Morphometric analysis of nodules in human onchocerciasis collected in communities of the southern Chiapas focus, México.

Human onchocerciasis is a disease that remains as an important public health problem. The morphometric and physical characteristics of 363 Onchocerca volvulus nodules collected in the major endemic focus of onchocerciasis in Southern Chiapas (Soconusco), was assessed. In the present work we found that treatment the morphometry of 363 onchocercal nodules preserved in a 67% glycerol solution was determined by measuring the length, width and thick of each nodule with a Vernier caliper. The mass was determined with an analytical balance and the volume by measuring the water displacement, while the specific gravity was calculated by dividing mass over the volume. Statistical analysis was calculated for each parameter. The results showed that the nodules were rather longer than wider or thicker. Morphometric characteristics were 9.87 +/-3.70 (mean +/- standard deviation), 7.52 +/- 2.81, and 4.62 +/-+/- 2.06 mm for length, width and thick respectively. In regard to the shape, 62.81% of the nodules showed a lenticular shape, while 18.18% were spherical and 19.01% were ovoid. Based on the distribution of frequencies of the length, the nodules were classified in three groups: the "small" (5.77 +/- 0.73 mm; n = 104, 28.65%), the "medium" group (9.86 +/- 2.05 mm; n = 203 nodules, 55.92%), and the group of the "big" ones (16.03 +/- 1.91 mm; n = 56, 15.43%). Moreover, the physical characteristics were: for the mass 0.33 +/- 0.24 g, the volume of displaced water was 0.28 +/- 0.26 ml, and the specific gravity was 1.10 +/- 0.55 g/ml. The results indicated that most of the Mexican Onchocerca nodules have a lenticular shape with average size of 10x7x5 mm, which is useful in the knowledge of the genus biodiversity and can be taken as a parameter in clinical or epidemiological trials, where onchocerciasis remains as a public health problem.

Influence of different processing procedures on the reproductive capacity of Trichinella spiralis in pork meat.

The aim of this work was to determine the influence of different processing procedures and preparations on the viability and infectivity of Trichinella spiralis ML. The muscles of limbs tongue and masseters of pigs experimentally infected were collected, splitted to pieces, and pooled. Five batches were used for the following processing procedures: (1) seasoning with "adobo", commercially acquired chilli and several other spices, (2) "wet-curing" by immersion of meat pieces in 3% brine during 24 hours, (3) cold storage without any further processing or preparation, (4) freezing to -20 degrees C and, (5) drying for 24 hours at 60 degrees C. Samples were stored at 4 degrees C for 15, 45, 60, 75, 90, 105 or 266 days after preparation. At the last-mentioned dates, ML were recovered and used to determine the reproductive capacity by infecting naïve mice. The state of meat conservation or spoilage respectively was tested by visual and tactile examination. In samples treated by freezing or drying no motile larvae were found after artificial digestion and, following inoculation of mice with larvae recovered from these groups, no ML were founded after 40 days of infection. After the artificial digestion of the cold stored samples, the ones seasoned with "adobo" and "wet-cured", a number of motile ML were consistently obtained. Initial reproductive capacity index was as of 80+/-0.5, then rates decreased to 60 - 70 between days 15 and 105 PT and dropped to 40+/-6.7 at day 266 for seasoned, 33+/-2.7 for cold-stored and 33+/-2.5 for cured samples. The influence of storage time (p=0.000005; factorial ANOVA) but not for processing procedure (p=0.724; factorial ANOVA) were statistically significant. The sensorial examination of the meat samples showed severe changes caused by spoilage in odour, texture and colour from day 45 of storage. Data reported from this trial proves that curing or flavoring do not inactivate the Trichinella Mexican strain, although cold storage for more than three months led to a partial decrease of the reproductive capacity. Freezing and drying seemed to be effective measures to eliminate the ML infectivity.

Study of the reproductive capacity of Trichinella spiralis recovered from experimentally infected mice under-dosed with albendazole or mebendazole.

The reproductive capacity of Trichinella spiralis recovered from experimentally infected mice under-dosed with albendazole (ALB) or mebendazole (MEB) was studied. Different groups of male C57/BL mice were infected with 10 +/- 0.5 muscular larvae (ML) per gram of body weight and treated with a single dose by oral (20 mg/kg) of ALB, MEB or praziquantel (PZQ) given at 5th day post infection (DPI), during the intestinal phase of infection. In other group of mice, treatment with the same drugs and dosage was for seven days, starting at day 45 PI through the stage of encapsulating larvae (parenteral phase of infection). A reduction of 72.9 to 89.9% in the parasitic load was observed in ALB or MEB treated groups but not in mice untreated or administered with PZQ. The recovered larvae were used to infect naïve mice and, after 45 DPI, a similar Reproductive Capacity Index (RCI) was observed between the different groups (P=0.323, one-way ANOVA), either from mice infected with larvae recovered from the intestinal treatments (RCI-ALB = 51.6 +/- 12.1 and RCI-MEB = 49.2 +/- 14.) or from the parenteral ones (RCI-ALB = 52.2 +/- 14.0 and RCI-MEB = 51.9 +/- 11.8). The RCI of non-treated ML was 59.5 +/- 7.7 and 57.9 +/- 15.9 for PZQ. This information is significant for practical strategies when under-dosage is dispensed.

Searching for antibodies against Trichinella spiralis in the sera of patients with fever of unknown cause.

Human cases of trichinellosis are often difficult to identify because the signs and symptoms of the disease, if the infection produces any at all, are non-specific, being similar to those observed in several other infectious diseases. In an investigation of Mexican patients with fever of unknown aetiology, attempts were made to develop a serodiagnostic test for the detection of antibodies specific for Trichinella spiralis. The excretory and secretory products of T. spiralis larvae (from the muscle tissue of experimentally infected rats) were used as the antigens in an enzyme-linked immuno-electrotransfer blot assay. The sera tested came from patients with fever of unknown cause (N=250), patients confirmed to have infectious or parasitic diseases other than trichinellosis (N=134) and 168 apparently healthy subjects. Overall, 4% of the samples from the febrile group, 1.8% of those from the healthy subjects but none of the sera from those with 'other diseases' reacted with the antigens of interest (of 45, 49 and 55 kDa). The results not only confirm that human infection with T. spiralis may be asymptomatic but also indicate that such infection may be mis-diagnosed.

Detection of circulating and fecal Trichinella spiralis antigens during experimental infection using monoclonal antibodies against the new born larvae.

Different assays to detect antigens of Trichinella spiralis during current infection have been standardized, although sensitivity values have been the main limitation to use them as routine diagnostic test. We report the production and use of monoclonal antibodies (MAbs) against the new born larvae which recognize both somatic and metabolic antigens from adult and muscular larvae (ML). We used two IgG3 MAbs (4B1, 4B2) and two IgG2a (2D3, 2D4) to detect antigens during experimental infection. All MAbs detect fecal antigens starting the second to third week post infection (wpi), although it was less clear with MAbs 4B1 and 4B2, while circulating antigens were detected from third to fourth wpi. Thus, the recognition of shared antigens among T. spiralis developmental stages can be used for early diagnosis of trichinellosis.

Serological evaluation of thin-layer immunoassay-enzyme-linked immunosorbent assay for antibody detection in human trichinellosis.

A new immunoenzymatic test, named the thin-layer immunoassay-enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 microl each of antigen (7 microg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis.

[Search for antibodies against Trichinella spiralis in free roaming rodents caught in a zoological park from Mexico City]. / Búsqueda de anticuerpos contra Trichinella spiralis en roedores de libre desplazamiento capturados en un zoológico de la Ciudad de México.

A serological survey to search for antibodies against T. spiralis was performed in free roaming rats (n = 64) and mice (n = 35) caught in a zoological park from Mexico City. Serum samples were analyzed by ELISA and immunoelectrotransfer blot assay (EIBT). None serum show positive absorbance values in ELISA nor recognized T. spiralis specific antigenic fractions in EIBT. However, two rat samples recognized three antigens of 31, 37 y 55 kDa, while one of them reacted with two additional antigens of 64 and 67 kDa. As it is known that the antigen epitope profiles varied among Trichinella species, it could be possible that in rats, there is 3% of antibody prevalence against Trichinella sp.; however, due that other organisms could induce the production of cross-reacting antibodies, such conclusion can not be supported at all. These results suggest that T. spiralis was not part of helminthological fauna in these rodents.

DIG-ELISA test in onchocerciasis. Serum antibodies and probability of infection.

An immunoenzymatic test (DIG-ELISA) was serologically evaluated for the serodiagnosis of onchocerciasis. Control and infected sera from the onchocerciasis endemic area of Mexico was collected and the donors assessed for onchocerciasis according to parasitological, clinical, and epidemiological data. The sera were submitted to the DIG-ELISA test using a crude extract prepared from O. volvulus adult worms which had been preserved in nodules in 67% glycerol. The test showed a 100% sensitivity with sera from 38 microfilariae carriers and 96% specificity with sera from 133 non-infected people living outside the endemic zone. In addition, seropositivity was 52.9% with samples from non-onchocercotic people living inside the endemic area, while 82.5 and 90.5% of sera from patients with clinical symptoms and subcutaneous nodules, respectively, were positive. A high rate (30%) of cross-reactivity with serum samples from people infected with Wuchereria bancrofti or Brugia timori was obtained, which is in contrast with the low seropositivity rates (7.4%) obtained with sera from patients infected with other parasites. These results suggest that DIG-ELISA test may be a useful serological test for antibody detection in onchocerciasis, especially for epidemiological surveillance of disease, but because of the high cross-reactivity observed, its use must be limited to areas where this parasitic infection does not coexist with other human filariasis. Finally, the arrangement of sera in groups according to the relative likelihood to have onchocerciasis seems to be a useful procedure for evaluation of serological tests.

Detection of serum antibodies in human Hymenolepis infection by enzyme immunoassay.

Although hymenolepiasis is the commonest cestode infection of man, there are no data available on the human immune response to this parasite. Thus, in order to determine if infection induces antibodies against Hymenolepis nana antigens, sera from 52 infected children were initially studied on Ouchterlony plates and then by enzyme-linked immunosorbent assay (ELISA), using a crude antigenic extract prepared from scolex and neck regions of adult worms. In addition, sera from persons with cysticercosis, taeniasis and other parasitoses, and normal human sera, were studied. Only one serum from the Hymenolepis group showed precipitin antibodies against H. nana antigen, while several were positive by ELISA. The sensitivity of the ELISA was 84.62% and its specificity was 100%. Very high cross-reactivity rates were obtained with taeniasis (70.6%) and cysticercosis (75%) sera. These results show that Hymenolepis infection in man induces a low but detectable humoral immune response. Although not useful for diagnostic purposes, this may be relevant to the serodiagnosis of other tissue cestode infections of man, since antibodies detected in serological tests used for the diagnosis of cysticercosis, and probably hydatidosis, could be induced by H. nana instead of Taenia solium or Echinococcus larvae.

Specific detection of human antibodies to Onchocerca volvulus.

Specific diagnosis of antibodies to Onchocerca was achieved through (1) the construction of direct and indirect ELISA systems, and (2) restricting ELISA assays to the IgG4 class. The direct ELISA was based on the isolation of a surface derived, low molecular weight surface antigen preparation containing two main antigens (M. wt. 16.2 and 12.8 kDA) as defined by Western blot analysis. The direct ELISA system detected antibodies in children of six years old, and may therefore be applicable to detecting reinvasion in OCP areas of Onchocerca volvulus control. The indirect ELISA system was a competitive binding ELISA-based assay using a monoclonal antibody recognising two Onchocerca components (M. wts. 15.6 and 25.9) on a Western blot. The direct and indirect ELISA systems were similarly specific and sensitive when evaluated in a preliminary survey. The direct ELISA system yielded a specificity and sensitivity of: 100% and 100% respectively, using Mexican endemic and Mexican intestinal nematode infection sera as positive and negative controls respectively: 91% and 96% respectively, using Venezuelan endemic and Venezuelan Mansonella ozzardi infection sera as positive and negative controls, respectively: 87% and 93% respectively, using African endemic and Papuan (New Guinea) Wuchereria bancrofti infection sera as positive and negative controls respectively: 93% and 93% respectively, using African endemic and Indian W. bancrofti infection sera as positive and negative controls respectively. Similar specificity and sensitivity levels were obtained when the same comparisons were made using the indirect (inhibition) ELISA assay. These values may be contrasted with the currently used PBS extract of O. volvulus which yielded specificities of less than 10% in all the above comparisons.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of surface molecules of adult Brugia parasites: cross-reactivity with antibody from Wuchereria, Onchocerca and other human filarial infections.

The extent of structural and immunological similarity between surface antigens of three species of Brugia filarial parasites was studied by lodogen-mediated surface labelling of adult worms of B. malayi, B. pahangi and B. timori. The close homology and cross-reactivity between these antigens reported in previous surface labelling studies with Bolton-Hunter reagent, was verified in this system. The surface antigens of adult B. pahangi are also recognised by antibody from patients with Wuchereria bancrofti, the major human lymphatic filariae, and by antibody to Loa loa and Mansonella perstans. Further experiments have begun to establish the boundaries of these cross-reactions: antibodies to nonfilarial nematodes such as Trichinella, Necator and Strongyloides does not recognise the adult surface antigens; however, although most anti-Onchocerca sera show little or no reaction to the major (29 kDa) surface antigen, there is consistent reactivity to the secondary 20 kDa antigen, and extensive recognition of a minor antigen of 15 kDa.

Human macrophages and T-lymphocyte subsets infiltrating nodules of Onchocerca volvulus.

The nature of the lymphoid infiltrate in nodules of Onchocerca volvulus was assessed using monoclonal antibodies to lymphoid cell surface markers. Although B cells were generally absent, T cells were present, but in variable amounts. The ratio of T4+ (helper phenotype) to T8+ (suppressor-cytotoxic phenotype) was usually in the normal peripheral blood range of about 3, although ratios ranging from 1 to 10 were seen in selected areas of the onchocercoma. The possibility of immunosuppression through dominance of T4+, Leu-8+ cells (suppressor-inducer phenotype) within the T4+ population was also excluded. The T cells did not tend to concentrate in close proximity to the parasite, and there was no general bias in favour of the T suppressor cell phenotype (T8) within the infiltrate. Macrophages and dendritic cells were consistently observed and consisted of three defined cell types in approximately equal proportions: normal, unactivated macrophages (HLA-DR-, acid phosphatase positive), activated macrophages (HLA-DR+, acid phosphatase positive) and cells of dendritic morphology (HLA-DR+, acid phosphatase negative). These results are discussed in relation to immune suppression in filariasis.

Successful application of the diffusion-in-gel-enzyme-linked immunosorbent assay (DIG-ELISA) in onchocerciasis.

The recently described DIG-ELISA test was evaluated on 38 sera from patients with onchocerciasis, using a soluble, crude extract of Onchocerca volvulus dissected out from glycerol-preserved nodules. Results showed that the diameters of the orange-yellow antibody-antigen reaction zones obtained with infected sera were significantly greater than those obtained with normal human sera. A close correlation was observed between the diameters of the reaction zones and antibody concentration (expressed as log of the serum dilution), indicating the potential utility of this test in the quantitative determination of serum antibodies. Because the test is easily performed, it can be used under field conditions when an immediate quantitative determination of serum antibodies is needed.

Identification of an antigen of Onchocerca volvulus of possible diagnostic use.

Adult Onchocerca volvulus worms obtained by enzyme digestion from nodules of infected Mexicans were radio-isotope labelled by the chloramine-T or Bolton-Hunter methods. No antigenic determinants were detected in extracts of worms labelled by the chloramine-T method but 3 antigens were detected in extracts of the Bolton-Hunter labelled worms. Two were present in such small amounts that it was impractical to investigate them further, but a major component of mol. wt 20 kDa was purified by gel filtration and used in a serological survey of inhabitants of villages in Southern Mexico. Using the 20 kDa antigen, which is superficially located on both sexes of O. volvulus, sera from both non-endemic and endemic regions were analysed by radio-immunoprecipitation of this antigen. In Southern Mexico, the average sensitivity of the test was 92%, and the specificity 98%. Whilst the 20 kDa antigen did not detect antibodies in the sera of Trinidadians infected with Wuncheria bancrofti or Mansonella ozzardi, this antigen detected high levels of antibodies in Indians exposed to W. bancrofti.

Quimioterapia de la onconcercosis: utilidad del mebendazol a nivel comunitario / Drug therapy of onchocercosis: use of mebendazole at communitary level

Resumen El mebendazol, compuesto que en un estudio controlado a nivel hospitalario demostró tener efecto sobre los adultos hembras y las microfilarias de Onchocerco volvulus. fue ahora evaluado en un estudio abierto comunitario para el tratamiento de la oncocercosis. Un total de 26 individuos de diferentes edades y sexos de una comunidad del foco endémico sur del estado de Chiapas, con diferentes densidades de microfilarias en la piel, fueron distribuidos en tres grupos de tratamiento: Grupo 1 formado por 11 hombres mayores de 14años que recibieron I g de mebendazol dos veces al día oralmente durante 4 semanas; Grupo 2 integrado por 6 mujeres mayores de 14 años que recibieron 100mg de dietilcarbamazina dos veces al día durante 4 semanas; y Grupo 3, de 9 menores de 14 aftas de ambos sexos, que recibieron 30 mg de mebendazol por Kg de peso al día. repartidos en dos tomas durante 4 semanas. En los grupos que recibieron mebendazol se observó una baja importante y sostenida en las densidades de microfiliarias, lo cual contrasta notablemente con lo observado en el grupo que recibió dietilcarbamazina. en quienes la baja de las densidades de microfilarias fue inmediata a la administración del medicamento (una semana) y a partir de los 6 meses se observ6 recuperación gradual en el número de microfilarias en la piel, persistiendo una alza lenta a los 14 meses posteriores al inicio del tratamiento. Los efectos secundarios se presentaron con más frecuencia y severidad en el grupo que recibió dietilcarbamazina que en los que se trataron con mebendazol. En ninguno de ellos se presentaron alteraciones oculares atribuibles al tratamiento. Estos resultados sugieren que el mebendazol es seguro y efectivo en el tratamiento de la oncocercosis en la comunidad y que puede ser preferible a la dietilcarbamazina

Comparative surface ultrastructure of adult Onchocerca volvulus recovered from human nodules by dissection or collagenase digestion.

The surface ultrastructure of male and female adult worms of Onchocerca volvulus obtained from human nodules by the technique of collagenase digestion has been compared with that of worms excised manually without the aid of enzyme treatment. No topographical differences have been identified.