Association of haemato-biochemical indices and blood composite ratios with microfilaridermia in Onchocerciasis patients.

BACKGROUND: Onchocerciasis causes chronic systemic inflammation. Several studies have used markers such as haemato-biochemical indices to predict the occurrence of systemic inflammation. This study assessed the variability and predictability of haemato-biochemical indices and blood composite ratios (BCRs) in microfilariae positive (MF+) and microfilariae negative (MF-) subgroups of onchocercomata participants. METHODS: One hundred and five (105) MF + and 34 MF- participants were retrospectively recruited into the study. Screening for the presence of O. volvulus microfilariae was done from skin snips taken from the left and right iliac crests of participants using established and approved protocols. Haematological and biochemical indices were measured using standard laboratory automated analyzers. Blood composite ratios (BCRs) were calculated as ratios of the absolute parameters involved. RESULTS: A significantly increased total WBC, absolute eosinophil, eosinophil percent and absolute basophil were observed in the MF + participants compared to MF- participants. Reduced gamma-glutamyl transferase (GGT) with increased estimated glomerular filtration rate (eGFR) was significantly associated with MF + participants compared to MF- participants. BCRs were significantly higher for eosinophil-to-neutrophil ratio (ENR), eosinophil-to-monocyte ratio (EMR), eosinophil-to-basophil ratio (EBR) and eosinophil-to-lymphocyte ratio (ELR) in MF + participants compared to MF- participants. After multivariate adjustment, onchocercomata participants with increased eosinophil counts (aOR = 13.86, 95% CI [2.07-92.90], p = 0.007), ENR x10 (aOR = 1.42, 95% CI [1.05-1.93], p = 0.025), EMR (aOR = 2.64, 95% CI [1.25-5.60], p = 0.011), EBR (aOR = 1.07, 95% CI [1.01-1.10], p = 0.020) and ELR x10 (aOR = 1.69, 95% CI [1.14-2.51], p = 0.009) were more likely to have microfilaridermia. CONCLUSIONS: Elevated eosinophil counts with higher ENR, EMR, EBR and ELR levels are significantly associated with microfilaridermia in onchocercomata participants. Combining BCRs with eosinophil count significantly led to an improvement in the conventional model for predicting microfilaridermia.

Microfilaruria of Dirofilaria immitis in a dog from Italy.

Dirofilaria immitis is a mosquito-borne nematode-causing canine heartworm disease, with adult worms localized in the pulmonary arteries and right heart. In rare cases, ectopic migration might occur, and adults and blood circulating microfilariae can be found in unusual organs or fluids (e.g., eyes, abdominal cavity, bone marrow, and urine). A 17-year-old mixed-breed female dog was presented in a private veterinary clinic in Italy for hematuria and dysuria. Physical examination showed cardiac mitral murmur with marked respiratory distress and cyanotic mucous membranes after handling. Abdominal ultrasounds revealed a non-specific chronic cystopathy, while the echocardiography showed enlargement of the right heart associated with tricuspid insufficiency and mitral regurgitation, with the presence of an adult filariae in the right ventricular chamber. Circulating microfilariae were observed in the blood smear and molecularly identified as D. immitis. Unusual microfilaruria was detected in the urine sediment. Data presented raise awareness about the occurrence of microfilariae in unusual locations, such as the bladder, suggesting the need of a thorough clinical and laboratory assessment where D. immitis is endemic.

Development of Acanthocheilonema viteae in Meriones shawi: Absence of microfilariae and production of active ES-62.

AIMS: ES-62 is a well-studied anti-inflammatory molecule secreted by L4-adult stage Acanthocheilonema viteae. We maintain the life cycle of A viteae using Meriones libycus as the definitive host. Here, we investigated whether the full life cycle could be maintained, and functional ES-62 produced, in a related jird species-Meriones shawi. METHODS AND RESULTS: Adult worms were produced in comparable numbers in the two species, but very few microfilariae (MF) were observed in the M shawi bloodstream. M shawi ES-62 produced ex vivo was functional and protective in a mouse model of arthritis. Myeloid-derived cells from naïve and infected jirds of both species were compared with respect to ROS production and osteoclast generation, and some differences between the two species in both the absence and presence of infection were observed. CONCLUSIONS: The life cycle of A viteae cannot be successfully completed in M shawi jirds but L3 stage worms develop to adulthood and produce functional ES-62. Preliminary investigation into jird immune responses suggests that infection can differentially modulate myeloid responses in the two species. However, species-specific reagents are required to understand the complex interplay between A viteae and its host and to explain the lack of circulating MF in infected M shawi jirds.

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.

The first case of periorbital human dirofilariasis in the Czech Republic.

Dirofilaria repens and Dirofilaria immitis are the most common filarial species affecting humans in Europe. Dirofilaria repens causes subcutaneous or ocular infection, whereas D. immitis is responsible mainly for the pulmonary form. In this report, we present the first human case of periorbital dirofilariasis in the Czech Republic. A 58-year-old woman suffered from an eyelid oedema, redness and pain in the left eye. After excising the parasite from her eyelid, all clinical symptoms disappeared. Based on the morphology and cytochrome oxidase I sequencing, the parasite was identified as D. repens. Histology revealed that the excised worm was female with absent microfilariae in uteri. With respect to the length of the incubation period and the sequence identity with a known Czech isolate, we concluded that D. repens was most likely of autochthonous origin.

Self-reactive IgG4 antibodies are associated with blocking of pathology in human lymphatic filariasis.

Lymphatic filariasis, a chronic disfiguring disease exhibits complex pathology. Based on different clinical manifestations, infected individuals are categorized into asymptomatic-carriers and chronic-patients. The mechanism behind differential clinical outcomes remains unclear. Roles of filaria-specific B cell responses in filariasis have been documented, whereas the contribution of B1 cell response and poly-specific IgG and IgA in the context of clinical filariasis is not deciphered. In this study, we measured the poly-specific IgG and IgA levels in different clinical categories of filariasis. Asymptomatic-carriers exhibited increased IgG4 antibodies against both filarial-antigens as well as auto-antigens compared to other clinical categories, although IgG against these auto-antigens remained lower. IgA levels against both filarial and auto-antigens were decreased in asymptomatic-carriers. A positive correlation between anti-filarial IgG4 and IgG4 against auto-antigens were observed, suggesting the synergistic role of poly-specific natural IgG4 with anti-filarial IgG4 in blocking the pathogenesis in asymptomatic microfilarial cases.

Therapeutic efficacy of doxycycline in domestic cats naturally infected with Brugia malayi in field condition.

OBJECTIVE: The aim of this study was to evaluate the efficacy of oral doxycycline treatment for Brugia malayi as measured by microfilarial and filarial DNA clearance in naturally infected domestic cats. METHODS: This study included 8 domestic cats that lived with families that resided in Tak Bai District of Narathiwat Province, which is located in Southern Thailand. The study area is a known B. malayi endemic area. All study cats received doxycycline treatment doses by their respective owners according to a previously described protocol. Briefly, doxycycline (VibraVet@) was given orally once a day during weeks 1-4, 10-11, and 16-17. Blood collections were performed at baseline before treatment, and then every month for 12 months after the initial dose of doxycycline to assess microfilaraemia by Giemsa stain, and filarial DNA detection by high-resolution melt (HRM) real-time polymerase chain reaction (PCR). RESULTS: One month after the start of doxycycline treatment, five of eight cats were negative for microfilaraemia, and 4 of those were negative for filarial DNA. All cats receiving doxycycline treatment were negative for microfilaria by Giemsa stain, and for filarial DNA by HRM real-time PCR within 8 months after receiving the initial dose of doxycycline treatment. CONCLUSION: Administration of oral doxycycline to domestic cats naturally infected with B. malayi in disease endemic areas can significantly reduce microfilaraemia at 1 month and filarial DNA was undetectable by 8 months after the initial dose of doxycycline treatment. No recurrence of microfilaraemia or filarial DNA was observed in study cats at 1 year after the start of doxycycline. Included cats appeared to tolerate doxycycline (VibraVet@) well, with no adverse drug reactions reported by any study cat owner.

Morphological and genetic variation of Wuchereria bancrofti microfilariae in carriers in Thailand, Lao PDR and Myanmar: evaluation using Giemsa-stained thick blood films.

There is geographical variation in the morphology and genetics of Wuchereria bancrofti, the major cause of human lymphatic filariasis. This study aims to compare morphological and genetic variation of W. bancrofti microfilariae recovered from carriers in Lao PDR, Myanmar and Thailand. Six morphological parameters (body length, cephalic space length and width, length of head to nerve ring, body width at nerve ring, Innenkȍrper length and number of column nuclei between the cephalic space and nerve ring) were evaluated from microfilariae in Giemsa-stained thick blood films. A portion of the cytochrome c oxidase subunit 1 gene of mitochondrial DNA was sequenced and analysed. Wuchereria bancrofti microfilariae showed a wide variation in their morphology and morphometry among three countries. Phylogenetic analysis confirmed that all microfilariae belonged to W. bancrofti. Higher mutation frequencies were observed in samples from Myanmar, relative to Thailand and Lao PDR. This study highlights the morphological disparities of microfilariae and genetic variability within W. bancrofti among three geographical locations. We found that reported morphometric differences between localities were less clear-cut than previously thought. Further studies are needed to determine the microfilarial periodicity in Lao PDR.

An assessment of mosquito collection techniques for xenomonitoring of anopheline-transmitted Lymphatic Filariasis in Ghana.

Monitoring vectors is relevant to ascertain transmission of lymphatic filariasis (LF). This may require the best sampling method that can capture high numbers of specific species to give indication of transmission. Gravid anophelines are good indicators for assessing transmission due to close contact with humans through blood meals. This study compared the efficiency of an Anopheles gravid trap (AGT) with other mosquito collection methods including the box and the Centres for Disease Control and Prevention gravid, light, exit and BioGent-sentinel traps, indoor resting collection (IRC) and pyrethrum spray catches across two endemic regions of Ghana. The AGT showed high trapping efficiency by collecting the highest mean number of anophelines per night in the Western (4.6) and Northern (7.3) regions compared with the outdoor collection methods. Additionally, IRC was similarly efficient in the Northern region (8.9) where vectors exhibit a high degree of endophily. AGT also showed good trapping potential for collecting Anopheles melas which is usually difficult to catch with existing methods. Screening of mosquitoes for infection showed a 0.80-3.01% Wuchereria bancrofti and 2.15-3.27% Plasmodium spp. in Anopheles gambiae. The AGT has shown to be appropriate for surveying Anopheles populations and can be useful for xenomonitoring for both LF and malaria.

The use of cytapheresis in the treatment of infectious diseases.

Cytapheresis (removal of cellular blood components) has been employed for treatment of infectious diseases since the 1960s. Techniques have included thrombocytapheresis (buffy coat apheresis) for loiasis, erythrocytapheresis for malaria and babesiosis, and leukocytapheresis for pertussis-associated lymphocytosis. Published data on these applications is largely limited to case level data and small observational studies; as such, recommendations for or against the use of cytapheresis in the treatment of infections have been extrapolated from these limited (and at times flawed) data sets. Consequently, utilization of cytapheresis in many instances is not uniform between institutions, and typically occurs at the discretion of treating medical teams. This review revisits the existing literature on the use of cytapheresis in the treatment of four infections (loasis, malaria, babesiosis, and pertussis) and examines the rationale underlying current treatment recommendations concerning its use.

New insights into the clinico-histopathological and molecular features of Pelecitus (Filarioidea: Onchocercidae) from a raptor bird.

Some Onchocercidae nematodes such as Pelecitus are parasites of medical and veterinary importance. The adult stage of Pelecitus has been reported infecting birds, and the microfilaria has been associated to human blindness. However, in some of these cases, the nematode was incompletely identified at the species level due to the scarcity of morphological taxonomic keys and, also, to the lack of molecular diagnostic analysis. Here, we report a new Pelecitus species in a crested caracara (Caracara cheriway) producing a severe tenosynovitis and microfilarial dermatitis. It is also the first record of Pelecitus in an American bird of prey. Clinical and histopathological features are described, contributing towards our understanding of the pathogenesis of Pelecitus and the health and conservation of wild bird populations. Our study also provides new information on the molecular diagnosis of this parasite and highlights the potential role of wild birds as Pelecitus reservoirs, and health risk for humans and wildlife.

Discovery of Specific Antigens That Can Predict Microfilarial Intensity in Loa loa Infection.

Antigen-based immunoassays are currently needed for point-of-care quantification of Loa loa microfilariae (mf). Coupling transcriptomic approaches with bioinformatic analysis, we have identified 11 specific putative proteins (coding mRNAs) with potential utility as biomarkers of patent (mf + ) L. loa infection. We successfully developed antigen capture immunoassays to quantify 2 (LOAG_14221 and LOAG_15846) of these proteins in individual plasma/serum samples. Of the 2 quantifiable circulating biomarkers, LOAG_14221 showed the highest degree of specificity, particularly with a monoclonal antibody-based immunoassay. Moreover, the levels of LOAG_14221 in L. loa mf + patients were positively correlated to the mf densities in the corresponding blood samples (r = 0.53 and P = 0.008 for polyclonal assay; r = 0.54 and P = 0.004 for monoclonal assay). Thus, LOAG_14221 is a very promising biomarker that will be exploited in a quantitative point-of-care immunoassay for determination of L. loa mf densities.

Sleeping site ecology, but not sex, affect ecto- and hemoparasite risk, in sympatric, arboreal primates (Avahi occidentalis and Lepilemur edwardsi).

BACKGROUND: A central question in evolutionary parasitology is to what extent ecology impacts patterns of parasitism in wild host populations. In this study, we aim to disentangle factors influencing the risk of parasite exposure by exploring the impact of sleeping site ecology on infection with ectoparasites and vector-borne hemoparasites in two sympatric primates endemic to Madagascar. Both species live in the same dry deciduous forest of northwestern Madagascar and cope with the same climatic constraints, they are arboreal, nocturnal, cat-sized and pair-living but differ prominently in sleeping site ecology. The Western woolly lemur (Avahi occidentalis) sleeps on open branches and frequently changes sleeping sites, whereas the Milne-Edward's sportive lemur (Lepilemur edwardsi) uses tree holes, displaying strong sleeping site fidelity. Sleeping in tree holes should confer protection from mosquito-borne hemoparasites, but should enhance the risk for ectoparasite infestation with mites and nest-adapted ticks. Sex may affect parasite risk in both species comparably, with males bearing a higher risk than females due to an immunosuppressive effect of higher testosterone levels in males or to sex-specific behavior. To explore these hypotheses, ectoparasites and blood samples were collected from 22 individuals of A. occidentalis and 26 individuals of L. edwardsi during the dry and rainy season. RESULTS: L. edwardsi, but not A. occidentalis, harbored ectoparasites, namely ticks (Haemaphysalis lemuris [Ixodidae], Ornithodoros sp. [Argasidae]) and mites (Aetholaelaps trilyssa, [Laelapidae]), suggesting that sleeping in tree holes promotes infestation with ectoparasites. Interestingly, ectoparasites were found solely in the hot, rainy season with a prevalence of 75% (N = 16 animals). Blood smears were screened for the presence and infection intensity of hemoparasites. Microfilariae were detected in both species. Morphological characteristics suggested that each lemur species harbored two different filarial species. Prevalence of microfilarial infection was significantly lower in L. edwardsi than in A. occidentalis. No significant difference in infection intensity between the two host species, and no effect of season, daytime of sampling or sex on prevalence or infection intensity was found. In neither host species, parasite infection showed an influence on body weight as an indicator for body condition. CONCLUSIONS: Our findings support that sleeping site ecology affects ectoparasite infestation in nocturnal, arboreal mammalian hosts in the tropics, whereas there is no significant effect of host sex. The influence of sleeping site ecology to vector-borne hemoparasite risk is less pronounced. The observed parasite infections did not affect body condition and thus may be of minor importance for shaping reproductive fitness. Findings provide first evidence for the specific relevance of sleeping site ecology on parasitism in arboreal and social mammals. Further, our results increase the sparse knowledge on ecological drivers of primate host-parasite interactions and transmission pathways in natural tropical environments.

Oleanolic acid from antifilarial triterpene saponins of Dipterocarpus zeylanicus induces oxidative stress and apoptosis in filarial parasite Setaria digitata in vitro.

Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-ß-D- glucopyranoside (1) (IC50 = 20.54 µM for adult worms, 19.71 µM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 µM for adult worms, 25.99 µM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 µM) and microfilaricidal (IC50 = 35.6 µM) activities. In adult female worms treated with 50 and 100 µM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.

Efficacy and tolerability of treatment with single doses of diethylcarbamazine (DEC) and DEC plus albendazole (ABZ) for three consecutive years in lymphatic filariasis: a field study in India.

Lymphatic filariasis (LF) affects 73 countries, causes morbidity and impedes socioeconomic development. We had found no difference in safety and micro (Mf) and macro filarial action of single-dose diethylcarbamazine (DEC) and DEC + albendazole (ABZ) in an F01 study done in India (year 2000). There was a programmatic need to evaluate safety and efficacy of multiple annual treatments (F02). Subjects (155) from the F01 study, meeting inclusion-exclusion criteria, were enrolled in F02 and treated with further two annual doses of DEC or DEC + ABZ. Efficacy was evaluated for Mf positivity by peripheral smear (PS) and nucleopore (NP) filter, circulating filarial antigen (CFA) and filarial dance sign (FDS) positivity and Mf count at yearly follow-up. Safety was assessed for 5 days after drug administration. Total of 139 subjects evaluated for efficacy (69 DEC and 70 DEC + ABZ group). Mf positivity prevalence declined progressively by 95% (PS), 66% (NP), and 95% (PS) and 86% (NP); CFA positivity prevalence declined by 15% and 9%; FDS by 100% each; Mf count declined by 75.5 and 76.9% with three annual treatment of DEC and DEC + ABZ, respectively. Addition of ABZ did not show any advantage over DEC given as three annual rounds for LF. DEC and DEC + ABZ were well tolerated. There was no correlation between result of CFA and FDS, (both claimed to be indicative of adult worm). Analysis of published studies and our data indicate that macrofilaricidal effect of DEC/DEC + ABZ may be seen in children and not adults, with three or more annual dosing.

[The 15 years' experience of diagnostic of human dirofilariasis.]

The article presents the results of studies of the clinical diagnostic laboratory of the E.I. Martcinovskii institute of medical parasitology, tropical and transmissive diseases related to diagnostic of human dirofilariasis brought in by Dirofilaria repens. During last 15 years, 165 cases were diagnosed. The most of dirofilaria (89.7%) migrated and localized superficially under skin or mucous membrane: on the head in 88 cases and out of them 43.6% in the area of eye-socket, in 25 cases on the body out of them 4 on genitalia, in 35 cases under skin of extremities. In 1 cases dirofilaria migrated to inner organs: 10 in lungs, 4 in pleura, 1 in small intestine, 1 in omentum. In case of pleuropulmonary localization the disease was taking its course under mask of tuberculoma malignant tumor, exudative pleurisy, neoplasm of obscure etiology.

Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro.

Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 µg/mL for hexane extract, 191.14 µg/mL for chloroform extract) and adult parasites (IC50 = 77.07 µg/mL for hexane extract, 259.87 µg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST), superoxide dismutase (SOD) and catalase activities, increased reactive oxygen levels (ROS) and lipid peroxidation were also observed indicating that an apoptotic event is induced by reactive oxygen species.