Microfilaria Counts Research Articles

An automated, high-resolution phenotypic assay for adult Brugia malayi and microfilaria

Brugia malayi are thread-like parasitic worms and one of the etiological agents of Lymphatic filariasis (LF). Existing anthelmintic drugs to treat LF are effective in reducing the larval microfilaria (mf) counts in human bloodstream but are less effective on adult parasites. To test potential drug candidates, we report a multi-parameter phenotypic assay based on tracking the motility of adult B. malayi and mf in vitro. For adult B. malayi, motility is characterized by the centroid velocity, path curvature, angular velocity, eccentricity, extent, and Euler Number. These parameters are evaluated in experiments with three anthelmintic drugs. For B. malayi mf, motility is extracted from the evolving body skeleton to yield positional data and bending angles at 74 key point. We achieved high-fidelity tracking of complex worm postures (self-occlusions, omega turns, body bending, and reversals) while providing a visual representation of pose estimates and behavioral attributes in both space and time scales.

Safety of Simparica Trio® (sarolaner, pyrantel, moxidectin) in heartworm-infected dogs

BackgroundAssessment of the safety of heartworm preventatives in dogs with pre-existing patent heartworm (Dirofilaria immitis) infections is necessary because rapid adult worm and microfilarial death can lead to severe clinical complications, including thromboembolism and anaphylactic shock in dogs. The aim of this study was to determine the clinical safety of Simparica Trio® (sarolaner, pyrantel, moxidectin) in heartworm-infected dogs and the degree of microfilaricidal and adulticidal activity of three consecutive monthly treatments of Simparica Trio.MethodsTwenty-four laboratory Beagle dogs were implanted with 10 male and 10 female D. immitis (ZoeKY isolate), and once infection was patent, they were randomized equally among three groups to receive no treatment, 1× or 3× the maximum recommended label dose of Simparica Trio. Dogs in the treated groups received Simparica Trio on days 0, 28 and 56. In-life assessments included body weight, physical examinations, clinical observations, daily general health observations, a quantitative estimate of food consumption and blood collections for pharmacokinetic (PK) analysis, microfilariae (MF) counts and D. immitis antigen testing. At the end of the study the heart, lungs and pleural and peritoneal cavities were examined for adult D. immitis worms.ResultsSimparica Trio was generally well tolerated. Emesis occurred at low frequency in all groups including control. Abnormal stool occurred occasionally in the 1× and 3× groups throughout the 3-month study. Fever (> 104 °F/40 °C) was recorded in one 1× and one 3× dog 1 day after the first dose and resolved by the following day. No severe hypersensitivity reactions occurred. The mean number of circulating microfilariae (MF) counts in the control group increased from 12,000/ml at study start (Day 0) to > 20,000/ml at Day 28 and remained > 20,000/ml for the duration of the study. The least squares means of circulating MF were reduced by 69.8% on Day 1 and 97.4% on Day 7 for the 1× group and remained at > 99% lower than the control group for the remainder of the study. Similarly, least squares means of circulating MF were reduced by 85.3% on Day 1 and 93.9% on Day 7 for the 3× group and remained > 98% lower than the control group for the remainder of the study. At the end of the study, the mean number of implanted adult worms recovered was < 10 per sex in all groups with 90%, 85% and 75% of live adult heartworms recovered in control, 1× and 3× treatment groups, respectively. Low numbers of dead adult worms were recovered in 1× and 3×, with none in control. Following each dose, the moxidectin and sarolaner AUC and Cmax had close to dose proportional increases.ConclusionsThis study demonstrated that Simparica Trio (sarolaner, pyrantel, moxidectin) was well tolerated when administered to heartworm-positive dogs at 1× and 3× the maximum recommended dose at 28-day intervals for 3 consecutive months. Simparica Trio significantly reduced microfilaria counts in both treatment groups, without significant clinical consequences. At the doses administered, Simparica Trio had minor adulticidal activity but resulted in no clinical sequelae.Graphical

Colorimetric and Real-Time Loop-Mediated Isothermal Amplification (LAMP) for Detection of Loa loa DNA in Human Blood Samples.

Loiasis, caused by the filarial nematode Loa loa, is endemic in Central and West Africa. Loa loa has been associated with severe adverse reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. Diagnosis of loiasis still depends on microscopy in blood samples, but this is not effective for large-scale surveys. New diagnostics methods for loiasis are urgently needed. Previously, we developed a colorimetric high-sensitive and species-specific LAMP for Loa loa DNA detection. Here, we evaluate it in a set of 100 field-collected clinical samples stored as dried blood spots. In addition, Loa loa-LAMP was also evaluated in real-time testing and compared with microscopy and a specific PCR/nested PCR. A simple saponin/Chelex-based method was used to extract DNA. Colorimetric and real-time LAMP assays detected more samples with microscopy-confirmed Loa loa and Loa loa/Mansonella perstans mixed infections than PCR/nested-PCR. Samples with the highest Loa loa microfilariae counts were amplified faster in real-time LAMP assays. Our Loa loa-LAMP could be a promising molecular tool for the easy, rapid and accurate screening of patients for loiasis in endemic areas with low-resource settings. The real-time testing (feasible in a handheld device) could be very useful to rule out high-microfilariae loads in infected patients.

Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon

BackgroundThe tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,β-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged.MethodsWe performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography–mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome.ResultsThe mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls.ConclusionsOn an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques.Graphical abstract

Fecundity of adult female worms were affected when Brugia malayi infected Mongolian gerbils were immunized with a multivalent vaccine (rBmHAXT) against human lymphatic filarial parasite

A multivalent recombinant fusion protein prophylactic vaccine, rBmHAXT developed against lymphatic filariasis (LF) demonstrated over 57% protection against challenge infection in rhesus macaque model. Currently, we do not know if the rBmHAXT vaccination has any effect on adult worms and/or on the fecundity of adult female worms. Thus, the major focus of this study was to determine the effect of rBmHAXT vaccination on Brugia malayi infected mongolian gerbils. We performed two sets of experiments. In the first set of experiment, gerbils were infected with 100 B. malayi L3. After confirming the establishment of infection, four rounds of DEC treatment and rBmHAXT vaccination was given. Results showed that following vaccination with rBmHAXT, the microfilaria (Mf) count was significantly decreased in all vaccinated animals compared to controls. At the end of these experiments, we collected and counted the established adult worms. There was a 36% reduction in the recovery of adult female worms, which might account for the low Mf load in vaccinated animals. In the second set of experiments, animals were vaccinated first with rBmHAXT followed by surgically implanting adult male or female B. malayi parasites into the peritoneal cavity to determine the effect of vaccination on each sex of the parasite. Our results show that the rBmHAXT vaccination has no effect on male adult worms compared to controls. However, there was 40% reduction in the Mf load in vaccinated animals that were transplanted with adult female worms. These findings suggested that the rBmHAXT vaccination has potential damaging effect on the fecundity of adult female worms. Scanning electron microscopy studies showed cuticular damage on the surface of adult female worms. These studies thus show that the rBmHAXT vaccination in infected gerbils has partial microfilaricidal effect and potentially affect the fecundity of adult female worms.

Short-course quinazoline drug treatments are effective in the Litomosoides sigmodontis and Brugia pahangi jird models

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16−18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10–14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days.

Prevalence of lymphatic filariasis in a tribal area of Maharashtra

Background: About 304 million persons are living in the known endemic areas and are exposed to the risk of filariasis. About 22 million persons are harboring microfilaria in their blood and 16 million persons show clinical manifestations. The objective of the study was to study the prevalence of lymphatic filariasis in a tribal area.Methods: A community based cross sectional study was carried out. Night blood smears were obtained between 9-11.30 pm by finger prick method and thick smears were prepared and usual staining procedure was performed. 451 people out of (enumerated) 565 could be covered excluding infants. Diethyl carbamazine (DEC) provocative test was performed the next day by administering 100 mg DEC orally and collecting blood sample after one hour with usual method. DEC could be administered to only 200 individuals in spite of utmost efforts to ensure cooperation.Results: Out of 451 persons examined, 20 (4.4%) were showing clinical manifestations. Overall microfilaria rate was 14.4%. Total endemicity rate was 17.3%. The lowest number of microfilaria was seen in 5 in 20 cumm of blood while the highest count was 78 in the 20 cumm of the blood. 13 smears (20%) showed that microfilaria count was in the range of 41-50. Maximum average infestation rate was 33.3 per 20 cumm of the blood. The most prominent clinical manifestation seen was hydrocele in males constituting 35% of the total clinical cases.Conclusions: The prevalence of filariasis in this tribal area was high. Mf rate was 14.4%. Endemicity rate was 17.3%. Hydrocele was the commonest clinical manifestation.

Effects of milbemycin oxime, combined with spinosad, when administered orally to microfilaremic dogs infected with adult heartworms (Dirofilaria immitis).

OBJECTIVE To evaluate the safety of PO administration of a milbemycin oxime (MBO) and spinosad product to heartworm (Dirofilaria immitis)-positive microfilaremic dogs. DESIGN Randomized, blinded, complete block trial. ANIMALS 32 purebred Beagles with a patent heartworm infection. PROCEDURES Dogs ranked by sex and microfilaria counts (range, 398 to 1,980 microfilaria/mL) were assigned to 4 groups of 8 to receive 3 treatments PO at 28-day intervals beginning on day 0: placebo (control group) or spinosad-MBO tablets containing MBO at the upper end of the label dose range (0.75 to 1 mg/kg [0.34 to 0.45 mg/lb]; 1× group) or 3 (3× group) or 5 (5× group) times that dose. Blood samples were collected at various points for adult heartworm antigen and Knott tests. Necropsies were performed on day 65, and recovered adult heartworms were counted. RESULTS 1 control dog died from heartworm-associated complications. Other adverse events included mild, transient emesis (1 dog in each of the 1× and 5× groups and 3 dogs in the 3× group). Similar adult heartworm counts (range, 13 to 41) were obtained for all 4 groups. Results of blood antigen and microfilaria tests were positive throughout the study, with 1 exception in each of the 3× and 5× groups. Mean microfilaria counts increased with time in the control group, whereas reductions from baseline in treated groups ranged from 61.5% to 96.4%. CONCLUSIONS AND CLINICAL RELEVANCE The evaluated MBO-spinosad formulation caused no severe adverse events when administered PO to microfilaremic dogs. Although microfilaria counts decreased following treatment, repeated monthly MBO treatments were incompletely microfilaricidal, suggesting MBO should not be used as a microfilaricide.

Microfilarial reduction following ProHeart\xae 6 and ProHeart\xae SR-12 treatment in dogs experimentally inoculated with a resistant isolate of Dirofilaria immitis

BackgroundEmerging resistance of heartworms (Dirofilaria immitis) to macrocyclic lactone (ML) preventives is an increasing concern for veterinarians, pet owners and animal health companies that supply heartworm preventives, with recent reports of resistant isolates identified from the Mississippi Delta region of the United States. Products that are effective in eliminating microfilariae (MF) in dogs harboring resistant heartworm infections could be important in reducing the spread of heartworm resistance. The current study was conducted to investigate the potential for ProHeart® 6 (PH 6; Zoetis) and ProHeart® SR-12 (PH 12; Zoetis) to reduce MF in dogs experimentally inoculated with an isolate of D. immitis (ZoeMo-2012) confirmed to be resistant to MLs.MethodsTwenty-three dogs with preexisting heartworm infections (via surgical transplantation) were randomly allocated to four groups based on pretreatment (Day −14) MF counts. On Day 0, dogs received a subcutaneous injection of either saline (placebo-treated control, 6 dogs), PH 6 (0.17 mg/kg, 6 dogs), PH 12 (0.5 mg/kg, 5 dogs) or a single oral dose of moxidectin powder in a gelatin capsule (0.25 mg/kg, 6 dogs). All dogs were bled for MF counts (modified Knott’s test) on Days 0 (pretreatment), 1, 3, 7, 14, 21, 28, 42, 56, and 84. Dogs in control and PH 6 groups were also bled for MF counts on Days 112, 140, and 168. No adverse events associated with treatment were observed for any dog.ResultsAverage reductions in MF counts compared with controls for PH 6 were 9.7% on Day 1, increasing to 75.0% on Day 7, and further to 86.5% on Day 28. On Day 42, average MF reduction increased to 90.3%. Reductions increased further over the next several months with reductions of 91.3, 96.8, 96.6, and 98.9% on Days 56, 84, 112, and 140, respectively. On Day 168, the reduction was 99.3% (P < 0.0001). Average reductions in MF counts compared with controls for PH 12 were 20.9% on Day 1, increasing to 78.9% on Day 7, and further to 91.2% on Day 28. On Day 84, the reduction was 96.9%. For dogs receiving a single oral moxidectin (0.25 mg/kg) on Day 0, reductions in MF were 86.3% on Day 1 and fluctuated between 74.4 and 83.6% through Day 28. On Days 42 and 56, percentage reductions were 87.1 and 81.8%, respectively, and 92.6% at the final time point (Day 84).ConclusionBoth PH 6 and PH 12 were highly effective in reducing the MF levels of a confirmed ML-resistant heartworm isolate following a single dose.

Blocking the transmission of heartworm (Dirofilaria immitis) to mosquitoes (Aedes aegypti) by weekly exposure for one month to microfilaremic dogs treated once topically with dinotefuran-permethrin-pyriproxyfen

BackgroundThis study assessed the influence of a topical ectoparasiticide (dinotefuran-permethrin-pyriproxyfen, DPP, Vectra®3D, Ceva Animal Health) on the acquisition of heartworm microfilariae by mosquitoes exposed to microfilaremic dogs weekly for 1 month.MethodsSix beagle dogs (9.2 ± 1.6 kg body weight) infected with Dirofilaria immitis were allocated to two groups of three dogs: an untreated control group and a DPP-treated group. Dogs were treated on Day 0 and exposed under sedation for 1 h to 80 ± 20 unfed Aedes aegypti. Each dog was exposed to mosquitoes released into mosquito-proof containers on Days −7 (pretreatment), 7, 14, 21 and 28. Up to 20 engorged mosquitoes were aspirated from the cage as soon as they were blood-fed. They were dissected and the blood from each midgut was stained for a microfilaria (MF) count. After each exposure, mosquitoes were classified as live, moribund or dead and engorged or nonengorged. The number of dead mosquitoes was recorded daily for 16 days, when the live mosquitoes were dissected to count the infective third-stage larvae (L3).ResultsPrior to treatment, 95% of the engorged mosquitoes in both groups had MF. After treatment, engorgement rates for the treated group were 0%, 2.3%, 2.7% and 2.2% for Days 7, 14, 21 and 28, respectively, with anti-feeding efficacy (repellency) of 100%, 98.0%, 95.8% and 97.0%, respectively. A total of 22 mosquitoes fed on treated dogs; most of them were dead within 24 h, and all were dead within 72 h. Only 2 unfed mosquitoes exposed to treated dogs survived the incubation period and no L3 were found in them. A total of 121 of the 132 (91.6%) surviving mosquitoes that had engorged on untreated dogs had an average of 12.3 L3 per mosquito (range, 0-39).ConclusionsDPP was more than 95% effective in inhibiting blood-feeding and killing both engorged and nonengorged mosquitoes exposed weekly to microfilaremic dogs for 28 days after treatment. Treatment with DPP was completely effective in killing the few mosquitoes that fed on the treated dogs before they lived long enough for the microfilariae to develop to L3 and, consequently, was completely effective in blocking the transmission of L3 to other animals. DPP can break the life cycle of D. immitis and prevent infected dogs and infected mosquitoes from being effective reservoirs and can slow down the spread of heartworms, even those resistant to macrocyclic lactone preventives.

Bivariate geostatistical modelling of the relationship between Loa loa prevalence and intensity of infection

Loiasis is a neglected tropical disease (NTD) caused by the parasitic roundworm Loa loa. A challenge faced by current multinational programmes to control two other diseases, namely, lymphatic filariasis and onchocerciasis, by mass administration of prophylactic medication to at‐risk communities is that individuals highly coinfected with Loa loa are at risk of developing serious adverse reactions to the medication. For this reason, understanding the geographical distribution of Loa loa prevalence and the distribution of microfilarial loads in communities has become of crucial importance. In this paper, we develop methodology to analyse data on microfilariae counts per millilitre of blood whilst allowing for spatial correlation. One feature of the data is the excess of zero counts, which makes the use of standard geostatistical methods for prevalence data inappropriate. This phenomenon, also known as zero inflation, is typical of count data from NTDs, whose endemic boundaries are often unknown, thus leading to the inclusion of disease‐free communities in the sampling frame. We introduce a bivariate geostatistical model in order to study the relationship between the distributions of prevalence and intensity of Loa loa infections at the community level. We show through a simulation study that the spatial model leads to more precise spatial predictions than the nonspatial approach used by Schlüter et al. (), and accordingly provide a geostatistical reanalysis of the Loa loa data.

Effect of 3 years of biannual mass drug administration with albendazole on lymphatic filariasis and soil-transmitted helminth infections: a community-based study in Republic of the Congo

The standard treatment strategy of mass drug administration with ivermectin plus albendazole for lymphatic filariasis cannot be applied in central Africa, because of the risk of serious adverse events in people with high Loa loa microfilaraemia. Thus, alternative strategies are needed. We investigated one such alternative strategy for mass drug administration for elimination of lymphatic filariasis and soil-transmitted helminth infections in Republic of the Congo. In 2012, we started a 3 year community trial of biannual mass administration of albendazole in a village in Republic of the Congo. All volunteering inhabitants aged 2 years or older were offered albendazole (400 mg) every 6 months. Infection with Wuchereria bancrofti was diagnosed with a rapid card immunochromatographic test for antigenaemia. People with antigenaemia were tested for microfilaraemia by night blood smears. Individuals were also tested for soil-transmitted helminth infections (ie, hookworm, Ascaris lumbricoides, Trichuris trichiura) with the Kato-Katz method. Assessment surveys were done at 12, 24, and 36 months. The main outcome measure was change in infection rates from baseline to year 3. Therapeutic coverage was more than 80% in all six rounds of mass administration of albendazole. Between 2012 and 2015, W bancrofti antigenaemia and microfilaraemia rates in the community fell significantly, from 17·3% (95% CI 14·7-20·0) to 4·7% (3·3-6·6; p<0·0001) and from 5·3% (3·9-7·1) to 0·3% (0·1-1·2; p<0·0001), respectively. The geometric mean microfilaria count in microfilaraemic people fell from 199·4 (120·4-330·5) per mL in 2012 to 39·1 (95% CIs not computed) per mL in 2015 (p=0·0095). Hookworm infection was undetectable after 1 year. Between 2012 and 2015, the number of A lumbricoides eggs expelled per g of faeces fell from 9844·6 (8209·0-11 480·0) to 724·4 (340·7-1114·2; p<0·0001), and of T trichiura eggs from 1107·4 (878·5-1336·3) to 366·0 (255·7-476·2; p<0·0001). Our findings strongly support WHO's provisional strategy of biannual mass administration of albendazole to eliminate lymphatic filariasis in areas where loiasis is co-endemic and ivermectin cannot be safely mass administered. Bill & Melinda Gates Foundation.

Molecular Epidemiology of Blood-Borne Human Parasites in a Loa loa-, Mansonella perstans-, and Plasmodium falciparum-Endemic Region of Cameroon.

The study of the interactions among parasites within their hosts is crucial to the understanding of epidemiology of disease and for the design of effective control strategies. We have conducted an assessment of infections with Loa loa, Mansonella perstans, Wuchereria bancrofti, and Plasmodium falciparum in eastern Cameroon using a highly sensitive and specific quantitative polymerase chain reaction assay using archived dried whole blood spots. The resident population (N = 1,085) was parasitized with M. perstans (76%), L. loa (39%), and P. falciparum (33%), but not with W. bancrofti. Compared with single infections (40.1%), coinfection was more common (48.8%): 21.0% had L. loa–M. perstans (Ll+/Mp+/Pf−), 2.7% had L. loa–P. falciparum (Ll+/Pf+/Mp−), 15.1% had M. perstans–P. falciparum (Mp+/Pf+/Ll−), and 10.0% had L. loa–M. perstans–P. falciparum (Ll+/Mp+/Pf+). Interestingly, those with all three infections (Ll+/Mp+/Pf+) had significantly higher L. loa microfilaria (mf) counts than either single Ll+ (P = 0.004) or double Ll+/Mp+ (P = 0.024) infected individuals. Of those infected with L. loa, the mean estimated counts of L. loa mf varied based on location and were positively correlated with estimated intensities of M. perstans mf. Finally, at a community level, heavy L. loa infections were concentrated in a few individuals whereby they were likely the major reservoir for infection.

A randomized controlled trial of increased dose and frequency of albendazole with standard dose DEC for treatment of Wuchereria bancrofti microfilaremics in Odisha, India.

Although current programmes to eliminate lymphatic filariasis have made significant progress it may be necessary to use different approaches to achieve the global goal, especially where compliance has been poor and ‘hot spots’ of continued infection exist. In the absence of alternative drugs, the use of higher or more frequent dosing with the existing drugs needs to be explored. We examined the effect of higher and/or more frequent dosing with albendazole with a fixed 300mg dose of diethylcarbamazine in a Wuchereria bancrofti endemic area in Odisha, India. Following screening, 104 consenting adults were randomly assigned to treatment with the standard regimen annually for 24 months (S1), or annually with increased dose (800mg albendazole)(H1) or with increased frequency (6 monthly) with either standard (S2) or increased (H2) dose. Pre-treatment microfilaria counts (GM) ranged from 348 to 459 mf/ml. Subjects were followed using microfilaria counts, OG4C3 antigen levels and ultrasound scanning for adult worm nests. Microfilarial counts tended to decrease more rapidly with higher or more frequent dosing at all time points. At 12 months, Mf clearance was marginally greater with the high dose regimens, while by 24 months, there was a trend to higher Mf clearance in the arm with increased frequency and 800mg of albendazole (76.9%) compared to other arms, (S1:64%, S2:69.2% & H1:73.1%). Although higher and/or more frequent dosing showed a trend towards a greater decline in antigenemia and clearance of “nests”, all regimens demonstrated the potential macrofilaricidal effect of the combination. The higher doses of albendazole did not result in a greater number or more severe side effects. The alternative regimens could be useful in the later stages of existing elimination programmes or achieving elimination more rapidly in areas where programmes have yet to start.

Safety and efficacy of 10% imidacloprid + 2.5% moxidectin for the treatment of Dirofilaria immitis circulating microfilariae in experimentally infected dogs

A controlled laboratory study was conducted to establish the safety and efficacy of 10% imidacloprid+2.5% moxidectin topical solution (Advantage Multi® for Dogs, Bayer HealthCare, Shawnee, KS) for the treatment of circulating Dirofilaria immitis microfilariae in dogs. Twenty beagles were experimentally infected with D. immitis via surgical implantation of 10 pairs of adult worms (Pepper strain, TRS Labs) from donor dogs on Day −82. Between Days −7 and −1, physical examinations were performed, chest radiographs were taken, and blood and urine samples were collected for microfilariae counts, serum chemistry, complete blood counts, and urinalysis. Each dog was required to have a mean pretreatment count of at least 300mf/ml of blood. On Day −1, all 20 dogs were randomized by mean pretreatment microfilarial counts to two study groups (10 animals/group). Animals in Group 1 were treated on Days 0 and 28 with 10% imidacloprid+2.5% moxidectin topical solution at the minimum label dose of 0.1ml/kg. Group 2 animals served as negative controls and were treated on Days 0 and 28 with mineral oil at an equivalent volume as for the study solution. All dogs were observed hourly for 8h after treatment, again at 12h, and then once daily on all other study days. Blood samples for microfilarial counts were collected daily for 3 days after treatment and then weekly for 6 weeks. The percentage reduction in microfilariae was determined by comparing the geometric mean number of circulating microfilariae remaining in Group 1 dogs with the mean counts remaining in control dogs. Group 1 mean microfilarial counts were reduced 93.1% three days following the first treatment and by >99% on Days 14 through 42. Group 1 had significantly fewer (p<0.05) microfilariae compared with Group 2 counts on Days 28 and 42. In addition, log-transformed geometric mean microfilarial counts were significantly different between the two groups (p<0.05) using separate repeated measures analysis of covariance for Days 2, 3, 7, 14, 21, 28, 35, and 42. No adverse events related to treatment were reported during the study. The results of this study demonstrate that 10% imidacloprid+2.5% moxidectin topical solution is efficacious for treatment of circulating D. immitis microfilariae in heartworm-positive dogs with no treatment-related adverse events observed.

Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic.

Emergence of new foci of filariasis in Madhya Pradesh, India

Lymphatic filariasis is a major vector-borne disease and India bears 81% of the filariasis burden in South East Asia. Screening and mass drug administration is carried out in 11 known endemic districts of Madhya Pradesh. However, regular monitoring is not carried out in non-endemic areas. Cross sectional surveys were carried out to examine blood samples for filariasis. Entomological surveys were conducted for infective mosquitoes. Altogether, 24 of 252 samples were positive for filariasis with a mean microfilaria count of 0.8. The entomological investigations showed 7.4% infection and 1.4 % infectivity rates in vectors. This study reveals the emergence of new foci of lymphatic filariasis in Madhya Pradesh. We conclude that filariasis surveys should be carried out in non-endemic areas of India to achieve the goal of elimination.

Case-control Studies on the Relationship between Onchocerciasis and Epilepsy: Systematic Review and Meta-analysis

ObjectiveA systematic review and meta-analysis of all available case-control studies on the relationship between onchocerciasis and epilepsy. Because age and level of onchocerciasis endemicity in the area of residence are major determinants for infection, an additional analysis was performed, restricted to studies achieving control of these confounding factors.Data sourcesMedical databases, the “African Neurology Database, Institute of Neuroepidemiology and Tropical Neurology, Limoges,” reference lists of relevant articles, commercial search engines, up to May 2012.MethodsWe searched for studies examining infection status with Onchocerca volvulus in persons with epilepsy (PWE) and without epilepsy (PWOE) providing data suitable for the calculation of pooled odds ratios (ORp) and/or standardized mean differences (SMD) using random-effects models.ResultsEleven studies providing data of qualitative skin biopsies for diagnosis of onchocerciasis were identified. Combined analysis on the total sample of 876 PWE and 4712 PWOE resulted in an ORp of 2.49 (95% confidence interval (95%CI): 1.61–3.86, p<0.001). When this analysis was restricted to those studies achieving control for age, residence and sex (367 PWE, 624 PWOE), an ORp of 1.29 (95% CI: 0.93–1.79; p = 0.139) was found. Presence of nodules for diagnosis of onchocerciasis was analyzed in four studies (225 PWE, 189 PWOE; ORp 1.74; 95%CI: 0.94–3.20; p<0.076), including two studies of the restricted analysis (106 PWE, 106 PWOE; ORp 2.81; 95%CI: 1.57–5.00; p<0.001). One study examined quantitative microfilariae counts in patients without preceding microfilaricidal treatment and demonstrated significantly higher counts in PWE than in PWOE.InterpretationOur results strengthen the hypothesis that, in onchocerciasis foci, epilepsy and infection with O. volvulus are associated. Analysis of indicators giving information on infection intensity, namely nodule palpation and quantitative microfilaria count in untreated patients, support the hypothesis that intensity of infection with O. volvulus is involved in the etiology of epilepsy.

Effect of Diethylcarbamazine (DEC) on prostaglandin levels in Wuchereria bancrofti infected microfilaraemics

Diethylcarbamazine (DEC) interferes with arachidonic acid metabolism for the clearance of microfilariae in Wuchereria bancrofti infected individuals. In this study, we have quantified the plasma concentrations of prostaglandin E2 (PGE2) and 6-keto-PGF1α, the end products of arachidonic acid metabolic pathway in microfilaraemics (DEC treated and untreated), and normal healthy individuals at pre- and 3,9,12,36, and 72h of post-DEC treatment. We have also determined the microfilariae counts at pre and post day 2 (36h) and day 3 (72h) of DEC treatment by membrane filtration technique. Significant reduction in PGE2 and 6-keto-PGF1α concentrations was found at 12h of DEC treatment. Rapid reduction in microfilarial counts was observed at 36h of post-DEC treatment. Higher levels of prostaglandins were found at pre-treatment hours in microfilaraemics compared to normal healthy individuals (P < 0.05). Our findings indicate that DEC inhibits prostaglandins for the clearance of microfilariae, and increased levels of prostaglandins in microfilaraemics may be contributed by the parasite or host upon stimulation.

Lymphatic Filariasis Survey in Some Social Welfare Hostels in the Twin Godavari Districts of Andhra Pradesh

A survey was conducted to look for the presence of Lymphatic.Filariasis (LF) disease and microfilaria (mf) among children residing in six Social Welfare Hostels (SWH) in the twin Godavari districts of Andhra Pradesh (AP). The study also elicited the personal protection measures taken by the students against mosquito bites and consumption of DEC (Diethyl-Carbamazine) during Mass Drug Administration (MDA). In none of the 678 (432 boys and 246 girls) students examined in both the districts, any disease manifestation was found. In East Godavari district, 4 boys in the age group of 8 to 12 years were found positive for Wuchereria bancrofti mf in the blood. No girl of the district was found positive for mf. Overall microfilaria rate (mfr) in SWH of East Godavari district was below 1 percent (0.87 percent). However, a rather high average mf count of 12.6 per 20 cu mm of blood was found. Microfilaria rate among SWH children of West Godavari district was calculated as 1.36 percent with average microfilaria count of 1.66 per 20 cu mm of blood. No student used bed net while sleeping at night. The DEC consumption rate during MDA in the SWH of West Godavari district was at a low 48.2 percent. Infection in wild Cx. quinquefasciatus mosquito was recorded from the study area. The study showed that there was still active transmission of L.F in the districts even after eleven rounds of MDA.