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.

A Trial of a Triple-Drug Treatment for Lymphatic Filariasis.

BACKGROUND: The World Health Organization has targeted lymphatic filariasis for global elimination by 2020 with a strategy of mass drug administration. This trial tested whether a single dose of a three-drug regimen of ivermectin plus diethylcarbamazine plus albendazole results in a greater sustained clearance of microfilariae than a single dose of a two-drug regimen of diethylcarbamazine plus albendazole and is noninferior to the two-drug regimen administered once a year for 3 years. METHODS: In a randomized, controlled trial involving adults from Papua New Guinea with Wuchereria bancrofti microfilaremia, we assigned 182 participants to receive a single dose of the three-drug regimen (60 participants), a single dose of the two-drug regimen (61 participants), or the two-drug regimen once a year for 3 years (61 participants). Clearance of microfilariae from the blood was measured at 12, 24, and 36 months after trial initiation. RESULTS: The three-drug regimen cleared microfilaremia in 55 of 57 participants (96%) at 12 months, in 52 of 54 participants (96%) at 24 months, and in 55 of 57 participants (96%) at 36 months. A single dose of the two-drug regimen cleared microfilaremia in 18 of 56 participants (32%) at 12 months, in 31 of 55 participants (56%) at 24 months, and in 43 of 52 participants (83%) at 36 months (P=0.02 for the three-drug regimen vs. a single dose of the two-drug regimen at 36 months). The two-drug regimen administered once a year for 3 years cleared microfilaremia in 20 of 59 participants (34%) at 12 months, in 42 of 56 participants (75%) at 24 months, and in 51 of 52 participants (98%) at 36 months (P=0.004 for noninferiority of the three-drug regimen vs. the two-drug regimen administered once a year for 3 years at 36 months). Moderate adverse events were more common in the group that received the three-drug regimen than in the combined two-drug-regimen groups (27% vs. 5%, P<0.001). There were no serious adverse events. CONCLUSIONS: The three-drug regimen induced clearance of microfilariae from the blood for 3 years in almost all participants who received the treatment and was superior to the two-drug regimen administered once and noninferior to the two-drug regimen administered once a year for 3 years. (Funded by the Bill and Melinda Gates Foundation; ClinicalTrials.gov number, NCT01975441 .).

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.

Setaria labiatopapillosa (Filarioidea, Nematoda) in Moroccan cattle: atypical localization and morphological characterization of females and microfilariae by light and scanning electron microscopy.

Filarioid nematodes are parasites of the tissues and tissue spaces of all vertebrates except fish. Females produce microfilariae that enter the host's blood circulation or skin and may cause ocular and neurological pathology, leading to important implications in veterinary and public health. The present work is the first investigation on Setaria labiatopapillosa conducted in Morocco to characterize the morphological features of both adult and microfilaria forms. Two adult female nematodes were found free in the thoracic cavity of a slaughtered 3.5-year-old (6 teeth) Moroccan enhanced cross-breed bull which was born and raised in Morocco. The worms were identified as S. labiatopapillosa by light microscope (LM) and scanning electron microscopy (SEM) on the basis of their characteristic features of the anterior and posterior parts of the worms. The two S. labiatopapillosa worms measured 90 mm and 105 mm in length and 0.55 and 0.64 mm in width, respectively. Microfilariae were detected in the fully developed eggs contained in the uterus of both nematodes. A detailed morphology of both the adult females and larvae of S. labiatopapillosa is described using LM and SEM. Although the origin of S. labiatopapillosa analyzed in the present study is unknown and there is currently no evidence that Setaria spp. have invaded Morocco, further surveillance is warranted to determine the incidence of setariasis, identify its vectors, and take appropriate measures to protect the livestock and cattle industry of the country.

Heterogeneous response of Wuchereria bancrofti-infected persons to diethylcarbamazine (DEC) and its implications for the Global Programme to Eliminate Lymphatic Filariasis (GPELF).

DEC or ivermectin (IVM) in combination with albendazole (ALB) has been the recommended strategy of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) since 2000. Despite effective population coverage (> 65%) with several rounds of MDA with DEC or combination of DEC plus ALB, microfilariae persist in few individuals and they continue to be the source of infection for transmitting LF. We report an individual's variability in response to DEC by defining the response as complete absence of microfilaria (mf) (post-treatment mf count = 0) and non-response as presence of mf (post-treatment mf count ≥ 1). We analyzed follow-up data on individual's response to treatment from two randomized clinical trials in which 46 microfilaremic individuals were treated with single-dose DEC (6 mg/kg body weight). They were classified into low, medium, and high mf density categories based on their pre-treatment mf counts. Of the 46 individuals, 65.2% have not responded throughout the 12-month post-treatment period. Application of a logistic regression model with fixed (age, gender, mf density, post-treatment time, and their interactions) and random (individual's response over time) effects indicated that treatment response is independent of age, gender, and time. The overall treatment response increases in low and decreases in high mf density categories. Furthermore, the estimates for the random coefficients model showed that there is a greater variability in response between individuals over post-treatment time. The results substantiate that individual variation in response to DEC exists which indicate the importance of studying the parasite as well as host genetic factors associated with DEC action.

Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview.

Acanthocheilonema reconditum is a filarial parasite transmitted by arthropods (fleas, lice, and ticks) that infect dogs. There is minimal published data available to date on potential haematological and biochemical changes associated with this parasitic infection. Study aims were (i) provide an overview of A. reconditum in Europe, (ii) define A. reconditum prevalence and risk factors in a specific dog population (hunting) from southern Italy, and (iii) assess the frequency of haemato-biochemical abnormalities associated with infection. Blood samples collected from 3020 dogs were tested by a modified Knott's technique to count and identify microfilariae. Eighty-four dogs were infected by A. reconditum (2.78%; 95% CI 2.19-3.37%). Microfilariae ranged from 1 to 212/ml. Based on clinical examination, all but six dogs with non-specific symptoms were healthy. Haematological abnormalities included leucocytosis (n = 15), with eosinophilia (n = 14) and monocytosis (n = 13). Serum biochemical abnormalities included increased total serum proteins (n = 19), albumins (n = 7), total globulins (n = 14), ALT (n = 1), and ALP (n = 1); one dog was hypoalbuminemic, and BUN was mildly increased in 2 dogs. Risk factors included the province origin (Napoli, OR=5.4, 95%CI: 2.1-14.0; Caserta, OR=5.1, 95%CI: 2.5-10.6), hunting wild mammals (OR=2.8, 95% 95%CI: 1.6-4.8), and ectoparasite infestation (OR=1.9, 95%CI: 1.1-3.1). There was a negative correlation between microfilaraemic load and decreased albumin level (-0.37; p=0.021). Our results showed that A. reconditum circulates within the hunting dog population of southern Italy, with seemingly low pathogenic potential.

Co-occurrence of Eutrombicula alfreddugesi and Oswaldofilaria chabaudi in Tropidurus torquatus and first report of microfilariae in the chigger mite: possible evidence of a lifecycle pathway?

While much attention has been paid to vector-borne filariasis, diseases that threaten millions of people in tropical and subtropical countries, the literature on host-parasite associations and transmission strategies of filarial nematodes in wildlife is scarce. Here, we report the co-occurrence of chigger mites (Eutrombicula alfreddugesi) and onchocercid nematodes (Oswaldofilaria chabaudi) parasitizing the lizard Tropidurus torquatus in the State of Minas Gerais, Brazil. Examination of chiggers established, for the first time, the occurrence of microfilariae in trombiculid mites (Trombiculidae). These larvae were morphologically similar to those recovered from adult females of O. chabaudi. The current evidence suggests that chiggers do not play a role in the transmission of filarioid nematodes, but rather act as accidental or dead-end hosts. Nevertheless, considering the polyphagous nature of trombiculid mites, similar to blood-sucking insects involved in the transmission of several infectious diseases, further studies may shed light on the potential role of chiggers as vectors of filarioids.

Elimination or Resurgence: Modelling Lymphatic Filariasis After Reaching the 1% Microfilaremia Prevalence Threshold.

The low prevalence levels associated with lymphatic filariasis elimination pose a challenge for effective disease surveillance. As more countries achieve the World Health Organization criteria for halting mass treatment and move on to surveillance, there is increasing reliance on the utility of transmission assessment surveys (TAS) to measure success. However, the long-term disease outcomes after passing TAS are largely untested. Using 3 well-established mathematical models, we show that low-level prevalence can be maintained for a long period after halting mass treatment and that true elimination (0% prevalence) is usually slow to achieve. The risk of resurgence after achieving current targets is low and is hard to predict using just current prevalence. Although resurgence is often quick (<5 years), it can still occur outside of the currently recommended postintervention surveillance period of 4-6 years. Our results highlight the need for ongoing and enhanced postintervention monitoring, beyond the scope of TAS, to ensure sustained success.

A Test-and-Not-Treat Strategy for Onchocerciasis in Loa loa-Endemic Areas.

BACKGROUND: Implementation of an ivermectin-based community treatment strategy for the elimination of onchocerciasis or lymphatic filariasis has been delayed in Central Africa because of the occurrence of serious adverse events, including death, in persons with high levels of circulating Loa loa microfilariae. The LoaScope, a field-friendly diagnostic tool to quantify L. loa microfilariae in peripheral blood, enables rapid, point-of-care identification of persons at risk for serious adverse events. METHODS: A test-and-not-treat strategy was used in the approach to ivermectin treatment in the Okola health district in Cameroon, where the distribution of ivermectin was halted in 1999 after the occurrence of fatal events related to L. loa infection. The LoaScope was used to identify persons with an L. loa microfilarial density greater than 20,000 microfilariae per milliliter of blood, who were considered to be at risk for serious adverse events, and exclude them from ivermectin distribution. Active surveillance for posttreatment adverse events was performed daily for 6 days. RESULTS: From August through October 2015, a total of 16,259 of 22,842 persons 5 years of age or older (71.2% of the target population) were tested for L. loa microfilaremia. Among the participants who underwent testing, a total of 15,522 (95.5%) received ivermectin, 340 (2.1%) were excluded from ivermectin distribution because of an L. loa microfilarial density above the risk threshold, and 397 (2.4%) were excluded because of pregnancy or illness. No serious adverse events were observed. Nonserious adverse events were recorded in 934 participants, most of whom (67.5%) had no detectable L. loa microfilariae. CONCLUSIONS: The LoaScope-based test-and-not-treat strategy enabled the reimplementation of community-wide ivermectin distribution in a heretofore "off limits" health district in Cameroon and is a potentially practical approach to larger-scale ivermectin treatment for lymphatic filariasis and onchocerciasis in areas where L. loa infection is endemic. (Funded by the Bill and Melinda Gates Foundation and others.).

Current trends in canine dirofilariosis in Austria-do we face a pre-endemic status?

A retrospective study based on cases of canine dirofilariosis presented to the University of Veterinary Medicine, Vienna or diagnosed by private practitioners throughout Austria, from 1998 to 2018 was conducted to investigate the long-term development and current state of canine dirofilarial infections in Austria. Included in this study were 146 dogs which were tested positive for D. immitis and/or D. repens. The most commonly used diagnostic methods and the probable geographical origins of the infections were evaluated and the treatment protocols applied were compared with each other and with the literature. The results show that most infections were found due to screening for common travel infections using antigen-ELISA or PCR-testing, or by the incidental finding of microfilariae. Remarkably, only 24.3% of all cases presented showed clinical signs indicating canine dirofilariosis. Regarding the origin and travel history of the dogs, thirteen different countries could be identified. The three treatment protocols used showed a similar outcome after 8 months of treatment and minor side effects, which is consistent with the literature. An alarming increase in reported infections with both D. immitis and D. repens in Austria was noted since 2014. The number of documented cases had almost tripled by 2018, raising severe concerns about the threat of it becoming endemic in Austria. Therefore, the existing recommendations in current guidelines regarding canine dirofilariosis should be widely publicised and more strictly enforced. Prophylactic measures for dogs travelling abroad and diagnostic and therapeutic strategies for dogs imported from endemic countries should be obligatorily established throughout Europe, to reduce the risk of further spread of canine filarial infections to non-endemic regions.

Diagnostic Identification and Differentiation of Microfilariae.

The morphologic similarities of the microfilariae and their infrequency in clinical specimens in settings of endemicity present challenges to clinical laboratories in maintaining competence for accurate identification and differentiation. We present here a review of the primary filarial nematodes causing human infection, including an illustrated key, which we hope will improve the diagnostic capabilities of hematologists, microbiologists, medical technologists, and similarly qualified laboratorians.

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.

Familial Aggregation and Heritability of Wuchereria bancrofti Infection.

BACKGROUND: The familial recurrence risk of lymphatic filariasis (LF) is unknown. This case study aimed to evaluate the familial susceptibility to infection with Wuchereria bancrofti and to microfilaremia in a village of the Republic of Congo. METHODS: The heritability and intrafamilial correlation coefficients were assessed for both W. bancrofti infection and microfilaremia by controlling for individual risk factors, environmental influence, and household effects. RESULTS: Pedigree charts were constructed for 829 individuals, including 143 individuals with a diagnosis of W. bancrofti circulating filarial antigens (CFAs) and 44 who also had microfilariae (MF). There was no intrafamilial correlation regarding CFA levels. However, the presence of MF (ρ = 0.45) and microfilarial density (ρ = 0.44) were significantly correlated among parent-offspring pairs. Heritability estimates for CFA positivity and intensity were 0.23 and 0.18, respectively. Heritability estimates were high for microfilarial positivity (h(2) = 0.74) and microfilarial density traits (h(2) = 0.81). CONCLUSIONS: Our study suggests that the acquisition of LF is mainly driven by environmental factors and habits and that genetic factors are moderately involved in the regulation of infection. By contrast, genetic factors play a major role in both the presence and intensity of microfilaremia.

Co-infections and environmental conditions drive the distributions of blood parasites in wild birds.

Experimental work increasingly suggests that non-random pathogen associations can affect the spread or severity of disease. Yet due to difficulties distinguishing and interpreting co-infections, evidence for the presence and directionality of pathogen co-occurrences in wildlife is rudimentary. We provide empirical evidence for pathogen co-occurrences by analysing infection matrices for avian malaria (Haemoproteus and Plasmodium spp.) and parasitic filarial nematodes (microfilariae) in wild birds (New Caledonian Zosterops spp.). Using visual and genus-specific molecular parasite screening, we identified high levels of co-infections that would have been missed using PCR alone. Avian malaria lineages were assigned to species level using morphological descriptions. We estimated parasite co-occurrence probabilities, while accounting for environmental predictors, in a hierarchical multivariate logistic regression. Co-infections occurred in 36% of infected birds. We identified both positively and negatively correlated parasite co-occurrence probabilities when accounting for host, habitat and island effects. Two of three pairwise avian malaria co-occurrences were strongly negative, despite each malaria parasite occurring across all islands and habitats. Birds with microfilariae had elevated heterophil to lymphocyte ratios and were all co-infected with avian malaria, consistent with evidence that host immune modulation by parasitic nematodes facilitates malaria co-infections. Importantly, co-occurrence patterns with microfilariae varied in direction among avian malaria species; two malaria parasites correlated positively but a third correlated negatively with microfilariae. We show that wildlife co-infections are frequent, possibly affecting infection rates through competition or facilitation. We argue that combining multiple diagnostic screening methods with multivariate logistic regression offers a platform to disentangle impacts of environmental factors and parasite co-occurrences on wildlife disease.

Detection of Dirofilaria repens microfilariae in a dog from Portugal.

Dirofilaria repens causes subcutaneous infection in dogs and cats, and is the main agent of human dirofilariosis in Europe. Detection of D. repens is described in a dog from Portugal, a finding that simultaneously represents the first presumable case of autochthonous infection with the parasite in any animal host species in the country. A mixed D. repens/Dirofilaria immitis infection (20:1 ratio) was found, with an overall density of 1267 microfilariae per millilitre of blood. Morphological features, including morphometry, and acid phosphatase histochemical staining confirmed identity of the two filarioids. Distribution of D. repens in Portugal should be further assessed at the population level both in vertebrate hosts and in vectors. Due to the zoonotic potential of the parasite, preventive measures such as the administration of microfilaricides and insect repellents should be put into practice to protect animals and public health.

Comparative Studies on Nucleic Acid Based Biosensors for Identification of Filarial Nematode.

Background: The microfilarial nematodes, found mainly in blood circulation, form a special group of human and veterinary parasitic diseases. Recently, the rapid nucleic acid based biosensors (NABs) have been established in our laboratory for the employment of a point-of-care diagnostic test. Herein, NABs involved in the exploitation of lateral flow dipstick (LFD), gold nanoparticle (AuNP) and turbidity in combination with PCR and LAMP amplification for detection of filarial nematodes. The validation of each NABs was investigated in comparison to that of standard detection methods using the same unknown blood specimens. Objective: To compare the sensitivity, specificity and accuracy of NABs with standard detection methods. Material and Method: In this study, the microfilariae of Dirofilaria immitis was used as the representative model for filarial nematode. The PCR and LAMP primers were designed and synthesized according to the specific nucleotide regions of a small subunit gene of the parasite as well as the DNA probes. The fifty unknown blood samples were sent as a gift from Prasu Arthorn Animal Hospital, Faculty of Veterinary Science, Mahidol University. The samples were tested by using PCR, PCRLFD, PCR-AuNP, LAMP, LAMP-LFD, LAMP-AuNP, LAMP-turbidity and film blood smear. The efficacy of NABs was compared to a standard film blood smear in terms of sensitivity, specificity and accuracy. Results: Upon detection of fifty unknown blood samples, LAMP-LFD assay presented 100% of sensitivity, specificity and accuracy. The data revealed that sensitivity, specificity and accuracy of NABs varied from 66.67-100.00% when compared to a film blood smear stained with Giemsa dye. The data clearly indicated that LAMP-LFD was a preferred choice for the use as a point-of-care NABs. Conclusion: NABs were highly sensitive performing as a selective diagnostic tool that could be applied particularly as the rapid screening tests for filarial nematodes hence as a recommended epidemiological survey.

Filariasis Presenting As Bone Marrow Suppression.

Lymphatic filariasis is caused by most commonly Wuchereria bancrofti in India. The diagnosis is made by demonstration of microfilariae in the peripheral blood, body fluids, fine needle aspirates and in bronchial brushings. Presence of microfilariae in the bone marrow is unusual and not been reported frequently. We are reporting here a case of a young male who had pancytopenia and was found to have microfilariae in the bone marrow.