Mirani V. Weerasooriya

Development of loop-mediated isothermal amplification method for detecting Wuchereria bancrofti DNA in human blood and vector mosquitoes

We have developed loop-mediated isothermal amplification (LAMP) method to detect Wuchereria bancrofti DNA. The sensitivity and specificity of LAMP method were equivalent to those of PCR method which detects SspI repeat sequence in W. bancrofti genomic DNA: both methods detected one thousandth of W. bancrofti DNA from one microfilaria (Mf), and did not cross-react with DNAs of Brugia malayi, B. pahangi, Dirofilaria immitis, human and Culex quinquefasciatus. We also examined the sensitivity of LAMP using the mimic samples of patients blood or blood-fed mosquitoes containing one W. bancrofti Mf per sample. The LAMP method was able to detect W. bancrofti DNA in 1000 μl of blood or in a pool of 60 mosquitoes, indicating its usefulness in detecting/monitoring W. bancrofti infection in humans and vector mosquitoes in endemic areas.

Social mobilisation, drug coverage and compliance and adverse reactions in a Mass Drug Administration (MDA) Programme for the Elimination of Lymphatic Filariasis in Sri Lanka

Background In Sri Lanka filariasis is endemic in Southern, Western and North Western provinces covering eight districts designated as implementation units in the Programme for the Elimination of Lymphatic Filariasis (PELF). Despite control activities over sixty years including multidose diethylcarbamazine, 6 mg/kg treatment microfilaria rates had persisted at low levels. Following systematic social mobilisation the first MDA with DEC albendazole combination was conducted in 2002. Methods We investigated the extent social mobilisation had reached the people, their drug compliance and adverse reactions. Three localities were selected from each district to pick target population samples for pre-tested questionnaire. Three teams each with six people visited one district each day. One team worked from three starting points in one locality. A member applied eight part questionnaire to one family member totalling 150–160 people from one locality. Questions included social mobilisation, drug compliance and adverse reactions. Results Information was disseminated by television, radio, banners and leaflets, to a lesser extent by people. Information reached more people in the periphery than in Colombo. 35.2% from Colombo municipality were unaware of the MDA. Drug coverage was 79.6%, home delivery 71.7% and delivery centres 7.9%. 35.6% in Colombo district and 53.4% from Colombo municipality did not receive drugs. Drugs were consumed by 71.4%. 28.6% who did not comply included 20.4% who did not receive them. 91.4% showed no adverse reactions, 7.5% were mild, 1.1% recovered with home remedies. Conclusion Drug compliance showed significant positive correlation with awareness of the MDA. Door to door delivery was more successful than delivery from centres. More delivery centres conveniently located would have rectified this disparity. Poor awareness and compliance in Colombo and urban areas could be rectified with separate strategy for urban areas. More time for MDA and trained adequate manpower would ensure coverage to achieve elimination.

The use of whole blood absorbed on filter paper to detect Wuchereria bancrofti circulating antigen

The Og4C3 enzyme-linked immunosorbent assay (ELISA) to detect circulating Wuchereria bancrofti antigen uses 50 microL of serum. In this study, a whole blood sample absorbed on filter paper was tested as a substitute for serum. Serum samples were obtained from 60 Sri Lankan subjects by venepuncture and finger-prick blood samples from the same individuals were directly absorbed on filter paper. Og4C3 ELISAs using serum and filter paper blood were compared. Despite the fact that the estimated amount of serum available for the ELISA with filter paper blood was only one-fifth of that available when serum was used, the 2 ELISAs gave almost identical results. Of the 39 positive serum samples, 38 were detected using filter paper blood. Employing the ELISA using filter paper blood, 619 people in Matara, Sri Lanka, were examined for antigenaemia. The positivity rate was 22.5%, 3.1 times higher than the rate of microfilaraemia detected by examination of 60 microL blood films.

Lymphoedema and its management in cases of lymphatic filariasis: the current situation in three suburbs of Matara, Sri Lanka, before the introduction of a morbidity-control programme

Abstract Using in-depth interviews, information on the current state of lymphoedema management was collected from 101 cases of lymphatic filariasis with lymphoedema in three suburbs of Matara. The interviews were conducted prior to the introduction of a programme of community home-based care (CHBC) that incorporates modern lymphoedema-management strategies. Thirty-two of the interviewees had severe lymphoedema (of grade III or above). The male interviewees had significantly more entry lesions than the female. Most of the subjects had suffered from episodes of limb pain with fever, although the incidence of these episodes appeared unrelated to the severity of the lymphoedema. The frequency of the episodes of limb-pain/fever in the 12 months prior to the interviews appeared unrelated either to the level of daily hygiene, which was generally poor, or to the frequency of bathing. Many (65%) of the subjects paid no attention to limb care when bathing, and 44% did not use footwear. Over 80% made no effort to keep their afflicted limbs elevated, and 95% did not exercise. Most of the female interviewees felt shameful of their condition and were, in consequence, less likely to attend government clinics, for treatment, than the male interviewees. The drug treatment that the interviewees had received was often inadequate, and most had stopped seeking treatment because they had not perceived any significant treatment-attributable improvement in their condition. Modern lymphoedema-management strategies (based on regular washing, careful drying, and treatment, with antifungal, antibiotic or emollient creams, of the affected limbs, limb elevation, exercise, and use of footwear) had not reached the study communities and the local physicians were not aware of them. When dermatology life-quality indexes (DLQI) were calculated for the interviewees, the 26 most impaired subjects gave scores of 5–15 (mean=8.6). The DLQI for these subjects will be regularly re-evaluated, as a measure of the effectiveness of the CHBC programme that has now begun.

Epidemiology of bancroftian filariasis in three suburban areas of Matara, Sri Lanka

The epidemiological parameters of bancroftian filariasis were investigated in three suburbs of Matara, within the south-western coastal belt of Sri Lanka where the disease is endemic. The overall prevalence of microfilaraemia and the geometric mean density of the microfilaraemias observed were 4.4% and 20.6 microfilariae/60 µl fingerprick blood, respectively. Prevalence was significantly lower in the female subjects than in the male, and in males aged <20 years than in older males. Overall, 9.5% of the subjects had the clinical manifestations of bancroftian filariasis (6.4% had filarial fever, 3.0% had elephantiasis and/or oedema, and 6.2% had hydrocele). The prevalence of elephantiasis/oedema was generally higher among the female subjects (4.2%) than among the male (1.4%), and an age-prevalence plot for this manifestation showed a linear increase in prevalence after the age of 40 years. Hydrocele also became commoner with increasing age, but this increase in prevalence began at the lower age of 20 years. More than 60% of the cases of elephantiasis/oedema but only 26.3% of the subjects found to have hydroceles experienced filarial fever attacks. The cases of fever and elephantiasis/oedema (but not those of microfilaraemia or hydrocele) were aggregated within households. However, the children whose mothers were microfilaraemic were much more likely to be microfilaraemic themselves (8.7%) than the children who had amicrofilaraemic mothers (2.8%), microfilaraemic fathers (0.0%) or amicrofilaraemic fathers (2.7%). The results of entomological surveys indicated that transmission of Wuchereria bancrofti occurred throughout the year in the study community.

Human infection with Wuchereria bancrofti in Matara, Sri Lanka : the use, in parallel, of an ELISA to detect filaria-specific IgG4 in urine and of ICT card tests to detect filarial antigen in whole blood

Abstract The ICT card test to detect circulating filarial antigen and an ELISA that detects filaria-specific urinary IgG4 were each used to screen 473 subjects from a community in Sri Lanka where Wuchereria bancrofti is endemic. When the ICT test was used as the gold standard, the ELISA was found to have a sensitivity of 91.2%. However, far more of the subjects were found ELISA-positive than ICT-positive (76.5% v. 31.1%). The youngest children studied (aged 1-10 years) were similar to the adult subjects in terms of the prevalence of antigenaemia (33.8%) and the prevalence (72.1%) and concentration of filaria-specific IgG4 in their urine. Therefore, especially as urine samples are easier, less painful and safer to collect than blood samples, the ELISA may be particularly useful to screen very young and school-age children, to estimate current levels of transmission in a particular area.

Multicentre evaluations of two new rapid IgG4 tests (WB rapid and panLF rapid) for detection of lymphatic filariasis

In the global effort to eliminate lymphatic filariasis (LF), rapid field-applicable tests are useful tools that will allow on-site testing to be performed in remote places and the results to be obtained rapidly. Exclusive reliance on the few existing tests may jeopardize the progress of the LF elimination program, thus the introduction of other rapid tests would be useful to address this issue. Two new rapid immunochromatographic IgG4 cassette tests have been produced, namely WB rapid and panLF rapid, for detection of bancroftian filariasis and all three species of lymphatic filaria respectively. WB rapid was developed using BmSXP recombinant antigen, while PanLF rapid was developed using BmR1 and BmSXP recombinant antigens. A total of 165 WB rapid and 276 panLF rapid tests respectively were evaluated at USM and the rest were couriered to another university in Malaysia (98 WB rapid, 129 panLF rapid) and to universities in Indonesia (56 WB rapid, 62 panLF rapid), Japan (152 of each test) and India (18 of each test) where each of the tests underwent independent evaluations in a blinded manner. The average sensitivities of WB rapid and panLF rapid were found to be 97.6% (94%–100%) and 96.5% (94%–100%) respectively; while their average specificities were both 99.6% (99%–100%). Thus this study demonstrated that both the IgG4 rapid tests were highly sensitive and specific, and would be useful additional tests to facilitate the global drive to eliminate this disease.

Prevalence and intensity of Wuchereria bancrofti antigenaemia in Sri Lanka by Og4C3 ELISA using filter paper-absorbed whole blood

In Sri Lanka 2741 people from Matara, an endemic area for Wuchereria bancrofti, were examined in 1996/97 for microfilariae by 60-microL blood smear and for circulating filarial antigens by Og4C3 ELISA using filter paper-absorbed whole blood. The overall prevalence of microfilaraemia was 3.4%, and that of antigenaemia 14.4%. The prevalence of antigen-positive and microfilaria-negative people was 11.3%. Analysed by age-group, antigenaemia prevalence was similar in all groups, and the average number of antigen units was already very high in the age-group < 10 years, indicating that the infection started in early childhood. Among those who were antigen positive, the microfilaria prevalence was lower in females than in males. Diethylcarbamazine treatment eliminated microfilariae in 78% of the positives. However, 17 months after the treatment, antigenaemia was still positive in 76% of those who were parasitologically cured.

Effects of 5 rounds of mass drug administration with diethylcarbamazine and albendazole on filaria-specific IgG4 titers in urine: 6-year follow-up study in Sri Lanka.

ELISA for filaria-specific IgG4 in urine (urine ELISA) was applied to children in 7 schools in Sri Lanka, before and after 5 rounds of annual mass drug administration (MDA). The pre-treatment IgG4 prevalence in 2002 was 3.20%, which decreased to 0.91% in 2003 after the first MDA (P<0.001), and finally to 0.36% in 2007 after the 5th MDA. Among 5-10 year-old children, the prevalence decreased from 3.37% in 2002 to 0.51% in 2003 (P=0.009). A pattern of IgG4 titer distribution according to age and its yearly change could also provide useful information in drug efficacy analysis. In 2008, new samples from eleven 2006/07 urine ELISA-positive students and their family members (total n=56) were examined by ICT antigen test, microfilaria test, and urine ELISA. No infection was confirmed among them. Urine ELISA will be useful in monitoring elimination/resurgence in a post-MDA low endemic situation.

Distribution of filarial elephantiasis and hydrocele in Matara district, Sri Lanka, as reported by local leaders, and an immunological survey in areas with relatively high clinical rates.

To eliminate lymphatic filariasis by means of mass drug administration, it is essential to have reliable data on the disease distribution and prevalence in targeted areas. In Matara district, Sri Lanka, self-administered questionnaires were mailed to 2105 local leaders questioning the presence and the numbers of elephantiasis and hydrocele cases. The information provided by them revealed that elephantiasis was clearly aggregated in the southern part of the district along the coast, while hydrocele was distributed rather evenly in the whole district, including Deniyaya region where no endemic filariasis had been known. To confirm active transmission of filariasis in Deniyaya, Wuchereria bancrofti antigen and filaria-specific urinary IgG4 antibody were measured with 2436 subjects. The positive rates for antigen and antibody were 0.6% and 4.3%, respectively. The titer analysis of IgG4 according to age revealed that the youngest IgG4 positive was 3 years old, and that in 10 years old or less, there were 16 positives out of 607 children examined (2.6%). It was concluded that filarial transmission at a low level was going on in the region.