Filarial Parasite Research Articles

Internalization of macromolecules into filarial parasites – Possible operation of host’s anti-fecundity immunity inside adult filarial nematodes

ObjectivesImmune effector mechanisms operating against large extracellular systemic pathogens, such as adult stages of filarial nematodes, are not clearly understood. The only immune effect on the adult worms appears to be directed towards embryos in adult worms. The role of IL-4 or TLR-4 in immune responses that inhibit embryogenesis in adult filarial worms has been well-documented. This host’s immune responses that control embryogenesis (anti-fecundity immunity) are considered critical and were addressed in the current study. The current investigation demonstrated the possible operation of anti-adult immunity that could be functional against large stage adults. MethodsAdult filaria worms were pulsed with macromolecules such as Concanavalin A, Human immunoglobulins, and Rabbit immunoglobulins for 6 hrs, and entry of these molecules was demonstrated using flow cytometry. Filaria worms were also preincubated with Latrunculin A, an endocytic inhibitor, to explain if macromolecules enter by endocytosis. ResultsUsing the flow cytometry approach, we could show the entry of immunoglobulins into the uterine cavity of female Setaria digitata, a cattle-dwelling filarial parasite. Immunoglobulins were observed to bind to the surface of intrauterine stages of female worms. We also demonstrated the presence of bovine immunoglobulins in different embryonic stages in situ. ConclusionsOverall, experimental evidence demonstrates the existence of the host’s immune molecules inside large-stage adult parasites. However, future studies are directed to understand the functional aspect of the presence of these effector molecules inside the adults.

First blood-meal record of Simulium asakoae (Diptera: Simuliidae) in Malaysia, with notes on its distribution in Asia and status as a potential vector.

Simulium asakoae Takaoka and Davies has been confirmed to bite humans and has been incriminated as a vector of blood protozoan parasites of the genera Leucocytozoon and Trypanosoma, as well as an unknown filarial parasite in Thailand. However, its attraction to humans has remained uninvestigated in Malaysia. Recently, 27 black flies were collected in Pahang, Malaysia, of which 25 were captured in CO2-baited Malaise traps and two were collected from humans during trapping activity. All specimens were morphologically identified as S. asakoae. Cytochrome c oxidase I sequences of the two specimens caught on humans showed 100% similarity with those of S. asakoae in the NCBI GenBank, confirming their morphological identity. Blood-meal analysis using a HAL·HARTM kit did not show the presence of domestic or wild animal DNA. However, human DNA was amplified from one engorged fly in the cytochrome b gene amplification assay, providing the first evidence of blood-feeding by S. asakoae in Malaysia.

Analysis of the role of Dirofilaria repens macrophage migration inhibitory factors in host-parasite interactions.

Dirofilaria repens is a zoonotic parasitic filarial nematode that infects carnivores and occasionally humans. Knowledge of the host-parasite molecular interactions enabling the parasite's avoidance of the host immune response in subcutaneous dirofilariasis remains limited. Parasitic orthologues of host macrophage migration inhibitory factor (MIF) are molecules potentially involved in this process. Complementary DNA encoding two D. repens MIF orthologues (rDre-MIF-1 and rDre-MIF-2) was cloned into a pET-28a expression vector. The recombinant proteins were produced in Escherichia coli and purified using affinity nickel chromatography. The reactivity of both recombinant proteins was analysed with infected dog and immunised mouse sera. Stronger antibody production was induced by rDre-MIF-1 in mice, as evidenced by significantly higher levels of anti-rDre-MIF-1 total IgG, IgG2 and IgE antibodies than of anti-rDre-MIF-2 immunoglobulins. Additionally, a significantly different level of antibodies specific to both proteins was noted between the sera of infected dogs and those of uninfected dogs. This study is the first attempt to characterise MIF orthologues from the filarial parasite D. repens, which may affect the immune response during infection.

Detection and characterization of vector-borne parasites and Wolbachia endosymbionts in greater one-horned rhinoceros (Rhinoceros unicornis) in Nepal

Vector-borne parasite infections affect both domestic and wild animals. They are often asymptomatic but can result in fatal outcomes under natural and human-induced stressors. Given the limited availability of molecular data on vector-borne parasites in Rhinoceros unicornis (greater one-horned rhinoceros), this study employed molecular tools to detect and characterize the vector-borne parasites in rescued rhinoceros in Chitwan National Park, Nepal. Whole blood samples were collected from thirty-six R. unicornis during rescue and treatment operations. Piroplasmida infections were first screened using nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA gene. Wolbachia was detected by amplifying 16S rRNA gene, while filarial nematodes were detected through amplification of 28S rRNA, COI, myoHC and hsp70 genes. Our results confirmed the presence of Theileria bicornis with a prevalence of 75% (27/36) having two previously unreported haplotypes (H8 and H9). Wolbachia endosymbionts were detected in 25% (9/36) of tested samples and belonged to either supergroup C or F. Filarial nematodes of the genera Mansonella and Onchocerca were also detected. There were no significant association between T. bicornis infections and the age, sex, or location from which the animals were rescued. The high prevalence of Theileria with novel haplotypes along with filarial parasites has important ecological and conservational implications and highlights the need to implement parasite surveillance programs for wildlife in Nepal. Further studies monitoring vector-borne pathogens and interspecies transmission among wild animals, livestock and human are required.

Detection of Wuchereria bancrofti infection in mosquitoes in areas co-endemic with Brugia malayi in Balasore district, Odisha, India

Lymphatic filariasis (LF) is a crippling and disfiguring parasitic condition. India accounts for 55% of the world’s LF burden. The filarial parasite Wuchereria bancrofti is known to cause 99.4% of the cases while, Brugia malayi accounts for 0.6% of the issue occurring mainly in some pockets of Odisha and Kerala states. The Balasore (Baleswar) district of Odisha has been a known focus of B. malayi transmission. We employed molecular xenomonitoring to detect filarial parasite DNA in vectors. In six selected villages, Gravid traps were used to collect Culex mosquitoes and hand catch method using aspirators was followed for collection of mansonioides. A total of 2903 mosquitoes comprising of Cx. quinquefasciatus (n = 2611; 89.94%), Cx. tritaeniorhynchus (n = 100; 3.44%), Mansonia annuliferea (n = 139; 4.78%) and Mansonia uniformis (n = 53; 1.82%) were collected from six endemic villages. The species wise mosquitoes were made into 118 pools, each with a maximum of 25 mosquitoes, dried and transported to the laboratory at VCRC, Puducherry. The mosquito pools were subjected to parasite DNA extraction, followed by Real-time PCR using LDR and HhaI probes to detect W. bancrofti and B. malayi infections, respectively. Seven pools (6.66%) of Cx. quinquefasciatus, showed infection with only W. bancrofti while none of the pools of other mosquito species showed infection with either W. bancrofti or B. malayi. Although the study area is endemic to B. malayi, none of the vectors of B. malayi was found with parasite infection. This study highlights the ongoing transmission of bancroftian filariasis in the study villages of Balasore district of Odisha and its implications for evaluating LF elimination programme.

Dual RNA-seq in filarial nematodes and Wolbachia endosymbionts using RNase H based ribosomal RNA depletion.

Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium Wolbachia as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80-90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and Wolbachia from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from Brugia malayi containing the Wolbachia symbiont (wBm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5'-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of Wolbachia mRNA reads, resulting in a 16-1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the Brugia malayi depletion probes can remove rRNA from the filarial worm Dirofilaria immitis and the majority of rRNA from the more distantly related free living nematode Caenorhabditis elegans. These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library.

Synthesizing environmental, epidemiological and vector and parasite genetic data to assist decision making for disease elimination.

We present a framework for identifying when conditions are favourable for transmission of vector-borne diseases between communities by incorporating predicted disease prevalence mapping with landscape analysis of sociological, environmental and host/parasite genetic data. We explored the relationship between environmental features and gene flow of a filarial parasite of humans, Onchocerca volvulus, and its vector, blackflies in the genus Simulium. We generated a baseline microfilarial prevalence map from point estimates from 47 locations in the ecological transition separating the savannah and forest in Ghana, where transmission of O. volvulus persists despite onchocerciasis control efforts. We generated movement suitability maps based on environmental correlates with mitochondrial population structure of 164 parasites from 15 communities and 93 vectors from only four sampling sites, and compared these to the baseline prevalence map. Parasite genetic distance between sampling locations was significantly associated with elevation (r = .793, p = .005) and soil moisture (r = .507, p = .002), while vector genetic distance was associated with soil moisture (r = .788, p = .0417) and precipitation (r = .835, p = .0417). The correlation between baseline prevalence and parasite resistance surface maps was stronger than that between prevalence and vector resistance surface maps. The centre of the study area had high prevalence and suitability for parasite and vector gene flow, potentially contributing to persistent transmission and suggesting the importance of re-evaluating transmission zone boundaries. With suitably dense sampling, this framework can help delineate transmission zones for onchocerciasis and would be translatable to other vector-borne diseases.

Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

Unveiling zoonotic threats: molecular identification of Brugia sp. infection in a lion.

Brugia malayi and B. pahangi, potential zoonotic pathogens transmitted by mosquitoes, are believed to primarily infect dogs and cats as reservoir hosts. Although previous studies have indicated nematode infections in lions, particularly in zoo environments where human contact with these reservoirs is possible, limited documentation exists regarding Brugia sp. infections in lions in Thailand. This study aims to investigate a case of Brugia infection in a lion from a zoo in Thailand. The blood sample was collected and examined from a female lion, using staining methods to morphologically identify microfilaria at the genus level. Subsequently, the PCR was employed targeting specific genes, including mitochondrial 12S rDNA, 18S rDNA, cytochrome oxidase I (COI) and Wolbachia surface protein (wsp), to confirm the species of the filarial nematode parasite. The genetic sequencing results revealed a high similarity (99-100%) to B. malayi for the 12S rDNA, 18S rDNA, COI and wsp genes. Phylogenetic analysis based on nucleotide sequences from the 12S rDNA, 18S rDNA, COI and wsp genes showed that the sequences from this study belong to different clusters. This marks the inaugural documentation of molecular identification of Brugia infection in a lion, signifying that lions could function as reservoirs for this parasite and present a potential public health risk in the region. Our research underscores the effectiveness of molecular techniques and phylogenetic analysis in discerning and comprehending the evolution of filarial parasites. Additionally, it emphasizes the significance of these methods in enhancing the diagnosis, control, and prevention of zoonotic filarial nematode infections.

A filarial parasite potentially associated with the health burden on domestic chickens in Japan

Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.

Immunomodulation of streptozotocin induced Type 1 diabetes mellitus in mouse model by Macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti

Helminth parasites modulate the host immune system to ensure a long-lasting asymptomatic form of infection generally, mediated by the secretion of immunomodulatory molecules and one such molecule is a homologue of human host cytokine, Macrophage migratory Inhibitory Factor (hMIF). In this study, we sought to understand the role of homologue of hMIF from the lymphatic filarial parasite, Wuchereria bancrofti (Wba-MIF2), in the immunomodulation of the Streptozotocin (STZ)-induced Type1 Diabetes Mellitus (T1DM) animal model. Full-length recombinant Wba-MIF2 was expressed and found to have both oxidoreductase and tautomerase activities. Wba-MIF2 recombinant protein was treated to STZ induced T1DM animals, and after 5 weeks pro-inflammatory (IL-1, IL-2, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and gene expressions were determined in sera samples and spleen respectively. Pro-inflammatory and anti-inflammatory cytokine levels were significantly (p<0.05) up-regulated and down-regulated respectively, in the STZ-T1DM animals, as compared to treated groups. Histopathology showed macrophage infiltration and greater damage of islets of beta cells in the pancreatic tissue of STZ-T1DM animals, than Wba-MIF2 treated STZ-T1DM animals. The present study clearly showed the potential of Wba-MIF2 as an immunomodulatory molecule, which could modulate the host immune system in the STZ-T1DM mice model from a pro-inflammatory to anti-inflammatory milieu.

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

BackgroundFilarial 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).ResultsWe 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.ConclusionsOur 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.

Molecular detection of Wuchereria bancrofti, Loa loa and Mansonella perstans from dried blood spots taken from pregnant women in rural Burkina Faso

Introduction: Human filariasis causes high morbidity and severe illness. There is a link between helminth infection and anemia. The objective of this study was to estimate the prevalence of blood-dwelling microfilariae among pregnant women in Burkina Faso using a molecular technique and attempt to find an association between anemia and filarial infection. Methods: A total of 1018 dried blood spot samples (DBS) were collected from pregnant women at the Health District of Nanoro. The DNA was isolated from DBS samples using a rapid and simple method. Afterward, the isolated DNA was assayed using the Filaria real-time PCR (F-RT-PCR) method. Results: Ten F-RT-PCR-positive samples were obtained as follows: two W. bancrofti (0.2%), four L. loa (0.39%), and four M. perstans (0.39%). No concomitant filarial infections were detected, as well as no coinfections between filarial disease and malaria. There was no link between the presence of W. bancrofti, L. loa, or M. perstans and anemia in pregnant women. Conclusions: The prevalence and intensity of human filariasis in this study were low for all of the samples in which microfilariae were detected. The F-RT-PCR can be a confirmatory test for diagnosis in remote areas due to its effectiveness in detecting and differentiating, both sensitively and specifically, a wide range of filarial parasites.

Anti-filarial efficacy of Centratherum anthelminticum: unravelling the underlying mechanisms through biochemical, HRAMS proteomics and MD simulation approaches.

Traditionally, Centratherum anthelminticum (CA) has been reported to be a potent anti-filarial, however no reports are available detailing its mechanism of action against filarial parasites. In this study, we have evaluated the anti-filarial activity of CA against lymphatic filarial parasites Setaria cervi using ex vivo biochemical, proteomics and in silico approaches. The motility and viability of the parasites decreased significantly after treatment with CA concentrations of ≥125 μg mL-1. An increase in lipid peroxidation (51.92%), protein carbonylation (48.99%), NADPH oxidase (88.88%) activity and decrease in the glutathione (GSH) (-39.23%), glutathione reductase (GR) (-60.17%), and glutathione S-transferase (GST) (-50.48%) activity was also observed after CA treatment. The proteomics analysis was performed by two-dimensional gel electrophoresis and high-resolution accurate mass spectrometry (HRAMS). In total, 185 proteins were differentially expressed (DEPs) following CA treatment. The major DEPs were mostly involved in tRNA processing, biosynthetic processes, metabolic activities, protein transport, the tricarboxylic acid cycle, protein translation, and stress response. The UPLC-ESI-MS/MS analysis of CA extract revealed the presence of 40 bioactive compounds. Further the docking analysis showed 10 CA bioactive compounds to have high binding affinity towards antioxidant proteins of filarial parasites. Additionally, MD simulation studies showed stable interactions (RMSF ≤ 10 Å) of 3-O-methylquercitin, quinic acid, gentisic acid, and vanillin with filarial antioxidant enzymes/proteins. To our knowledge, this is the first report detailing the molecular mechanism of anti-filarial activity of CA, which can be further evaluated for the development of new anti-filarial formulations.

Detection of Wuchereria bancrofti through Molecular Xenomonitoring of Putative Mosquito Vectors in the Philippines

The advancement of molecular methods resulted in the application of molecular xenomonitoring (MX) as a complementary tool to detect the presence of circulating LF parasites in mosquito vectors. In the Philippines, a number of endemic areas are still undergoing evaluations by the World Health Organization (WHO) and Department of Health – Philippines (DOH). This study investigated the presence of filarial parasites in Oriental Mindoro (Region 4B) and Sultan Kudarat (Region 12), Philippines through MX. All collected and identified mosquito vectors were subjected to a real-time PCR assay targeting the Wuchereria bancrofti long-dispersed repeat Wb-LDR gene. This study detected the presence of W. bancrofti parasites in two species of mosquito vectors: (1) Aedes poicilius collected in Barangay Sta. Clara, Kalamansig, Sultan Kudarat (n=10 pools; 10.00%); and (2) Culex quinquefasciatus from Barangay Poblacion II, Victoria, Oriental Mindoro (n=344 pools; 0.29%) and Barangay Sta. Maria, Kalamansig, Sultan Kudarat (n=96 pools; 1.04%). These results support transmission assessment survey findings of continuous presence of LF parasites in the two endemic provinces of Oriental Mindoro and Sultan Kudarat. This study also suggests that further refinement of MX may produce broader applicability in the control and elimination of mosquito-borne diseases.

A case of pie (pulmonary infiltrates and eosinophilia)

ABSTRACT Tropical eosinophilia is a rare but clinically significant condition caused by an exaggerated immune response to filarial parasites, predominantly Wuchereria bancrofti and Brugia malayi. We present a case of a 19-year-old male who presented with a 2-month history of cough, peripheral eosinophilia, elevated serum immunoglobulin E levels, and diffuse pulmonary infiltrates on imaging. The patient, returning from a tropical region, was diagnosed with tropical eosinophilia based on clinical presentation and laboratory findings. Prompt initiation of diethylcarbamazine therapy and albendazole resulted in the resolution of symptoms and radiographic abnormalities. This case highlights the importance of considering tropical eosinophilia in the differential diagnosis of respiratory symptoms in individuals with a history of travel to endemic regions and underscores the necessity of early diagnosis and treatment to prevent long-term complications.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

Platelet functions in lymphatic filariasis patients

Filariasis is a chronic disease where parasitic worms survive in human hosts even for decades and lead to complications like lymphedema and elephantiasis. Despite the persistent existence of filarial parasites in human hosts, fatal and thrombotic complications are not known, unlike other parasitic diseases like malaria. This suggests that filarial parasites might be affecting the host's platelet functions. This study was conducted to examine platelet functions in confirmed filariasis patients and healthy controls. Results showed that filariasis patients had larger platelets, inhibited aggregation, and slower speed of aggregation, compared to controls. However, in vivo markers of platelet activation and degranulation (beta thromboglobulin and soluble P-selectin) were not affected. Observations suggested that there is increased platelet turnover, cellular apoptosis and inhibited platelet functions in filariasis patients compared to controls. Platelet function inhibition was not associated with the duration of disease, lymphedema-affected organs, or gender of patients. This study confirms that filarial parasites modulate platelet functions in human hosts.

Studi filariasis berbasis pemeriksaan laboratorium di Kota Semarang

Background: Filariasis is a disease caused by parasitic nematodes of Filariae. Filariasis is caused by filarial worm species, namely Wuchereria bancrofti, Brugia malayi, and Brugia timori. This disease is transmitted through the bite of infected mosquitoes (Anopheles, Aedes, and Culex). Symptoms of filariasis in men or women include damage to the lymphatic system swelling of the arms, legs, and genitals, which causes pain, decreased productivity, and social problems. Filarial diagnosis through examination of blood smears to detect filarial parasites in the blood, accompanied by hematological and clinical chemical examinationsObjective: This study aimed to describe filariasis through blood tests (blood smears, hematological examinations, and clinical chemistry examinations) on filariasis respondents using a cross-sectional approach.Method: This type of descriptive research uses a cross-sectional approach. The research subjects were 6 filarial respondents in Pedurungan District, Semarang City. The examination was conducted at the Tologosari Wetan and Kulon Health Centers on September 12-18, 2020. Secondary data collection was obtained from capillary blood examination using thin blood smears, automatic hematological examination using a hematology analyzer, and clinical chemistry examination using the spectrophotometer method with a spectrophotometer. The research data was processed and analyzed using Univariate analysis using maximum, minimum, and average values.Results: Hematological examination for hemoglobin level of 12,5 g%, leukocytes 3200 cells/ml, platelets 137.00 cells, erythrocytes 4,07x106 cells, lymphocytes 35 cells, neutrophils 73 cells, eosinophils 1 cell, monocytes 9 cells, MCH 30,78 pg, MCV 86,48 fl, MCHC 36,24 g/l and clinical chemistry examination results for cholesterol 145,7 g/dl and triglycerides 104,6 g/dl. Meanwhile, on examination of thin blood smears, microfilariae were found.Conclusion: The results of the blood smear evaluation showed an abnormality in the size of the red blood cells in the form of microcytic (smaller red blood cell size), and filarial findings were found. In contrast, hematology and clinical chemistry examination results obtained normal results.

Wing geometric morphometrics and DNA barcoding to distinguish three closely related species of Armigeres mosquitoes (Diptera: Culicidae) in Thailand

Armigeres subalbatus, a mosquito species widely found in Thailand and other Asian countries, serves as a vector for filarial parasites, affecting both humans and animals. However, the surveillance of this vector is complicated because of its morphological similarity to two other species, Armigeres dohami and Armigeres kesseli. To differentiate these morphologically similar species, our study employed both wing geometric morphometrics (GM) and DNA barcoding, offering a comprehensive approach to accurately identify these closely related Armigeres species in Thailand. Our GM analyses based on shape demonstrated significant accuracy in differentiating Armigeres species. Specifically, the outline-based GM method focusing on the 3rd posterior cell exhibited an accuracy rate of 82.61%, closely followed by the landmark-based GM method with 81.54%. Both these GM techniques effectively distinguished Ar. subalbatus from Ar. dohami and Ar. kesseli. Regarding DNA barcoding, our investigation of pairwise intra- and interspecific divergences revealed a "barcoding gap". Furthermore, the results of species confirmation using both species delimitation methods including the automatic barcode gap discovery method (ABGD) and the Multi-rate Poisson tree process (mPTP) were consistent with those of morphological identification, sequence comparisons with the GenBank and Barcode of Life Data System (BOLD) databases, and the neighbor-joining tree construction. These consistent results emphasize the efficacy of DNA barcoding in the precise identification of Armigeres species.