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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.

Background Dirofilaria immitis , D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. Methods We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S -based pan-filarial and 16S -based pan- Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum , D. immitis and D. repens , and a duplex qPCR targeting Wolbachia of D. immitis and D. repens . The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms ( D. immitis and Angiostrongylus vasorum ) and pan-filarial cox 1 and pan- Wolbachia fts Z PCRs to identify other filarial parasites and their Wolbachia , respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearmanʼs correlation was used to assess the association between filarial species and the strain of Wolbachia . Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. Results The detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10 −1 to 1.5 × 10 −4  mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia . Compared to the true positives, the approach showed excellent agreement ( k  = 0.98–1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial ( k  = 0.6) and a weak ( k  = 0.15) agreements before and after thermal pre-treatment of sera, respectively. Conclusions The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

Since the launch of the Global Programme to Eliminate Lymphatic Filariasis, more than 70% of the endemic countries have implemented mass drug administration (MDA) to interrupt disease transmission. The monitoring of filarial infection in sentinel populations, particularly schoolchildren, is recommended to assess the impact of MDA. A key issue is choosing the appropriate tools for these initial assessments (to define the best intervention) and for monitoring transmission. This study compared the pre-MDA performance of five diagnostic methods, namely, thick film test, Knott's technique, filtration, Og4C3-ELISA, and the AD12-ICT card test, in schoolchildren from Brazil. Venous and capillary blood samples were collected between 11 pm and 1 am. The microfilarial loads were analyzed with a negative binomial regression, and the prevalence and associated 95% confidence intervals were estimated for all methods. The accuracies of the AD12-ICT card and Og4C3-ELISA tests were assessed against the combination of parasitological test results. A total of 805 schoolchildren were examined. The overall and stratified prevalence by age group and gender detected by Og4C3-ELISA and AD12-ICT were markedly higher than the prevalence estimated by the parasitological methods. The sensitivity of the AD12-ICT card and Og4C3-ELISA tests was approximately 100%, and the positive likelihood ratios were above 6. The specificity of the Og4C3-ELISA was higher than that of the AD12-ICT at different prevalence levels. The ICT card test should be the recommended tool for monitoring school-age populations living in areas with ongoing or completed MDA.

Filariasis, a neglected tropical disease caused by roundworms, is a significant public health concern in many tropical countries. Microscopic examination of blood samples can detect and differentiate parasite species, but it is time consuming and requires expert microscopists, a resource that is not always available. In this context, artificial intelligence (AI) can assist in the diagnosis of this disease by automatically detecting and differentiating microfilariae. In line with the target product profile for lymphatic filariasis as defined by the World Health Organization, we developed an edge AI system running on a smartphone whose camera is aligned with the ocular of an optical microscope that detects and differentiates filarias species in real time without the internet connection. Our object detection algorithm that uses the Single-Shot Detection (SSD) MobileNet V2 detection model was developed with 115 cases, 85 cases with 1903 fields of view and 3342 labels for model training, and 30 cases with 484 fields of view and 873 labels for model validation before clinical validation, is able to detect microfilariae at 10x magnification and distinguishes four species of them at 40x magnification: Loa loa , Mansonella perstans , Wuchereria bancrofti , and Brugia malayi . We validated our augmented microscopy system in the clinical environment by replicating the diagnostic workflow encompassed examinations at 10x and 40x with the assistance of the AI models analyzing 18 samples with the AI running on a middle range smartphone. It achieved an overall precision of 94.14%, recall of 91.90% and F1 score of 93.01% for the screening algorithm and 95.46%, 97.81% and 96.62% for the species differentiation algorithm respectively. This innovative solution has the potential to support filariasis diagnosis and monitoring, particularly in resource-limited settings where access to expert technicians and laboratory equipment is scarce.

The Brugia Test Plus (BT+) is a new rapid diagnostic test for Brugia species which detects human IgG4 antibodies specific for the immunogenic Brugia protein BmR1. The aim of this study was to evaluate the BT+ assay with several types of sample-matrices: whole blood, plasma, and dried blood spots (DBS) from individuals living in Belitung Timur, a Brugia malayi endemic area in Indonesia. Night blood was collected from residents living in four presumed endemic villages, while DBS were collected from schoolchildren living in those four villages. The sensitivity of BT+ was measured by comparing the BT+ results to microscopic examination for microfilaria (Mf). The sensitivity of BT+ with whole blood under field conditions was 84.9% (95% CI 68.1–94.9, n = 33), and with EDTA plasma under laboratory conditions was 95.9% (95% CI 88.5–99.1, n = 73). Specificity was not assessed as it is impossible do so for an antibody test in endemic areas. In Mf-negative individuals, BT+ detected IgG4 antibodies in 204 out of 1,547 adult plasma samples (13.2%) and 7 out of 146 plasma samples collected from children aged 10–16 (4.8%). Detection of IgG4 antibodies in DBS collected from first and second grade schoolchildren (age 6–8) showed that only 1 out of 244 schoolchildren was positive (0.1%). Three individual readers were responsible for reading the BT+ . Statistical analysis showed high agreement among those readers (Kappa agreement value above 0.9). Laboratory technicians found the BT+ is simple to perform, easy to interpret the results, and appreciated the small volume of blood required (5 µL). This study has demonstrated that the novel BT+ is feasible for teams to implement and achieves good sensitivity, making it well suited for monitoring and evaluating the progress of the brugian- filariasis elimination program.

With the evolution of the Coronavirus Disease 2019 (COVID-19) pandemic, the number of patients brought to medical attention has increased. This has led to the unmasking of many coexisting occult infections and comorbidities such as tuberculosis, dengue, human immunodeficiency viral infection, diabetes, and hypertension. We report the first case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, unveiling the diagnosis of asymptomatic filariasis. A 37-year-old gentleman presented with shortness of breath, fever, and cough. He was found to have COVID-19 pneumonia. During his stay, microfilaria of Wuchereria bancrofti was detected incidentally on a blood smear exam. Consequently, the patient received appropriate treatment for both conditions. In order not to miss relevant concomitant diagnoses, it is prudent to keep a broad differential diagnosis when faced with SARS-CoV-2–infected patients; this is especially true when atypical symptoms are present or in areas endemic with other infections.

Filariasis is recognised as a global public health threat, particularly in tropical and subtropical regions. It is caused by infection with a nematode parasite of the superfamily Filarioidea, including Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, and Onchocerca lupi. Three main types of filariasis have been classified: lymphatic filariasis, subcutaneous filariasis, and serous cavity filariasis. The symptoms exhibited by individuals afflicted with filariasis are diverse and contingent upon several variables, including the species of parasite, the host’s health and immune response, and the stage of infection. While many classical parasitological techniques are considered indispensable tools for the diagnosis of parasitic infections in humans, alternative methods are being sought due to their limitations. Novel tests based on host–parasite interactions offer a rapid, simple, sensitive, and specific diagnostic tool in comparison to traditional parasitological methods. This article presents methods developed in the 21st century for the diagnosis of filariasis caused by invasion from W. bancrofti, B. malayi, O. volvulus, and O. lupi, as well as techniques that are currently in use. The development of modern diagnostic methods based on molecular biology constitutes a significant advancement in the fight against filariasis.

Filarial nematodes of the genus Brugia include parasites that are significant to both human and veterinary medicine. Accurate diagnosis is essential for managing infections by these parasites and supporting elimination programs. Traditional diagnostic methods, such as microscopy and serology, remain vital, especially in resource-limited settings. However, advancements in molecular diagnostics, including nucleic acid amplification tests, offer enhanced sensitivity and specificity. These techniques are becoming increasingly field-friendly, expanding their applications in diagnostics. By refining existing methods, developing novel biomarkers, and understanding the zoonotic potential of various Brugia species, it is possible to improve control measures and better support elimination efforts.

Background The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. Methods The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were ‘spiked’ with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. Results In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi , 15 as Dirofilaria immitis and 7 as| D. repens . Conclusions We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.

Background Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus ( sensu lato ), are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection. Methods Dermal punch biopsies were opportunistically collected from municipal shelter and client-owned dogs. Multiple skin samples collected from interscapular and head regions were tested by saline sedimentation to recover live microfilariae for morphometric identification and by PCR to amplify a 330 bp region of the filarioid 12S rRNA gene. Also, ticks observed on surveyed dogs were collected, identified to species level, and tested for filarioid DNA. Results A total of 496 saline sedimentations were performed on 230 shelter and 20 client-owned dogs. Cercopithifilaria bainae infections were identified in 2.6% (6/230) of shelter dogs by morphometry of microfilariae in sedimentations and/or amplification of DNA from skin. DNA sequences amplified from PCR positive skin samples were 99–100% identical to C. bainae reported in Italy. All skin samples from client-owned dogs were negative for filarioid infection by saline sedimentation and PCR. A total of 112 ticks, comprised of four species, were collected. Two of 72 R. sanguineus ( s.l .), both engorged females found attached to a C. bainae infected dog, harbored C. bainae DNA (99–100% identity). One attached R. sanguineus ( s.l .) male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae . Conclusions The distribution of C. bainae is more widespread than previously known. To our knowledge, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.