Mosquito identification and molecular xenomonitoring of lymphatic filariasis in selected endemic areas in Giza and Qualioubiya governorates, Egypt

Lymphatic filariasis is a vector-borne health problem that has been focally endemic in Egypt for centuries. The chief vectors of transmission are Culicinae species. Control measures in the form of mass drug administration of DEC citrate treatment have been implemented in Nile delta for almost a decade. This study aimed to identify the prevalent mosquito species in endemic areas in Giza and Qualioubiya governorates and to monitor Wuchereria bancrofti infection by detecting the parasite DNA in collected mosquitoes. Adult mosquitoes were collected using light traps hung indoors. Microscopic examination was performed to identify and examine the morphologic characters of mosquitoes. Female Culex mosquitoes were subjected to semi-nested PCR to detect filarial DNA targeting repetitive DNA sequences [pWb12 repetitive region] specific for W.bancrofti

The results revealed 3 species of mosquitoes Culex pipiens, Culex pusillus and Culex quinquefasciatus with the predominance of Culex pipiens [85.7%]. Wuchereria bancrofti DNA was not detected in any of the collected mosquito pools. With the progress of elimination programme in Nile Delta, follow up studies with larger sample size are recommended as the predominance of Culex pipiens the main lymphatic filariasis vector remains a risk of transmission in such areas

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.

Survey of Bancroftian filariasis infection in humans and Culex mosquitoes in the western Brazilian Amazon region: implications for transmission and control

Introduction The aim of this work was to identify possible lymphatic filariasis foci in the western Brazilian Amazonian that could be established from the reports of Rachou in the 1950s. The study was conducted in three cities of the western Brazilian Amazon region - Porto Velho and Guajará-Mirim (State of Rondônia) and Humaitá (State of Amazonas). Methods For human infection evaluation thick blood smear stained with Giemsa was used to analyze samples collected from 10pm to 1am. Polymerase chain reaction (PCR) was used to examine mosquito vectors for the presence of Wuchereria bancrofti DNA. Humans were randomly sampled from night schools students and from inhabitants in neighborhoods lacking sanitation. Mosquitoes were collected from residences only. Results A total 2,709 night students enrolled in the Program for Education of Young Adults (EJA), and 935 people registered in the residences near the schools were examined, being 641 from Porto Velho, 214 from Guajará-Mirim and 80 from Humaitá. No individual examined was positive for the presence of microfilariae in the blood stream. A total of 7,860 female Culex quinquefasciatus specimens examined were negative by PCR. Conclusions This survey including human and mosquito examinations indicates that the western Amazon region of Brazil is not a focus of Bancroftian filariasis infection or transmission. Therefore, there is no need to be included in the Brazilian lymphatic filariasis control program. .

Desenvolvimento de sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral / Development systems based on nested-PCR and conventional single tube for the diagnosis of visceral leishmaniasis

A leishmaniose visceral ocorre em países dos cinco continentes e quando não tratada pode levar a óbito. Para se evitar esse desfecho, são essenciais o diagnóstico precoce e tratamento adequado. Com o objetivo de contribuir na pesquisa de novos diagnósticos para leishmaniose visceral, esse trabalho propôs desenvolver sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral. A partir de uma revisão na literatura em busca de alvos moleculares utilizados no diagnóstico dessa parasitose, foram selecionados os alvos subunidade menor do RNA ribossômico (ssu rRNA) e espaçador transcrito interno 1 (ITS-1), que compõem o DNA do agente etiológico Leishmania infantum, para o desenvolvimento das nested-PCR. Foi também escolhido o alvo kDNA, o mais aplicado nas abordagens de PCR, para comparações com os sistemas desenvolvidos. Após otimizar todas as PCR com DNA genômico de L. infantum, esses sistemas foram avaliados em amostras de sangue, soro e urina de indivíduos com suspeita de leishmaniose visceral dos hospitais de referência da cidade do Recife - PE. Para utilização da urina, foram avaliados quatro protocolos de extração de DNA e identificou-se que a extração por fenol-clorofórmio, com modificações, foi a de melhor desempenho. Na avaliação com amostras biológicas, as PCR simples e nested-PCR com os alvos ssu rRNA e ITS-1 não tiveram boa sensibilidade ao se usar sangue, e não foram capaz de amplificar DNA do parasito em soro e urina. Esses sistemas desenvolvidos não podem ser usados para o diagnóstico da leishmaniose visceral. No entanto, a kDNAPCR apresentou bons resultados quando avaliada com urina. Mais estudos devem ser feitos para avalia-la como um diagnóstico seguro para esse tipo de amostra biológica. Esse trabalho representa um ponto de início para posteriores estudos que objetivem o aprimoramento e validação da nested-PCR único tubo para o diagnóstico da leishmaniose visceral.

Acquired alpha 1-antitrypsin deficiency in tropical pulmonary eosinophilia.

Background & objectives: Observation of an increased frequency of an intermediate deficiency of serum alpha1-antitrypsin (α1-AT) in patients with Tropical Pulmonary Eosinophilia (TPE) was earlier reported. Though the possibility of existence of an acquired deficiency was suggested, without phenotyping a hereditary α1-AT deficiency in TPE could not totally be ruled out. In this study, we have done Pi (Protease inhibitor) phenotyping to investigate the possibility of association of any heterozygous (or homozygous) α1-AT deficiency in patients with TPE. Methods: Serum a1antitrypsin (α1-AT) was measured in 103 patients (Group A) with TPE, 99 patients with pulmonary eosinophilia who had associated intestinal worm infestation (Group B) and 43 healthy volunteers who served as controls. In 19 α1-AT deficient patients (9 of Group A and 10 of Group B), α1-AT level was measured before and after treatment. In 58 patients with TPE and in 5 controls, phenotyping was done. Results: Fifteen patients of Group A and 16 from Group B showed intermediate α1-AT deficiency (150 mg % or less. None of the control subjects had α1-AT deficiency (<200 mg%). After treatment with DEC and/or deworming, in 19 patients there was a significant (P < 0.001) rise in α1-AT levels. Results of phenotyping showed that all had M1 or M2 allele and none had S or Z variant (either homozygous or heterozygous) thus ruling out any underlying genetic cause for the observed α1-AT deficiency. Interpretation & conclusions: The observed α1-AT deficiency may be due to the chronic inflammation in TPE and associated oxidative stress. However, in such α1-AT deficient patients with TPE and those with worm infested pulmonary eosinophilia, faecal α1-AT concentration and faecal α1-AT clearance should be routinely estimated to rule out the possibility of any intestinal protein loss.

Assessment of family and neighbors of an individual infected with Wuchereria bancrofti from a non-endemic area in the city of Maceió, Brazil

The family and neighbors of a patient infected with W. bancrofti microfilariae were assessed aiming to evaluate the occurrence of cases of lymphatic filariasis in a non-endemic area in the city of Maceió, in the Brazilian state of Alagoas. The patient had previously lived in an endemic focus; however, he has been living in an area where the parasite has never been detected for the past ten years. Female ingurgitated Culex quinquefasciatus mosquitoes captured in the houses of the microfilaremic individual and of his neighbors in the non-endemic region were also examined by polymerase chain reaction (PCR) technique. The thick blood smear examination, blood membrane filtration, and rapid immunochromatography (antigen search) revealed no infected individuals in the family of the microfilaremic individual. All 334 neighbors undergoing the thick blood smear examination were negative for W. bancrofti microfilariae. In 478 ingurgitated C. quinquefasciatus mosquitoes examined by PCR, no W. bancrofti DNA was detected. The microfilaremic individual had a microfilaremia considered very low according to WHO standards (4 microfilariae/mL of blood). As the vectorial infection depends on microfilaremia, the patient's low parasite load did not determine the contamination of other individuals in the area. Our data have shown that the long-term residence of the microfilaremic individual in the non-endemic region was not sufficient to start a new transmission focus of lymphatic filariasis in Maceió.

Avaliação da acurácia de diferentes testes laboratoriais no diagnóstico da filariose em crianças e adolescentes / Evaluation of the accuracy of different laboratory tests for the diagnosis of filariasis in children and adolescents

Apesar de a filariose linfática (FL) ser considerada uma doença de adultos, estima-se que 22 milhões de crianças 15 anos de idade estejam infectadas no mundo. A Organização Mundial da Saúde tem recomendado o monitoramento da infecção nessa população. Este estudo teve como objetivo comparar a acurácia dos métodos parasitológicos e imunológicos no diagnóstico da filariose bancroftiana em escolares. O estudo foi desenvolvido em escolares, com idade entre 4 e 15 anos, residentes em três bairros do município de Olinda - Pernambuco, Brasil. As amostras de sangue capilar e venoso foram coletadas entre 23:00 e 1:00 hora da manhã. Em seguida, as amostras de sangue venoso foram guardadas para posterior realização das técnicas de filtração, concentração de Knott, Og4C3 - ELISA. Amostras de sangue capilar foram obtidas para confecção das lâminas de gota espessa e realização do teste rápido de imunocromatografia (ICT). As médias de densidade de microfilaremia foram calculadas em escala logarítmica. A acurácia dos testes foi avaliada em relação ao padrão-ouro (filtração). A especificidade do Og4C3 e ICT foi estimada utilizando a equação de Staquet et al. (1981). Um total de 805 escolares foi examinado. As médias de antigenemia filarial foram mais elevadas entre as crianças residentes nos bairros de alto da conquista e alto da bondade. As cargas parasitárias e os níveis de antigenemia não variaram com idade e sexo. A prevalência de microfilaremia pela técnica de filtração, de 5,2 por cento, foi a mais elevada em relação às demais técnicas parasitológicas. A prevalência de antigenemia filarial pelo teste Og4C3 foi de 17,4 por cento. Na comparação da acurácia dos testes em relação ao padrão-ouro, as técnicas de gota espessa e Knott apresentaram valores de sensibilidade de 85,2 por cento, inferiores aos testes ICT e Og4C3, que foi de 100 por cento. Conclui-se que as técnicas de filtração e Og4C3 são as mais apropriadas para a avaliação de transmissão em áreas com programas de eliminação em andamento.

Subperiodic, asymptomatic microfilaremia in an adult male from Mysore: a nonendemic area.

Wuchereria bancrofti is found throughout tropics and subtropics like Asia, Pacific islands, Africa, areas of South America and Caribbean basin. In all these areas, except Pacific islands, microfilaria occurs in the periodic form, in which case the microfilaria are found in large numbers in the peripheral blood during night. In the Pacific islands, they occur in the subperiodic form, i.e., microfilaria are present in the peripheral blood at all times and reach the maximum level of parasitemia in the afternoon. Microfilaria of Wuchereria bancrofti and Brugia malayi occurring in India displays a nocturnal periodicity, appearing in large numbers at night. This is the biological adaptation to the nocturnal biting habits of the vector mosquitoes. The maximum density in blood is reported between 10 PM and 2 AM. Here is a case report of asymptomatic microfilaremia showing subperiodicity, which is very unusual in India.

Disseminated Microfilaremia Associated with Lung Cyst and Empyema: An Autopsy Report

Clinical manifestations of extralymphatic disease caused by filariasis are varied and range from symptoms due to tropical pulmonary eosinophilia to hematuria, proteinuria, splenomegaly, and rarely arthritis. Disseminated microfilaremia in association with loculated lung cyst and empyema is of rare occurrence and to the best of our knowledge has not been documented in the literature so far. We report here a case of disseminated microfilaremia due to Wuchereria bancrofti infection accompanied by a lung cyst and empyema in a 21-year-old Indian man.

RT-PCR assay for the detection of infective (L3) larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus. MATERIAL & METHODS: Subtracted probe development technique was employed for the identification of infective stage (L3) specific genes. The subtracted cDNA was labeled by non-radioisotopic method and used for screening cDNA library of L3 stage larvae of W. bancrofti constructed in UniZap XR. Recombinants were probed and identified from the library. The inserts of the recombinant clones were purified and sequenced. Primers were designed based on the sequence information of three recombinant clones for detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay. Preliminary laboratory evaluation was carried out to assess the sensitivity and specificity of WbL31 RT-PCR assay. RESULTS: cDNA library of L3 stage of W. bancrofti constructed in UniZap XR vector, constituted 5 x 10(5) phages with 80-90% recombinant phages and the size of inserts varied from 0.1 to 1.0 kb. When subtracted cDNA was random prime labeled and used for screening cDNA library of L3 stage of W. bancrofti constructed in UniZap XR, 18 clones were identified from the library. Three genes were found up-regulated in the L3 stage, out of which WbL31 (cuticular collagen) was found to be useful in detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay with high specificity and sensitivity (98-100%). CONCLUSION: Present paper marks first report on the development of an infective stage-specific RT-PCR assay (WbL31 RT-PCR assay) to detect L3 stage W. bancrofti in the vector. This assay will have potential application in assessing the transmission of infection and hence in decision-making related to elimination programme.

Giant penoscrotal filariasis.

A 42 year old male individual having a hugely distended scrotum and barely perceptible penis, unable to maintain his livelihood on account of his handicap and socially withdrawn for the fear of humiliation, got admitted into the Surgery department of Mymensingh Medical College Hospital in August 5, 2007. The person had been carrying the so called curse for the last four years. On the basis of clinical, serological and haematological examinations, diagnosis was established as lymphatic filariasis causing elephantiasis of the scrotum. After assessment for operative feasibility, the patient underwent an operation in August 20, 2007 during which, ligation of both the spermatic cords, excision of the scrotum along with the testicles and reposition of the penile shaft into the anterior pelvic wall in the subcutaneous plane (Fleying procedure) was performed. The scrotum measured about 36 inches in diameter and weighed 40 kgs after excision. Histopathological examination of the scrotal skin confirmed the diagnosis. The patient was released without any complication, on the 11th post operative day after removal of stitches, with indwelling urethral catheter. He was prescribed Doxicycline for the next 8 weeks, a drug which has been found to be effective in treating filariasis in a number of studies, and acts by killing a symbiotic bacteria necessary for the survival of the worm. Follow up after 3 months was uneventful.

Microfilariae in bone marrow aspiration smears, correlation with marrow hypoplasia: a report of six cases.

Microfilariae have been reported in various cytological preparations, however there are very few case reports describing microfilariae in bone marrow aspirates. We report six such cases with the interesting finding of marrow hypoplasia in five of these cases.

Filariasis in Ghatampur Tahsil of Kanpur Nagar District: indications of high endemicity locus.

A study in the 26 villages surveyed, the mf rate was observed to vary from 6.4% to 17.8%, the disease rate ranged from 1.9% to 10% and total infection rate from 8.2% to 26.4%. The median microfilaraemia density among positives was 10 and 90% of persons had density below 60 and in 10% above this level. The mf rate among those who never used bednets while sleeping was found to be 11.8%, 15.7% higher than 10.2% among those who ever used bednets (8.7% in regular users and 10.7% among irregular users) to protect from mosquitoes bites (p < 0.05). The lymphatic disease was found to be 3.8%; 3.7% in males and 4.1% in females. Of the males, 16.3% had acute disease, 51.8%) hydroceles of varying grades and 32% edema of different grades including elephantiasis. Of the females with lymphatic disease, 25.6% acute disease, 62.8% edema including elephantiasis and 11.6% had mastitis. The study indicates that area is endemic for filariasis and needs control programmes.

Lymphatic filariasis in rural areas of Patna District, Bihar. A challenge ahead.

Patna district was endemic for lymphatic filariasis (LF). During November 2004, a lymphatic filariasis survey was carried out in seven randomly selected villages from four PHCs of Patna district. Of 1878 night blood smears (NBS) examined, 117 were found positive for W. bancrofti infection (mf rate 6.2%). Microfilaria carriers were detected from all surveyed villages. In all areas prevalence of microfilaria generally increased with age to maximum 15-34 years and then decline within most age-groups. More males (6.4%) were affected than the females (5.8%). All microfilaria (mf) carriers were residents of Patna district. Over all disease rate was 9.1%. Out of 171 diseased individuals, 121 persons were having hydrocele (6.4%). Disease rate was higher in males (12.0%) than females (4.1%). The mean mf density was 11.7. There was no significant difference of mean mf density in males and females. Vector density ranged from 205 to 780 per ten man hours. The survey indicates that the filariasis situation remained unchanged since last fifty years and still is one of the major public health problem in surveyed areas.

A juvenile filarial worm, Wuchereria bancrofti, extracted from the vitreous of the eye: the first report in the world literature.

A live worm was extracted from the vitreous of the eye of a patient in Sri Lanka. Based on the details of its length, width, oesophagus and vulva it was identified as Ajuvenile female Wuchereria bancrofti.

Filariose linfática em Belém, Estado do Pará, Norte do Brasil e a perspectiva de eliminação / Lymphatic filariasis in Belém, Pará State, North of Brazil and the perspective of elimination

Com o objetivo de caracterizar a situação epidemiológica da filariose linfática em Belém-PA foram analisados dados dos inquéritos hemoscópicos de 1951 a 2003. As informações do período de 1951 a 1994 foram coletadas de relatórios disponibilizados pela Fundação Nacional de Saúde. Os dados de 1995 a 2003 foram obtidos através de inquéritos realizados em 62 bairros, dos oito distritos administrativos da cidade. Observou-se uma queda apreciável ao longo dos anos nos índices de microfilarêmicos. As percentagens de parasitados nas décadas de 1950, 1960, 1970, 1980 e 1990, foram respectivamente: 8,2 por cento, 2,6 por cento, 0,7 por cento, 0,16 por cento e 0,02 por cento. Em 2001, foi diagnosticado um único microfilarêmico, interrompendo uma série de dois anos sem registro de exames positivos na cidade. Em 2002 e 2003, inquéritos hemoscópicos e entomológicos foram realizados, simultaneamente, não sendo detectados indivíduos microfilarêmicos ou mosquitos infectados. Para manter essa tendência, medidas de vigilância devem ser observadas, a fim de detectar e tratar precocemente pacientes, para evitar o risco de ressurgimento dos focos, aparentemente já controlados.

A focus of lymphatic filariasis in a tea garden worker community of central Assam.

A survey for lymphatic filariasis was conducted among tea garden workers of central Assam. Of the 656 night blood samples examined, 31 were found positive for Wuchereria bancrofti parasite (microfilaria rate 4.7%). Microfilaria rate was higher in male (7.3%) than females (2.1%). Culex quinquefasciatus was incriminated as vector mosquito.