Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans.

BACKGROUND: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. 'hongkongensis'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott's test (MKT). RESULTS: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. 'hongkongensis'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. CONCLUSIONS: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT' small and portable MinION™ means that such methods could be deployed for field use.

Canine filaria species in selected lymphatic filariasis endemic and non-endemic areas in Sri Lanka.

Subperiodic brugian filariasis and dirofilariasis show a rising trend in Sri Lanka posing a threat to public health. As information was limited on canine filaria species in Sri Lanka, we studied the filaria parasites among dog populations in lymphatic filariasis (LF) endemic and non-endemic regions by microscopy and molecular methods. Thick blood smears (TBSs) were performed among 295 dogs presenting to veterinary clinics for surgical or sterilization procedures in Galle (LF endemic) and Mullaitivu (LF non-endemic) districts, of which 55.6% were positive for any microfilariae. We identified Dirofilaria repens (50.8%) and Brugia spp. (20.6%) by microscopy, which, included mono-infections (D. repens 35.3% and Brugia spp. 5%) and co-infections (15.6%). Infections in Galle and Mullaitivu were 61% and 44.9% respectively. The brugian filariasis rate was significantly higher among canines in LF endemic Galle district (29.9%) than in Mullaitivu (LF non-endemic) (1.1%) (P < 0.001), while D. repens infections were comparable in both districts. Genomic DNA extracted from 10% of microfilariae positive TBSs was amplified using pan-filarial primers targeting the internal-transcriber-spacer region-2 (ITS-2). Sequencing of amplicons confirmed the presence of D. repens (89.28%), Brugia pahangi (7.14%) and B. malayi (3.57%) infections. The phylogeny constructed and analysed in MEGA X indicated genetic variability among D. repens and B. pahangi isolates from Sri Lanka. With this study, we were able to report B. pahangi infections for the first time in Sri Lanka.

Pyruvate produced by Brugia spp. via glycolysis is essential for maintaining the mutualistic association between the parasite and its endosymbiont, Wolbachia.

Human parasitic nematodes are the causative agents of lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), diseases that are endemic to more than 80 countries and that consistently rank in the top ten for the highest number of years lived with disability. These filarial nematodes have evolved an obligate mutualistic association with an intracellular bacterium, Wolbachia, a symbiont that is essential for the successful development, reproduction, and survival of adult filarial worms. Elimination of the bacteria causes adult worms to die, making Wolbachia a primary target for developing new interventional tools to combat filariases. To further explore Wolbachia as a promising indirect macrofilaricidal drug target, the essential cellular processes that define the symbiotic Wolbachia-host interactions need to be identified. Genomic analyses revealed that while filarial nematodes encode all the enzymes necessary for glycolysis, Wolbachia does not encode the genes for three glycolytic enzymes: hexokinase, 6-phosphofructokinase, and pyruvate kinase. These enzymes are necessary for converting glucose into pyruvate. Wolbachia, however, has the full complement of genes required for gluconeogenesis starting with pyruvate, and for energy metabolism via the tricarboxylic acid cycle. Therefore, we hypothesized that Wolbachia might depend on host glycolysis to maintain a mutualistic association with their parasitic host. We did conditional experiments in vitro that confirmed that glycolysis and its end-product, pyruvate, sustain this symbiotic relationship. Analysis of alternative sources of pyruvate within the worm indicated that the filarial lactate dehydrogenase could also regulate the local intracellular concentration of pyruvate in proximity to Wolbachia and thus help control bacterial growth via molecular interactions with the bacteria. Lastly, we have shown that the parasite's pyruvate kinase, the enzyme that performs the last step in glycolysis, could be a potential novel anti-filarial drug target. Establishing that glycolysis is an essential component of symbiosis in filarial worms could have a broader impact on research focused on other intracellular bacteria-host interactions where the role of glycolysis in supporting intracellular survival of bacteria has been reported.

What does not kill them makes them stronger: larval environment and infectious dose alter mosquito potential to transmit filarial worms.

For organisms with complex life cycles, larval environments can modify adult phenotypes. For mosquitoes and other vectors, when physiological impacts of stressors acting on larvae carry over into the adult stage they may interact with infectious dose of a vector-borne pathogen, producing a range of phenotypes for vector potential. Investigation of impacts of a common source of stress, larval crowding and intraspecific competition, on adult vector interactions with pathogens may increase our understanding of the dynamics of pathogen transmission by mosquito vectors. Using Aedes aegypti and the nematode parasite Brugia pahangi, we demonstrate dose dependency of fitness effects of B. pahangi infection on the mosquito, as well as interactions between competitive stress among larvae and infectious dose for resulting adults that affect the physiological and functional ability of mosquitoes to act as vectors. Contrary to results from studies on mosquito-arbovirus interactions, our results suggest that adults from crowded larvae may limit infection better than do adults from uncrowded controls, and that mosquitoes from high-quality larval environments are more physiologically and functionally capable vectors of B. pahangi. Our results provide another example of how the larval environment can have profound effects on vector potential of resulting adults.

Immunology of lymphatic filariasis.

The immune responses to filarial parasites encompass a complex network of innate and adaptive cells whose interaction with the parasite underlies a spectrum of clinical manifestations. The predominant immunological feature of lymphatic filariasis is an antigen-specific Th2 response and an expansion of IL-10 producing CD4(+) T cells that is accompanied by a muted Th1 response. This antigen-specific T-cell hyporesponsiveness appears to be crucial for the maintenance of the sustained, long-standing infection often with high parasite densities. While the correlates of protective immunity to lymphatic filariasis are still incompletely understood, primarily due to the lack of suitable animal models to study susceptibility, it is clear that T cells and to a certain extent B cells are required for protective immunity. Host immune responses, especially CD4(+) T-cell responses clearly play a role in mediating pathological manifestations of LF, including lymphedema, hydrocele and elephantiasis. The main underlying defect in the development of clinical pathology appears to be a failure to induce T-cell hyporesponsiveness in the face of antigenic stimulation. Finally, another intriguing feature of filarial infections is their propensity to induce bystander effects on a variety of immune responses, including responses to vaccinations, allergens and to other infectious agents. The complexity of the immune response to filarial infection therefore provides an important gateway to understanding the regulation of immune responses to chronic infections, in general.

First molecular detection and genetic diversity of Hepatozoon sp. (Apicomplexa) and Brugia sp. (Nematoda) in a crocodile monitor in Nakhon Pathom, Thailand.

The crocodile monitor (Varanus salvator) is the most common monitor lizard in Thailand. Based on habitat and food, they have the potential to transmit zoonoses, with a high possibility of infecting ectoparasites and endoparasites. Diseases that could infect crocodile monitors and be transmitted to other animals, including humans. This research aims to identify and evaluate the phylogenetic relationships of Hepatozoon sp. and sheathed microfilaria in crocodile monitors. The phylogenetic analyses of Hepatozoon, based on 18S rRNA, and sheathed microfilaria, based on the COX1 gene, revealed that the Hepatozoon sp. were grouped with H. caimani, while sheathed microfilaria were grouped together with B. timori. This study provides insights into the genetic diversity and host-parasite interactions of hemoparasites in crocodile monitors in Thailand.

An Overview of Management Considerations for Mongolian Gerbils (Meriones unguiculatus), Cats (Felis catus), and Dogs (Canis familiaris) as Hosts for Brugia Infection.

Lymphatic filariasis is a mosquito-borne parasitic infection affecting an estimated 51.4 million people. Brugia malayi and Brugia pahangi are used in research because common nonprimate research species such as Mongolian gerbils (Meriones unguiculatus), cats (Felis catus), and dogs (Canis familiaris) can maintain the life cycle of these species of filarial nematodes. Although overall care and management of animals infected with Brugia spp. is relatively straightforward, there are some unique challenges and special considerations that must be addressed when managing a research colony infected with these parasites. In this review, we discuss our experience, share insight into biosafety and clinical management, and describe the expected clinical signs associated with Brugia infection in gerbils, cats, and dogs.

Surveillance and Selective Treatment of Brugia malayi Filariasis Eleven Years after Stopping Mass Drug Administration in Belitung District, Indonesia.

Brugia malayi is the major cause of lymphatic filariasis (LF) in Indonesia. Zoophilic B. malayi was endemic in Belitung district, and mass drug administration (MDA) with diethylcarbamazine (DEC) and albendazole ceased after five annual rounds in 2010. The district passed three transmission assessment surveys (TAS) between 2011 and 2016. As part of the post-TAS3 surveillance of the national LF elimination program, we collected night blood samples for microfilaria (Mf) detection from 1,911 subjects more than 5 years of age in seven villages. A B. malayi Mf prevalence ranging from 1.7% to 5.9% was detected in five villages. Only 2 (5%) of the total 40 Mf-positive subjects were adolescents aged 18 and 19 years old, and 38 (95%) Mf-positive subjects were 21 years and older. Microfilarial densities in infected individuals were mostly low, with 60% of the subjects having Mf densities between 16 and 160 Mf/mL. Triple-drug treatment with ivermectin, DEC, and albendazole (IDA) was given to 36 eligible Mf-positive subjects. Adverse events were mostly mild, and treatment was well tolerated. One year later, 35 of the treated Mf-positive subjects were reexamined, and 33 (94%) had cleared all Mf, while the anti-Bm14 antibody prevalence remained almost unchanged. Results indicate that in B. malayi-endemic areas, post-TAS3 surveillance for Mf in the community may be needed to detect a potential parasite reservoir in adults. Selective treatment with IDA is highly effective in clearing B. malayi Mf and should be used to increase the prospects for LF elimination if MDA is reintroduced.

Laboratory detection of strongyloidiasis: IgG-, IgG4 - and IgE-ELISAs and cross-reactivity with lymphatic filariasis.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG4 and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG4 , IgG, IgE and IgG (IVD) assays were 76.9%, 84.6%, 7.7% and 84.6%, respectively, while the specificities were 92.7%, 81.8%, 100% and 83.6%, respectively. If filariasis samples were excluded, the specificities of the IgG4 -ELISA and both IgG-ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG- and IgG4 -ELISAs (r = 0.4828; P = 0.0125). IgG- and IgG- (IVD) ELISAs (r = 0.309) were positively correlated, but was not significant (P = 0.124). Meanwhile there was no correlation between IgG4 - and IgG- (IVD) ELISAs (r = 0.0042; P = 0.8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG4 and the optical densities of anti-Strongyloides IgG4 -ELISA (r = 0.4544, P = 0.0294). In conclusion, the detection of both anti-Strongyloides IgG4 and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

An unusual case of Brugia sp. infection in a dog from Alberta, Canada.

Brugia is a vector-transmitted nematode that is commonly known for its zoonotic significance of causing lymphatic filariasis in Asia and Oceanic regions of the world. In addition to public health concerns, Brugia species have been known to infect domestic animals, including dogs and cats. However, information is scarce regarding genus Brugia in North America, and rare infections have been noted in domestic cats, humans, and other wild mammals. Herein, we document the first reported case of a Brugia species infection in a dog from North America and the first molecular characterization of the species in question. A three-year-old German Shepard from Alberta, Canada presented to a local veterinary clinic with a facial subcutaneous nodule that was surgically excised. Histopathology identified an enlarged buccal lymph node containing small foci of eosinophilic and granulomatous inflammation within the cortex and capsule. This inflammation was associated with adult filarioid nematodes localized within lymphatic vessels or adjacent connective tissue. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue, and PCR targeting the Hha1 repeat and the partial cytochrome oxidase c subunit 1 (cox1) of the mitochondrial DNA confirmed parasite identity as Brugia sp. While we can rule out B. beaveri being the causative agent, we cannot exclude B. lepori infection or a third uncharacterized Brugia species. Veterinarians and physicians should be made aware of North American Brugia infections and their possible health concerns.

The effects of size and synthesis methods of gold nanoparticle-conjugated MαHIgG4 for use in an immunochromatographic strip test to detect brugian filariasis.

This study describes the properties of colloidal gold nanoparticles (AuNPs) with sizes of 20, 30 and 40 nm, which were synthesized using citrate reduction or seeding-growth methods. Likewise, the conjugation of these AuNPs to mouse anti-human IgG(4) (MαHIgG(4)) was evaluated for an immunochromatographic (ICG) strip test to detect brugian filariasis. The morphology of the AuNPs was studied based on the degree of ellipticity (G) of the transmission electron microscopy images. The AuNPs produced using the seeding-growth method showed lower ellipticity (G ≤ 1.11) as compared with the AuNPs synthesized using the citrate reduction method (G ≤ 1.18). Zetasizer analysis showed that the AuNPs that were synthesized using the seeding-growth method were almost monodispersed with a lower polydispersity index (PDI; PDI≤0.079), as compared with the AuNPs synthesized using the citrate reduction method (PDI≤0.177). UV-visible spectroscopic analysis showed a red-shift of the absorbance spectra after the reaction with MαHIgG(4), which indicated that the AuNPs were successfully conjugated. The optimum concentration of the BmR1 recombinant antigen that was immobilized on the surface of the ICG strip on the test line was 1.0 mg ml(-1). When used with the ICG test strip assay and brugian filariasis serum samples, the conjugated AuNPs-MαHIgG(4) synthesized using the seeding-growth method had faster detection times, as compared with the AuNPs synthesized using the citrate reduction method. The 30 nm AuNPs-MαHIgG(4), with an optical density of 4 from the seeding-growth method, demonstrated the best performance for labelling ICG strips because it displayed the best sensitivity and the highest specificity when tested with serum samples from brugian filariasis patients and controls.

Diethylcarbamazine mediated potentiation of emodepside induced paralysis requires TRP-2 in adult Brugia malayi.

Human and veterinary filarial nematode infections are a major health concern in tropical countries. They are transmitted by biting insects and mosquitoes. Lymphatic filariasis, a group of filarial infections caused by Brugia spp. and Wucheria bancrofti affect more than 120 million people worldwide. Infected individuals develop swollen limbs and disfigurement, leading to an inability to work and ostracization from society. Control and prophylaxis for these infections involve mass drug administration combinations of anthelmintics including diethylcarbamazine (DEC). DEC has actions on microfilariae, but its effects on adult worms are less pronounced. The SLO-1 (BK) channel activator, emodepside, kills adults of many filarial species. However, the in vivo efficacy of emodepside is suboptimal against B. malayi, possibly due to reduced bioavailability in the lymphatic system. Expressing different slo-1 splice variants in B. malayi also affects sensitivity to emodepside. This study explores the potentiation of emodepside mediated paralysis by DEC in adult female B. malayi. Worminator motility measurements show that co-application of DEC and emodepside increases the potency of emodepside 4-fold. The potentiation of the emodepside effect persists even after the worms recover (desensitize) from the initial effects of DEC. RNAi knock-down demonstrates that the DEC-mediated potentiation of emodepside requires the presence of TRP-2 channels. Our study demonstrates that the addition of DEC could enhance the effect of emodepside where bioavailability or activity against a specific species may be low.

IgG4 antibodies against Bm14 as an evaluation tool of mass drug administration in a co-endemic area of Brugia timori and Wuchereria bancrofti.

To evaluate the success of mass drug administration for lymphatic filariasis, WHO has recommended two rapid tests, Brugia Rapid (BR) to detect the presence of IgG4 antibodies against Brugia sp and Filariasis Test Strip (FTS) to detect antigens of Wuchereria bancrofti. As a country co-endemic for Brugia sp. and W. bancrofti, Indonesia needs a single diagnostic tool that can detect the exposure to both species. This study aimed to evaluate the efficacy of mass drug administration by measuring Bm14-specific IgG4 levels in blood samples of the population living in a co-endemic area of B. timori and W. bancrofti in Southwest Sumba Regency. A total of 132 plasma samples obtained before and one year after DEC-albendazole administration, which have been previously tested with BR and FTS, were examined for IgG4 against Bm14 using enzyme-linked immunosorbent assay. The results showed that before treatment all 32 individuals (100%) with BR+/ FTS+ were also positive for Bm14-specific IgG4, while in BR+ or FTS+ group there were >90% samples detected positive. At one year after treatment, positive results for Bm14-specific IgG4 were still detected in 96.9% samples with BR+/ FTS+, 78.8% samples with BR+/ FTS- and 82.9% samples with BR-/ FTS+. On the other hand, the BR-/ FTS- group also had high rate of Bm14-specific IgG4 positivity either before treatment (62,5%) and at one year after treatment (43.8%). The lowest decrease of Bm14-specific IgG4 positivity at one year after treatment was shown in the double positive group (3.1%), while the highest was in the double negative group (18.7%). The measurement of IgG4 against Bm14 has the potential as a sensitive diagnostic tool to evaluate the success of MDA in the areas co-endemic for B. timori and W. bancrofti.

Characterization of the genetics and epidemiology of Brugia sp. in domestic dogs in Chad, Africa.

Neglected tropical diseases pose a threat to domestic animal health, as domestic animals can serve as reservoirs for certain zoonotic parasitic infections, including Guinea worm (Dracunculus medinensis) and lymphatic filariasis. Surveillance for these parasites in domestic animals is needed to understand infection prevalence and transmission cycles, with the goal of instituting appropriate interventions. The goal of this research was to report our finding of Brugia sp. infection in dogs from Chad, Africa, and to characterize the genetics and epidemiology of the parasite. During a recent Chadian canine pathogen surveillance project, we identified Brugia sp. infections in a total of 46 out of 428 dogs (10.7%) sampled at three time points in 2019-2020. We found high levels of sequence similarity to B. malayi and B. pahangi based on amplification of 18S rRNA, 5.8S rRNA, and ITS-2 regions. Phylogenetic analysis of 18S rRNA gene sequences placed the Chadian Brugia sp. in a clade with other Brugia spp. but grouped it separately from both B. malayi and B. pahangi. Analysis of Hha I sequences showed the greatest similarity with B. patei, a parasite previously reported from dogs, cats, and wildlife hosts in Kenya. Epidemiologic analysis using generalized linear regression modeling found significantly higher odds of Brugia sp. detection among dogs in villages in southern Chad compared to those in the northern region. Further, within the northern region, there were higher odds of detection in the dry season, compared to the wet season, which is consistent with the ecology of a presumably mosquito-borne parasite. The same 428 dogs were tested for Dirofilaria immitis antigen using a commercial assay (IDEXX SNAP 4Dx) at the earliest time point of the study, with 119 dogs testing positive. However, no association was noted between Brugia infection and a dog being positive for Di. immitis antigen, with only seven of the 119 Di. immitis antigen-positive dogs being Brugia-positive. This is the first report of Brugia sp. in domestic dogs in Chad and additional research is needed to definitively identify the species present, elucidate transmission, and understand potential risks to canine and human health.

Lymphatic filariasis in children: clinical features, infection burdens and future prospects for elimination.

Lymphatic filariasis (LF), a common parasitic infection in tropical countries, causes lymphoedema of limbs, hydrocele and acute attacks of dermato-lymphangio-adenitis. Recent advances in diagnosis have helped to recognize that LF infection is often acquired in childhood. Newly available diagnostic techniques like sensitive antigen and antibody assays, Doppler ultrasonography and lymphoscintigraphy have helped to understand the subclinical pathology caused by this infection, which was hitherto generally believed to be irreversible. Recent studies indicate that drugs used in the mass drug administration (MDA) programme under GPELF are capable of reversing the sub-clinical lymphatic damage in children and provide benefits other than interruption of transmission. Albendazole and ivermectin used in MDA are effective against soil-transmitted helminthic infections common in children in LF endemic areas. Thus MDA had other 'beyond LF' benefits in treated children including increased appetite, weight gain, greater learning ability and concentration, better school attendance and prevention of anaemia. MDA should no longer be viewed as a measure for interrupting transmission alone. Recent findings of reversibility of early lymphatic pathology in treated children indicate that both MDA and 'foot-hygiene' measures are effective strategies in preventing and managing morbidity. Programme managers should effectively utilize this information to strengthen their advocacy efforts to achieve high and sustainable coverage in MDA.

"Wonders unconceived": reflections on the birth of medical entomology.

Prior to Patrick Manson's discovery in 1877 that the mosquito Culex fatigans was the intermediate host of filariasis, the association of insects with disease and the nature of disease transmission was almost entirely speculation. Manson's work was incomplete, however, because it showed the manner in which the mosquito acquired the infection from humans, but failed to show the way in which the mosquito passed the infection to humans. That pathogens were transmitted by the bite of an infected female mosquito was later proven experimentally with bird malaria by Manson's protégé, Ronald Ross. In 1898 Ross demonstrated that the infective stage of the malarial parasite was injected into the host when the mosquito released saliva into the wound prior to injesting blood. Insects were suspected as carriers of disease for centuries, yet it was not until the late 1870s that the uncritical acceptance of folk beliefs was supplanted by research-based scientific medicine. Why did it take so long? The answer lies in the fact that early medicine itself was imprecise and could not have pursued the subject with any hope of useful results until the last quarter of the 19th century. A better understanding of the nature of the disease process (germ theory of disease) and improved technology (microscopes and oil-immersion lenses with greater resolving power, and synthetic tissue stains) were indispensable for revealing the nexus between those partners in crime: insects and parasites.

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

The Course of Brugia malayi Microfilaremia in Experimentally Infected Cats.

As one of the causative agents of lymphatic filariasis in humans, Brugia malayi has been established as the laboratory model of choice for studying this infection owing to its viability in small animal hosts, with the domestic cat being significant among these. The usefulness of individual feline infections is highly dependent on the levels of circulating microfilariae in the blood; thus, characterizing the course of microfilaremia benefits our understanding of this model. In B. malayi-endemic regions, cats are also known reservoirs of infection, and describing microfilaremia in a controlled setting may improve transmission modeling. We followed the course of B. malayi infection in 10 experimentally infected cats from inoculation to ultimate resolution. Seven cats developed patency, with a peak microfilaria concentration of 6525/mL. In addition, to identify cellular responses with potential value as predictors of patency, we measured the peripheral blood leukocyte counts during the first 8 months of infection and tested for correlations with lifelong microfilaria production. No strong relationships were observed, though cell values did appear to shift with the maturation phases of the parasite. The data we present reflect the course of microfilaremia in an important laboratory model under controlled conditions.

Recombinant antigens used as diagnostic tools for lymphatic filariasis.

Lymphatic filariasis (LF) is a parasitic disease caused by the worms Wuchereria bancrofti, Brugia malayi, or Brugia timori. It is a tropical and subtropical illness that affects approximately 67 million people worldwide and that still requires better diagnostic tools to prevent its spread and enhance the effectiveness of control procedures. Traditional parasitological tests and diagnostic methods based on whole protein extracts from different worms are known for problems related to sample time collection, sensitivity, and specificity. More recently, new diagnostic tools based on immunological methods using recombinant antigens have been developed. The current review describes the several recombinant antigens used as tools for lymphatic filariasis diagnosis in antigen and antibody capture assays, highlighting their advantages and limitations as well as the main commercial tests developed based on them. The literature chronology is from 1991 to 2021. First, it describes the historical background related to the identification of relevant antigens and the generation of the recombinant polypeptides used for the LF diagnosis, also detailing features specific to each antigen. The subsequent section then discusses the use of those proteins to develop antigen and antibody capture tests to detect LF. So far, studies focusing on antibody capture assays are based on 13 different antigens with at least six commercially available tests, with five proteins further used for the development of antigen capture tests. Five antigens explored in this paper belong to the SXP/RAL-2 family (BmSXP, Bm14, WbSXP-1, Wb14, WbL), and the others are BmShp-1, Bm33, BmR1, BmVAH, WbVAH, BmALT-1, BmALT-2, and Wb123. It is expected that advances in research with these antigens will allow further development of tests combining both sensitivity and specificity with low costs, assisting the Global Program to Eliminate Lymphatic Filariasis (GPELF).