Analysis of diagnostic test outcomes in a large loiasis cohort from an endemic region: Serological tests are often false negative in hyper-microfilaremic infections.

BACKGROUND: The parasitic disease loiasis is associated with significant morbidity and mortality. Individuals with hyper-microfilaremia (greater than 20,000 microfilariae per mL of blood) may suffer from serious treatment-related or spontaneous adverse events. Diagnosing loiasis remains complex and primarily relies on direct parasite detection. In this study, we analyzed the performance of various diagnostic tests and the influence of parasitological and clinical factors on test outcomes in samples from individuals living in an endemic region. METHODS: Data and samples were collected from rural Gabon. Loiasis was defined as either detectable microfilaremia, or a positive history of eyeworm as assessed by the RAPLOA questionnaire. Diagnostic testing included a quantitative PCR (qPCR) for detection of Loa loa DNA in blood samples, an in-house crude L. loa antigen IgG ELISA, and a rapid test for antibodies against the Ll-SXP-1 antigen (RDT). Sensitivity and specificity were determined for each test and factors potentially influencing outcomes were evaluated in an exploratory analysis. RESULTS: ELISA, RDT and qPCR results were available for 99.8%, 78.5%, and 100% of the 1,232 participants, respectively. The ELISA and RDT had only modest diagnostic accuracy. qPCR was specific for L. loa microfilaremia and Cycle threshold values correlated with microfilarial density. Anti-L. loa IgG levels were highest in occult loiasis, and antibody levels correlated inversely with L. loa microfilarial density as did RDT line intensities. Only 84.6% and 16.7% of hyper-microfilaremic individuals tested positive by ELISA (11/13) and RDT (2/12), respectively. CONCLUSION: None of the tests demonstrated high sensitivity and specificity for loiasis. Indirect diagnostic assays were characterized by low specificity. Additionally, hyper-microfilaremic individuals often tested negative by RDT and ELISA, indicating that these tests are not suitable for individual case management in endemic populations.

A Novel, Highly Sensitive Nucleic Acid Amplification Test Assay for the Diagnosis of Loiasis and its Use for Detection of Circulating Cell-Free DNA.

Mass drug administration programs targeting filarial infections depend on diagnostic tools that are sensitive and specific. The coendemicity of Loa loa with other filarial species often hampers the control programs. LL2634 was identified as the most promising target among several highly repeated targets, with sensitivity between 500 ag and 1 fg of genomic DNA. Using DNA from infected individuals, LL2643 quantitative polymerase chain reaction (qPCR) was positive in all individuals. LL2643 was detected in plasma-derived circulating cell-free DNA (ccfDNA) from 48 of 53 microfilariae-positive patients. Detection of ccfDNA in urine was possible, but it occurred rarely among those tested. Importantly, LL2643 ccfDNA became undetectable within 1 month following diethylcarbamazine (DEC) treatment and remained negative for at least a year. LL2643 offers a more sensitive and specific target for detection of L. loa infection and would be easily configurable to a point-of-contact assay. Clinical Trials Registration. NCT00001230 and NCT00090662.

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.

Integrated xenosurveillance of Loa loa, Wuchereria bancrofti, Mansonella perstans and Plasmodium falciparum using mosquito carcasses and faeces: A pilot study in Cameroon.

BACKGROUND: Community presence of loiasis must be determined before mass drug administration programmes for lymphatic filariasis and onchocerciasis can be implemented. However, taking human blood samples for loiasis surveillance is invasive and operationally challenging. A xenosurveillance approach based on the molecular screening of mosquitoes and their excreta/feces (E/F) for Loa loa DNA may provide a non-invasive method for detecting the community presence of loiasis. METHODS: We collected 770 wild mosquitoes during a pilot study in a known loiasis transmission area in Mbalmayo, Cameroon. Of these, 376 were preserved immediately while 394 were kept in pools to collect 36-hour E/F samples before processing. Carcasses and E/F were screened for L. loa DNA. To demonstrate this method's potential for integrated disease surveillance, the samples were further tested for Wuchereria bancrofti, Mansonella perstans, and Plasmodium falciparum. RESULTS: Despite limited sample numbers, L. loa DNA was detected in eight immediately-stored mosquitoes (2.13%; 95% CI 1.08 to 4.14), one carcass stored after providing E/F (0.25%; 95% CI 0.04 to 1.42), and three E/F samples (estimated prevalence 0.77%; 95% CI 0.15 to 2.23%). M. perstans and P. falciparum DNA were also detected in carcasses and E/F samples, while W. bancrofti DNA was detected in E/F. None of the carcasses positive for filarial worm DNA came from pools that provided a positive E/F sample, supporting the theory that, in incompetent vectors, ingested parasites undergo a rapid, complete expulsion in E/F. CONCLUSIONS: Mosquito xenosurveillance may provide a useful tool for the surveillance of loiasis alongside other parasitic diseases.

Comparison of three PCR-based methods to detect Loa loa and Mansonella perstans in long-term frozen storage dried blood spots.

OBJECTIVES: Loa loa and Mansonella perstans are two very common filarial species in Africa. Although microscopy is the traditional diagnostic method for human filariasis, several polymerase chain reaction (PCR) methods have emerged as an alternative approach for identifying filarial parasites. The aim of this study is to compare three molecular methods and decide which is the most suitable for diagnosing human loiasis and mansonellosis in non-endemic regions using dried blood spot (DBS) as a medium for sample collection and storage. METHODS: A total of 100 DBS samples, with their corresponding thin and thick blood smears, were selected for this study. Microscopy was used as the reference method to diagnose and calculate the microfilaraemia. Filarial DNA was extracted using the saponin/Chelex method and the DNA isolated was assayed by Filaria-real time-PCR, filaria-nested PCR, and cytochrome oxidase I PCR. All PCR products were subsequently purified and sequenced. The statistical values for each molecular test were calculated and compared. RESULTS: Overall, 64 samples were identified as negative by all tests and a further 36 samples were positive by at least one of the methods used. The sensitivity and specificity were similar for the different molecular methods, all of which demonstrated good agreement with microscopy. CONCLUSIONS: Based on this study, and from a practical point of view (single and short amplification round), the optimal technique for diagnosing filarial infection in non-endemic regions is filaria-real time-PCR, which presents high sensitivity and specificity and is also able to detect a wide range of human filariae.

Comparison of six methods for Loa loa genomic DNA extraction.

OBJECTIVES: Good-quality and sufficient DNA is essential for diagnostics and vaccine development. We aimed to compare six DNA extraction techniques applied to Loa loa microfilariae in order to evaluate the purity and integrity of extracts in terms of quality and quantity. METHODS: The microfilariae were purified via a Percoll gradient procedure with blood from hyper-microfilaremic individuals (> 30,000 microfilaria [mf]/ml). DNA extraction was carried out in duplicate at a rate of 350,000 mf/tube for each technique: phenol/chloroform, commercial Qiagen kit, salting out, Tris-EDTA, methanol, and cetyltrimethylammonium bromide (CTAB). The integrity, purity, concentration, and quality of the DNA extracts were successively verified by agarose gel electrophoresis, spectrophotometry (A260/A280 and A260/A230 wavelength ratio), Qubit fluorometry, and endonuclease and polymerase activity. The six techniques were compared on the basis of the following parameters: concentration, purity, efficiency, effectiveness, integrity, safety of the technique, as well as cost and duration of the protocol. RESULTS: The ratios of the optical densities of the extracts A260/A280 and A260/A230 were, respectively: phenol/chloroform (1.82; 1.11), Qiagen (1.93; 1.36), salting-out (1.9; 2.04), Tris-EDTA (1.99; 1.183), methanol (2.126; 1.343), and CTAB (2.01; 2.426). The DNA yield was: phenol/chloroform (3.920 µg), Qiagen (10.280 µg), salting-out (10.390 µg), Tris-EDTA (0.5528 µg), methanol (0.1036 µg), and CTAB (1.115 µg). Endonuclease and polymerase activity was demonstrated by digestion of DNA and through amplicons obtained via polymerase chain reaction assays with phenol/chloroform, Qiagen, and salting-out extracts. CONCLUSION: The phenol/chloroform, Qiagen, and salting-out DNA extracts were all of good quality. Salting out had the best yield followed by Qiagen and then phenol/chloroform. Endonuclease and polymerase activity was effective in all three extracts despite the presence of some contaminants. These methods are therefore suitable for the extraction of DNA from Loa loa microfilariae. Tris-EDTA and methanol did not show adequate sensitivity, while CTAB was found to be unsuitable.

Geostatistical modelling enables efficient safety assessment for mass drug administration with ivermectin in Loa loa endemic areas through a combined antibody and LoaScope testing strategy for elimination of onchocerciasis.

The elimination of onchocerciasis through community-based Mass Drug Administration (MDA) of ivermectin (Mectizan) is hampered by co-endemicity of Loa loa, as individuals who are highly co-infected with Loa loa parasites can suffer serious and occasionally fatal neurological reactions from the drug. The test-and-not-treat strategy of testing all individuals participating in MDA has some operational constraints including the cost and limited availability of LoaScope diagnostic tools. As a result, a Loa loa Antibody (Ab) Rapid Test was developed to offer a complementary way of determining the prevalence of loiasis. We develop a joint geostatistical modelling framework for the analysis of Ab and Loascope data to delineate whether an area is safe for MDA. Our results support the use of a two-stage strategy, in which Ab testing is used to identify areas that, with acceptably high probability, are safe or unsafe for MDA, followed by Loascope testing in areas whose safety status is uncertain. This work therefore contributes to the global effort towards the elimination of onchocerciasis as a public health problem by potentially reducing the time and cost required to establish whether an area is safe for MDA.

Molecular characterization and genetic diversity of Loa loa parasites responsible of a long-delayed filarial infection in an immigrant patient inhabited in Paris.

A 21-year-old young boy who lived alone since one year and a half ago in Paris was referred due to severe vertigo. He is originally from Ivory Coast but lived from 2011 to 2017 in Douala city in west of Cameroon. Beside vertigo, he complained from headache, sudden abdominal pain and edema in both left and right forearms for about two years. General examination demonstrated a healthy condition with no subcutaneous nodules and swelling on any other part of the body, not splenomegaly or lymphadenopathy. Moreover, the eyes were normal with clear lens. Blood count analysis revealed a hypereosinophilia (2670*106/L, N: <500*106/L). A couple of direct and May-Grunwald-Giemsa stained smears, analyzed by microscopy revealed the semitransparent cylindrical worms with almost 300 µm length and 45 µm width identified as Loa loa. The identity of the worm was then confirmed by bidirectional sequencing of 450 bp fragment of internal transcribed spacer 1 (ITS1-rDNA). Based on Neighbor-Joining phylogenetic tree, our isolate was clustered tightly with other few Loa species from Gabon in the same clade. No hybrid was observed among processed sequences since all species groups were discriminated separately. In the current case, he was originally from Ivory Coast but absence of medical and epidemiological evidences as well as the residency of our patient for 6 years in Cameroon made us suspicious that the patient has been most likely infected by L. loa worms in this country. The patient was treated by a couple of ivermectin (200 µg/kg for 3 days) and diethylcarbamazine (3 mg/kg, 2 times per day for 4 weeks) and a favorable evolution was observed within few weeks. Regarding at least one year and a half interval between the probable Loa loa infection in Cameroon and diagnosis, Loa loa worms are competent to persist in the human host for several years. Consequently, the clinicians should be aware of this parasitosis among the travelers or immigrants coming from endemic regions in Africa.

Validation of loop-mediated isothermal amplification for the detection of Loa loa infection in Chrysops spp in experimental and natural field conditions.

BACKGROUND: The mass drug administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that has, in turn, led to reduction of infection levels in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission as it provides a potential alternative or indirect strategy for addressing the problem of co-endemic loiasis and lymphatic filariasis through the Onchocerciasis Elimination Programme and it further reflects the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, the sensitivity of this method is compromised when the intensity of infection is low. The loop-mediated isothermal amplification (LAMP) assay of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. The aim of this study was to validate the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions. METHODS: Two sets of 18 flies were fed on volunteers with either a low (< 10 mf/ml) or high (> 30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed using LAMP for the detection of L. loa infection. In addition, a total of 9270 flies were collected from the north-west, east, and south-west regions (SW 1 and 2) of Cameroon using sweep nets and subjected to microscopy (7841 flies) and LAMP (1291 flies plus 138 nulliparous flies) analyses. RESULTS: The LAMP assay successfully detected parasites in Chrysops fed on volunteers with both low and high microfilariaemic loads. Field validation and surveillance studies revealed LAMP-based infection rates ranging from 0.5 to 31.6%, with the lowest levels in SW 2 and the highest infection rates in SW 1. The LAMP assay detected significantly higher infection rates than microscopy in four of the five study sites. CONCLUSION: This study demonstrated the potential of LAMP as a simple surveillance tool. It was found to be more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

Divergent Evolution of a Protein-Protein Interaction Revealed through Ancestral Sequence Reconstruction and Resurrection.

The postsynaptic density extends across the postsynaptic dendritic spine with discs large (DLG) as the most abundant scaffolding protein. DLG dynamically alters the structure of the postsynaptic density, thus controlling the function and distribution of specific receptors at the synapse. DLG contains three PDZ domains and one important interaction governing postsynaptic architecture is that between the PDZ3 domain from DLG and a protein called cysteine-rich interactor of PDZ3 (CRIPT). However, little is known regarding functional evolution of the PDZ3:CRIPT interaction. Here, we subjected PDZ3 and CRIPT to ancestral sequence reconstruction, resurrection, and biophysical experiments. We show that the PDZ3:CRIPT interaction is an ancient interaction, which was likely present in the last common ancestor of Eukaryotes, and that high affinity is maintained in most extant animal phyla. However, affinity is low in nematodes and insects, raising questions about the physiological function of the interaction in species from these animal groups. Our findings demonstrate how an apparently established protein-protein interaction involved in cellular scaffolding in bilaterians can suddenly be subject to dynamic evolution including possible loss of function.

Molecular Epidemiology of Mansonella Species in Gabon.

Mansonella perstans, a filarial nematode, infects large populations in Africa and Latin America. Recently, a potential new species, Mansonella sp "DEUX," was reported. Carriage of endosymbiotic Wolbachia opens treatment options for Mansonella infections. Within a cross-sectional study, we assessed the prevalence of filarial infections in 834 Gabonese individuals and the presence of the endosymbiont Wolbachia. Almost half of the participants (400/834 [48%]) were infected with filarial nematodes, with Mansonella sp "DEUX" being the most frequent (295/400 [74%]), followed by Loa loa (273/400 [68%]) and Mansonella perstans (82/400 [21%]). Being adult/elderly, male, and living in rural areas was associated with a higher risk of infection. Wolbachia carriage was confirmed in M. perstans and Mansonella sp "DEUX." In silico analysis revealed that Mansonella sp "DEUX" is not detected with currently published M. perstans-specific assays. Mansonella infections are highly prevalent in Gabon and might have been underreported, likely also beyond Gabon.

Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis.

The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis.

Increasing evidence of low lymphatic filariasis prevalence in high risk Loa loa areas in Central and West Africa: a literature review.

In West and Central Africa, there is a need to establish the prevalence of Wuchereria bancrofti in areas that are co-endemic for Loa loa, in order to implement the appropriate strategies to scale-up interventions for the elimination of lymphatic filariasis (LF). Due to the risk of severe adverse events (SAEs) to ivermectin in individuals with high L. loa microfilaraemia, the current strategy recommended by the World Health Organization (WHO) is twice yearly mass drug administration (MDA) with albendazole, supplemented by vector control targeting the Anopheles vectors. Defining W. bancrofti prevalence in areas co-endemic with L. loa is complicated by the cross-reactivity of rapid diagnostic immunochromatographic card tests (ICT), widely used for LF mapping, in individuals with high L. loa microfilaraemia. This has probably resulted in the overestimation of LF prevalence, triggering the implementation of MDA strategies, which may be unnecessary and wasteful of the limited resources for elimination programme implementation. Here we review the literature and present historical evidence, which uniformly highlight low or no prevalence of W. bancrofti infection and/or clinical LF cases across five Central African countries, in more than 30 different geographical areas covering 280 individual sites and > 22,000 individuals tested within high risk L. loa areas. This highlights the very limited information available on LF prevalence in L. loa areas, and potentially has major policy implications, which could shift the focus towards revised mapping criteria to verify low or no W. bancrofti prevalence in high risk L. loa areas. In this situation, revising the current WHO strategy from MDA, to focus more on ensuring high and effective vector control, through insecticide treated/long-lasting impregnated bednets (ITNs/LLINs), integration of point-of-care test-and-treat options into health systems, and consolidating closer links with the malaria control programme may be a more effective and appropriate use of the limited resources and drug donations available for LF elimination.

Protein Translation Enzyme lysyl-tRNA Synthetase Presents a New Target for Drug Development against Causative Agents of Loiasis and Schistosomiasis.

Helminth parasites are an assemblage of two major phyla of nematodes (also known as roundworms) and platyhelminths (also called flatworms). These parasites are a major human health burden, and infections caused by helminths are considered under neglected tropical diseases (NTDs). These infections are typified by limited clinical treatment options and threat of drug resistance. Aminoacyl-tRNA synthetases (aaRSs) are vital enzymes that decode genetic information and enable protein translation. The specific inhibition of pathogen aaRSs bores well for development of next generation anti-parasitics. Here, we have identified and annotated aaRSs and accessory proteins from Loa loa (nematode) and Schistosoma mansoni (flatworm) to provide a glimpse of these protein translation enzymes within these parasites. Using purified parasitic lysyl-tRNA synthetases (KRSs), we developed series of assays that address KRS enzymatic activity, oligomeric states, crystal structure and inhibition profiles. We show that L. loa and S. mansoni KRSs are potently inhibited by the fungal metabolite cladosporin. Our co-crystal structure of Loa loa KRS-cladosporin complex reveals key interacting residues and provides a platform for structure-based drug development. This work hence provides a new direction for both novel target discovery and inhibitor development against eukaryotic pathogens that include L. loa and S. mansoni.

A Step Toward Eradication of Human Filariases in Areas Where Loa Is Endemic.

Mass drug administration (MDA) programs have achieved remarkable success in limiting the pathology and transmission of the human parasitic infections onchocerciasis and lymphatic filariasis. The full implementation of MDA campaigns for filariasis elimination has been stymied by the unacceptable incidence of severe adverse events observed following drug treatment of a subset of individuals who harbor high loads of Loa loa microfilaria. Extending MDA strategies to regions where loiasis is coendemic could be done confidently if a simple, inexpensive, and rapid diagnostic method was available that could accurately identify individuals who have L. loa microfilarial loads above the risk threshold and could thus be excluded from treatment. A recent paper in mBio reports the discovery of an antigen unique to L. loa microfilaria that can be detected in blood and urine and may form the basis for such an assay. Further work will reveal whether this discovery will smooth the path to achieve filariasis eradication.

Molecular Epidemiology of Blood-Borne Human Parasites in a Loa loa-, Mansonella perstans-, and Plasmodium falciparum-Endemic Region of Cameroon.

The study of the interactions among parasites within their hosts is crucial to the understanding of epidemiology of disease and for the design of effective control strategies. We have conducted an assessment of infections with Loa loa, Mansonella perstans, Wuchereria bancrofti, and Plasmodium falciparum in eastern Cameroon using a highly sensitive and specific quantitative polymerase chain reaction assay using archived dried whole blood spots. The resident population (N = 1,085) was parasitized with M. perstans (76%), L. loa (39%), and P. falciparum (33%), but not with W. bancrofti Compared with single infections (40.1%), coinfection was more common (48.8%): 21.0% had L. loa-M. perstans (Ll(+)/Mp(+)/Pf(-)), 2.7% had L. loa-P. falciparum (Ll(+)/Pf(+)/Mp(-)), 15.1% had M. perstans-P. falciparum (Mp(+)/Pf(+)/Ll(-)), and 10.0% had L. loa-M. perstans-P. falciparum (Ll(+)/Mp(+)/Pf(+)). Interestingly, those with all three infections (Ll(+)/Mp(+)/Pf(+)) had significantly higher L. loa microfilaria (mf) counts than either single Ll(+) (P = 0.004) or double Ll(+)/Mp(+) (P = 0.024) infected individuals. Of those infected with L. loa, the mean estimated counts of L. loa mf varied based on location and were positively correlated with estimated intensities of M. perstans mf. Finally, at a community level, heavy L. loa infections were concentrated in a few individuals whereby they were likely the major reservoir for infection.

Ancient horizontal transfers of retrotransposons between birds and ancestors of human pathogenic nematodes.

Parasite host switches may trigger disease emergence, but prehistoric host ranges are often unknowable. Lymphatic filariasis and loiasis are major human diseases caused by the insect-borne filarial nematodes Brugia, Wuchereria and Loa. Here we show that the genomes of these nematodes and seven tropical bird lineages exclusively share a novel retrotransposon, AviRTE, resulting from horizontal transfer (HT). AviRTE subfamilies exhibit 83-99% nucleotide identity between genomes, and their phylogenetic distribution, paleobiogeography and invasion times suggest that HTs involved filarial nematodes. The HTs between bird and nematode genomes took place in two pantropical waves, >25-22 million years ago (Myr ago) involving the Brugia/Wuchereria lineage and >20-17 Myr ago involving the Loa lineage. Contrary to the expectation from the mammal-dominated host range of filarial nematodes, we hypothesize that these major human pathogens may have independently evolved from bird endoparasites that formerly infected the global breadth of avian biodiversity.

Mansonella, including a Potential New Species, as Common Parasites in Children in Gabon.

BACKGROUND: Like other tropical African countries, Gabon is afflicted by many parasitic diseases, including filariases such as loiasis and mansonellosis. This study aimed to assess the prevalence of these two filarial diseases in febrile and afebrile children using quantitative real-time PCR and standard PCR assays coupled with sequencing. METHODOLOGY/PRINCIPAL FINDINGS: DNA from blood specimens of 1,418 Gabonese children (1,258 febrile and 160 afebrile) were analyzed. Overall, filarial DNA was detected in 95 (6.7%) children, including 67 positive for M. perstans (4.7%), which was the most common. M. perstans was detected in 61/1,258 febrile children (4.8%) and 6/160 afebrile children (3.8%, P = 0.6). Its prevalence increased statistically with age: 3.5%, 7.7% and 10.6% in children aged ≤ 5, 6-10 and 11-15 years, respectively. M. perstans prevalence was significantly higher in Koulamoutou and Lastourville (12% and 10.5%, respectively) than in Franceville and Fougamou (2.6% and 2.4%, respectively). Loa loa was detected in seven febrile children including one co-infection with M. perstans. Finally, 21 filarial DNA positive were negative for M. perstans and Loa loa, but ITS sequencing could be performed for 12 and allowed the identification of a potential new species of Mansonella provisionally called "DEUX". Mansonella sp. "DEUX" was detected only in febrile children. CONCLUSIONS/SIGNIFICANCE: Further study should be performed to characterize Mansonella sp. "DEUX" and evaluate the clinical significance of mansonellosis in humans.

Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25-30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.

Loa loa and Onchocerca ochengi miRNAs detected in host circulation.

A combination of deep-sequencing and bioinformatics analysis enabled identification of twenty-two microRNA candidates of potential nematode origin in plasma from Loa loa-infected baboons and a further ten from the plasma of an Onchocerca ochengi-infected cow. The obtained data were compared to results from previous work on miRNA candidates from Dirofilaria immitis and O. volvulus found in host circulating blood, to examine the species specificity of the released miRNA. None of the miRNA candidates was found to be present in all four host-parasite scenarios and most of them were specific to only one of them. Eight candidate miRNAs were found to be identical in the full sequence in at least two different infections, while nine candidate miRNAs were found to be similar but not identical in at least four filarial species.