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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

An open label, randomized clinical trial to compare the tolerability and efficacy of ivermectin plus diethylcarbamazine and albendazole vs. diethylcarbamazine plus albendazole for treatment of brugian filariasis in Indonesia.

Improved treatments for lymphatic filariasis (LF) could accelerate the global elimination program for this disease. A triple drug combination of the anti-filarial drugs ivermectin, diethylcarbamazine (DEC) and albendazole (IDA) has been shown to be safe and effective for achieving sustained clearance of microfilariae (Mf) of the filarial parasite Wuchereria bancrofti from human blood. However, the triple drug combination has not been previously been evaluated for treatment of brugian filariasis, which accounts for about 10% of the global LF burden. This hospital-based clinical trial compared the safety and efficacy of IDA with that of the standard treatment (DEC plus albendazole, DA) in persons with Brugia timori infections on Sumba island, Indonesia. Fifty-five asymptomatic persons with B. timori Mf were treated with either a single oral dose of IDA (28 subjects) or with DEC plus albendazole (DA, 27 subjects). Participants were actively monitored for adverse events (AE) for two days after treatment by nurses and physicians who were masked regarding treatment assignments. Passive monitoring was performed by clinical teams that visited participant's home villages for an additional five days. Microfilaremia was assessed by membrane filtration of 1 ml night blood at baseline, at 24h and one year after treatment. IDA was more effective than DA for completely clearing Mf at 24 hours (25/28, 89% vs. 8/27, 30%, P < 0.001). By 12 months after treatment, only one of 27 IDA recipients had Mf in their blood (4%) vs. 10 of 25 (40%) in persons treated with DA (P = 0.002). Approximately 90% of participants had antibodies to recombinant filarial antigen BmR1 at baseline. Antibody prevalence decreased to approximately 30% in both treatment groups at 12 months. About 45% of persons in both treatment groups experienced AE such as fever, muscle aches, lower back, joint and abdominal pain. These were mostly mild and most common during the first two days after treatment. No participant experienced a severe or serious AE. This study showed that IDA was well-tolerated and significantly more effective for clearing B. timori Mf from the blood than DA. Larger studies should be performed to further assess the safety and efficacy of IDA as a mass drug administration regimen to eliminate brugian filariasis. Trial Registration: NCT02899936.

Morphological characteristics of microfilariae in blood smears of the common treeshrew Tupaia glis (Mammalia: Scandentia) in Gemas, Negeri Sembilan, Malaysia.

Some filarial nematodes, such as Wuchereria bancrofti, Brugia malayi, and Brugia timori, cause lymphatic diseases in humans in the tropics, whereas other filarial parasites from wild animals cause zoonotic diseases in humans worldwide. To elucidate the prevalence and diversity of filarial parasites in Malaysia, we investigated the filarial parasites from wild animals in Gemas, Negeri Sembilan. To find adult filarial parasites, we dissected 26 animals, which included five frogs, one skink, one snake, two birds, six common treeshrews, and 11 rats. Then, we examined microfilariae in the blood smears and skin snips obtained from each animal. We found two types of microfilariae in the blood smears of common treeshrews: one was very similar to Malayfilaria sofiani and the other closely resembled Brugia tupaiae. These findings indicate an additional distribution of these filarial parasites in Gemas.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 1. Amide Linked Analogs.

A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good in vitro activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series, 8a (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound 8a was effective in killing Brugia malayi, B. pahangi, and Litomosoides sigmodontis worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of 8a at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both 8a and the positive control, flubendazole.

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.

Association of a PD-L2 Gene Polymorphism with Chronic Lymphatic Filariasis in a South Indian Cohort.

Lymphatic filariasis (LF) is a parasitic infection, caused by three closely related nematodes, namely Wuchereria bancrofti, Brugia malayi, and Brugia timori. Previously, we have shown that lysate from B. malayi microfilariae induces the expression of interleukin (IL)-10 and programmed death-ligand (PD-L) 1 on monocytes, which lead to inhibition of CD4+ T-cell responses. In this study, we investigated associations of IL-10 and programmed cell death (PD)-1 pathway gene polymorphisms with clinical manifestation in LF. We evaluated the frequency of alleles and genotypes of IL-10 (rs3024496, rs1800872), IL-10RA (rs3135932), IL-10RB (rs2834167), PD-1 (rs2227982, rs10204525), PD-L1 (rs4143815), PD-L2 (rs7854413), and single-nucleotide polymorphisms (SNPs) in 103 patients with chronic pathology (CP), such as elephantiasis or hydrocele and 106 endemic normal (EN) individuals from a South Indian population living in an area endemic for LF. Deviations from the Hardy-Weinberg equilibrium were tested, and we found a significant difference between the frequency of polymorphisms in PD-L2 (rs7854413; P < 0.001) and IL-10RB (rs2834167; P = 0.012) between the CP and the EN group, whereas there were no significant differences found among IL-10, IL-10RA, PD-1, and PD-L1 SNPs. A multivariate analysis showed that the existence of a CC genotype in PD-L2 SNP rs7854413 is associated with a higher risk of developing CP (OR: 2.942; 95% confidence interval [CI]: 0.957-9.046; P = 0.06). Altogether, these data indicate that a genetically determined individual difference in a non-synonymous missense SNP of PD-L2 might influence the susceptibility to CP.

Comparison of the Impact of Annual and Semiannual Mass Drug Administration on Lymphatic Filariasis Prevalence in Flores Island, Indonesia.

We compared the impact of annual and semiannual mass drug administration (MDA) on the prevalence of Brugia timori and Wuchereria bancrofti in Flores Island. Two villages (Paga, B. timori only; Lewomada, co-endemic) received annual MDA with diethylcarbamazine/albendazole and a larger village (Pruda, co-endemic) received semiannual MDA. Infection parameters (microfilariae [Mf], antibodies to recombinant filarial antigen BmR1 [Brugia Rapid (BR)], and a test for W. bancrofti antigenemia [immunochromatographic test (ICT)]) were assessed before and after treatment. The crude Mf prevalence in Pruda decreased after five semiannual treatments from 14.2% to 1.2%, whereas the Mf prevalence in the other two villages decreased after three annual treatments from 3.9% to 0% and from 5% to 0.3%, respectively. ICT positivity prevalence in Pruda and Lewomada decreased from 22.9% and 6.5% to 7% and 0.8%, respectively, whereas BR antibody prevalence in Pruda, Lewomada, and Paga decreased from 28.9%, 31.7%, and 12.5% to 3.6%, 4.1%, and 1.8%, respectively. Logistic regression analysis indicated that that Mf, BR, and ICT prevalence decreased significantly over time and that for the Mf and ICT outcomes the semiannual treatment had higher odds of positivity. Model-adjusted prevalence estimates revealed that apparent differences in treatment effectiveness were driven by differences in baseline prevalence and that adjusted prevalence declined more rapidly in the semiannual treatment group. We conclude that in this setting, annual MDA was sufficient to reduce Mf prevalence to less than 1% in areas with low to moderate baseline prevalence. Semiannual MDA was useful for rapidly reducing Mf prevalence in an area with higher baseline endemicity.

Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes.

BACKGROUND: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack. CONCLUSIONS/SIGNIFICANCE: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

Cervical Lymphatic Filariasis in a Pediatric Patient: Case Report and Database Analysis of Lymphatic Filariasis in the United States.

Lymphatic filariasis is a mosquito-borne parasitic infection caused by Wuchereria bancrofti and Brugia spp. Commonly seen in tropical developing countries, lymphatic filariasis occurs when adult worms deposit in and obstruct lymphatics. Although not endemic to the United States, a few cases of lymphatic filariasis caused by zoonotic Brugia spp. have been reported. Here we present a case of an 11-year-old female with no travel history who was seen in our clinic for a 1-year history of painless left cervical lymphadenopathy secondary to lymphatic filariasis. We review the literature of this infection and discuss the management of our patient. Using the National Inpatient Sample (NIS), the largest publicly available all-payer inpatient care database in the United States, we also examine the demographics of this infection. Our results show that chronic lymphadenopathy in the head and neck is the most common presenting symptoms of domestic lymphatic filariasis. Diagnosis is often made after surgical lymph node excision. Examination of the NIS from 2000 to 2014 revealed 865 patients admitted with a diagnosis of lymphatic filariasis. Most patients are in the mid to late sixties and are located on the eastern seaboard. Eight hundred and twenty six cases (95.5%) were likely due to zoonotic Brugia spp. and 39 (4.5%) due to W. bancrofti. Despite being rare, these data highlight the need to consider filariasis in patients presenting with chronic lymphadenopathy in the United States.