Recent advances in the application of molecular biology in filariasis.

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

Ivermectin and diethylcarbamazine trials in leaf monkeys (Presbytis cristatus) infected with Wuchereria kalimantani.

Clinical trials of Ivermectin in single oral doses of 200, 400, and 1,000 mg/kg body weight or in multiple doses of 200 mg/kg body weight for 5 consecutive days were performed in leaf monkeys (Presbytis cristatus) infected with Wuchereria kalimantani. Optimal microfilaricidal effect occurred at 200 mg/kg body weight. The drug was less effective than diethylcarbamazine in this animal model for human filariasis but had no adverse effects.

Adverse reactions to a single dose of diethylcarbamazine in patients with Brugia malayi infection in Riau Province, West Indonesia.

A study on the adverse reactions, occurring after treating microfilaremic patients infected with B. malayi, revealed that all reacted to a single oral dose of DEC (5 mg/kg). The major reactions were fever, headache, anorexia, abdominal pain, muscle and joint pains, nausea and vomiting. There seemed to be no association between the time of fever onset and microfilarial density, but the number of cases observed was too small to make any firm conclusion. There was a tendency for more severe reactions to occur in patients with higher microfilaria counts. Local reactions, probably due to destruction of adult worms, were seen in 3 patients. The reactions were serious enough to necessitate the patients spending approximately 48 hours in bed.

Further studies on the development of Wuchereria bancrofti in laboratory animals.

In an attempt to obtain a small animal laboratory model for Wuchereria bancrofti Mongolian gerbils and hamsters were infected with third-stage larvae of strains of the parasite from Indonesia and China. Gerbils were necropsied at 7 to 591 days and larvae recovered up to 219 days. The third molt occurred on or before 10 days and worms found after 14 days were in the fourth stage. More worms were recovered early in the infection (7 to 30 days) than in older infections. The worms were usually recovered from the testes, pelt and carcass and heart and lungs. An adult male worm was recovered on one occasion from the lung of a gerbil at 92 days. The parasite was also found to develop in hamsters with results similar to those in gerbils. Gerbils and hamsters were given antithymocyte sera before and after infection and more worms were recovered and the worms larger in size in some treated animals. Continued studies with geographic strains of W. bancrofti in various animals should be encouraged in order to find a small laboratory animal model for the parasite.

Human intestinal parasites in Karakuak, West Flores, Indonesia and the effect of treatment with mebendazole and pyrantel pamoate.

A survey for intestinal parasites and mass-treatment with a combination of mebendazole and pyrantel pamoate were conducted in Karakuak, West Flores in 1977. A total of 198 stool specimens from 104 males and 94 females ranging in age from less than 1 to 70 years were examined and 72% harbored one or more intestinal parasites. Ascaris lumbricoides (43%) and Entamoeba histolytica (21%) were the most common, followed by Entamoeba coli (19%), hookworm (18%), Iodamoeba bütschlii (8%), Giardia lamblia (5%) and Trichuris trichiura (4%). Other intestinal parasites infrequently found were: Entamoeba hartmanni (2%), Chilomastix mesnili (2%), Endolimax nana (1%), Enterobius vermicularis (1%) and a heterophyid sp. (1%). A combination of mebendazole base at 200 mg/day and pyrantel pamoate salt at 60 mg/day for three consecutive days was 100% effective.

A study on vectors of Bancroftian filariasis in West Flores, Indonesia.

A brief survey was conducted in a filariaendemic village of Robek, West Flores, Indonesia, to identify the mosquito fauna, to determine the vector of W. bancrofti, and to update information on the bionomics of An. subpictus. A total of 6 genera and 22 species were collected. Five species of culicines collected, have not been previously recorded from Flores. Dissections of wild caught mosquitoes consisted of 592 anophelines, and 70 culicines did not reveal any filarial infection. An. subpictus was a potential vector of bancroftian filariasis in the Robek area, as 11.3% of this species that fed on carriers were found to harbour W. bancrofti larvae. Larvae of An. subpictus were collected in lagoons, rice fields, swamps and ground pools. Breeding sites are exposed to sunlight, contain fresh or brackish water, with or withour vegetation. They have been found primarily associated with An. aconitus, An. annularis, An. barbirostris, Cx. vishnui and Cx. bitaeniorhynchus. The time for a female to become fully engorged with human blood is 3-10 minutes. This species is a night biter, attacking man and animals from twilight to dawn. Its peak of biting activity is between 0100-0300 hrs.

Clinical features of timorian filariasis among immigrants to an endemic area in West Flores, Indonesia.

Fifty-nine persons, who immigrated into a Brugia timori endemic area from non-filarial areas on the island of Flores, Indonesia were examined for filariasis after residing in the village for 2 to 10 years. Six persons had B. timori microfilaremia and 31 had filarial disease. The disease seems to affect immigrants from non-filarial areas severely within a relatively short period. Among those residing in the village, for 2 years, the microfilaria rate was 5% and the elephantiasis rate 21%. Selective treatment using 50 mg diethylcarbamazine per Kg body weight was given to all microfilaria (Mf) positive persons. Approximately one year later the Mf-rate by finger stick and Nuclepore filtration was 9% and 18% respectively. There was indirect indications that the Mf-rate might increase with the passage of time. However, the total filarial disease rate remained constant during the one year period. The relationship between these findings and American servicemen exposed to filariasis during World World II is briefly discussed.

The association of clinical filariasis and Wuchereria bancrofti infections in Jakarta.

In a study carried out in Jakarta, Indonesia between 1969--1977, patients with clinical manifestations of filariasis were examined for microfilaremia. Finger blood (3 X 20 microliter) were examined by conventional methods, venous blood by membrane filtration, urine and hydrocele fluid by concentration methods. A total of 1,422 patients were examined and 182 (12.8%) were positive by blood smear; however, examination by membrane filtration and concentration of urine and hydrocele fluid did not appreciably increase the association between clinical manifestations and Wuchereria bancrofti infection. Approximately 16% of the patients without patent infection were indirectly associated with filariasis, since they co-habitated with persons with microfilaremia. Results of these studies showed that more than 60% of the patients were without patent infection. Factors that may be responsible for these findings are briefly discussed.

Malayan filariasis in Central Sulawesi (Celebes), Indonesia.

Seven villages in Central Sulawesi were surveyed in September 1971 and finger blood samples from 3,658 persons examined for microfilariae. Periodic, nocturnal Brugia malayi was endemic in all seven villages with microfilarial rates (Mf-rate) of 10-42% (av. 25%). The median microfilarial density (MfD50) averaged 5.4 and varied from 0.8 to 9.4 microfilariae per 20 microliter of blood. Males had higher Mf-rate and MfD50 value than females and in males Mf-rates and MfD50 values increased with age. In the female population, however, neither Mf-rates or MfD50 values varied significantly with age. Clinical manifestations of filariasis were found in 12% of 2,412 persons examined and only 2% had elephantiasis. Clinical signs of disease with the exception of elephantiasis, showed good correlations with Mf-rates but not with MfD50 values. Transmigrants experienced clinical manifestations of filariasis earlier and more often than the indigenous population.

Experimental Brugia timori and Wuchereria bancrofti infections in certain species of mosquitoes.

Laboratory reared Aedes aegypti (black eye and Jakarta strains), Aedes togoi, (Taiwan), Aedes albopictus, (Jakarta), wild caught Anopheles barbirostris, (Java) and Mansonia uniformis, (Jakarta) were fed on a carrier with mixed infection of Brugia timori and Wuchereria bancrofti. B. timori and W. bancrofti were able to develop in A. aegypti (black eye) and A. togoi, with development proceeding more rapidly for of B. timori than W. bancrofti. Both species of parasites were readily distinguishable in each of their developmental stages. A. barbirostris from Java was able to support development of B. timori as well as A. barbirostris from Flores. B. timori and W. bancrofti did not develop in M. uniformis, A. aegypti (Jakarta strain) and A. albopictus.

Brugia malayi in seven villages in South Kalimantan, Indonesia.

Seven villages in South Kalimantan were visited in 1971 and night peripheral blood smears from 2,764 people examined for microfilariae. Brugia malayi was found endemic in all villages with microfilarial rates of 12--46% (average 25%) and the median microfilarial density (MfD50) of 6 to 15 microfilariae per 20 microliter of blood. The microfilariae showed a typical subperiodic pattern. The disease was more common in males than females and the prevalence increased with age. Clinical manifestations of filariasis were found in 20% of 1,099 persons examined. Mansonia species are considered important vectors and cats important reservoir hosts. In addition to B. malayi, Dirofilaria repens and an unknown microfilaria were found in cats in the area and strains of the B. malayi and D. repens have been established in the laboratory.

Parasitological and clinical survey of bancroftian filariasis in Kepu District, Central Jakarta, Indonesia.

A blood and clinical survey for bancroftian filariasis was made in the Kepu District of Central Jakarta in June 1976. About 90% of 614 inhabitants in the survey area was examined. The microfilarial rate was 4.5%; 5.8% in males and 3.3% in females. Most of the carriers were asymptomatic. The microfilarial density was low. There were 15 persons (2.7%) with signs and symptoms of filariasis; 11 males and 4 females. The total filariasis infection rate was 6.7%. The percentage of infected persons increased with years of residency. Persons living in poorly built houses had approximately 9 times higher microfilarial rate and 5 times higher disease rate than those living in modern houses.

Brugia timori: experimental infection in some laboratory animals.

Experimental infection with Brugia timori of 7 jirds (Merionesunguiculatus), 4 cats and 2 monkeys (Macaca fascicularis) is described. Although no microfilariae were detected by examining 20 microliter samples of tail blood of jirds, adult worms were recovered from 6 of the 7 jirds at autopsy 69-141 days following infection. Some worms were gravid and microfilariae were found in visceral blood of 2 animals. The adult recovery rate in jirds was 16%; the male to female ratio was 1:3. In cats patent infection developed in 95-105 days but microfilaraemias were of low level and transient. No parasites were recovered from monkeys.

Toxoplasma antibodies in Indonesian and Chinese medical students in Jakarta.

The presence of antibodies to Toxoplasma gondii in medical students of Indonesian and Chinese ancestry was determined by the indirect hemagglutination test with titers of 1:256 or greater considered positive. The sera from 90 Indonesian and 203 Chinese male (146) and female (147) students between 20 and 30 years of age were tested and positive titers detected in the sera of 16 (18%) Indonesians and 14 (7%) of the Chinese. There were no difference between the presence of positive titer and between males and females of either ethnic groups. Both groups denied eating raw or partly cooked meats and no correlation could be found between the ownership of domestic cats, the eating of exotic meats and positive titers.

Parasitology survey in the Palu Valley, central Sulawesi (Celebes), Indonesia.

A survey was undertaken in the Palu Valley, Central Sulasesi to determine whether schisto somiasis japonica was endemic in the area and to determine the prevalences rates of intestinal and blood parasites. Seven villages along the Palu River drainage system with an estimated popualtion of 18,700 were surveyed and 2,433 stools, 3,651 blood smears and 1,167 sera were collected and examined.

Studies on filariasis in the Pacific. 4. The application of the membrane filter concentration technique to a survey of Wuchereria bancrofti filariasis in Kepu district, Jakarta, Indonesia.

A blood survey for filariasis was carried out in the Kepu District of Jakarta. Examination of stained 20 c.mm finger-prick blood thick films and membrane filter concentration (MFC) of 1 ml venous blood. The youngest age group examined, 5-9 years old revealed a mf rate of 17% of which 71% were of a low density character detected by MFC only. The mf rate appears to remain relatively constant from the 10-14 year old group onward, approximately 35%, but at all age groups at least half the infections were of low density. The highest mf rate and density was observed present in the 15-20 year old group.