Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

Acometimento ocular em pacientes com mansonelose / Ocular manifestations in mansonelliasis

OBJETIVOS: Avaliar as alterações oculares observadas em indivíduos com mansonelose residentes na área rural do município de Coari, AM, na região do médio Amazonas. MÉTODOS: Foi conduzido um estudo de corte transversal envolvendo 543 moradores residentes na área rural do município de Coari, na margem direita do rio Solimões. Todos os indivíduos foram submetidos a exame de gota espessa para pesquisa de microfilária e os indivíduos positivos foram submetidos a exame oftalmológico. Lesões oculares suspeitas foram biopsiadas e enviadas para exame histopatológico. RESULTADOS: Mansonella ozzardi foi encontrada no sangue periférico em 103 (18,9 por cento) indivíduos. Noventa e cinco pacientes com mansonelose foram examinados, observando ceratite puntata em 12 (11,7 por cento) destes, ceratite numular em um indivíduo (1 por cento) e esclerosante em outro (1 por cento). Biópsia de pele foi negativa em todos os indivíduos. Biópsia de conjuntiva e limbo foi realizada em cinco pacientes com mansonelose e biópsia de córnea em outros três pacientes. Duas biópsias de conjuntiva e limbo e uma biópsia de córnea foram enviadas para exame histopatológico, não sendo observadas microfilárias ou alterações sugestivas da presença destas nas peças estudadas. O restante das biópsias foi enviado para pesquisa de DNA do parasita por PCR, não se encontrando microfilária. CONCLUSÕES: Associação entre infecção por Mansonella ozzardi e lesões corneanas sem etiologia aparente foi verificada neste estudo, mas a ausência de achados específicos no exame histopatológico e a não identificação de microfilárias por PCR em lesões suspeitas põem em dúvida a o acometimento corneano na mansonelose. Estudos mais amplos de populações com alta prevalência de M. ozzardi e a realização de outros testes para identificação de microfilárias em tecido ocular são necessários para investigar a patogenicidade da microfilária ao tecido ocular.

PURPOSE: To evaluate eye manifestations in mansonelliasis patients from Coari, AM, rural area on the middle Amazonas River. METHODS: Cross-sectional study consisted of evaluation of 543 subjects from Coari rural area on Solimões River right margin. All subjects were submitted to blood test for microfilaria and the subjects who had a positive test also were submitted to skin biopsies and ophthalmologic examination. Histopathology and PCR research for microfilaria were done on eye biopsies of suspect lesions. RESULTS: Mansonella ozzardi was found in peripheral blood of 103 (18.9 percent) subjects. Ninety-five mansonelliasis patients were examined. Punctate keratitis was observed in 12 of them, nummular keratitis in one subject and sclerosing keratitis in another one. Skin biopsies were negative in all subjects. Conjunctival and limbal biopsies were done in five mansonelliasis patients and corneal biopsy in another three patients. Conjunctival and limbal biopsies of two patients and corneal biopsy of one patient showed no microfilaria or alterations due to its presence on histopathology. PCR showed no evidence of microfilaria in the other biopsies. CONCLUSION: Association between Mansonella ozzardi infection and corneal lesions with no evident etiology was observed in this study, but the lack of conclusive findings on histopathology and PCR make us doubt the corneal involvement in mansonelliasis. Large studies of high mansonelliasis prevalence populations and extensive employment of microfilaria identification tests in ocular tissue are needed to evaluate the microfilaria corneal pathogenicity.

Susceptibility of Mansonia uniformis to Brugia malayi microfilariae from infected domestic cat.

Microfilariae of Brugia malayi is transmitted to man and other susceptible hosts via mosquito. The transmission of B. malayi from cat to man by Ma. uniformis bite has never been reported. The Ma. uniformis mosquito is the normal vector for Wuchereria bancrofti but has never been reported as a vector for B. malayi, or a susceptible host for the growth and development of the microfilariae of B. malayi. The purpose of this study was to examine the development of B. malayi in Mansonia uniformis after feeding on the blood of an infected cat in the laboratory. The B. malayi infected cat was identified using PCR with the primers Bm-1/Bm-2 on DNA (at 10 ng/50 microl) extracted from the WBC of the cat. W. bancrofti was employed as a negative control. The sensitivity of the B. malayi DNA detection by PCR was 0.0001 ng. Adult Ma. uniformis mosquitos at the ages of 5, 10, and 15 days, 100 mosquitos in each group, were fed on the infected cat blood. Recovery of third stage microfilariae was found to be the highest in the 5-day old mosquito group (48%), followed by the 10- and 15-day old mosquito groups (32% and 18%, respectively). The mean number of B. malayi microfilariae found in thorax, head, and abdomen of the mosquitos were composed. The 5-day old (40.3%) and 10-day old (41.9%) mosquitos were significantly more susceptible to microfilariae than the 15-day old mosquitos (17.8%) (p-values using the Scheffe method: 0.027 and 0.039, respectively). There was no significant difference in the mean number of microfilariae in the thorax (p = 0.482) by age, but the mean numbers of microfilariae in the heads, and abdomens were significantly different by age between the 5- and10-, and the 15-day old mosquitos (p < 0.001 and p = 0.004, respectively).