Unusual Localization of Blood-Borne Loa loa Microfilariae in the Skin Depends on Microfilarial Density in the Blood: Implications for Onchocerciasis Diagnosis in Coendemic Areas.
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ABSTRACT: Background
The diagnostic gold standard for onchocerciasis relies on identification and enumeration of (skin-dwelling) Onchocerca volvulus microfilariae (mf) using the skin snip technique (SST). In a recent study, blood-borne Loa loa mf were found by SST in individuals heavily infected with L. loa, and microscopically misidentified as O. volvulus due to their superficially similar morphology. This study investigates the relationship between L. loa microfilarial density (Loa MFD) and the probability of testing SST positive.Methods
A total of 1053 participants from the (onchocerciasis and loiasis coendemic) East Region in Cameroon were tested for (1) Loa MFD in blood samples, (2) O. volvulus presence by SST, and (3) Immunoglobulin (Ig) G4 antibody positivity to Ov16 by rapid diagnostic test (RDT). A Classification and Regression Tree (CART) model was used to perform a supervised classification of SST status and identify a Loa MFD threshold above which it is highly likely to find L. loa mf in skin snips.Results
Of 1011 Ov16-negative individuals, 28 (2.8%) tested SST positive and 150 (14.8%) were L. loa positive. The range of Loa MFD was 0-85 200 mf/mL. The CART model subdivided the sample into 2 Loa MFD classes with a discrimination threshold of 4080 (95% CI, 2180-12 240) mf/mL. The probability of being SST positive exceeded 27% when Loa MFD was >4080 mf/mL.Conclusions
The probability of finding L. loa mf by SST increases significantly with Loa MFD. Skin-snip polymerase chain reaction would be useful when monitoring onchocerciasis prevalence by SST in onchocerciasis-loiasis coendemic areas.
SUBMITTER: Niamsi-Emalio Y
PROVIDER: S-EPMC8201578 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The specificity of skin snips for onchocerciasis diagnoses is considered to be almost 100%. Our molecular methods revealed that microfilariae emerging from skin snips collected from highly microfilaremic Loa loa-infected individuals were largely misidentified as Onchocerca volvulus. This has important implications for onchocerciasis diagnostic testing in Loa-endemic areas.
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Project description:BackgroundA test-and-not-treat (TaNT) strategy has been developed to prevent people with high concentrations of circulating Loa loa microfilariae (>20 000 microfilariae per mL) developing serious adverse events after ivermectin treatment during mass drug administration to eliminate onchocerciasis. An important question related to cost and programmatic issues is whether annual retesting is required for everyone. We therefore aimed to investigate changes in L loa microfilarial densities during TaNT campaigns run 18 months apart.MethodsIn this observational cohort study, we assessed the participants of two TaNT campaigns for onchocerciasis. These campaigns, which were run by a research team, together with personnel from the Ministry of Health and community health workers, were done in six health areas (in 89 communities) in Okola health district (Cameroon); the first campaign was run between Aug 10, and Oct 29, 2015, and the second was run between March 7, and May 26, 2017. All individuals aged 5 years and older were invited to be screened for Loa loa microfilaraemia before being offered ivermectin (unless contraindicated). L loa microfilarial density was measured at the point of care using the LoaScope. All those with a L loa microfilarial density of 20 000 microfilariae per mL or less were offered treatment; in the first 2 weeks of the 2015 campaign, a higher exclusion threshold of 26 000 microfilariae per mL or less was used. At both rounds of the intervention, participants were registered with a paper form, in which personal information were collected. In 2017, we also recorded whether each individual reported participation in the 2015 campaign. The primary outcome, assessed in all participants, was whether L loa microfilarial density was above or below the exclusion threshold (ie, the criteria that guided the decision to treat).FindingsIn the 2015 TaNT campaign, 26 415 people were censused versus 29 587 people in the 2017 TaNT campaign. All individuals aged 5 years and older without other contraindications to treatment (22 842 people in 2015 and 25 421 people in 2017) were invited to be screened for L loa microfilaraemia before being offered ivermectin. In 2015, 16 182 individuals were examined with the LoaScope, versus 18 697 individuals in the same communities in 2017. 344 (2·1%) individuals were excluded from ivermectin treatment because of a high L loa microfilarial density in 2015, versus 283 (1·5%) individuals in 2017 (p<0·0001). Records from 2017 could be matched to those from 2015 for 6983 individuals (43·2% of the 2015 participants). In this cohort, in 2017, 6981 (>99·9%) of 6983 individuals treated with ivermectin in 2015 had L loa microfilariae density below the level associated with neurological serious adverse events.InterpretationIndividuals treated with ivermectin do not need to be retested for L loa microfilaraemia before the next treatment, provided that they can be re-identified. This adjusted approach will enable substantial cost savings and facilitate reaching programmatic goals for elimination of onchocerciasis in areas that are co-endemic for loiasis.FundingBill & Melinda Gates Foundation, Division of Intramural Research (National Institute of Allergy and Infectious Diseases, US National Institutes of Health).
| S-EPMC8150319 | biostudies-literature
Project description:Co-infection with loiasis remains a potential problem in control programs targeting filarial infections. The effects of many anti-parasitic drugs often administered to Loa loa infected people are not well documented. This study compared the in vitro activity of several of these drugs on the viability of L. loa microfilariae (mf).Human strain L. loa mf were isolated from baboon blood using iso-osmotic Percoll gradient, and cultured in RPMI 1640/10% FBS with antimalarial drugs (mefloquine, amodiaquine, artesunate, chloroquine and quinine), anthelmintics (ivermectin, praziquantel, flubendazole and its reduced and hydrolyzed metabolites), two potential trypanocidal agents (fexinidazole and Scynexis-7158) and the anticancer drug imatinib. The drug concentrations used varied between 0.156 ?g/ml and 10 ?g/ml. Mf motility (CR50 = 50% immotility) and a metabolic viability assay (MTT) were used to assess the effects of these drugs on the parasites.Mf in control cultures showed only a slight reduction in motility after 5 days of culture. Active inhibition of Loa loa motility was seen with mefloquine and amodiaquine (CR50 values of 3.87 and 4.05 ?g/ml, respectively), immobilizing > 90% mf within the first 24 hours: mefloquine killed the mf after 24 hours of culture at concentrations ? 5 ?g/ml. SCYX-7158 also induced a concentration-dependent reduction in mf motility, with > 50% reduction in mf motility seen after 5 days at 10 ?g/ml. The anticancer drug imatinib reduced mf motility at 10 ?g/ml from the first day of incubation to 55% by day 5, and the reduction in motility was concentration-dependent. Praziquantel and fexinidazole were inactive, and FLBZ and its metabolites, as well as ivermectin at concentrations > 5 ?g/ml, had very minimal effects on mf motility over the first 4 days of culture.The considerable action of the anti-malarial drugs mefloquine and amodiaquine on Loa mf in vitro highlights the possibility of repurposing the existing anti-infectious agents for the development of drugs against loiasis. The heterogeneity in the activity of anti-parasitic agents on Loa loa mf supports the need for further investigation using animal models of loiasis.
| S-EPMC5883330 | biostudies-literature
Project description:BackgroundSuitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa.MethodsIn vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval.ResultsOf the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility.ConclusionsThe findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.
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Project description:A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human alpha-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.
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