Morphological variations in microfilaria of Wuchereria bancrofti in cytology smears: a morphometric study of 32 cases.

OBJECTIVE: Microfilaria of Wuchereria bancrofti has been described in many cytological specimens, where typical blood film morphology has been used for evaluation. However, these studies have not documented the morphological variations in microfilaria in cytological smears. In the present study, cytological findings in 32 clinically unsuspected cases of filariasis were reviewed with emphasis on morphological details and image morphometric measurements. STUDY DESIGN: A retrospective analysis of 32 cases of clinically unsuspected filariasis diagnosed by cytology from April 2001 to March 2011 was carried out. RESULTS: All microfilariae were characterized as W. bancrofti and showed a wide variation in their length (202 to 300 µm) and width (6.2 to 8.4 µm). Terminal and subterminal swellings were seen in one of the cases causing diagnostic confusion with Brugia malayi. Microfilariae were shorter and wider in May-Grünwald-Giemsa stain than in Papanicolaou-stained smears. CONCLUSIONS: Natural variations in the size of microfilariae of W. bancrofti are the probable reason for the range of these findings. The overlapping features with microfilaria of B. malayi might be related to subspecies variations in W. bancrofti. Fixation, degeneration and staining procedure also seem to influence the morphological features. This morphometric study highlights the morphological disparities of microfilaria and the differential diagnostic considerations.

Ultrastructural analysis of Wuchereria bancrofti (Nematoda: Filarioidea) body wall.

Bancroftian filariasis constitutes the principal mosquito-borne nematode infection of humans and the surface of adult of Wuchereria bancrofti seems to be especially important in the intricate interplay between host and parasite. The study of the parasite's surface structure might help to understand the localization and function of various organelles. W. bancrofti adult worms were recovered from untreated patients during hydrocele repair surgery and studied by transmission electron microscopy. The body wall of adult parasite is composed of cuticle, hypodermis and muscular layer. Cuticle is the external layer and shows transverse cuticular striation. It is composed by an epicuticle, cortical layers, median layer, fibrous layers and basal layer. The epicuticle is the most external cuticular layer and appears as a single laminar electron-dense layer. The cortical external region is more electron-dense and granular in appearance than the inner cortical layer. Electron-dense structures, called bosses are randomly distributed filling the cuticular striation. The median layer is formed by an electron-dense and continuous thick line. The fibrous layer is subdivided in inner and external layers connected by projections. The basal layer includes a large quantity of membranous projections directed toward the hypodermis. The hypodermis is a syncytium where some cellular organelles are observed. The somatic musculature is meromyarian. The muscle fibers consist of contractile and non-contractile regions and the contractile region is composed of myofilaments separated by dense body. This is the first study of W. bancrofti adult worms obtained from untreated patients and studied by transmission electron microscopy.

Ultrastructural analysis of microfilariae of Wuchereria bancrofti obtained from persistent carriers after repeated courses of diethylcarbamazine.

Single dose of diethylcarbamazine (DEC) used in control programs is effective in breaking the transmission of filariasis. In order to investigate the effect of aggressive therapy on Wuchereria bancrofti (Wb) microfilariae, DEC was given to 29 patients who were positive for the circulating filarial antigen (CFA) assay but did not have clinical manifestations of filariasis, at 6 mg/kg/day for 12 days and again six months later using the same dosing regimen. For each patient, microfilarial density and serum CFA were followed up for two years. Ultrastructural analyses on Wb microfilariae obtained after repeated treatment with DEC were also performed. Microfilaremia and antigenemia decreased significantly after 12 months but returned to the initial levels after 24 months. This could indicate, as shown by other authors, that aggressive repeated therapy with DEC alone is ineffective in eradicating adult W. bancrofti, particularly in infected but asymptomatic individuals. The objective of the present study was to analyze the microfilaremic and antigenemic behavior and ultrastructural changes caused by different DEC concentrations in vitro in Wb microfilariae obtained from individuals who were sensitive and refractory to treatment. After in vitro treatment of the microfilariae using 5 and 10 microg/ml of DEC for 1h, ultrastructural analysis revealed low levels of cell damage compared with embryos obtained from individuals from a different area who had never received DEC treatment before. The results obtained suggest that microfilariae from patients who receive repeated aggressive therapy are less sensitive to DEC in vitro.

Molecular evidence for apoptosis in microfilariae of Wuchereria bancrofti induced by diethylcarbamazine.

Apoptosis, a programmed cell death, is characterized by chromatin condensation, numerous vacuoles, reduction in cell volume, and endonuclease cleavage DNA degradation detected in gel electrophoresis as nucleosomal ladder. Here we report that diethylcarbamazine induces DNA fragmentation in microfilariae of Wuchereria bancrofti revealed by ligation-mediated polymerase chain reaction and by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling at the light and electron transmission level.

Comparative analysis of a chemotherapy effect on the cuticular surface of Wuchereria bancrofti adult worms in vivo.

Wuchereria bancrofti is the main species responsible for human lymphatic filariasis and remains a major public health problem in tropical countries around the world. Diethylcarbamazine (DEC) has been used for decades in control programs as an effective microfilaricide, although its efficacy in killing adult worms is only around 50% and its direct mode of action is unclear. Recently, in an attempt to control and eliminate lymphatic filariasis, WHO has recommended albendazole (ALB), a broad-spectrum anthelminthic combined with DEC or ivermectin for mass treatment. Some studies have shown that DEC alone blocks oogenesis, fertilization in adult worms, and loss of the microfilarial sheath of several filarial species, whereas ALB is thought to target nematode tubulin. So far, the direct effect of ALB in combination with DEC has not been described in W. bancrofti adult worms. Therefore, the purpose of this study is to investigate by scanning electron microscopy if DEC coadministered with ALB can induce in vivo morphological alterations of the W. bancrofti adult worm surface obtained from a patient in whom the adult worm remained alive, checked serially by ultrasonography for 2 months after antifilarial treatment. Our analysis demonstrates that worms presented morphologic alterations in some regions suggesting cuticular surface damage. On the other hand, adult worms that were recovered from a patient treated with DEC alone after a single dose did not show such any abnormalities.

Identification and location of albumin-like antigens in third-stage larva of W. bancrofti, in adult forms of Litomosoides chagasfilhoi and in the free-living nematode Caenorhabditis elegans.

Antigens resembling those of host proteins have been identified on the surface of several filarial parasites, such as immunoglobulins and serum albumins. The origin of albumin-like antigens on filarial parasites remains unclear. Several authors suggested that they have been adsorbed, or that they were metabolic waste products from nutritional utilization of human albumin, or perhaps a contamination with human products. This study searched for human albumin-like antigens by Western blot and ultrastructural analyses on filarial parasites, third stage of W. bancrofti and adult females of Litomosoides chagasfilhoi, and on the free-living Caenorhabditis elegans nematode. Our results showed approximately 67kDa proteins recognized by anti-human albumin antibodies on extracts and excretory-secretory (ES) products of the third-stage W. bancrofti. Similar albumin-like proteins were also detected on the filarial parasite L. chagasfilhoi and on C. elegans extracts. The immunocytochemistry analysis showed human albumin-like antigens on similar tissues of these nematodes. These results provide evidence that these proteins have antigenic similarity and similar distribution in nematodes tissues. Our observations suggest that albumin-like antigens presented on filarial parasites are not acquired from the host, but rather are shared antigenic determinants found even in the third-stage larvae recovered from the invertebrate host.

Some morphological aspects of Wuchereria bancrofti uterus after treatment with diethylcarbamazine.

Confocal and EM analyses revealed that some female Wuchereria bancrofti, obtained from volunteers that received recommended diethylcarbamazine dose regimens, showed few or no embryos. Furthermore, inside the gravid uterus of female W. bancrofti treated with DEC we observed a finely granular, electron-dense material organised as strings of pearls, approximately 70 nm in maximal length surrounding intra-uterine microfilariae and apparently secreted by the embryo. Over the eggshells a similar material was also observed, possibly secreted by the uterine wall. The surface of intra-uterine microfilariae presented a material with identical electron-density to the scattered structures observed inside the egg. Similarly, the sheath of blood microfilariae of W. bancrofti also showed electron-dense projections, with shape and size similar to that observed inside the uterus.

The ultrastructure of infective larvae (L3) of Wuchereria bancrofti after treatment with diethylcarbamazine.

Although the large use of diethylcarbamazine (DEC), as the major anti-filaricide drug, its mechanism of action remains a matter of controversy. Several authors defend the hypothesis that DEC has no direct effect on nematodes. This study demonstrated that infective larvae (L3) of Wuchereria bancrofti treated in vitro with DEC presented several behaviour and morphological changes. The first alteration produced by treatment for 2 h with 3, 5, 10 microg/ml of DEC was the reduction of motility. Larvae treated with 5, 10 microg/ml DEC showed severely affected organelles, formation of several vacuoles, mainly in neurocytes and in the muscle cells, and dissolution of cytoplasm. Some larvae showed extreme cellular disorganization with abundance of large and dense mitochondria and numerous large vacuoles containing residual organelles. Lamellar bodies, probably related to an assembly of hipodermal membranes, were also observed in some damaged larvae. Thus, undoubtedly in vitro treatment with concentrations of DEC similar to therapeutic conditions, which are 1-5 microg/ml (Hawking, 1979), had a direct effect on infective larvae of W. bancrofti by causing, primarily neuromuscular alterations with subsequent damage to organelles.

The effects of diethylcarbamazine on the ultrastructure of microfilariae of Wuchereria bancrofti in vivo and in vitro.

An ultrastructural study of microfilariae of Wuchereria bancrofti was performed after treatment in vitro and in vivo with diethylcarbamazine citrate (DEC). The morphological alterations produced by treatment in vitro with 5 micro g/ml of DEC were the loss of microfilarial sheaths and lysis of the cytoplasm, with the destruction of all organelles and the formation of several vacuoles, the contents of which presented various degrees of electron-density, or showed an empty appearance. Some of these vacuoles seemed to be extruding from the cytoplasm as apoptotic bodies and others presented organelles inside. Similar alterations were observed after in vivo treatment. At 40 min after treatment of a microfilaremic individual with DEC, almost all microfilariae observed had lost their sheaths; and, in some of them, remains of the microfilarial sheath on the larval surface could be detected. Numerous vacuoles were observed, mainly in the hypodermis and somatic cells, showing organelles inside or an empty appearance. Condensed chromatin was also observed in some somatic cells. At 1 h after treatment of a microfilemic individual with DEC, microfilariae presented drastic morphological alterations, with large vacuoles within somatic cell cytoplasm and complete lysis of all cellular organelles. Therefore, both treatments with DEC in vitro and in vivo had a direct mechanism of action on the microfilariae of W. bancrofti, including organelle damage and apoptosis.

Diethylcarbamazine induces loss of microfilarial sheath of Wuchereria bancrofti.

Light microscopy analyses of microfilariae of Wuchereria bancrofti treated with DEC revealed a striking loss of the microfilarial sheath. However, no effect was observed on microfilariae of Litomosoides chagasfilhoi treated with DEC. For quantitative analyses microfilariae of W. bancrofti were processed for SEM. Controls, which have not received DEC, had 29.8% of exsheathed microfilariae. Conversely, the number of exsheathed microfilariae increased as increased DEC concentrations: 5 microg/ml (75.9%), 10 microg/ml (80.1%), and 50 microg/ml (87.7%). After DEC treatment some of sheathed microfilariae showed a wrinkled surface, and in some microfilariae, sheaths were observed being liberated almost intact from the larvae surface. But, frequently residues of the lost sheath over the surface were also observed. No damage was observed in the microfilariae cuticle. The present work shows quantitative data on the loss of the microfilarial sheath of W. bancrofti after treatment with DEC. Since no loss of microfilarial sheath was observed in microfilariae of L. chagasfilhoi submitted to the same conditions, DEC may present different mechanisms of action for distinct filarial species.

Immunocytochemical localization of antigens recognized by human antisera in infective larvae of Wuchereria bancrofti.

Ultrathin sections of L3 of Wuchereria bancrofti embedded in hydrophilic resin were incubated with antisera pools from individuals (1) asymptomatic microfilaremic with different microfilaria (mf) densities (1-100, 101-500, and >1,000 mf/ml); (2) chronic with hydrocele or lymphedema; and (3) with no evidence of microfilaremia or clinical filariasis but residing in an endemic area. The groups of microfilaremic subjects studied presented differences relative to the intensity of labeling, with the density of gold particles per square micrometer proportional to microfilaremia. Incubation of ultrathin sections of W. bancrofti L3 larvae in the presence of antisera from patients exhibiting chronic obstructive lymphatic pathology of hydrocele and from individuals with clear clinical evidence of lymphedema exhibited a strong reaction in the same tissues. Except for the endemic normal group, all groups studied showed reactivity against epitopes in all tissues of infective larvae of W. bancrofti. The cuticle presented an intense labeling, suggesting a possible target structure for immune response.

The effects of diethylcarbamazine on the ultrastructure of microfilariae of Wuchereria bancrofti.

An ultrastructural study of microfilariae of Wuchereria bancrofti was performed after in vitro treatment with diethylcarbamazine. One of the first morphological alterations produced by treatment with 5, 10 or 50 microg/ml of DEC was the loss of microfilarial sheaths. Drastic effects of DEC were only detected when microfilariae were exsheathed. Microfilariae treated with 5, 10 or 50 microg/ml DEC showed severely affected organelles, formation of several vacuoles mainly in the hypodermis, and cytolysis. Some microfilariae showed extreme cellular disorganization with abundance of electron-dense degenerating organelles, numerous large vacuoles, and nuclear condensation. Lamellar bodies probably related to an assembly of endoplasmic reticulum membranes were observed in some damaged microfilariae. Thus, in vitro treatment with concentrations of DEC similar to therapeutic conditions had direct effect by causing a loss of the microfilarial sheath of W. bancrofti with subsequent damage of organelles and apoptosis.

Localisation and distribution of Wuchereria bancrofti antigens recognised by antisera from tropical pulmonary eosinophilia and from individuals with intestinal helminths.

Serological analyses of sera from patients with a typical picture of filarial tropical pulmonary eosinophilia (TPE) and sera from patients from a region non-endemic for filariasis harbouring intestinal helminths, as Ascaris lumbricoides and Strongyloids stercoralis, revealed equally high titers of IgG4, usually considered diagnostic for filariasis. Ultrathin sections of adult worms of W. bancrofti embedded in the hydrophilic resin L.R. White were incubated with sera from patients with a typical picture of filarial tropical pulmonary eosinophilia (TPE) and sera from patients of a region that was non-endemic for filarial TPE but endemic for intestinal helminths. Both groups had a similar pattern of labelling, except that the labelling intensity was higher with the sera of patients with filarial TPE. The present study indicates relevant epitopes recognised by sera from TPE-patients and also individuals with intestinal helminths in all tissues of adult and intra-uterine microfilaria of W. bancrofti, instead of being localised in a specific nematode region. These findings suggest that people from areas not endemic for filariasis, but who harbour intestinal helminths, also share antifilarial antibodies in their serum that recognise antigens of adult worms and intrauterine microfilaria of W. bancrofti.

Ultrastructural characterization of intracellular bacteria of Wuchereria bancrofti.

Ultrastructural observations on the structure and distribution of endosymbiotic bacteria within the tissues of Wuchereria bancrofti are described. In female worms the organisms were observed in the lateral cords of the hypodermis, oocytes, developing eggs and in intrauterine microfilariae. Organisms were also detected in blood microfilariae and in the intestine of third-stage larvae. Bacteria were not observed in male worms.

Fine structure of intrascrotal lymphatic vessels infected by Wuchereria bancrofti adult worms.

Lymphangiectasia represents a basic phenomenon of acute and chronic pathology in lymphatic filariasis, and the prevalence or degree of lymphatic dilation caused by filarial worms is considered an indirect measurement of the altered lymphatic function. We examined the morphological alterations of intrascrotal lymphatic vessels surgically removed from a volunteer infected by adult worms of Wuchereria bancrofti. Scanning electron microscopy revealed lymphatic vessels with an irregular endothelium and adherent flattened lymphocytes and macrophages in variable proportions. On transmission electron microscopy the lymphatic vessels showed a thin endothelium which had an irregular contour and projected several cytoplasmic processes into the lumen. Numerous micropinocytotic vesicles and collagen fibers were abundant and disorganized. The hyperplastic endothelial cells and the subendothelial fibrosis suggest that abnormal changes in these cells may play a crucial role in the development of lymphangiectasia.

Wolbachia bacteria of filarial nematodes.

The finding that the intracellular bacteria of filarial nematodes are related to the Wolbachia symbionts of arthropods has generated great interest. Here, Mark Taylor and Achim Hoerauf review recent studies by several groups on the structure, distribution and phylogeny of these endosymbionts, and discuss the potential role for these bacteria in filarial disease and as a target for chemotherapy.

Immunocytochemical localization and distribution of human albumin in Wuchereria bancrofti adult worms.

To determine whether albumin is present on adult worms of Wuchereria bancrofti, thin sections of resin-embedded parasites were incubated with a specific antiserum to human albumin. With the exception of the epicuticle, all layers of the cuticle and the hypodermis were intensely labeled. Concentration of gold particles was observed within infoldings of the hypodermal membrane. Moderate labeling of the thin basement membrane that lines the pseudocelomic cavity and the gonoduct was also observed. Within the uterus, ovular membranes labeled intensely; groups of organized particles were seen below ovular membranes and also within invaginations of microfilarial embryos. In contrast, few gold particles were seen on the surface of mature intrauterine microfilariae. No labeling was observed in control sections incubated with antiserum preadsorbed with purified human albumin. The findings suggest that human albumin may be essential for the nutrition and development of W bancrofti microfilariae.

Scanning electron microscopic study of microfilaria and the third stage larva of Wuchereria bancrofti.

The surface structures of microfilaria and of the third stage larva of Wuchereria bancrofti were studied by scanning electron microscopy. Distinct features were observed that could be used for differentiating species of this parasite. Specifically, the sheath of microfilariae of W. bancrofti projected beyond the head. The head region of the microfilaria was composed of a cephalic cap with hook, mouth and amphidial opening, and its cuticle showed annulation. Spines were absent at the first transverse annulation, and the tail end showed a slight constriction. In the infective stage larva, characters which are used for differentiating species, such as the two bubble-like ventro-lateral papillae and one dorso-terminal papilla were rather similar to each other in size, but the grooves seen around the base were absent. A previously unreported feature of the third stage larva of W. bancrofti that was discovered in this study is a papilliform process on the left side of the posterior region, between the anus and the tail end.