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.

Successful treatment of Brugia pahangi in naturally infected cats with ivermectin.

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 µg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.

A case report of Brugian filariasis outside an endemic area in Thailand.

A 2-year-old boy living outside the endemic area of lymphatic filariasis in Surat Thani Province, Thailand, developed a high fever. To investigate the cause of his presenting symptoms, blood was collected and microfilariae were detected and identified as Brugia malayi using thick blood smear staining. The sources of the infection were investigated. Microfilariae from two domestic cats residing in the boy's village were detected and identified as B. pahangi using a high-resolution melting real-time polymerase chain reaction analysis. The possible sources of this cryptic infection are discussed.

Seasonal distribution and environmental parameters associated with Brugia pahangi and Dirofilaria immitis in naturally infected dogs in Bangkok and vicinity, Thailand.

Dirofilaria immitis and Brugia pahangi are vector-borne parasites found in dogs and cats, including Thailand. In order to evaluate the effects of season and environmental parameters on the prevalence of these parasites, this retrospective study was conducted in 2019. A total of 79,506 canine blood samples were examined. B. pahangi was found in 0.55% of samples (438/79,506; 95% CI 0.50-0.61) while D. immitis was detected in 0.43% (345/79,506; 95% CI 0.39-0.48). One-way ANOVA found no effect of seasonal conditions on prevalence. For B. pahangi, the parameters rainfall, relative humidity and sunshine hours showed associations with p ≤ 0.20 and were included in multiple logistic regressions resulting in adjusted odds ratios of 0.53, 1.31 and 0.55, respectively. For D. immitis, only average temperature showed p ≤ 0.20, resulting in an odds ratio of 0.42. In conclusion, Thailand has environmental parameters that do not change very much during the year, so they might not affect the prevalence of two filarial nematodes. However, the threat of B. pahangi and D. immitis should not be ignored, especially in subtropical regions where their vectors are abundant. Both owners and veterinarians should be concerned about filarial prevention and control of D. immitis and B. pahangi.

Filariasis of the breast caused by Brugia pahangi: A concomitant finding with invasive ductal carcinoma.

Extralymphatic filariasis is an uncommon phenomenon that can be caused by several lymphatic filarial species, including zoonotic filaria of animal origins. In this study, we report a case of a 64-year-old Thai woman who presented with a lump in her left breast that was diagnosed with invasive ductal carcinoma. At the same time, a small nodule was found in her right breast, via imaging study, without any abnormal symptoms. A core needle biopsy of the right breast nodule revealed a filarial-like nematode compatible with the adult stage of Brugia sp. A molecular identification of the nematode partial mt 12rRNA gene and ITS1 suggested the causative species as closely related to Brugia pahangi, a zoonotic lymphatic filaria of animals such as cats and dogs. The sequence of the partial mt 12rRNA and ITS1 gene in this patient was 94% and 99% identical to the previously reported sequence of mt 12rRNA and ITS1 genes of B. pahangi. The sequence of ITS1 gene is 99% similar to B. pahangi microfilaria from infected dogs in Bangkok, which was highly suspected of having a zoonotic origin. As far as we know, this is the first case report of B. pahangi filariasis presented with a breast mass concomitantly found in a patient with invasive ductal carcinoma. This raised serious concern regarding the zoonotic transmission of filariasis from natural animal reservoirs.

Differential detection of Brugia malayi and Brugia pahangi by real-time fluorescence resonance energy transfer PCR and its evaluation for diagnosis of B. pahangi-infected dogs.

A real-time fluorescence resonance energy transfer PCR combined with melting curve analysis was developed for differentiating Brugia malayi and Brugia pahangi DNA in host blood using one set of primers and fluorophore-labeled hybridization probes specific for HhaI repetitive DNA. The differentiation of both species was based on their melting temperatures (Tm). The mean Tm +/- SD of B. malayi and B. pahangi were 56.18+/-0.21 and 52.49+/-0.07, respectively. The method was used for the molecular detection of B. pahangi in infected dog blood samples. The diagnostic sensitivity, specificity, accuracy,and positive and negative predictive values of this method were 100%. The detected mean difference of the Tm might allow the simple discrimination of two related species. This method is fast, sensitive, allows for a high throughput, can be performed on very small volumes, and has potential for diagnosis of B. pahangi-infected dogs in endemic areas as well as for large epidemiological investigations.

Intraspecies variation of Brugia spp. in cat reservoirs using complete ITS sequences.

The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites.

Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1.

This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA sequencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host.

High resolution melting real-time PCR for rapid discrimination between Brugia malayi and Brugia pahangi.

OBJECTIVE: To identify two closely related Brugia malayi and B. pahangi in cat reservoirs by using high resolution melting real-time PCR (HRM real-time PCR). MATERIAL AND METHOD: HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 5 Brugia specimens by using five sets of specific primers for HhaI repetitive region (HR), small heat shock protein (SHP), small subunit ribosomal DNA (18S rDNA), internal transcribed spacer region (ITS), and trans-spliced leading Exon I gene (SLX1). RESULTS: HRM analysis of ITS and SLX clearly generated 2 profiles of B. malayi and B. pahangi while those of HR, 18S rDNA, and SHP could classify B. pahangi. CONCLUSION: HRM is a simple and rapid method for identification of two closely related B. malayi and B. pahangi in which it can detect both parasites within 30 min after real-time PCR detection. This assay is probe-free HRM and reduces a risk of PCR carryover. It does not require multiplex methods and DNA sequencing; therefore, HRM provides a new approach for genetic screening and rapid detection of closely related species in a clinical laboratory.

Confirmation of elimination of lymphatic filariasis by an IgG4 enzyme-linked immunosorbent assay with urine samples in Yongjia, Zhejiang Province and Gaoan, Jiangxi Province, People's Republic of China.

A sensitive and specific IgG4 enzyme-linked immunosorbent assay (ELISA) with urine samples has been reported. To confirm elimination of bancroftian filariasis, the ELISA was used in a study conducted in Yongjia County and Gaoan City, People's Republic of China, where filariasis elimination was declared, with 10,409 students 5-16 years of age. The antibody positive rates were 0.08% in Yongjia and 0.34% in Gaoan. All positive samples were re-examined and found to be negative. Our results show that this ELISA is practical and useful for confirmation of the elimination of filariasis. If similar results are obtained in different filariasis-endemic countries, this method may be useful in global filariasis elimination programs.

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes.

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is an emerging tool for routine identification of bacteria, archaea and fungi. It has also been recently applied as an accurate approach for arthropod identification. Preliminary studies have shown that the MALDI-TOF MS was able to differentiate whether ticks and mosquitoes were infected or not with some bacteria and Plasmodium parasites, respectively. The aim of the present study was to test the efficiency of MALDI-TOF MS tool in distinguishing protein profiles between uninfected mosquitoes from specimens infected by filarioid helminths. Aedes aegypti mosquitoes were engorged on microfilaremic blood infected with Dirofilaria immitis, Brugia malayi or Brugia pahangi. Fifteen days post-infective blood feeding, a total of 534 mosquitoes were killed by freezing. To assess mass spectra (MS) profile changes following filariae infections, one compartment (legs, thorax, head or thorax and head) per mosquito was submitted for MALDI-TOF MS analysis; the remaining body parts were used to establish filariae infectious status by real-time qPCR. A database of reference MS, based on the mass profiles of at least two individual mosquitoes per compartment, was created. Subsequently, the remaining compartment spectra (N = 350) from Ae. aegypti infected or not infected by filariae were blind tested against the spectral database. In total, 37 discriminating peak masses ranging from 2062 to 14869 daltons were identified, of which 17, 11, 12 and 7 peak masses were for legs, thorax, thorax-head and head respectively. Two peak masses (4073 and 8847 Da) were specific to spectra from Ae. aegypti infected with filariae, regardless of nematode species or mosquito compartment. The thorax-head part provided better classification with a specificity of 94.1% and sensitivity of 86.6, 71.4 and 68.7% of D. immitis, B. malayi and B. pahangi respectively. This study presents the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes. Considering that the results might vary in other mosquito species, further studies are needed to consolidate the obtained preliminary results before applying this tool in entomological surveillance as a fast mass screening method of filariosis vectors in endemic areas.

Tissue migration capability of larval and adult Brugia pahangi.

Infection with mosquito-born filarial nematodes occurs when hosts are bitten by a vector carrying the infective third stage larvae (L3) of the parasites. These larvae, deposited on the skin by the feeding mosquito, are presumed to enter the skin via the vector-induced puncture wound. Larvae of Brugia spp. must then migrate from the entry site, penetrate various skin layers, and locate a lymphatic vessel that leads to their lymphatic predilection site. We have recently established an intradermal (ID) infection model using B. pahangi and the Mongolian gerbil, allowing us to investigate the migratory capability ofB. pahangi. Larval and adult parasites recovered from the peritoneal cavities of gerbils were capable of establishing an infection following ID (larvae) or subcutaneous (adult) injection. Third and fourth stage larvae both migrated away from the injection site within hours, although data suggest they localize to different lymphatic tissues at 3 days postinfection (DPI). Immature adult (28 day) B. pahangi also migrated away from their SC inoculation site within 7 DPI. Mature (45 day) adult B. pahangi displayed little migration away from the SC infection site, suggesting tissue migration may be limited to developing stages of the parasite.

Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis.

Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method to detect and differentiate a broad range of filarial species in a single PCR. The first internal transcribed spacer 1 (ITS1) along with the flanking 18S and 5.8S ribosomal DNA (rDNA) were isolated and cloned from Wuchereria bancrofti, Brugia malayi, and Brugia pahangi. Sequence analysis identified conserved sites in the 18S and 5.8S rDNA sequence that could be used as universal priming sites to generate ITS1-distinctive PCR products that were useful for distinguishing filariae at the genus level. The addition of a digestion of the ITS1 PCR product with the restriction endonuclease Ase I generated a fragment profile that allowed differentiation down to the species level for W. bancrofti, B. malayi, B. pahangi, Dirofilaria immitis, and D. repens. The PCR-RFLP of ITS1 rDNA will be useful in diagnosing and differentiating filarial parasites in human, animal reservoir hosts, and mosquito vectors in disease-endemic areas.

Assessment of Blood Collection from the Lateral Saphenous Vein for Microfilaria Counts in Mongolian Gerbils (Meriones unguiculatus) Infected with Brugia pahangi.

The NIH guidelines for survival bleeding of mice and rats note that using the retroorbital plexus has a greater potential for complications than do other methods of blood collection and that this procedure should be performed on anesthetized animals. Lateral saphenous vein puncture has a low potential for complications and can be performed without anesthesia. Mongolian gerbils (Meriones unguiculatus) are the preferred rodent model for filarial parasite research. To monitor microfilaria counts in the blood, blood sampling from the orbital plexus has been the standard. Our goal was to refine the blood collection technique. To determine whether blood collection from the lateral saphenous vein was a feasible alternative to retroorbital sampling, we compared microfilaria counts in blood samples collected by both methods from 21 gerbils infected with the filarial parasitic worm Brugia pahangi. Lateral saphenous vein counts were equivalent to retroorbital counts at relatively high counts (greater than 50 microfilariae per 20 µL) but were significantly lower than retroorbital counts when microfilarial concentrations were lower. Our results indicate that although retroorbital collection may be preferable when low concentrations of microfilariae need to be enumerated, the lateral saphenous vein is a suitable alternative site for blood sampling to determine microfilaremia and is a feasible refinement that can benefit the wellbeing of gerbils.

Rapid Differentiation of Filariae in Unstained and Stained Paraffin-Embedded Sections by a High-Resolution Melting Analysis PCR Assay.

BACKGROUND: Apart from infection with human filariae, zoonotic filariasis also occurs worldwide, and the numbers of cases have been increasing steadily. Diagnosis of intact filariae in tissues or organs depends on histological identification. The morphology of parasites in tissue-embedded sections is poor and shows high levels of homoplasy. Thus, the use of morphological characteristics in taxonomic studies is difficult and may not allow a specific diagnosis. METHODS: Here we report the use of real-time PCR with high-resolution melting analysis (HRM) to detect and identify Brugia malayi, Brugia pahangi, Wuchereria bancrofti, and Dirofilaria immitis in paraffin-embedded sections. Assay specificity was determined using other tissue-dwelling parasites, Angiostrongylus cantonensis, Gnathostoma spinigerum, and Cysticercus cellulosae. We also developed a quick paraffin removal protocol. RESULTS: Both human and animal filariae in formalin-fixed paraffin-embedded sections (FFPES) were diagnosed and identified rapidly, whereas other parasites were negative. There was no difference in the melting temperature of products amplified from filarial DNA obtained from unstained FFPES and Hematoxylin & Eosin-stained sections. Therefore, the DNA extraction protocols developed in this study could be used for real-time PCR with HRM. CONCLUSIONS: We report the successful application of a HRM-PCR assay to differentiate four filarial parasites in FFPES, thus providing the pathologist with an effective alternative diagnostic procedure. Furthermore, the quick paraffin removal protocol developed could shorten the duration and number of steps required for paraffin removal using a standard protocol.

Aedes (Gymnometopa) mediovittatus (Diptera: Culicidae) as an experimental vector of Brugia pahangi and B. malayi (Spirurida: Filariidae).

To test the susceptibility of Aedes (Gymnometopa) mediovittatus to infection with Brugia pahangi and Brugia malayi, females originating from the suburbs of San Juan, Puerto Rico, were fed on infected gerbils (Meriones unguiculatus). On average, 39.2% of the Ae. mediovittatus females became infected with L3 larvae of B. pahangi and 47.4% with B. malayi. The average number of infective L3 larvae of B. pahangi and B. malayi dissected from mosquitoes was 2.6 +/- 1.2 and 2.9 +/- 1.0, respectively. The largest number of L3 in a single mosquito was 16. After 10 d of development in the mosquitoes, L3 larvae of both Brugian species were found in greatest number in the thorax, in lesser number in the head/proboscis, and in least number in the abdomen. Ae. mediovittatus may serve as a useful laboratory model for the study of genetic susceptibility and refractoriness of mosquito vectors to filarial parasites.

Effect of gamma radiation on Brugia L3 development in vivo and the kinetics of granulomatous inflammation induced by these parasites.

Previous studies have shown that the downregulation of parasite-specific cellular immune response in Brugia-infected jirds requires viable worms but is not dependent on microfilariae (MF) for either induction or maintenance of this phenomenon. To clarify further which life cycle stages induce filarial hyporesponsiveness, jirds were infected intraperitoneally with third stage larvae (L3) exposed to 0, 15, 25, 35, 45, or 90 krad of gamma radiation to differentially alter L3 development. Necropsies were performed at 7, 14, 28, and 118 days postinoculation (DPI). The degree of parasite development, intraperitoneal inflammation, and pulmonary granulomatous inflammation (PGRN) to parasite antigen-coated beads embolized in the lungs were monitored at the time of necropsy. Parasite survival and worm lengths were inversely related to the irradiation dose. Gamma radiation at 35, 45, or 90 krad prevented larval molt to the adult stage. Some parasites irradiated with 15 or 25 krad developed beyond fourth stage larvae (L4) to infertile adult females. The PGRN peaked at 14 DPI in all infected groups. Downregulation of the PGRN occurred after 14 DPI in groups that received nonirradiated L3 or L3 irradiated with 15 krad. No significant decrease of the PGRN occurred in groups that received parasites irradiated with more than 15 krad. Significant peritoneal inflammation as indicated by an increase in macrophages occurred only in jirds that received nonirradiated L3. These data demonstrate the importance of the adult stages in inducing downmodulation in the absence of MF and suggest that the L4 may also play a role in the induction of this phenomenon. An alternate conclusion is that parasite burden and not developmental stage is important in the induction of this hyporesponsive state.

Protective immunity against multiple challenges of Brugia pahangi in Mongolian gerbils induced by drug-abbreviated infection.

Protective immunity against multiple challenge infections was examined in Mongolian gerbils after a drug-abbreviated infection with Brugia pahangi. The gerbils treated with mebendazole (MBZ) during the late prepatent period (7-9 weeks of postinfection) were challenged with 5 inoculations of 50 infective larvae of B. pahangi at 4-week intervals. The worm burden was significantly reduced 68.6% (19.0 in average number) to that of controls (60.6) and was accompanied with enhanced eosinophil responses 1 week after each challenge. MBZ-treated gerbils suppressed microfilaremia almost completely after the challenge infections.

Plasma levels of diethylcarbamazine and their effects on implanted microfilariae of Brugia pahangi in rats.

Plasma level of diethylcarbamazine (DEC) was measured by using gas chromatography and was compared to the changes of microfilaremia after an intraperitoneal injection with 200 mg/kg of DEC in rats. The microfilaremia was induced artificially by an intravenous implantation with 2 x 10(5) Brugia pahangi microfilariae (mf) 1 day before DEC treatment. The rats treated with DEC showed a rapid and significant decrease in mf number in the circulation within 30 min, continued for 4 hr, and then increased rapidly. DEC seemed to cause transient but significant suppression of microfilaremia of B. pahangi in rats directly.

Recent advances in the application of molecular biology in filariasis.

Monitoring of filarial parasites in the host and vector has traditionally depended on morphological identification. Recently, species-specific DNA probes have been developed for Brugia malayi, Brugia pahangi and Wuchereria bancrofti. Repeated DNA sequences are useful in developing DNA probes because they evolve more rapidly then coding sequences and their high copy number increases the sensitivity of detection. The Hhal repeated DNA family represents 12% of the total B. malayi DNA. This DNA family is present in species of Brugia (B. malayi, B. timori and B. pahangi) but not W. bancrofti. Sequence analysis of the repeated DNA in B. malayi and B. pahangi has allowed construction of two species-specific DNA probes. These probes were used in a double blind field study in Indonesia. Microfilariae (mf) from infected cats and humans were identified by classical morphological methods and DNA probes. Agreement was found in 98.6% of the 642 samples tested by the two different techniques. Besides mf identification DNA probes can be used to determine the species of infective larvae (L3s) in infected mosquitos. This is useful because the L3s have similar morphology. DNA probes for the identification of W. bancrofti have recently been developed and are in the initial stages of testing in China (Piessens, personal communication) and Egypt (Williams, personal communication). An alternative approach for identification of infected individuals is to detect specific parasite antigens in circulation. A WHO initiative to use either an antigen or antibody assay to replace night blood is presently underway. This approach, if successful would not require the presence of microfilariae, but could detect occult infections.