Efficacy of oral and transepidermal application of diethylcarbamazine citrate in Brugia malayi infected cats

This experimental study showed that daily oral administration of DEC at levels of 5 to 15 mg/kg/day with food for 3 weeks decreased the level of both developing and adult B. malayi in infected cats. There were no adverse reactions due to the medication. Topical application of 5% DEC in skin cream or in mineral oil appears to be effective in killing developing B. malayi in cats.     

Effect of diethylcarbamazine citrate on fourth stage and adult Brugia malayi in cats.

Sixty-three experimental and 58 control cats were infected with Brugia malayi so that the developing and adult worms localized in the regional lymphatics of the hind legs. At 20 days after infection when Brugia were in the 4th larval stage, and at 8 weeks when worms were young adults, cats were divided into groups to test the efficacy of diethylcarbamazine citrate (DEC) at various dosage levels. At 100 mg total DEC/kg no 4th-stage larvae were seen in 5 cats compared with a mean of 20.4 living larvae in each of 5 controls. At this level of DEC, 2 of 5 cats had 1 and 2 adult worms while 4 of 4 controls had a mean of 23.2 living adult worms. At 50 and 25 mg/kg there was a substantial reduction of both 4th stage and adult worms when compared to controls. At 10 mg/kg, 4 of 6 cats had 4th-stage larvae but at a lower level (mean = 7.0) than in 6 controls (mean = 23.2). No reduction of either 4th-stage larvae or adult worms was seen at 1 mg/kg. This study establishes the efficacy of DEC against 4th-stage and adult Brugia malayi in cats, although considerably higher levels of the drug were required than the level previously determined to kill 3rd-stage larvae. It appears that the cat-B. malayi model will be an effective method to compare the efficacy of drugs against adult lymphatic-dwelling filariae.     

Threshold of transmission of Brugia malayi by Anopheles sinensis

In an area of central China where Brugia malayi is transmitted by Anopheles sinensis, three villages were followed for 4 years without any control measures. Diethylcarbamazine (DEC) had been used in a control programme reducing the parasite rate in Shuiwa from 12.72 to 0.59%, in Gubomen from 3.18 to 1.55% and in Moshi from 5.62 to 2.81% (although this fell the following year to 2.23%) up until the start of the trial. The village populations, the human biting rate, the parous and the gonotrophic cycle were comparable for all the areas, yet the microfilariae rate dropped to 0.18% in Shuiwa, 0.31% in Gubomen, but not in Moshi where it increased from 2.23 to 2.43%. This indicated that Shuiwa and Gubomen were below the threshold of transmission. Due to the small number of positive cases remaining, the density measurements showed less change, but a separate study on 44 individuals followed for 6 years revealed that those above 13 microfilariae per 60 mm3 remained unchanged or increased, while those below progressively decreased their densities. The threshold of transmission of B. malayi by An. sinensis was considered to be between 1.55 and 2.23% of the population, providing no individual had a higher density than 12 microfilariae per 60 mm3.     

Effect of treatment with diethylcarbamazine on immune responses to filarial antigens in patients infected with Brugia malayi

We studied the effect of treatment with diethylcarbamazine (DEC) on immune responses to parasite antigens in humans infected with Brugia malayi. In vitro lymphocyte proliferative responses to microfilarial antigens increased in patients who became amicrofilaremic after treatment with DEC. No changes in reactivity were observed in amicrofilaremic individuals who were given DEC or in a small number of patients who remained microfilaremic after treatment. Reactions to other antigens (PPD and SKSD) were not affected by drug treatment. Serum titers of antibodies to the sheath of B. malayi microfilariae did not significantly change during the period of observation. These findings indicate that DEC partially reverses the state of cellular unresponsiveness to parasite antigens associated with patient filarial infections.     

The efficacy of a single-oral-dose administration of ivermectin and diethylcarbamazine on the treatment of feline Brugia malayi

The combination of ivermectin and diethylcarbamazine (DEC) have been shown to be superior to either drug alone for the suppression of Brugia malayi in humans, but their efficacy against infection with B. malayi in cats has never been investigated. Fourteen asymptomatic microfilaremic (1-200 microfilariae/20 microl blood) cats received oral doses of ivermectin (400 microg/kg body weight) and DEC (6 mg/kg body weight) as a single treatment. A two-month post-treatment examination revealed that 87-100% of the microfilariae in each subject had been cleared, with two of the subjects being amicrofilaremic. A further reduction in microfilarial levels was observed until the final follow-up, at 8 months post-treatment, when the mean clearance rate was 99% and 12 out of the 14 subjects (86%) were amicrofilaremic. The combination of ivermectin and DEC demonstrated a microfilaricidal effect superior to that of either drug used alone, both in the initial rapid clearance of microfilariae, and in sustaining the effect for 8 months. This finding has important implications for the control of brugian lymphatic filariasis in the cat reservoir.     

The effect of diethylcarbamazine citrate on incidence and recovery rates of Brugia malayi microfilaremia in Sabah, Malaysia

Mass drug administration via 3 modes of delivery reduced the incidence and prevalence rates and intensity of Brugia malayi infection in 3 rural villages in the Bengkoka Peninsula, Sabah, in 1982-1983. A dosage of 6 mg diethylcarbamazine citrate (DEC-C)/kg body weight was administered either daily or weekly (total of 6 doses, 36 mg/kg body weight), and impact on B. malayi cases were comparable in the 3 villages. A total of 384 people participated in the DEC-C regimens, and all pregnant women and children under 2 years were excluded from the study. Bekessy's method of estimation of incidence and recovery rates was applied to data on B. malayi microfilaremia before drug administration. Treatment with DEC-C by any of the 3 modes of delivery drastically reduced the number of episodes of patent microfilaremia, incidence and prevalence, and median microfilarial density. Reduction was sustained for at least 18 to 24 months after treatment.     

Effect of diethylcarbamazine citrate on Brugia malayi infections in cats following daily, weekly, or monthly administration

Cats with patent infections of Brugia malayi were treated by intraperitoneal injection of diethylcarbamazine citrate (DEC) for 6 consecutive days, weekly for 6 consecutive weeks or monthly for 3 months. Each cat received a total of 100 mg DEC per kg. At necropsy 7 months after infection, no living worms were recovered from any of eight cats treated weekly and only one of nine cats treated daily had a single living Brugia. Five of nine cats treated monthly and six of eight untreated controls had one or more living worms. Cats treated weekly showed a larger decline in microfilariae than those of the other treated groups. The mean microfilariae level of untreated controls increased 2-fold. At necropsy, gross appearance of regional lymphatics in daily and weekly treated cats resembled those of uninfected controls more closely than those in cats treated monthly or untreated. Differences in degree of histological changes between groups of infected cats were not apparent. Weekly administration of DEC appeared to be the most effective regimen; monthly treatment was less effective.     

The vectors of Brugia malayi in Southern Thailand.

Mansonia uniformis, with an infective rate of 0.02, was incriminated as the vector of periodic Brugia malayi in Pattani province. Mansonia bonneae and Ma. dives, with infective rates of 0.18 and 0.20 respectively, were the vectors of B. malayi in Narathiwat, where the microfilarial periodicity was the subperiodic form.     

Control of bancroftian filariasis by diethylcarbamazine medicated common salt in Karaikal, Pondicherry, India

A trial of diethylcarbamazine (DEC) mixed in crystal common salt at a concentration of 0.15 to 0.2 per cent was carried out for 46 months (1982 to 1986) in Karaikal town and five commune panchayats of Pondicherry for control of bancroftian filariasis. Comparison of pre and post trial surveys showed 97.6 per cent reduction of microfilaria rate. No microfilaria or disease case was found in less than 5 years age group in the post trial survey. There was about 72 per cent reduction of disease rate. In the post trial substantial reduction of disease manifestation was found in 40 years and below age groups. Vector Culex quinquefasciatus density, filaria infection and infectivity rates were monitored only in Karaikal town. Infective vector mosquitoes were found in 1982 and 1983 only but afterwards no infective mosquito was encountered.     

Experimental Brugia malayi infections in the rhesus monkey.

Twenty-eight rhesus monkeys in 3 groups were exposed to single (Group I), double (Group II), and multiple (Group III) inoculations with B. malayi infective larvae. Infections were monitored by microfilarial and blood counts, selected biochemical tests, IFA responses, and records of body temperature and lymphadenopathy before and/or after treatment with DEC. As a whole, the highest microfilaraemia levels were observed in Group II and lowest in Group III monkeys. Eosinophilia was a common occurrence but reached the highest mean levels in Group III. Intermittent fevers and lymph node enlargements were observed in all groups of monkeys and the occurrence of these appeared to be correlated. No definite pattern of antibody production was discernable among groups, but an inverse relationship existed between microfilaraemia and detectable microfilarial antibodies. Treatment with DEC produced a microfilaraemia-taxic effect within the initial half hour and responses to treatment varied according to individuals. Although post-treatment reinfection appeared to cause lymphoid responses and tissue eosinophilia, no substantial resistance to reinfection was observed.     

In vitro development of Brugia pahangi and Brugia malayi in cultured mosquito thoraces.

In vitro cultivation of Brugia pahangi and subperiodic Brugia malayi one-day old larvae to infective stage larvae (L3) within thoraces excised from Aedes aegypti (Black Eye, Liverpool) and Anopheles quadrimaculatus was attempted. The mosquito thoraces were excised under aseptic conditions, 24 h after a blood meal on either B. pahangi- or B. malayi-infected jirds. The excised thoraces were washed aseptically and inoculated into a diphasic media. A nutrient agar base was overlaid with either Grace's insect cell culture medium or Schneider's Drosophila medium or a mixture (1:1) of these two media. Each overlay medium contained a 1 x concentration of antibiotic/antimycotic mixture plus 20% fetal bovine serum. The excised thoraces provided the intracellular milieu for development of Brugia larvae. In Grace's or Schneider's insect tissue culture medium alone, the filaria larvae of both species developed only to the second larval stage after 12 days; whereas, in a mixture (1:1) of Grace's and Schneider's media, some one-day old larvae of both Brugia species developed to the infective larval (L3) stage after 12 days. However, large numbers of both species of larvae developed to the infective larval stage when, prior to providing an infective blood meal, the mosquitoes of both species were fed 1 x concentration of antibiotic/antimycotic mixture in a 10% sucrose solution containing 0.1% p-aminobenzoic acid for 6 days. These results showed for the first time that if one-day old Brugia larvae are confined intracellularly in excised thoraces, they can then develop in insect tissue culture media without adding a feeder layer of mosquito cells or conditioning the media with mosquito cell lines.     

A high performance liquid chromatographic method for the estimation of diethylcarbamazine content in medicated salt samples

A simple and reproducible method for the estimation of diethylcarbamazine citrate (DEC) by high performance liquid chromatography (HPLC) in DEC-medicated salt was developed. HPLC analysis was conducted with a mobile phase containing acetonitrile/phosphate buffer (20 mM KH(2)PO(4,) adjusted to pH 3.2 with 10% ortho-phosphoric acid) in the ratio of 1:9 and at a flow rate of 1.5 ml/min. A Phenomenex C8 column (15 cmx4.6 mm) of 5 microm particle size was used for the analysis. Analysis was done at UV 210 nm, 0.02 a.u.f. and 40 degrees C. The coefficient of variation was <10% in the range of 1-25 microg/ml and the minimum detectable level was 0.5 microg/ml. The quality of DEC-medicated salt prepared by two methods was analyzed by using the HPLC method. In spray drying method, 29 and 71% of the samples and in rotating drum method, 9 and 12% of samples were found to contain DEC at 0.15-0.25% and >0.25%, respectively. Thus, this quick and simple HPLC method for the estimation of DEC could play a vital role in checking the quality of the DEC medicated salt used for the control of filariasis.     

Cultivation of sexually mature Brugia malayi in vitro

Sexually mature male and female Brugia malayi were developed from third stage larvae after 60 days in the in vitro culture system described by Franke and others in 1987 (Am J Trop Med Hyg 37: 370-375). Between 75 and 100 days in culture, many worms produced living microfilariae. Each gravid female produced 200-1,500 microfilariae/day.     

Brugia malayi: ivermectin inhibits the exsheathment of microfilariae.

Brugia malayi-infected microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg of body weight injected subcutaneously. Susceptible Aedes aegypti mosquitoes were fed on treated jirds 24 hours later. Mosquitoes fed on untreated jirds served as controls. Infected mosquitoes were dissected at 1, 3, 24, 48, 72, and 96 hr after the blood meal, and differential counts of sheathed microfilariae, exsheathed microfilariae, and cast sheaths were performed using fluoresceinated wheat germ agglutinin. Microfilariae failed to exsheath in mosquitoes fed on ivermectin-treated jirds. Microfilariae from ivermectin-treated jirds also did not exsheath in vitro in the presence of 10 mM CaCl2, whereas 85-90% of sheathed microfilariae from untreated jirds exsheathed in vitro. In addition, sheathed microfilariae from untreated jirds, when pretreated in vitro with ivermectin at 0.25, 0.5, or 1 microgram/ml, lost their ability to exsheath in vitro in the presence of 10 mM CaCl2. However, ivermectin treatment had no effect on exsheathing of microfilariae when incubated with papaya protease. Thus, ivermectin appears to inhibit the intrinsic exsheathing process of microfilariae in the mosquito host, thereby blocking their development and further transmission of infection.     

Comparative assessment of the in vitro sensitivity of Brugia malayi infective larvae to albendazole, diethylcarbamazine and ivermectin alone and in combination

The antifilaricidal drugs ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB), used alone or in combinations against infective third-stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain), were tested in vitro for sensitivity, for 7 days. IVM alone reduced larval motility at concentrations of 10(-7), 10(-6), and 10(-5) M on day 3. DEC alone also had this effect at concentrations of 10(-6). 10(-5), and 10(-4) M on day 2. ALB alone did not have this effect throughout the experiment, at various concentrations. However, it had greater effect when used in combination with either DEC or IVM. The result also indicated that DEC or IVM, when used in combination with ALB at concentrations of 10(-6) M/10(-6) M, and 10(-5) M/10(-5) M was effectively better than each drug used alone at those concentrations. When both drug combinations were compared, ALB/DEC seemed to be more effective than ALB/IVM at a concentration of 10(-6) M/10(-6) M on day 3. Although IVM and DEC can reduce larval motility when used alone or in combination with ALB, they cannot kill these larvae in an in vitro cultivation, even at a high concentration (10(-5) M).     

Relevance of anti-BmR1 IgG4 antibodies in children from an area endemic for Brugia malayi infection in Kerala, India

Brugian filariasis prevalent mostly in South-East Asian countries including India contributes to a small but significant proportion of the socioeconomic burden due to lymphatic filariasis. Along with bancroftian filariasis, brugian filariasis has been targeted for elimination globally. The lack of a reliable daytime diagnostic test has been seen as an important barrier to the successful implementation and monitoring of elimination programmes in brugia endemic areas. We evaluated an anti-BmRI-IgG4 antibody test namely, 'Brugia Rapid' in a large study meant to understand the clinical and pathological manifestations of brugian filariasis in children. We found the test superior to traditional night blood screening for microfilaraemia. Although an antibody detection test, we found it to be a reliable indicator of brugian infection. Among the 100 children studied extensively, 94% of the microfilaraemics, 86% of those showing filarial dance sign indicating presence of, live adult worms and 78% having abnormal lymphatics on lymphoscintigraphy were IgG4 positive. Coupled with its advantages like ease of use any time of the day, high sensitivity and specificity, this test may be the ideal tool to assist programme managers in their efforts to eliminate lymphatic filariasis where brugian infections are found.     

Field and laboratory observations on Coquillettidia crassipes in relation to transmission of Brugia malayi in Peninsular Malaysia

Field observations were made on Coquillettidia crassipes during a study of Mansonia in a swamp forest ecotype in Tanjong Karang. There was an increase in abundance in July consistent with the increase in abundance of Mansonia and an increase in rainfall. The biting cycle showed a dramatic early peak during the period 1900-2000 hours. The probability of daily survival through one day for the first three gonotrophic cycles was 0.770, 0.722 and 0.759. Two of the 54 Cq. crassipes dissected were infective, with two and 25 L3 larvae of Brugia. Both subperiodic B. malayi and B. pahangi developed into L3 larvae in laboratory bred Cq. crassipes. The index of experimental infection was higher for B. pahangi. Mansonia bonneae and Ma. uniformis showed higher indices of experimental infection than Cq. crassipes for subperiodic B. malayi. It is concluded that in an endemic area with a high density of Cq. crassipes it could act as a secondary vector of Brugian filariasis.