An allele specific PCR assay for screening for drug resistance among Wuchereria bancrofti populations in India.

Background & objectives: Albendazole, a commonly used anthelminthic drug that targets the polymerization of α- and β-tubulin dimer is currently co-administered with the antifilarial drug, diethylcarbamazine citrate (DEC) in the ongoing Global Programme for Elimination of Lymphatic Filariasis (GPELF). The experience in veterinary field has shown that there can be a rapid development of resistance to this drug, which therefore, needs to be monitored regularly in GPELF. Hence, we investigated the nucleotide polymorphism in the albendazole-binding domain of the isotype 1 β-tubulin gene from several populations of Wuchereria bancrofti and developed an AS-PCR assay useful in screening for sensitive/resistance alleles among parasite populations and also evaluated its utility. Methods: For studying the polymorphism of isotype 1 β-tubulin gene, a 475 bp fragment spanning exon 5 and 6 of the gene was amplified and sequenced from the genomic DNA of W. bancrofti collected from six geographic regions of India. An allele specific (AS) PCR for screening albendazole sensitivity/resistance was developed and a total of 55 mf samples from blood smears on slides collected from Thiruvannamalai, Thanjavur and Puducherry were screened. Selective therapy with DEC was in place in three areas, mass drug administration (MDA) with DEC alone was implemented in four areas, while DEC plus albendazole was administered in one district. Results: The analysis of the nucleotide sequence of the fragment from 20 W. bancrofti populations showed the domain to be highly conserved. An allele-specific PCR assay developed was used to detect sensitive/ resistance alleles among 55 isolates of W. bancrofti and no albendazole resistance alleles were detected among the populations tested. Interpretation & conclusion: The drug-binding domain of isotype 1 β-tubulin gene of W. bancrofti from different geographical locations was highly conserved. The AS-PCR developed showed potential application as a tool for monitoring albendazole sensitivity/resistance alleles among W. bancrofti populations, in areas where combination therapy of DEC-albendazole is being mass administered in the LF elimination programme.

Enhanced recovery of fourth stage larvae of Wuchereria bancrofti from mongolian gerbil, Meriones unguiculatus & their in vitro maintenance.

Earlier attempts to produce different stages of W. bancrofti, such as fourth stage larvae (L4), in small animal models have yielded very low recovery rates. In order to enhance the recovery of L4, two routes of inoculating a small animal, M. unguiculatus, with infective larvae (L3) viz., intraperitoneal and intrathoracic routes, were compared. On day 17 post-inoculation, higher percentage (23-25%) of L4 were recovered from animals inoculated intrathoracically compared to that from animals inoculated intraperitoneally (2-8%). Also, comparatively higher proportion of worms (75-92%) remained within the intrathoracic region, unlike in the intraperitoneal region (50-80%). A few worms (1-4%) could be recovered even on 31 days post-inoculation from animals inoculated intrathoracically. When the L4 produced in animals were cultured in modified Frank's medium, all of them survived for 15 days and 50 per cent survived till the 25th day. The higher yield and ease of recovery from the thoracic cavity makes this route of inoculation a suitable method for production of L4. In vitro maintenance of L4 for prolonged period is significant with respect to excretory/secretory products or for drug screening.

Microfilarial periodicity of Wuchereria bancrofti and man landing periodicity of the vector Culex quinquefasciatus say in Matara, Sri Lanka.

OBJECTIVE: To compare the microfilarial periodicity of Wuchereria bancrofti, with the man landing periodicity of the vector Culex quinquefasciatus in Matara, Sri Lanka. DESIGN: Periodicity was estimated using a statistical method. 60 microliters finger prick (FP) blood was smeared from a single subject every 2 hours for 24 hours of the day to make 12 samples. Smears were stained with Giemsa and the microfilariae (mff) counted. Man landing catches of mosquitoes were made inside a bedroom of a house in the same area on a sleeping volunteer during the night, between 18.00 and 06.00 hours. Each hourly catch was placed in separate paper cups. Hourly C. quinquefasciatus taken were counted. SUBJECTS: 10 asymptomatic microfilaria (mf) carriers. RESULTS: The individual mf peaks in the 10 carriers varied from 22.00 to 04.00 hours. Using the statistical method the parameter k showing the mf peak hour was 1.19 estimating the peak mf density at 01.11 hours. The influence of different times of blood collection on false negatives among the very low density carriers was estimated by the periodicity curve. It would be desirable to collect blood during the estimated time interval when the mf count was 80% of the peak count, between 21.55 and 04.27 hours in Matara. The results of 25 all-night mosquito landing catches gave a peak activity hours of k as 7.78, corresponding to 01.47 hours. CONCLUSION: The close agreement in the peak hours of mf density and vector activity suggests a perfect adaptation between parasite and vector for optimum transmission.

Current status of filariasis in Indonesia.

Filariasis in Indonesia is widely distributed. Three species consisting of 5 ecologically different types have been identified infecting man. Compared to older data, infection rates are much lower, partly due to environmental change and partly as a result of control programs. Various dosage treatments have given good results. The higher dosage treatment gave severe reactions especially in brugian filariasis. Pockets of high endemicity can still be found in remote rural areas. Therefore a weekly low dosage treatment of 40 weeks through the Primary Health Care approach has been adopted. Filariasis research in Indonesia at present is concentrating on the use of biotechnological tools, especially for diagnostic and vector identification purposes, and to understand better the pathophysiology. Treatment trials with new drugs such as Ivermectin and DEC are being conducted both in man and experimental animals.

Current status of filariasis in Malaysia.

The Filariasis Control Program was established more than 30 years ago in the country and the disease is still a public health problem in some states. Since 1983, a total of 17 filariasis control teams were formed throughout the country to carry out filariasis control work. The teams conduct house and population censuses, nocturnal mass blood surveys and treatment of microscopically confirmed cases. Individual case follow-up is being carried out after 3-5 months while the locality is resurveyed after about 2-3 years. During the years 1988 to 1990, there appeared to be a decreasing trend in the number of filariasis cases detected countrywide. In 1991, brugian filariasis accounted for 92% of the cases detected. The microfilaria rate (MFR) also showed a decreasing trend countrywide for the years 1988 (0.57%) to 1990 (0.35%) but there was an increase in 1991 although it remained well below the 5% MFR targeted in the program objective, In 1991, the filariasis control teams and the district multi-purpose teams collected a total of 167, 151 blood slides out of which 871 were found to be positive for microfilaria. To determine the true endemicity of filariasis in the country, the malaria district multi-purpose teams are also utilized to assist in probe surveys in new areas of the district. Two species of filarial worms, namely Brugia malayi and Wuchereria bancrofti, and the mosquito vectors belonging to the Anopheles and Mansonia genera are involved in the transmission of filariasis in Malaysia. Monkeys and domestic cats are the reservoir hosts for the subperiodic strain of B. malayi.

Filariasis and its control in Fujian, China.

Epidemiological survey of filariasis in Fujian Province, China showed that malayan filariasis, transmitted by Anopheles lesteri anthropophagus was mainly distributed in the northwest part and bancroftian filariasis with Culex quinquefasciatus as vector, in middle and south coastal regions. Both species of filariae showed typical nocturnal periodicity. Involvement of the extremities was not uncommon in malayan filariasis. In contrast, hydrocele was often present in bancroftian filariasis, in which limb impairment did not appear so frequently as in the former. Hetrazan treatment was administered to the microfilaremia cases identified during blood examination surveys, which were integrated with indoor residual spraying of insecticides in endemic areas of malayan filariasis when the vector mosquito was discovered and with mass treatment with hetrazan medicated salt in endemic areas of bancroftian filariasis. At the same time the habitation condition was improved. These factors facilitated the decrease in incidence. As a result malayan and bancroftian filariasis were proclaimed to have reached the criterion of basic elimination in 1985 and 1987 respectively. Surveillance was pursued thereafter and no signs of resurgence appeared.

In vitro differentiation of Wuchereria bancrofti (Filariidae)

Wuchereria bancrofti microfilariae were isolated from blood of infected individuals and cultured in vitro under several conditions. RPMI 1640 and TC-199 media supplemented with fetal or human serum were able to support the microfilariae for periods up to 35 days at 37-C (viability > 85%). In contrast, in minimal essential medium the microfilariae did not survive for more than 48h. In culture kept at 28-C, where viability was much lower (approximately 10% on day 15), microfilariae differentiated into type IV larvae ("sausage form). The in vitro maintainance of microfilarial larval forms is particularly important in the case of W. bancrofti due to the absence of an experimental model for the disease

Estimation of fecundic life span of Wuchereria bancrofti from longitudinal study of human infection in an endemic area of Pondicherry (south India).

The fecundic life span of adult female W. bancrofti was estimated by longitudinal study of microfilaraemia in a cohort of population (7,525) in Pondicherry. The estimation was based on a deterministic model, using the rate of loss in infection. The life span of the parasite was 10.2 yr without chemotherapy, while it was reduced to 5.3 yr following diethyl-carbamazine therapy. The analysis of mean microfilarial counts in microfilaraemic persons without chemotherapy indicates that the rate of production of microfilaria by the adult female is stable at least for a period of five years.