Integrated Histological and Molecular Analysis of Filarial Species and Associated Wolbachia Endosymbionts in Human Filariasis Cases Presenting Atypically in Thailand.

Atypical presentations of filariasis have posed diagnostic challenges due to the complexity of identifying the causative species and the difficulties in both diagnosis and treatment. In this study, we present the integrative histological and molecular analysis of seven atypical filariasis cases observed in regions of nonendemicity of Thailand. All filariasis cases were initially diagnosed based on histological findings. To confirm the causative species, molecular characterization based on both filarial mitochondrial (mt 12S rRNA and COI genes) and nuclear ITS1 markers was performed, together with the identification of associated Wolbachia bacterial endosymbionts. Among the cases studied, Brugia pahangi (N = 3), Brugia malayi (N = 1), Dirofilaria sp. "hongkongensis" (N = 2), and a suspected novel filarial species genetically related to Pelecitus copsychi (N = 1) were identified. By targeting the 16S rRNA gene, Wolbachia was also molecularly amplified in two cases of infection with Dirofilaria sp. "hongkongensis." Phylogenetic analysis further revealed that the detected Wolbachia could be classified into supergroups C and F, indicating the high genetic diversity of this endosymbiont in Dirofilaria sp. "hongkongensis." Furthermore, this study demonstrates the consistency between histological findings and species identification based on mitochondrial loci rather than on the nuclear ITS1. This suggests the utility of mitochondrial markers, particularly COI, as a highly sensitive and reliable diagnostic tool for the detection and differentiation of filarial species in clinical specimens. Precise identification of the causative species will facilitate accurate diagnosis and treatment and is also essential for the development of epidemiological and preventive strategies for filariasis.

Pseudofilariasis, a Presenting Sign of Alkaptonuria.

PURPOSE: To report the association of Pseudofilariasis as a presenting sign of Alkaptonuria. METHOD: Case Report. RESULTS: A 49-year-old Indian man was referred because of wormlike objects in his left conjunctiva. Ocular and family history was non-contributory. He had not been to India in 15 years. Past medical history revealed hypertension, hypercholesterolemia, arthritis, and a myocardial infarct. He had undergone two stents, bilateral Achilles tendon repairs and bilateral knee replacements. ROS showed longstanding back stiffness and pain. On ocular examination the vision was 20/25 in each eye and positive findings were in the left eye bulbar conjunctival which showed stationary black vermiform (filarial in appearance) foreign bodies along with 2 small corneal limbal pigmented deposits. Conjunctival biopsy showed dilated lymph channels with interstitial proteinaceous material of a light brown color consistent with Ochronotic pigment; hence diagnostic of Alkaptonuria. CONCLUSIONS: Pseudofilariasis may be a presenting sign of Alkaptonuria and occur years before a clinical diagnosis is made. Filariasis is always involves white worms and never black. Knowing the ophthalmic signs of this rare disease may lead to an accurate diagnosis earlier thusly avoiding unnecessary tests and examinations.

Edge Artificial Intelligence (AI) for real-time automatic quantification of filariasis in mobile microscopy.

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.

A Giant Scrotal Neurofibroma in a Child Masquerading as Filariasis: Uncommon Presentation of a Common Disease.

Neurofibroma of the scrotum is a very uncommon benign neoplasm, specifically when it affects teenagers and is not associated with neurofibromatosis type I. To the best of our knowledge, only a couple of cases of neurofibroma in children have been documented. Here, we report a case study of a 17-year-old boy who had a giant scrotal lump for ten years masquerading clinically as filariasis. A provisional diagnosis of benign nerve sheath neoplasm was made based on cytology findings. The lump was surgically removed from the patient, and a histopathological and immunohistochemistry examination established the diagnosis of neurofibroma. The combined clinical, preoperative cytological, histological, and immunohistochemistry findings were not presented in the literature in any of the formerly documented cases of scrotal neurofibroma. The current case expands the spectrum of differential diagnoses for scrotal tumours that clinicians have previously observed.

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.

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.

Performance of Field's Stain Compared with Conventional Giemsa Stain for the Rapid Detection of Blood Microfilariae in Gabon.

Filarial infections caused by Loa loa and Mansonella perstans are a considerable public health burden in rural regions of Central Africa. Rapid diagnostic tools for the detection of microfilariae in the blood are needed. Field's stain is a rapid staining technique for microscopic slides originally established for malaria diagnostics. It requires less than 1 minute of staining compared with conventional staining protocols requiring at least 15 to 20 minutes for staining and could thus significantly accelerate diagnostics for human filariasis. Here we evaluated Field's stain as a rapid staining technique in comparison to Giemsa stain for the detection of microfilariae in peripheral blood. Blood smears were collected from 175 participants residing in the region of Lambaréné and Fougamou, Gabon. Each participant's samples were stained in parallel with Field's stain and conventional Giemsa stain. Slides were then microscopically assessed and compared for qualitative and quantitative results by a blinded assessor for the two endemic filarial blood pathogens M. perstans and L. loa. Field's stain shows excellent diagnostic performance characteristics for L. loa microfilariae compared with Giemsa staining. Concordance was favorable for M. perstans although lower than for L. loa. Field's stain offers a rapid alternative to Giemsa stain for detection of L. loa microfilariae in thick blood smears. This could help accelerate diagnostics of blood filarial pathogens in mass screening programs or resource constrained health care institutions with high patient load.

A rare case of ocular filariasis caused by Brugia malayi.

Extra-lymphatic manifestation of filariasis is uncommon and may usually be clinically misdiagnosed. The extra lymphatic presentation of Brugia malayi may due to the site of entry of the infective larvae, and only a few cases have been proven for the causative species. We report here a 59-year-old woman presented with swollen right conjunctiva and complaint of migratory swelling at her eyelid, which has turned out to be ocular filariasis by B. malayi in Chantaburi province, Thailand. This report highlights the increasing cases of B. malayi as a causative agent of ocular filariasis in human.

Characterization of a novel microfilarial antigen for diagnosis of Wuchereria bancrofti infections.

BACKGROUND: Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori. The Global Program to Eliminate LF uses mass drug administration (MDA) of anti-filarial drugs that clear microfilariae (Mf) from blood to interrupt transmission by mosquitos. New diagnostic tools are needed to assess the impact of MDA on bancroftian filariasis, because available serologic tests can remain positive after successful treatment. METHODOLOGY/PRINCIPAL FINDINGS: We identified Wb-bhp-1, which encodes a W. bancrofti homologue of BmR1, the B. malayi protein used in the Brugia Rapid antibody test for brugian filariasis. Wb-bhp-1 has a single exon that encodes a 16.3 kD protein (Wb-Bhp-1) with 45% amino acid identity to BmR1. Immunohistology shows that anti-Wb-Bhp-1 antibodies primarily bind to Mf. Plasma from 124 of 224 (55%) microfilaremic individuals had IgG4 antibodies to Wb-Bhp-1 by ELISA. Serologic reactivity to Wb-Bhp-1 varied widely with samples from different regions (sensitivity range 32-92%), with 77% sensitivity for 116 samples collected from microfilaremic individuals outside of sub-Saharan Africa. This variable sensitivity highlights the importance of validating new diagnostic tests for parasitic diseases with samples from different geographical regions. Individuals with higher Mf counts were more likely to have anti-Wb-Bhp-1 antibodies. Cross-reactivity was observed with a minority of plasma samples from people with onchocerciasis (17%) or loiasis (10%). We also identified, cloned and characterized BmR1 homologues from O. volvulus and L. loa that have 41% and 38% identity to BmR1, respectively. However, antibody assays with these antigens were not sensitive for onchocerciasis or loiasis. CONCLUSIONS: Wb-Bhp-1 is a novel antigen that is useful for serologic diagnosis of bancroftian filariasis. Additional studies are needed to assess the value of this antigen for monitoring the success of filariasis elimination programs.

Cytological diagnosis of microfilaria coexistent with suspected malignancy of breast and ovary in a young pregnant female.

Filariasis is a major health issue of tropical and subtropical regions and is endemic in India. It is rarely seen in cytological smears, exfoliative scrapings or in effusions. We present the case of a 29-year old female with filaria found on cytological examination of both breast and ovary.

Microfilaria Positification Test Using Real-Time PCR Technique with HRM (High-Resolution Melting).

Microscopic examination results in patients with filariasis are often not identified by the presence of microfilaria, so it needs to be checked by Polymerase Chain Reaction (PCR). One PCR method uses High-Resolution Melting (HRM). The purpose of this study was to compare qPCR-HRM with microscopic examination methods to determine the types of microfilaria found in patients with filariasis. 19 samples were examined using a microscopic method and qPCR-HRM. The results of microscopic examination found no type of microfilaria and in qPCR-HRM identified B.malayi and W.bancrofti with peak temperature melting 78.1-78.7 ℃ and 80.2-80.8 ℃. The results of the study based on the comparison of two methods show that the types of microfilaria W.bancrofti and B.malayi can be found using qPCR-HRM.

Ovarian Filariasis: Diagnosis by detection of microfilariae in follicular fluid, a case report.

Filariasis is a common parasitic infection in India. It is rare to find neglected cases of Filariasis nowadays. We reported the presence of microfilaria species in the follicular fluid of an egg donor undergoing an ovum pick up procedure. She was a 23-year-old egg donor who underwent stimulation using the GnRH antagonist protocol. Antagonist protocol is one of the standard protocols used for controlled ovarian hyperstimulation as a part of the IVF/ICSI(in-vitro fertilization / intracytoplasmic sperm injection) procedure where GnRH antagonist (cetrorelix) is used to suppress the endogenous LH surge. Her baseline investigations were normal, with no significant history suggestive of any worm infestations. During the ovum pickup procedure, follicular fluid revealed the presence of worm-like structures suggestive of larvae of some parasites. The follicular fluid was sent to the microbiology department along with the blood sample to confirm the parasite species. The parasite was found to be the larvae of W. Bancroft. The oocytes were of poor quality and were discarded. The patient was treated with Diethylcarbamazine citrate. There are so many reports about scrotal Filariasis, but rare literature quotes ovarian Filariasis.

Cytological diagnosis of microfilariae in clinically unsuspected cases: A retrospective review of 12 cases.

BACKGROUND: Filariasis is a major health problem in India. Despite the high prevalence, microfilariae are rarely found in cytology smears. Most of the cases are incidentally found, solely or in association with other pathologies. AIMS AND OBJECTIVES: This study was undertaken to analyse the prevalence and cytological findings of cases of incidentally found microfilariae in cytology smears (fine needle aspiration cytology [FNAC]/exfoliative cytology) from different parts of the body. MATERIALS AND METHODS: This was a retrospective study over 3 years, in which cases of microfilariae in aspirates from swelling of different locations, body fluids, and pap smears were reviewed, and the clinicopathological data analysed. RESULTS AND ANALYSIS: Out of 11 530 cases of FNAC, 8700 cases of fluid cytology, and 9000 of conventional cervicovaginal smears, 12 cases (0.04%) of incidental findings of microfilariae were documented in cytology smears. The cases were diagnosed from lymph node (one case), hand (one case), scrotal area (one case), axilla (one case), breast (one case), subcutaneous tissue (three cases), urine (three cases), and Pap smear (one case). We found eosinophilia in one case (8.3%) of filarial lesions. We found two cases of incidental findings of microfilariae in association with malignant lesions. CONCLUSION: Cytology smear examination can play an important role in diagnosing occult filariasis in clinically unsuspected cases in association with other pathologies.

An unusual case of genital filariasis of the testicular tunics in an Italian patient.

At least 27 million men present with urogenital manifestations of genital filariasis (GF). Although there is a large burden of GF in residents in endemic regions, infection in short-term travellers and in non-endemic areas is rare. We report the case of a 75-year-old Italian man referred to our institution for a testicular discomfort. Clinical examination and ultrasound detected a mass of 40×18 mm in the scrotum without signs of varicocele, hydrocele and testicular or epididymal abnormalities. After ineffective medical treatment, the patient underwent surgical excision. Histology demonstrated filarial granuloma with thick cuticles pathognomonic of an adult worm in the testicular tunics. Surgery has a fundamental role in localized cases of GF to remove nematodes from the fibrotic nodules. The present manuscript describes a singular case of GF observed in Italy in a non-immigrant patient with evidence of remnants of an adult worm in the testicular tunics. SIMILAR CASES PUBLISHED: 11.

Membranoproliferative glomerulonephritis: A rare presentation of occult filariasis.

Parasitic infections do not usually present with rapidly progressive renal failure but can provoke glomerular lesions which are mostly proliferative. In filarial infection, glomerular involvement is usually mild and transient, and presentation with renal failure is rare. We report occult filariasis presenting as rapidly progressive renal failure due to immune-complex mediated membranoproliferative glomerulonephritis. Our patient responded to treatment with diethylcarbamazine and a short course of steroid. This case highlights the importance of thorough workup to identify the cause and consideration of filariasis in an endemic area.

Filariasis presenting as isolated pleural effusion: A case report and mini review.

Isolated pleural effusion is a rare manifestation of filariasis that mimics tuberculosis, especially in endemic regions. We describe a case of lymphocytic and exudative pleural effusion showing microfilaria on pleural fluid cytology. A retrospective review of all cases of filarial pleural effusion reported after 2000 was conducted to evaluate the association between filariasis and pleural effusion as well as to screen the features that can help in accurate detection of these patients. The analysis suggested a causal association between the parasite and the development of pleural effusion with a high sensitivity of pleural fluid cytology for diagnosis.