This first detailed study of SERA in a malaria-hyperendemic region suggests that natural human IgG3 anti-SE47' might be associated with immunity to malaria.

Low birthweight associated with maternal anaemia and Plasmodium falciparum infection during pregnancy, in a peri-urban/urban area of low endemicity in Uganda.

A cross-sectional study of pregnant women was conducted at Nsambya Hospital in Kampala, to investigate the prevalence and effect of Plasmodium falciparum infections during pregnancy, in a peri-urban/urban location. Overall, 544 pregnant women were recruited when they presented at the labour ward for delivery. After giving informed consent, each subject answered a questionnaire and underwent a physical examination, and peripheral-blood samples were obtained. After each uncomplicated delivery, samples of placental and cord blood were obtained from the placenta and infant, respectively, and infant birthweights were recorded. Smears were prepared from the blood samples and checked for parasites. Only 46 and 36 of the 537 women investigated were positive for P. falciparum infection in their peripheral and placental blood, respectively. Plasmodium falciparum was the only parasite encountered. The prevalences of low birthweight and maternal parasitaemia and the intensities of maternal infection were each greater in primigravidae than secundi- or multi-gravidae. Despite the low prevalence of parasitaemia in this population, P. falciparum infection in the primigravidae was a significant contributor to their ill health, leading to low birthweights in their infants.

Human IgG subclass antibodies to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) and predominance of the MAD20 allelic type of MSP1 in Uganda.

OBJECTIVE: To determine the natural human humoral immune responses to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)), a malaria candidate vaccine antigen and to determine the prevalence of MAD20 and K1 alleles of P. falciparum MSP1. DESIGN: Community based cross-sectional study. SETTING: Atopi Parish, Apac District, Uganda, 1995. SUBJECTS: Three hundred and seventy four Ugandans between <1 and 70 years old provided serum samples. MAIN OUTCOME MEASURES: IgG subclass antibodies by ELISA; MAD20 and K1 allelic types of MSP1 by PCR. RESULTS: Both the prevalence and the mean concentration of serum IgG1, and to a lesser extent IgG3, antibodies increased with age. IgG2 or IgG4 antibodies were virtually nonexistent. The cross-reactivity between the 4 sequence variants (E-KNG, E-TSR, Q-KNG and Q-TSR) of MSP1(19) was confirmed; however, a minority of sera preferentially recognised the KNG but not the TSR variants. All 33 P. falciparum isolates from different parts of Uganda carried the E-TSR (Mad20) allelic type and 3 isolates were mixed infections with E-TSR (MAD20) and Q-KNG (K1) allelic types, confirming the rarity of the K1 allele in Uganda. CONCLUSION: There is a robust IgG1 antibody response to the malaria vaccine candidate antigen MSP1(19) which begins at an early age. Future cohort studies are necessary to estblish the impact of these antibodies on clinical immunity to malaria. The MAD20 allelic type of MSP1 id predominant in Ugandan P. falciparum isolates.

Plasmodium falciparum malariometric indices in Apac district, northern Uganda.

OBJECTIVE: To establish Plasmodium falciparum malariometric indices in a field study site in Apac district, northern Uganda. DESIGN: A community-based cross sectional survey. SETTINGS: Atopi Parish, Apac district, Uganda, 1995. SUBJECTS: One thousand two hundred and thirty four volunteers aged below one and ninety years. MAIN OUTCOME MEASURES: P. falciparum parasitaemia rates and parasite density, splenomegaly, bednet use and chloroquine consumption. INTERVENTIONS: All subjects with P. falciparum positive smears were treated with chloroquine. RESULTS: The population prevalence of parasitaemia was 62.1% with the predominant species being P. falciparum (100%) and P. malariae in the minority (3.5%); P. ovale was not seen. The prevalence of parasitaemia in subjects older than 20 years and in those under ten years was 36% and 85%, respectively. The geometric mean parasite density started to decline by the age of six years. The splenomegaly rate in subjects over the age of 12 years and in those under nine years was 19.8% and 63.1%, respectively. Bednet use and chloroquine consumption was low. Interestingly, the reported use of chloroquine in the week immediately preceding the study was more frequent in children under two years old than in the rest of the population. CONCLUSION: Malaria transmission in Atopi Parish in northern Uganda is hyperendemic and age-related acquired anti-parasite immunity seems to appear by seven years of age.

[Comparative analysis of 2 diagnostic methods of human loiasis: IgG4 serology and nested PCR]. / Analyse comparative de deux méthodes diagnostiques de la loase humaine: sérologie IgG4 et PCR nichée.

By evaluating the diagnostic methods developed in our laboratory, the prevalence of loaiosis was estimated among 201 individuals from the province of Haut Ogooué in Gabon using IgG4 serology and nested-PCR. The study showed that the prevalence of loaiosis was higher than that described using standard microscopy. IgG4-based ELISA (Enzyme Linked Immunosorbant Assay) using crude extract of Loa loa microfilariae showed that 80% (35/44) of microfilaraemic individuals (MF') and 56% (88/157) of amicrofilaraemics (AMF) presented antibodies. By contrast, L. loa specific DNA amplified by nested-PCR was detected in all MF and in 68% (106/157) of AMF. Among the 201 samples tested, 95 (47%) gave positive results in both tests. These results indicate that the presence of IgG4 antibodies directed against crude extract of L. loa microfilariae is not linked to the positivity of nested-PCR assay (chi 2 for paired data = 8.78; P < 0.02). We conclude that the PCR assay is more sensitive than the detection of IgG4 antibodies (directed against crude extract of L. loa microfilariae) in detecting loaiosis, and particularly occult loaiosis (infection without circulating microfilariae).

Sequence conservation of repeat 3 region of the gene coding for the 15 kDa polyprotein within human and simian Loa loa.

The human and simian strains of Loa loa microfilariae are morphologically identical even though their periodicities vary. When using primate models (Mandrillus sphinx) of human loaisis for vaccination trials, the absence of any ongoing simian L. loa infection must be demonstrated. Nested primers derived from a human strain of L. loa (targeted on the repeat 3 region of the gene encoding the 15 kDa polyprotein; 15r3) amplified at 366 bp sequence from simian L. loa genomic DNA and blood lysates from mandrills infected with simian L. loa. This nested-PCR assay has been tested on 12 amicrofilaremic (AMF) mandrills (without filarial microfilariae) and was positive in four mandrills. The nested-PCR product derived from simian L. loa genomic DNA and from three of four AMF mandrills has been sequenced. No difference was observed between the four sequences, which, in addition, were 99.18% identical to the 15r3 of human L. loa. Therefore, the 15r3 sequence is conserved within human and simian L. loa. These results suggest that the four PCR-positive mandrills without circulating microfilariae had occult simian L. loa infections. The study demonstrates the ability of a nested-PCR assay to identify animals naturally infected with simian L. loa.

Use of polymerase chain reaction for accurate follow-up of Loa loa experimental infection in Mandrillus sphinx.

Mandrills (Mandrillus sphinx) experimentally infected with human Loa loa usually remain microfilaremic for a long period of time. Nevertheless some control their microfilaremia while still harboring adults worms, and therefore become occult-infected. A nested polymerase chain reaction (PCR) assay, targeted on the repeat 3 region of the gene coding for the L. loa 15-kD protein (15r3-PCR), has been evaluated in mandrills infected with third-stage larvae (L3) of L. loa. The results of this assay were negative during the prepatency period (4 months after inoculation), but became positive when microfilariae appeared in the blood, and remained positive in all mandrills, even in those that became amicrofilaremic. These results show that the positivity of the 15r3-PCR assay is linked to the appearance of microfilariae in peripheral blood and demonstrated that L. loa-specific DNA can be detected in blood from occult-infected mandrills.

[Relationship between the intensity of Loa loa filariasis transmission and prevalence of infections]. / Relation entre intensité de la transmission de la filaire Loa loa et prévalence des infections.

Filarial loiasis differs from other filariases in that most infected subjects are amicrofilaremic. This difference raises the notion of occult infection. The aim of this study was to evaluate the relationship between the intensity of transmission and incidence of infection. For this purpose we determined the incidence of loiasis both microscopically and by PCR in 201 subjects from three villages in the province of Haut Ogooue in Gabon. Intensity of transmission, expressed in ATP (annual transmission potential) in these villages was estimated to be 250 infecting larvae per individual per year (L3/man/yr) in Moyabi, 180 L3/man/yr in N'dokaye, and 43,000 L3/man/yr in Okoumbi. Although there was no significant difference between the three villages with regard to the incidence of microfilaremia (21 p. 100 and 22 p. 100), the incidence of occult infection, i.e., positive PCR in amicrofilaremic subjects, was 45 p. 100 in Moyabi, 79 p. 100 in N'dokaye and 80 p. 100 in Okoumbi. The overall incidence of loiasis was 57 p. 100 in Moyabi and 85 p. 100 in both N'dokaye and Okoumbi. These findings demonstrate that the incidence of loiasis is correlated with the intensity of transmission (p < 0.001), especially in children. Taking this information into account will improve control of Loa loa in endemic areas.

Human occult loiasis: improvement in diagnostic sensitivity by the use of a nested polymerase chain reaction.

The development of control strategies for loiasis is of crucial importance in endemic areas and depends heavily on the accurate identification of occult-infected individuals. A polymerase chain reaction (PCR) and nested polymerase chain reaction (nested PCR) were developed and based on sequences of the repeat 3 region (15r3) of the gene encoding a Loa loa 15-kD protein. The assays was performed on 20 blood samples from occult-infected subjects and 30 from field-collected amicrofilaremic individuals. The size of the initial PCR product was 396 basepairs (bp). When this initial amplification using primers 15r3(1) and 15r3(2) was carried out for 30 cycles, the PCR products from three of the 20 occult-infected and five of the 30 amicrofilaremic individuals were visualized after electrophoresis by staining the gel with ethidium bromide. Subsequent Southern blotting and hybridization with the specific probe revealed hybridization in 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples but only after two days of exposure of the blot to the x-ray film. When the nested PCR was carried out (product size = 366 bp, primers 15r3(3) and 15r3(4)), 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples that were positive by Southern hybridization of the initial PCR products were strongly positive by staining with ethidium bromide. Qualitative Southern blotting of the nested PCR products using the same probe previously described confirmed the ethidium bromide staining results after a very short exposure time of 4 hr. These results demonstrate that the nested PCR amplification product is specific and that its sensitivity in detecting occult loiasis is 95%. This approach has significant promise for the screening of large human populations for active loiasis without the requirement for blotting and hybridization of the PCR products.

Human occult loiasis: field evaluation of a nested polymerase chain reaction assay for the detection of occult infection.

A nested polymerase chain reaction (nested PCR) assay, targeted on the repeat 3 region (15r3) of the gene coding for a Loa loa 15 kD polyprotein, was developed to detect L. loa infection. The assay has a sensitivity of 95% and is 100% specific with regard to sympatric filarial parasites: Mansonella perstans, Onchocerca volvulus and Wuchereria bancrofti. In this field study in a mixed filarial (L. loa and M. perstans) endemic region of Gabon, 157 L. loa amicrofilaraemic blood samples (AMF; diagnosed by leucoconcentration followed by standard microscopic examination) from the residents from four villages were screened by the 15r3-nested PCR assay. The assay detected 106 occult infected subjects among the 157 AMF individuals (68%), including 59 of 87 adults (68%) and 47 of 70 children (67%). In each village the prevalence of occult infection was, respectively, 38%, 52%, 79% and 80% for Moyabi, Djoutou, N'djokaye and Okoumbi. The annual transmission potential (ATP) of loiasis has been estimated to be 250 infective larvae (L 3) per man per year for Moyabi and Djoutou, 1800 for N'djokaye and 433000 L3/man/year for Okoumbi. This implies a correlation between occult infection of loiasis and the intensity of transmission. By contrast, the prevalence of L. loa microfilariae was 21% for Okoumbi, 22%, for N'djokaye and 19% for Djoutou and Moyabi. These results show that the prevalence of loiasis in this region of Gabon is higher than previously described by standard microscopic examination and that the application of this assay will be significant in the development of control strategies for loiasis.

IgG4 serology of loiasis in three villages in an endemic area of south-eastern Gabon.

Human filariasis due to Loa loa differs from other filariasis in that the majority of infected subjects are without circulating microfilariae (occult loiasis). In search for alternative diagnostic methods, which do not depend on circulating microfilariae or the (rather infrequent) eye-passage of adult worms, it was shown earlier that IgG4 antibodies directed against Loa loa adult worm antigen are apparently a good marker of occult loiasis and specific with regard to the sympatrically occurring Mansonella perstans. In this study we evaluated an IgG4 antibody-based ELISA using crude extract of Loa loa microfilariae (which is easier to obtain than adult worm) to estimate the prevalence of loiasis in 3 villages in South-East Gabon. Of 222 examined individuals (80 children < 16 years, 142 adults) 44 (20%) carried Loa loa microfilariae and 170 (77%) M. perstans. Using the mean OD-value + 1 standard deviation of 9 sera from patients solely infected with M. perstans (from the Gambia, where Loa loa is not endemic) as a cut-off, 35 of the 44 microfilaraemic Loa loa patients and 2 of the 9 Gambian controls were positive. This shows that our method had a sensitivity of 80% and a specificity of 78%. Among the remaining 178 subjects who had no microfilariae of Loa loa, as many as 97 (55%) had significant levels of specific IgG4 antibodies against Loa loa, suggesting that they carried occult loiasis. The mean IgG4 level in these putatively occult loiasis patients was slightly but significantly lower than in microfilaraemic subjects (P < 0.03). In conclusion, despite the limited sensitivity and specificity of our method, IgG4- ELISA at present is a very useful tool in estimating the real prevalence of loiasis in epidemiological surveys and at the individual level can confirm the diagnosis of L. loa amicrofilaraemic subjects with clinical signs suggesting loiasis.

Detection of Loa loa-specific DNA in blood from occult-infected individuals.

Accurate and specific diagnosis of human loiasis is of crucial importance in an endemic area where two-thirds of infected individuals are without circulating microfilariae (occult loiasis). By using the polymerase chain reaction (PCR) and specific primers to the repeat 3 region (15r3) of the gene coding for Loa loa 15-kDa polyprotein antigen, DNA was amplified from total blood lysate of occult-infected subjects. A 396-bp DNA fragment was specifically detected. We tested the specificity of this method by qualitative hybridization to PCR products using blood lysates of the following subjects: (1) from Gabon (80 individuals residing in L. loa endemic area where loiasis exists sympatrically with Mansonella perstans); (2) from Togo (12 individuals infected with Onchocerca volvulus and M. perstans); (3) from Tahiti (12 individuals infected with Wuchereria bancrofti); and (4) from Mali (12 individuals infected with O. volvulus and M. perstans). Samples from Gabon included 60 L. loa amicrofilaremics and 20 L. loa occult-infected subjects. Qualitative hybridization carried out at 50 degrees C on PCR products, using a 15r3-specific oligonucleotide probe, revealed hybridization with L. loa-infected samples from Gabon and four samples from Togo after 2 days exposure to the film. The positive samples from Togo were characterized by the use of nested PCR. Three nested PCR products have been sequenced. No differences were observed between the three sequences and they are 99.72% identical to L. loa 15r3. None of bancroftian-infected individuals from Tahiti, nor O. volvulus- and M. perstans-infected individuals from Mali reacted after 1 week's exposure (overexposure) to the film. This allows us to conclude first that our 15r3 PCR assay is specific for L. loa and secondly that L. loa infections occur in Togo. The sensitivity of this 15r3 PCR assay was further investigated with occult patients and field-collected amicrofilaremic samples. We found that 19 of the 20 occult-infected individuals were positive on Southern hybridization, whereas 35/60 amicrofilaremics were positive. These results have shown that the sensitivity of this assay in detecting unequivocal, parasitologically proven occult loiasis was 95%, while the specificity with regard to the sympatric M. perstans was 100%.

T helper responsiveness in human Loa loa infection; defective specific proliferation and cytokine production by CD4+ T cells from microfilaraemic subjects compared with amicrofilaraemics.

The proliferation and cytokine profiles of peripheral blood mononuclear cells (PBMC) from microfilaraemic (Mf+) subjects infected by Loa loa in response to antigens of several parasitic stages were compared with those from amicrofilaraemic (Mf-) individuals. While a strong lymphoproliferative response and consistent levels of both Th1 (IL-2, interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-5) type cytokines were observed in response to adult worm (AW) and microfilariae (Mf) antigen in Mf- individuals, Mf+ subjects were characterized by a T cell unresponsiveness, including proliferation, cytokine production and IL-2 mRNA expression. Conversely, T cell responsiveness to mitogens and non-specific antigen were similar in the two endemic populations. Depletion of lymphocyte subpopulations indicated that T CD4+ were mainly involved in the specific cellular response. In contrast to other cytokines, IL-10 was produced in response to all parasitic stages, in both Mf+ and Mf- patients. Neutralization of IL-10 did not restore cytokine production in Mf+ patients, while B7 mRNA expression was similar between Mf+ and Mf- subjects in response to Mf antigen, suggesting that IL-10 was not the only factor responsible for T cell unresponsiveness. Mf+ patients have lower Mf antigen-specific IgG levels compared with Mf-, and there is a significant correlation between Mf antigen-specific antibodies and IL-5 responses. These findings suggest that Mf- status is correlated with T helper responsiveness, including proliferation and production of both Th1- and Th2-type cytokines, whereas Mf+ status is characterized by unresponsiveness of the same cell population, induced and/or maintained by microfilariae.

Experimental infection of a nonhuman primate with Loa loa induces transient strong immune activation followed by peripheral unresponsiveness of helper T cells.

Human infection with the parasite Loa loa is characterized by a good adaptation between the parasite and its host. One portion of the human population harbors only adult worms in subcutaneous tissues, whereas another portion also harbors the L1 microfilarial stage in peripheral circulation. This study was undertaken to understand the mechanisms by which the parasite evades or modulates host immunological attack. The cellular responses, based on T-cell proliferation, to the production of various cytokines (interleukin-2 [IL-2], gamma interferon [IFN-gamma], IL-4, and IL-5) and to expression of cytokine (IL-2, IFN-gamma, IL-4, IL-5, IL-10, and IL-12) mRNAs were investigated during the experimental infection with human parasite L. loa of a nonhuman primate which has been shown to display a spectrum of disease similar to that found in humans. Our results indicate that a T-cell unresponsiveness occurs when female worm products are released into the peripheral circulation, preceded by a transient period of strong T-cell proliferation, cytokine production, and cytokine mRNA expression. In the unresponsive state, only IL-10 mRNA is expressed, suggesting a role for IL-10 in down-regulation and maintenance of unresponsiveness. Taken together, these results indicate that both IL-10 production, which is known to inhibit B7 expression on monocytes, and the massive release of female products in the blood where T cells encounter antigens presented by nonactivated B lymphocytes, which lack costimulatory signals, should contribute to the inactivation of T cells.

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.

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.

Evaluation of Onchocerca volvulus-specific IgG4 subclass serology as an index of onchocerciasis transmission potential of three Gabonese villages.

The major objective of this study was to evaluate the usefulness of IgG4 ELISA and Western blot analysis, using a crude extract of Onchocerca volvulus adult worms as antigens, for diagnosing onchocerciasis in a Gabonese paediatric population with mixed filarial infections. The subjects had loaisis, streptocercosis or mansonellosis in addition to onchocerciasis. Control sera from loaisis or mansonellosis subjects residing outside the endemic zone were used to provide the cut-off point for positive results. The IgG4 ELISA had a specificity of 96% but a lower sensitivity of 78.7%. It detected 25 onchocerciasis cases out of 65 individuals who were negative on parasitological examination. Furthermore, the ELISA provided a more accurate picture of onchocerciasis transmission in a village with very low skin microfilarial load. A 27.5-kD antigen was identified on Western blots as a marker of onchocerciasis. The paediatric population provided a reliable window for assessing the parasitologic and serologic parameters in the three villages with disparate levels of onchocerciasis transmission.