Importância do método de gota espessa de sangue no diagnóstico de filárias simpátricas no Amazonas, Brasil / The importance of thick blood film method for sympatric filarial diagnosis in Amazon basin

Esse trabalho relata os métodos de diagnósticos para filárias humanas no sangue, referindo-se a importância do método de gota espessa em relação aos métodos de filtração em membrana de policarbonato e Knott na diferenciação das espécies de microfilárias, principalmente em áreas de ocorrência de mais de uma espécie, como em regiões do Amazonas. Lâminas com microfilárias de Mansonella ozzardi foram montadas e fotografadas pelos diferentes métodos de diagnósticos. O método da gota espessa de sangue é o mais confiável, pois permite visualizar com nitidez o espaço cefálico e caudal, disposição dos núcleos caudais e formato da cauda, que são características morfológicas que diferenciam as espécies de microfilárias sanguíneas que ocorrem no Amazonas.

In this work, the visual diagnostic methods for human filarias in the blood are evaluated. The thick blood film method is more faithful to identify the microfilariae species if compared to policarbonate membrane filtration and Knott methods, especially in areas where more than one species occur, as in Amazon basin. Slides with Mansonella ozzardi microfilariae were mounted and photographed by different diagnosis methods. The thick blood film method is much easier to see the diagnostic morphological characters as cephalic and caudal space, pattern of nucleation of the tail and tail shape that can separate the blood Amazonian microfilariae species.

Detection of IgG antibodies of Brugian filariasis with crude male and female antigens of Dirofilaria immitis.

Crude antigens from male and female Dirofilaria immitis were used to detect antibody to Brugian filariasis in humans by indirect ELISA. Both antigens were tested with 42 cases of Brugian filariasis, 131 cases of 20 heterologous infections and 35 healthy controls. The results--using male and female antigens--showed sensitivity of 88.1% and 88.1%, and specificities of 64.1% and 51.8%, respectively. Cross-reaction from other helminthic infections using crude male antigen gave false-positives with 48 sera from 13 heterologous diseases at the threshold value of 0.180, while the female antigen gave 63 sera from 15 diseases, at 0.309. Serum antibodies from patients with other helminthic infections--gnathostomiasis, strongyloidiasis, hookworm infections, trichinellosis, capillariasis, angiostrongyliasis, ascariasis, trichuriasis, toxocariasis, neurocysticercosis, cystic echinococcosis, taeniasis and opisthorchiasis--resulted in false-positives with both male and female antigens. One each of sparganosis and paragonimiasis heterotremus sera cross-reacted with only crude female antigen and their OD values were close to the threshold value. Although crude male antigen showed better specificity than crude female antigen, both female and male worms are sources of antigens needed for further purification. This study provides baseline data for further serodiagnosis of Brugian filariasis using dirofilaria antigen.

Decline of brugian filariasis in Cherthala taluk, Alappuzha district, Kerala.

A total of 4492 persons from 5 panchayats and 1 town were investigated from the Brugia malayi most endemic taluk of Cherthala, Alappuzha district of Kerala state. The urban area in Cherthala taluk only revealed mf carriers; mf rate was 0.13%. Rural areas in Cherthala taluk were free from infection. Microfilaria rate had declined by 99.5% and disease rate by 90.7% in Cherthala compared to 1934 prevalence. Shedding of sheath by B. malayi microfilariae was recorded for the first time in India. The youngest person with microfilaria and disease manifestation was 4 1/2 and 9 years respectively. All the 3 major vectors, Mansonia annulifera, Ma.uniformis and Culex quinquefasciatus were prevalent throughout. Complete disappearance of brugian filariasis from this taluk is a distinct possibility. The reasons for the drastic decline are discussed.

Short-term effects of treatment with 300 mg oral-dose diethylcarbamazine on nocturnally periodic Wuchereria bancrofti microfilaremia and antigenemia.

Seven microfilaremic Myanmar patients were treated with a single 300 mg dose of diethylcarbamazine (DEC) orally, as part of a case-finding survey in Ranong Province, Southern Thailand. This was conducted in order to evaluate the short-term effects of single-dose DEC on Wuchereria bancrofti microfilaremia and antigenemia during a 12-week course of treatment. Analysis of microfilarial periodicity on initial treatment revealed the microfilarial peak density (k) was at 52 minutes after midnight (0052). The periodicity index was then 103.26%. Single-dose DEC treatment did not affect the k values. A linear model of W. bancrofti microfilarial density reduction predicts a sharp decrease in the mean microfilarial density 2 weeks after DEC intake (Z = -2.197, p = 0.028). Over a longer period, a non-linear model predicts an increase in the mean microfilarial density to pre-treatment levels, having little or no macrofilaricidal effects. We reconfirmed the existence of nocturnally periodic W. bancrofti infection in Myanmar migrants in Ranong Province, and the short-term microfilaricidal activity of 300 mg single-dose DEC treatment used for biannual mass treatment and the DEC provocative test. Without an adequate DEC treatment dose, recrudescence can occur. A rational approach to the management of introduced nocturnally periodic W. bancrofti in Myanmar migrants, who came for short periods of stay in transmission-prone areas, is needed.

A baseline study of rural Bancroftian filariasis in southern India.

Night mass blood surveys were carried out for parasitological evidence of Bancroftian filariasis in 45 rural areas belonging to 9 National Filaria Control Program (NFCP) zones of East Godavari and West Godavari districts of Andhra Pradesh, India during the period 1998 to 2001. Mf prevalence range between 2.9 to 10.2%, and mf intensities in 20 mm3 blood samples ranged from 1-281. The present study explains the trend of microfilaria dynamics in the rural population, where mass drug delivery has been implemented since 1997, and anti-larivicidal and adulticidal control measures have not been adopted.

The preferential site of adult Wuchereria bancrofti: an ultrasound study of male asymptomatic microfilaria carriers in Pondicherry, India.

BACKGROUND: The traditional method of detection of microfilaria in night blood specimens for the diagnosis of Wuchereria bancrofti infection is being replaced with circulating filarial antigen in day blood specimens, which has a high sensitivity. However, both methods are indirect tests to detect the presence of adult worms in vivo. Localization of adult worms in vivo in their natural habitat may help in understanding better the end-point of drug treatment, the adulticidal action of antifilarial drugs, and in locating the site of lymphatic pathology. We used ultrasound examination to assess the preferential location of adult worms in an area endemic for lymphatic filariasis. METHODS: Ultrasound examination was done in 36 asymptomatic male carriers of Wuchereria bancrofti microfilaria to detect the location of adult worms. Both sides of the scrotum (root of the scrotum, epididymis, spermatic cord, testis and the adjoining area), lymphatic vessels and inguinal, popliteal, axillary and epitrochlear lymph nodes were examined using a 7.5 MHz probe in real-time B mode. RESULTS: The 'filaria dance sign (FDS)' suggesting the presence of adult worms was observed in 22 carriers (61%). The preferential site of location of the adult worms was the intrascrotal juxtatesticular lymphatic vessels in 'nests' along the lymphatic vessels of the epididymis, spermatic cord and paratesticular region. The number of nests varied between 1 and 4 with a mean size of 0.3 cm2. In 95% of cases, localization of the worms was unilateral. The mean microfilaria (SD) count-positive cases for those with the filarial dance sign (264 [199]) was significantly higher (p<0.05) than for the negative cases (171 [196]). CONCLUSION: Ultrasound visualization of adult worms of Wuchereria bancrofti in vivo is possible and confirms the concept that the worms have their own territory and reside in 'nests'. The preferential site of localization of the adult worms in men is the intrascrotal juxtatesticular lymphatic vessels.

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis.

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.

IgG- and IgG4-detected antigens of Dirofilaria immitis adult worms for bancroftian filariasis by enzyme-linked immunoelectrotransfer blot.

In Thailand, Wuchereria bancrofti filariasis has persisted along the border between Thailand and Myanmar, its dynamic distribution caused by the infected transmigrants between neighboring countries, and the availability of susceptible mosquito vectors. Dirofilaria immitis adult worm was used as a source of antigens, excretory-secretory (ES) and partial surface extracts, to detect human filariasis. ES products showed several stained bands with Coomassie brilliant blue ranging from 14.5-93 kDa and mostly being glycoproteins as shown by concurrent reaction with Concanavalin A, except those at 18, 16 and 14.5 kDa which stained only with Coomassie brilliant blue. Surface proteins of 33.5-91.5 kDa were stained with Coomassie brilliant blue and showed smear bands with Concanavalin A. By enzyme-linked immunoelectrotransfer blot, Bancroftian filariasis sera gave specific reactions with glycoprotein ES antigens at MW 20.5 kDa against anti-human IgG. A prominent band of 18 kDa appeared consistently with the IgG4-ES antigen system. Surface extracts reacting with IgG and IgG4 were considered to be unsuitable as antibodies from all cases of filariasis could not detect any bands.

ICT filariasis test: a new screening test for Bancroftian filariasis.

Bancroftian filariasis can be detected by using the ICT Filariasis test kit which is composed of specific polyclonal and monoclonal antibodies to Wuchereria bancrofti antigen. Chromatographic reaction with serum or plasma shows a result within 5 minutes. When compared with 454 thick blood films (standard smear method) within the same study, the ICT Filariasis test had sensitivity = 100%, specificity = 96.37%, efficiency = 96.70%, predictive value positive (PVP) = 70.70%, predictive value negative (PVN) = 100%. Compared with 454 membrane filtration technic (MFT), the MFT had sensitivity = 95.10%, specificity = 99.50%, efficiency = 99.12%, PVP = 95.10%, PVN = 99.50%. When we compared capillary tube technic (CAP) with TBF, CAP showed sensitivity = 85.40%, specificity = 100%, efficiency = 98.68%, PVP = 100%, PVN = 98.60%. With the convenience, high sensitivity-efficiency, lack of cross-reactions, no night blood collection, single reagent and rapidity of the test, the ICT Filariasis test can be recommended for screening of Bancroftian filariasis, and is suitable for the confirmation of suspected cases in the field where microscopic diagnosis is not available.

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.

Prevalence of Bancroftian filariasis in Burdwan District, west Bengal: a comparative study between colliery and non-colliery areas.

The present study reports results of a survey of bancroftian filariasis in Burdwan district West Bengal. 85,974 persons were examined of which 46,221 were inhabitants of the colliery areas of the district and rest 39,753 were from non-Colliery areas, including urban and rural areas of the district. The clinicopositivity, endemicity, microfilaria(mf)-positivity, mf-clinicopositivity, and mf-density were determined. It was found that all these epidemiological and parasitological parameters were significantly higher in the colliery area as compared to non-colliery area. The high vector density in the colliery area may account for such significantly higher prevalence of filariasis in this area. From a differential count of neutrophils, eosinophils and lymphocytes in the mf-positive cases, it was revealed that neutrophil percentage was significantly (P < 0.01) decreased while eosinophils and Lymphocytes increased. The decrease of neutrophils was correlated ((Correlation co-efficient, r = -0.78) with the increase of lymphocytes, indicating an imbalance in the immune-system of the infected persons. The geographical and geological factors resulting in high vector density are considered responsible for the significantly higher rate of bancroftian filariasis in the Colliery area.

Recent advances in diagnostic techniques in filariasis.

Accurate diagnosis of human filarial infections still remains a problem for clinicians and co-ordinators of filariasis control programs. Diagnosis of filariasis is based on parasitological, histopathological, clinical and immunological approaches. No significant advances have been made for the first three approaches although some refinements in their use and interpretation of results have occurred. For the immunological approach, intradermal tests and antibody detection assays using crude parasite extracts generally lack specificity and/or sensitivity to discriminate between past and present filarial infections in humans. Antigen detection assays would therefore provide a more accurate indication of active filarial infections. Several monoclonal antibodies to various stages of lymphatic filarial parasites have been developed and appear potentially useful for filarial antigen detection.

Daily diethylcarbamazine for the treatment of Brugia malayi microfilaria carriers.

BACKGROUND. Diethylcarbamazine is effective for the treatment of brugian filaria carriers. However, the 12-day course with a daily dose of 6 mg per kg body weight is rarely completed. The World Health Organization has indicated the need for better regimes to control filariasis. METHODS. We evaluated the long term effects of three different dosage regimes of diethylcarbamazine--6, 9 and 12 mg per kg body weight--to a total of 72 mg on 93 microfilaria carriers of Brugia malayi. RESULTS. All cases treated with the 6 mg daily dose showed complete clearance of microfilaria immediately after the treatment. However, at the 9 and 12 mg daily doses only 91% and 85% of cases respectively showed complete clearance. There was a close association between the daily dose and the proportion of cases developing side-effects. The mean initial microfilaria counts were significantly higher in those who had side-effects than in those who did not. An 11-month follow up showed that with the 6 mg daily dose the recurrence rate of microfilaraemia was 29% while with the 12 mg daily dose it was 67%. Four per cent of patients on a daily dose of 6 and 9 mg discontinued treatment while 21% of those on the 12 mg daily dose did so. CONCLUSION. The 6 mg daily dose of diethylcarbamazine was the most effective and best accepted regime.

A field survey on the role of low-density microfilaraemia cases in the transmission of filariasis.

In former filariasis endemic areas, where the disease has been basically controlled, a few cases of low-density microfilaraemia remain. A survey was carried out in Deqing County, Zhejiang Province, from September 1981 to 1986 in order to determine whether such cases play a role in the continuation of transmission. The results of parasitological and entomological investigations for two consecutive years revealed that after the implementation of intervention measures, the mean microfilaraemia rate in the population fell to about 0.5% and the mean microfilaria density to about 4.2 mf/60 microliter of blood in previously endemic areas of malayan filariasis. Although there were considerable numbers of An. sinensis biting humans, infective larvae could be found in only two positive mosquitoes out of 5,484 dissected, and no new microfilaraemic cases were detected in the 1983 and 1986 follow-up blood examinations, indicating that transmission had already been interrupted. Two volunteers with a microfilaraemia of 3-5 mf/60 microliter of blood were exposed to two batches of An. sinesis in August 1981. The engorged mosquitoes were dissected eight days later. Even though the infection rate of An. sinensis having fed on low-density microfilaraemic cases was as high as 16.8%, the intensity of infection was extremely low, being 1.1 mf/mosquito. From the transmission dynamics point of view, infected mosquitoes carrying very few infective larvae have no practical significance in the transmission of filariasis. It is suggested that the treatment of persons with low-density microfilaraemia (with 5 mf/60 microliters of blood) in areas with low microfilaria rates (less than 1%) need not to be considered as essential.

Filariasis en Colombia: presencia de Dipetalonema perstans en la Comisaria del Guainia / Filariasis in Colombia: presence of Dipetalonema perstans in the Comisaria del Guainia

Al examinar 75 muestras de sangre (metodo de Knott) colectadas en Puerto Inirida, Coco y Pajuil en la Comisaria del Guainia, Colombia, se descubrieron 26 portadores de micofilarias. En 18 personas habia unicamente Mansonella ozzardi; 3 tenian infeccion con M. ozzardi; 3 tenian infeccion con M. ozzardi y Dipetalonema perstans; y cinco solo con D. perstans. La infeccion por M. ozzardi se observo en blancos y en indigenas de las tribus curripacos, puinaves, tukanos, guananos y salivas, pero D. perstans se encontro solamente entre los curripacos. El numero de microfilarias (mf) circulantes fue bajo, 73% de los portadores tenian menos de 200 mf/ml de sangre; las personas que presentaron unicamente D. perstans tenian menos de 310 mf/ml. Estos resultados confirman la presencia de D. perstans en Colombia y sugieren que su prevalencia y distribucion en la Comisaria del Guainia y areas cercanas pueden ser mas altas de lo que se ha sospechado hasta el momento

Parasitological and entomological studies on filariasis in seven villages, Serian District, Sarawak, East Malaysia.

A total of 82 persons have been found to be positive for microfilaria a sub-periodic Brugia malayi out of 1,613 examined in seven villages in Serian District. This represents an average microfilaria infection rate of 5.1% with a range of zero to 10.7%. It is found that males are more predominantly affected than females with a ratio of 3:1. It is also confirmed that Mansonia dives and M. bonneae are possible vectors for the transmission of the disease although Anopheles species cannot be ruled out owing to the small number of specimens examined. The study is confined to areas where indoor DDT spraying has been done since 1960 and only in two of the areas it has been terminated in 1966. Yet to be published data shows higher filaria infection rate than any of these villages.