Human filariasis in travelers and migrants: a retrospective 25-year analysis at the Institute of Tropical Medicine, Antwerp, Belgium

Background Information on human filariasis in international travelers is scarce. We describe the epidemiology, clinical presentation and outcome of these infections in a reference travel clinic over the past decades. Methods We reviewed all cases of filariasis diagnosed at the Institute of Tropical Medicine, Antwerp, Belgium, from 1994 to 2018. Diagnosis was obtained by either parasitological methods (confirmed) or strict clinical case definitions (probable). We assessed the characteristics of cases at diagnosis and response to therapy within three to 12 months. Results A total of 320 patients (median age: 41 years; 71% males) were diagnosed with 327 filarial infections (Wuchereria bancrofti = 6; Onchocerca volvulus = 33, Loa loa = 150, Mansonella perstans = 130; unspecified species = 8). Diagnosis was confirmed in 213/320 (67%) patients. European long-term travelers accounted for 166 patients (52%) and visitors/migrants from tropical countries for another 110 (34%). Central Africa was the likely region of acquisition for 294 (92%) patients. The number of filariasis cases decreased from 21.5/year in average in the nineties to 6.3/year in the last decade, when loiasis became predominant. Cases reported symptoms in > 80% of all filarial infections but mansonellosis (45/123 single infections; 37%). Lymphatic filariasis and onchocerciasis cases responded well to conventional therapy. However, 30% of patients with loiasis and mansonellosis experienced treatment failure (with diethylcarbamazine and levamisole-mebendazole, respectively). Conclusions The burden and species distribution of filariasis in travelers evolved in the past decades. Most presentations were symptomatic. Case management would benefit from more effective therapies for loiasis and mansonellosis.

Diagnostic performance of capillary and venous blood samples in the detection of Loa loa and Mansonella perstans microfilaraemia using light microscopy

Background Loa loa and Mansonella perstans–the causative agents of loiasis and mansonellosis—are vector-borne filarial parasites co-endemic in sub-Saharan Africa. Diagnosis of both infections is usually established by microscopic analysis of blood samples. It was recently established that the odds for detecting Plasmodium spp. is higher in capillary (CAP) blood than in venous (VEN) blood. In analogy to this finding this analysis evaluates potential differences in microfilaraemia of L. loa and M. perstans in samples of CAP and VEN blood. Methods Recruitment took place between 2015 and 2019 at the CERMEL in Lambaréné, Gabon and its surrounding villages. Persons of all ages presenting to diagnostic services of the research center around noon were invited to participate in the study. A thick smear of each 10 microliters of CAP and VEN blood was prepared and analysed by a minimum of two independent microscopists. Differences of log2-transformed CAP and VEN microfilaraemia were computed and expressed as percentages. Furthermore, odds ratios for paired data were computed to quantify the odds to detect microfilariae in CAP blood versus in VEN blood. Results A total of 713 participants were recruited among whom 52% were below 30 years of age, 27% between 30–59 years of age and 21% above 60 years of age. Male-female ratio was 0.84. Among 152 participants with microscopically-confirmed L. loa infection median (IQR) microfilaraemia was 3,650 (275–11,100) per milliliter blood in CAP blood and 2,775 (200–8,875) in VEN blood (p<0.0001), while among 102 participants with M. perstans this was 100 (0–200) and 100 (0–200), respectively (p = 0.44). Differences in linear models amount up to an average of +34.5% (95% CI: +11.0 to +63.0) higher L. loa microfilaria quantity in CAP blood versus VEN blood and for M. perstans it was on average higher by +24.8% (95% CI: +0.0 to +60.5). Concordantly, the odds for detection of microfilaraemia in CAP samples versus VEN samples was 1.24 (95% CI: 0.65–2.34) and 1.65 (95% CI: 1.0–2.68) for infections with L. loa and M. perstans, respectively. Conclusion This analysis indicates that average levels of microfilaraemia of L. loa are higher in CAP blood samples than in VEN blood samples. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was higher in CAP than in VEN blood which may pre-dispose CAP blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in CAP blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in CAP and VEN blood above the limit of detection of 100 microfilariae/ml. Yet, it cannot be excluded that the study was underpowered to detect a moderate difference.

Aplicación de la técnica LAMP para la detección de Loa loa y Mansonella perstans

Las filariosis son enfermedades endémicas de regiones tropicales ocasionadas por nematodos filiformes trasmitidos por la picadura de insectos. Producen elevada morbilidad. La loaosis (Loa Loa) y la mansonelosis (Mansonella perstans) afectan globalmente a 10 y 100 millones de personas, respectivamente. El diagnóstico de certeza es parasitológico, pero no detecta infecciones precoces o bajas microfilaremias y hay que considerar la periodicidad de las microfilarias en sangre. Las técnicas moleculares, como la PCR, tienen gran sensibilidad y especificidad, pero son caras, técnicamente complejas y requieren infraestructura no disponible en zonas endémicas de escasos recursos. La tecnología LAMP (loop-mediated isothermal amplification) presenta ventajas sobre la PCR como mayor rapidez, escaso equipamiento, más tolerante a inhibidores y los resultados pueden observarse colorimétricamente. En este trabajo se aplica y valora la tecnología LAMP para la detección de ADN de Loa loa y M. perstans en 22 muestras de sangre almacenadas en papel de filtro de individuos residentes en Guinea Ecuatorial. Las muestras se analizaron microscópicamente, mediante qPCR y LAMP. Los métodos moleculares resultaron más sensibles que la microscopía. El LAMP resultó más sensible que la qPCR para la detección de ADN de Loa loa y M. perstans.

Genomes of Wolbachia endosymbionts from the human filarial parasites Mansonella perstans and Mansonella ozzardi

Mansonella ozzardi and Mansonella perstans, filarial parasites infecting millions of people worldwide, harbor their unique obligate endosymbionts, the alpha-proteobacteria WolbachiawMoz and wMpe, respectively. Currently, little is known about these Wolbachia and no genome sequences are available. In the current study, high quality draft genomes of wMoz and wMpe were assembled from complex clinical samples using a metagenome assembly and binning approach. These represent the first genomes from supergroup F Wolbachia originating from human parasites and share features characteristic of filarial as well arthropod Wolbachia, consistent with their position in supergroup F. Metagenomic data analysis was also used to estimate Wolbachia titers, which revealed wide variation in levels across different clinical isolates, addressing the contradicting reports on presence or absence of Wolbachia in M. perstans. These findings may have implications for the use antibiotics to treat mansonellosis. The wMoz and wMpe genome sequences provide a valuable resource for further studies on symbiosis, evolution and drug discovery.

Limitations of PCR detection of filarial DNA in human stools from subjects non-infected with soil-transmitted helminths

The standard techniques for diagnosis of human filariasis are the microscopic examination of blood smears or skin biopsies, which are relatively invasive and poorly sensitive at low levels of infection. Recently, filarial DNA has been detected in fecal samples from non-human primates in Central Africa. The aim of this study was to demonstrate proof-of-concept of a non-invasive molecular diagnosis technique for human filariasis by targeting fragments of 12S rDNA, Cox1, ITS1 and LL20-15kDa ladder antigen-gene by conventional PCR in DNA extracted from stool samples of 52 people infected with Mansonella perstans and/or Loa loa. Of these, 10 patients were infected with soil-transmitted helminths (Trichuris trichiura and/or Ascaris lumbricoides), and none were positive for Necator americanus. Interestingly, no filarial gene fragments were detected in the stools of any of the 52 patients. Future studies should evaluate whether a co-infection with soil-transmitted helminths causing gastrointestinal bleeding and likely allowing (micro)filaria exit into the digestive tract, may facilitate the molecular detection of filarial DNA fragments in stool samples.

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.

Comparison of Three PCR-Based Methods to Detect Loa loa and Mansonella perstans in Long-Term Frozen Storage Dried Blood Spots

Background: Loa loa and Mansonella perstans are two filarial species very common in Africa, with overlapping geographic distribution in some areas. Microscopy is the traditional diagnostic method for human loiasis and mansonellosis, but is a time-consuming, labor intensive and tedious. Polymerase chain reaction (PCR) methods have emerged as an alternative approach for identification of filarial parasites. Dried blood spot (DBS) has been reported as a convenient way to keep DNA for epidemiological investigations and diagnosis of infectious diseases, and does not require venipuncture. The finding of a highly sensitive DNA extraction method for filarial nematodes is also required for a good molecular performance. The aim of this study was to compare three different molecular methods to diagnose human loiasis and mansonellosis using DBS as a medium of sample collection and storage. The saponin/Chelex method for extracting filarial DNA was also applied. Methods: A total of 100 clinical samples were randomly selected for this study. Microscopy was used as the reference method for diagnosing and calculating the microfilaraemia. Filarial DNA was extracted using saponin/Chelex method from DBS. DNA isolated was assayed by three different molecular methods: qPCR, Filaria-Nested PCR, and cytochrome oxidase I PCR. All PCR-products were subsequently purified and sequenced. Statistical values for each molecular test were computed and compared.Results: Overall, 64 samples were identified as negative by all the tests and 36 samples were positive by at least one of the methods tested. Microscopy detected 27 positive samples, meanwhile qPCR, Filaria-Nested PCR and COI PCR detected 30, 31 and 33 positive samples, respectively. The best overall results were obtained with COI PCR protocol (sensitivity 92.6%; specificity 89.0%; kappa index 76.3%). Conclusions: Despite the good statistical values obtained for COI PCR, this method needs the sequencing of the fragment obtained to identify the filarial species; thus the optimal technique to diagnose filarial infection was qPCR, it was very similar in terms of sensitivity and specificity compared to microscopy and for its capacity to detect a wide range of human filariae. It is an appropriate method for filarial diagnosis in non-endemic settings.

Complete Mitochondrial Genome Sequence of Mansonella perstans

ABSTRACT The 13,647-bp complete mitochondrial genome of Mansonella perstans was sequenced and is syntenic to the mitochondrial genome of Mansonella ozzardi. Phylogenetic analysis of the mitochondrial genome is consistent with the known phylogeny of ONC5 group filarial nematodes.