Bancroftian microfilariasis in association with pulmonary tuberculosis. Report of a case with diagnosis by fine needle aspiration

A 25-year-old man presented with clinical and radiologic features suggestive of pulmonary tuberculosis. Since the examination of Ziehl-Neelsen-stained sputum smears for acid-fast bacilli was repeatedly negative, a transthoracic fine needle aspiration biopsy was performed. Papanicolaou-stained smears of the aspirate showed microfilariae of Wuchereria bancrofti and a tuberculous exudate but no acid-fast bacilli or classic granulomas. Subsequent sputum samples did show acid-fast bacilli, while a nocturnal peripheral blood sample showed microfilariae.     

Characterization of a muscle-associated antigen from Wuchereria bancrofti.

A recombinant clone, WbN1, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbN1 indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbN1 fused to the maltose-binding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbN1 antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbN1 is related to myosin or a similar myofibrillar protein.     

Dirofilaria repens diagnosed by the presence of microfilariae in fine needle aspirates: a case report

BACKGROUND: Dirofilariasis due to Dirofilaria repens with viable microfilariae outside the worm have not been reported before. CASE: A 40-year-old truck driver from rural Shiraz, Iran, had a firm mass, 2.5 x 2.5 cm, at the dorsolateral aspect of the right forearm. Fine needle aspiration (FNA) was performed on 2 occasions. Several microfilariae with blunt heads, pointed posterior ends and empty caudal spaces resembling microfilariae of Wuchereria bancrofti but longer were seen. Since Iran is a nonendemic area for lymphatic filariae and the patient had a history of contact with a dog, with the impression of dirofilariasis, the mass was excised, and the presence of adult worms in tissue sections confirmed the diagnosis. CONCLUSION: This case ofsubcutaneous dirofilariasis was diagnosed by detecting microfilariae in FNA smears and was confirmed on histopathology.     

Role of fine needle aspiration cytology in detection of microfilariae: report of 2 cases

BACKGROUND: Filariasis is a major public health problem in developing countries, and the diagnosis is conventionally made by demonstrating microfilariae in the peripheral blood smear. However, microfilariae have been incidentally detected in fine needle aspirates of various lesions in clinically unsuspected cases of filariasis with absence of microfilariae in the peripheral blood. CASES: In case 1, a 21-year-old woman presented with multiple left axillary lymphadenopathy of 3 months' duration. In case 2, a 32-year-old woman presented with a thyroid nodule of 7 months' duration. Fine needle aspiration smears from both cases showed sheathed microfilariae of Wuchereria bancrofti. In both cases, microfilariae could not be demonstrated in the peripheral blood smears and the blood eosinophil counts were within normal limits. The histopathologic examination showed neither microfilariae nor adult worm. CONCLUSION: Although microfilariae in cytologic material are considered incidental findings, these cases illustrate the value of routine fine needle aspiration cytology in the detection of asymptomatic and clinically unsuspected cases of bancroftian filariasis. Absence of microfilariae in the peripheral blood does not exdude filarial infection.     

New tools for diagnosis and monitoring of bancroftian filariasis parasitism: the Polynesian experience

Bancroftian filariasis is endemic in French Polynesia and control programs with diethylcarbamazine, started in the 1950s, led to a sharp reduction of the microfilaria prevalence. Consequently, the control program was interrupted in 1982. Ten years later, however, the incidence of the parasitism again reached pre-control levels (20-30% microfilaremia in some islands), indicating that the adult worms (for which no diagnostic tool was available) had persisted. Apart from research on chemotherapy strategies, the Institut Malardé has been actively involved in developing and evaluating more-powerful diagnostic tools than the unique detection of microfilariae by blood smear examination. These include: (1) the detection of adult worm circulating antigens in humans, and (2) the detection of Wuchereria bancrofti larvae in mosquitoes, using DNA probes. In this paper, Luc Nicolas reviews the available diagnostic tools to detect W. bancrofti and their implementation in epidemiological areas, based on the Polynesian experience.     

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema vileae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanoi and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection offilarial infections, is discussed.     

Filarial-specific IgG4 response correlates with active Wuchereria bancrofti infection

The filarial-specific humoral immune response of adult residents of two areas of Papua New Guinea, differing in transmission of Wuchereria bancrofti infection was compared. The majority of residents of the village of Bonahoi, in an area where transmission of filariasis had been interrupted by a 20-year insecticide spray program to control malaria, showed no parasitologic signs of active W. bancrofti infection and were negative for both circulating phosphorylcholine Ag and peripheral blood microfilariae. In contrast, adult residents of the village of Nanaha were in an area exposed to infection, and were phosphorylcholine-Ag- and microfilariae-positive. The antibody response of these two groups to both adult worm excretory/secretory (ES) Ag and somatic antigen extract was examined to determine which components of the filarial-specific immune response were dependent on active infection. Identification of these immune responses may point to immunologic methods to evaluate control programs for lymphatic filariasis. Adults from Bonahoi were found to have significant immune responses to [35S] methionine-labeled ES Ag by immunoprecipitation and to adult somatic antigen extracts by ELISA and by immunoblotting. This result is consistent with the fact that these individuals were previously exposed to and/or infected with W. bancrofti. Similarly, residents of the endemic village had detectable immune responses to these Ag irrespective of if they were microfilaremic. The most striking immunologic difference observed between the two groups was that residents of Bonahoi had a dramatically reduced filarial-specific IgG4 antibody response to both adult somatic Ag and adult ES Ag. These data suggest that longitudinal measurement of filarial-specific IgG4 levels may be a useful seroepidemiologic indicator of changes in W. bancrofti infection status.     

Age-grading and growth of Wuchereria bancrofti (Filariidea: Onchocercidae) larvae by growth measurements and its use for estimating blood-meal intervals of its Polynesian vector Aedes polynesiensis (Diptera: Culicidae)

Growth in length and width of Wuchereria bancrofti (Filariidea: Onchocercidae) larvae developing in its Polynesian vector Aedes polynesiensis (Diptera: Culicidae) was analysed using a mathematical approach to objectively extract patterns. L1 had a U-shaped growth in length, while widths followed an S-shaped function. L2 had an S-shaped growth in length and width. Growth in length of L3 was also S-shaped, while widths had an asymptotic size following a period of rapid shrinkage. The greatest difference between length and width was in stage 3 where the length was over 75 times greater than the width. The ratio of length to width was approximately 50 for microfilariae and only 10 for the L1 ('sausage') stage. Characteristic mean length (and width) were approximately 280(7) microm for microfilariae, approximately 181 microm for L1 at their smallest, and approximately 1584(22) microm for L3 infective larvae. There was a great increase in length during stage 2 from approximately 322(27) to approximately 982(31) microm. Stage duration decreased with increasing temperature while growth rate increased, giving steeper growth curves. There was no effect of temperature on size, except for L3, which were shorter when mosquitoes were reared at higher temperature. It appears that larval growth is a continuous process from microfilariae to the young L3 stage, and continuously modifies the larval parasite aspect, even within each stage. Thus, information on larval shape may be used as an age indicator and in some cases, may give an estimation on time elapsed since infection of the vector.An important demographic parameter used in most mathematical models describing transmission of parasites by insect vectors is the length of the gonotrophic cycle of the vector, i.e. the time interval between two successive blood-meals. Usual methods for computing such a parameter are based on mark-recapture techniques. However, reliable estimates need substantial capture rates, which are not always possible. This paper presents another approach in which marked mosquitoes are those naturally infected by W. bancrofti. For one mosquito, the time since infection is simply the age of the developing larval parasite. Our method first expresses the age of larval parasite as a fraction of total development time (from microfilariae entering the vector to L3 larvae) using a regression model based on measurements of the parasite's length and width. This fraction of development is then converted to a chronological age since infection, using a back-calculation procedure involving ambient temperatures and growth rates of W. bancrofti larvae in the vector. The method is applied to wild caught Ae. polynesiensis in French Polynesia to compute the length of the gonotrophic cycle. This mosquito species comes to bite approximately 3, 6-7 and 9 days after a first infectious blood-meal. Then the length of the gonotrophic cycle may be of 3-4 days.     

Quinone analogue irrecoverably paralyses the filarial parasites in vitro

2,3-Dimethoxy-5-methyl-1,4-benzoquinone (Q0), an analogue of ubiquinone, irreversibly paralyses the adult and microfilariae of the cattle filarial parasite Setaria digitata. The same concentration of Q0 that paralyses the microfilariae of S. digitata also paralyses the microfilariae of the human filarial parasite Wuchereria bancrofti within the same duration. Thus the experiments done in the model S. digitata system can well be extended to the human filarial system. A drug at the level of the quinone-centered energy generating system, perhaps an analogue of quinone like Q0, can inactivate the filarial parasites and may prove to be an effective drug to control filariasis.     

Analysis of polymorphism of 18S rRNA gene in Wuchereria bancrofti microfilariae

The polymorphism of the 18S rRNA gene in Wuchereria bancrofti microfilariae (mf) collected from three different zones in India was analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The RFLPs of the amplified products obtained after digestion with restriction enzymes Ssp I, Msp I and Hha I showed no difference in the banding patterns among the mf isolates from different endemic zones. Further the sequencing of PCR products did not show any difference in the nucleotide sequence either. The phylogenetic analysis of the sequences of W. bancrofti mf isolates from different endemic zones has shown branching with the earlier reported sequences of W. bancrofti and its close relative Brugia malayi.     

Cytologic detection of Wuchereria bancrofti microfilariae in urine collected during a routine workup for hematuria

A 23-year-old Guyanese man experienced intermittent, total, painless, gross hematuria for a month for which he sought medical attention at the Kings County Hospital Center in Brooklyn. Hematuria was accompanied by weakness but not by frequency of urination or burning on urination. Catheterized urine at the time of cystoscopy and each of two subsequent voided specimens examined cytologically contained sheathed microfilariae. Distinguishing features of the microfilariae were well demonstrated with the Papanicolaou stain. The well-stained nuclei, which did not extend into the clear zone, and the distinct, pale-stained sheath led to the positive identification of the microfilariae as Wuchereria bancrofti. The Papanicolaou stain may well be the stain of choice for the identification of microfilariae in the blood. The excellent detail obtained with this routine cytologic stain is as good as that with Giemsa, which does not stain the sheath.     

The invasion of the midgut of the mosquito Culex (Culex) quinquefasciatus Say, 1823 by the helminth Litomosoides chagasfilhoi Moraes Neto, Lanfredi and De Souza, 1997

The Litomosoides chagasfilhoi helminth was studied as a model for microfilaria invasion of the midgut of Culex quinquefasciatus mosquito, vector of Wuchereria bancrofti helminth, causative agent of the human filariasis. Histology and transmission and scanning electron microscopy were utilized to show the topography of mosquito midgut invasion by the helminth. An analysis of midguts dissected at different time points after a blood meal demonstrated that the microfilariae interacted and crossed the peritrophic matrix and the midgut epithelium of C. quinquefasciatus. The microfilariae invaded preferentially the mosquito abdominal midgut and the invasion process occurred between 2 and 3h after the blood feeding. In some cases, microfilariae caused an opening in the midgut that separated the epithelial cells, while in others cases, the worms caused the detachment of cells from the epithelium. Ultimately, L. chagasfilhoi crossing activity appeared to damage the midgut. It was also observed that the microfilariae lost their sheaths during their passage through the fibrous material of the peritrophic matrix, before they reached the midgut epithelium. Since the exsheathment process is necessary for the continuity of larvae development, it seems that the passage through the peritrophic matrix is an important step for the parasite's life cycle. This experimental model revealed details of the interaction process of helminthes within the vector midgut, contributing to the knowledge of factors involved in the vector competence of C. quinquefasciatus as a vector of filariasis.     

Setaria cervi: in vitro released collagenases and their inhibition by Wuchereria bancrofti infected sera

In vitro released products of adult Setaria cervi females, microfilariae and extracts showed considerable amounts of collagenase activity. On the basis of per mg protein released in vitro, the products of both microfilariae and adult females exhibited comparable activity but this was much higher than that of extract of microfilariae and adult females. Two collagenase enzymes with molecular masses of 50 kDa and 70 kDa were separated using DEAE-sepharose CL6B and Sephadex G-100 column chromatography. The 50 kDa and 70 kDa collagenase exhibited pH optima of 5.2 and 7.0, respectively. Considering specific activity, the 50 kDa enzyme was found to contribute about ten times more collagenase activity as compared to the 70 kDa enzyme. An inhibition study revealed obvious differences between them. Thiol group inhibitors such as N-ethylmaleimide and leupeptin inhibited the 50 kDa enzyme but this was strongly activated by dithiothreitol, a thiol group stabilizer. Alternatively, the 70 kDa enzyme showed a sensitivity to a metal chelator and a serine group inhibitor indicating its metalloserine protease nature. The antifilarial drug diethylcarbamazine did not demonstrate any inhibition under in vitro conditions. Both enzymes were significantly inhibited by antibody IgG separated from Wuchereria bancrofti infected human sera, showing a possible immunoprotective role.     

Microfilariae of Wuchereria bancrofti in cytologic smears

Microfilariae of Wuchereria bancrofti were observed in cytologic material in 35 cases. The material included cervicovaginal smears (17 cases), effusions (14), urine (2), bronchial washings (1) and ovarian cyst fluid (1). The initial diagnosis was made from the cytologic smear in all cases; none had clinical filariasis. Symptomatic vaginal bleeding in 9 of the 17 cases with microfilaria-positive cervicovaginal smears was reflected in the large numbers of red blood cells found in the smear. Blood eosinophilia was present in 11 of 19 cases investigated. Eosinophils were seen in the smears in 20 cases. In the majority of the cases of effusions with microfilariae the effusions were malignant. Significant adherence of inflammatory cells and macrophages to microfilariae was present in 7 of the 35 cases. The significance of these findings is discussed.     

Microfilarial surface carbohydrates as a function of developmental stage and ensheathment status in six species of filariids

The lectin-binding properties of microfilariae of Onchocerca volvulus, O. lienalis, Brugia pahangi, Wuchereria bancrofti, Dirofilaria immitis, and Monanema (= Ackertia) marmotae share a number of characteristics. Carbohydrates specific for lectins are associated with the egg shell or sheath. N-acetyl-D-glucosamine is the predominant carbohydrate associated with the ensheathed forms with lesser quantities of D-galactose and/or alpha-lactose and D-galactosamine. The density of these carbohydrates on the sheath surface diminishes as the larvae undergo normal growth and development. Similar carbohydrates are not found on the cuticle as exsheathed microfilariae show virtually no ability to bind lectins.     

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti.

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2. 4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.     

An analysis of genetic diversity and inbreeding in Wuchereria bancrofti: implications for the spread and detection of drug resistance

Estimates of genetic diversity in helminth infections of humans often have to rely on genotyping (immature) parasite transmission stages instead of adult worms. Here we analyse the results of one such study investigating a single polymorphic locus (a change at position 200 of the beta-tubulin gene) in microfilariae of the lymphatic filarial parasite Wuchereria bancrofti. The presence of this genetic change has been implicated in benzimidazole resistance in parasitic nematodes of farmed ruminants. Microfilariae were obtained from patients of three West African villages, two of which were sampled prior to the introduction of mass drug administration. An individual-based stochastic model was developed showing that a wide range of allele frequencies in the adult worm populations could have generated the observed microfilarial genetic diversity. This suggests that appropriate theoretical null models are required in order to interpret studies that genotype transmission stages. Wright's hierarchical F-statistic was used to investigate the population structure in W. bancrofti microfilariae and showed significant deficiency of heterozygotes compared to the Hardy-Weinberg equilibrium; this may be partially caused by a high degree of parasite genetic differentiation between hosts. Studies seeking to quantify accurately the genetic diversity of helminth populations by analysing transmission stages should increase their sample size to account for the variability in allele frequency between different parasite life-stages. Helminth genetic differentiation between hosts and non-random mating will also increase the number of hosts (and the number of samples per host) that need to be genotyped, and could enhance the rate of spread of anthelmintic resistance.     

周期型马来丝虫微丝蚴寿命的观察

将实验感染周期型马来丝虫的长爪沙鼠的微丝蚴蚴阳性腹腔稀释液,移注于正常沙鼠腹腔内,微丝蚴除能在腹腔内长期生存外,还可出现于外周血液中,其在外周血液内末次阳性检出时间最长可超过32周,在腹腔液内末次阳性检出时间最长为77周,故马来微丝蚴在沙鼠外周血液中的最长寿命不短于7.5月,而在腹腔液内的最长寿命可超过1.5年以上。     

Suppression of Con A induced lymphocyte transformation by sera from filarial patients with clinical manifestations

Reduced lymphocyte transformation to Wuchereria bancrofti microfilariae excretory-secretory antigen and Con A were observed in clinical filarial patients. Pre-incubation of normal human peripheral blood mononuclear cells with sera from filarial patients with clinical manifestations such as hydrocele and elephantiasis suppressed Con A induced responses. Effect of fractionated clinical filarial serum on Con A induced lymphocyte transformation showed that the inhibitory activity was associated with high molecular weight serum fraction.