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Loa loa antigen detection by ELISA: a new approach to diagnosis.

Circulating immune complexes associated Loa loa antigen in sera of 27 subjects with Loa loa (13 'occult' and 14 microfilaraemic infections) was detected with Polyethylene glycol enzyme linked immunosorbent assay (PEG-ELISA) in a controlled study. Loa loa antigen was detected in all 27 sera. There was no correlation between antigen levels and microfilaraemia density. Cross reaction with Mansonella perstans was found. Although this serological test is able to detect all Loa loa infections, its use is limited by cross reaction with other filariasis. It is hoped that sensitivity will be improved by using monoclonal antibodies.

Adolescent

Cloning and characterization of a Loa loa-specific repetitive DNA.

A Loa loa EcoRI genomic library in lambda gt11 was screened with 32P-labeled L. loa DNA and 1 repetitive clone, LL20, was isolated. An 800-bp Rsa I fragment of LL20, which is L. loa specific, was subcloned into pUC19 and the recombinant plasmid was designated pRsa4. While the 3.8-kb Eco RI fragment of LL20 cross-hybridized to other filarial DNA under low stringency conditions, the 800-bp fragment of pRsa4 was L. loa specific under the same conditions. Further characterization of the insert of pRsa4 was therefore carried out. Its lower limit of detection is 800 pg of L. loa genomic DNA, it has a low copy number (50-100) and an interspersed distribution in the genome. As a probe it does not distinguish between simian and human L. loa DNA. The nucleotide sequence contains 69% A + T and 31% G + C and shows no notable internal repeats.

Animals

Lymphadenitis caused by Loa loa.

The World Health Organization's Collaborating Centre for the Histopathology of Filarial Diseases of Man contains specimens of lymph node from 13 patients infected by the filarial nematode Loa loa. Ten of these nodes have distinctive microscopic features characterized by distended sinuses that contain histiocytes and eosinophils and by atrophy of lymphoid follicles. Less striking features include fibrosis of capsule and trabeculae, dilated lymphatic vessels of capsule and medulla, and inflammatory cell infiltrates. We believe that these changes, although in themselves nonspecific, are characteristic of lymphadenitis caused by Loa loa. These 10 lymph nodes were removed from the inguinal region--one from each of 10 native Zairians . At the time of herniorrhaphy the nodes in eight patients were found to be enlarged and were removed for diagnosis. Seven of the 10 patients were infected with Dipetalonema perstans as well as Loa loa, and one of these seven had three filarial infections--L. loa, D. perstans and D. streptocerca . Lymph nodes from other patients infected by other filariae that were available for study at the AFIP did not have these histopathologic features.

Adult

Filariasis due to Loa loa and Mansonella perstans: distribution in the region of Okondja, Haut-Ogooué Province, Gabon, with parasitological and serological follow-up over one year.

The prevalence of Loa loa and Mansonella perstans filariasis has been determined in 6 rural villages in eastern Gabon. Between 18.9 and 27.2% of people carry L. loa microfilariae with an overall microfilarial rate of 25.1%. The microfilarial rate for M. perstans was more variable, between 33.3 and 62.2% (average 49.1%). No significant difference was seen in the microfilarial rate with age over 15 years for either parasite, but men were infected more frequently than women. Anti-L. loa antibody titres were measured, using a homologous microfilarial antigen in ELISA. Taking the parasitological and immunological evaluations together, only 10% of the sample population appear to be free of these filarial infections. L. loa and M. perstans microfilaraemia and corresponding serology were also investigated twice in 150 people at a one-year interval. 99.1% of the cases who had no circulating L. loa microfilaria in March 1984 still did not show any 12 months later. Similarly, 97.1% of the untreated, microfilaraemic cases still harboured this parasite a year later. The same was not observed for M. perstans, since microfilariae appeared or disappeared in 26.7% of the cases. This suggests different dynamics for the two filarial infections. Variation in individual anti-L. loa antibody titres was low. The possibility of a genetic influence on the expression of loiasis is discussed.

Adolescent

Loa loa microfilariae aspirated during oocyte retrieval.

Loa loa is a filarial parasite which is mainly confined to West and Central Africa. Although considered as mildly pathogenic for people living in endemic areas, its appearance in other places might be a cause for concern. We report here the unusual finding of Loa loa microfilariae in the follicular aspirate of a 35-year-old black women during oocyte retrieval for in-vitro fertilization. Most likely these microfilariae were aspirated, along with follicular fluid, from the small blood vessels around the ovarian follicles, which are frequently lacerated during ovarian puncture. Despite the spectacular microscopic view of vigorous moving worms trapped on the cumulus cells, the presence of Loa loa microfilariae does not seem to prevent fertilization and cleavage completely.

Adult

Age of appearance of IgG, IgM, and IgE antibodies specific for Loa loa in Gabonese children.

IgG, IgM, and IgE antibodies against the filaria Loa loa were measured in umbilical cord blood and in blood from young Gabonese children by an ELISA technique using a homologous metabolic antigen. For children in eight consecutive age groups and adults the percentage of the population positive for each of the antibody classes was determined. The number of children with maternal IgG decreased until one year of age when new synthesis began to become apparent. IgM antibodies were detected only after six months, probably indicating an early infancy as opposed to a fetal infection. The percentage of individuals positive for IgM or IgE reached a peak between two and three years old, followed by a slight decline. Over half of the individuals over one year of age had IgM antibody against L. loa, indicating long-term synthesis of this class of immunoglobulin in many people. In the first two years of life, IgE antibodies were usually accompanied by L. loa-specific IgM. This specific IgE did not appear to trigger the synthesis of nonspecific IgE. By the age of two, 95% of the population had some antibodies against L. loa and by five the percentage of individuals positive for each antibody class had reached adult levels.

Adult

Antibody-dependent cell-mediated immune reactions to Loa loa microfilariae in amicrofilaraemic subjects.

Antibody-mediated mechanisms that could be important in controlling microfilaraemia in Loa loa infected amicrofilaraemic adults (mf-ve) were studied. These subjects were selected as having a verified ocular passage of an adult L. loa but being amicrofilaraemic and without recent diethylcarbamazine treatment. Sera from 37 mf-ve subjects were compared to 14 sera from heavily (greater than 4000 mf/ml) infected subjects (mf+ve) and 9 sera from Caucasian control subjects for their reactions with L. loa mf (mf). Many mf-ve sera (22/37) were strongly positive in immunofluorescence (IFAT) on living mf. Mf+ve sera were negative, or only weakly positive, and Caucasian sera were negative. Clinical signs were not significantly different between IFAT reactive and non-reactive mf-ve subjects. Approximately half of the IFAT positive, mf-ve sera were also able to agglutinate mf; no other sera were active in this test. Titres ranged from log2 3-6 and in most cases, 9/11, the agglutination reaction was mercaptoethanol-sensitive. Antibody-dependent cellular adherence was studied using mf and leukocytes from uninfected donors. Using cryopreserved mf many heat-inactivated mf-ve sera gave strong reactions with obvious adherence by 4 h and few motile mf remained by 16 h but when fresh mf were employed these reactions were weak. However, addition of complement to many (10/11) mf-ve sera considerably enhanced adherence to fresh mf. The effect of various treatments on the complement source indicated a role for both the classical and alternative pathways. The cells attached to mf were mainly neutrophils (83%) with some eosinophils (15%) and few mononuclear cells (2%). The common occurrence of antibodies able to mediate complement-dependent adherence of polymorphonuclear leukocytes to L. loa mf in the sera of mf-ve subjects may indicate that such a mechanism is important in controlling microfilaraemia in vivo.

Adult

Loa loa: development to the infective stage in an American deerfly, Chrysops atlanticus.

Chrysops atlanticus, an American species of deerfly collected along the Mississippi Gulf Coast, will support the development of the microfilaria of human Loa loa to the infective stage. Development takes place in the fat body of the fly and requires 9 to 10 days of development and pattern of morphogenesis of the parasite in C. atlanticus is virtually identical to that described in natural African vectors. Chrysops atlanticus will support the development of large numbers of L. loa to the infective stage without apparent ill effects. Two uninfected patas nonkeys each were given 75 third-stage larvae obtained from experimentally infected C. atlanticus. Both animals developed patent infections in approximately 5 months, clearly demonstrating that the entire life cycle of Loa loa can be maintained in the laboratory outside endemic areas.

Animals

Macaca fascicularis, a nonpermissive host for the human filarial parasite Loa loa.

The ability of the filarial nematode Loa loa to infect 2 species of primates was studied. The primate species selected were closely related to species known to be susceptible. A mandrill (Mandrillus sphinx) and 6 cynomolgus monkeys (Macaca fascularis) were infected by subcutaneous injection of third-stage larvae of human L. loa from Gabon. The mandrill developed microfilaremia with an estimated prepatent period of 147 days, but microfilariae were not detected in any of the cynomolgus monkeys. Thus, mandrills appear permissive to human L. loa, whereas cynomolgus monkeys are not. Serum antibody responses were examined on western blots of adult L. loa antigens. Preinfection sera from all animals gave no reactions, but, after infection, sera from cynomolgus monkeys reacted more intensely and with more antigens than mandrill sera. Antibodies were still detectable in cynomolgus monkeys 15 mo postinfection. These reactions were compared with those found using human infection sera. Reactions with the cynomolgus monkey sera resembled those found with resistant endemic and amicrofilaremic human sera.

Animals

[Cameroon swelling; infection by the filaria Loa loa].

Recurrent skin edemata due to an infestation with the filaria Loa loa are called Cameroon or Calabar swellings. These are tense elastic, circumscribed, indolent swellings, which are just slightly red coloured and disappear within 24 to 48 hours. Intervals of several weeks or months may occur. One should think of Loa loa disease seeing travellers return from Loa endemic areas (West Africa) with recurrent, transitory edema and eosinophilia. The stay in these areas may have been several years before! Treatment and prophylaxis: Diethylcarbamazine.

Cameroon

Identification of a surface antigen on Loa loa microfilariae the recognition of which correlates with the amicrofilaremic state in man.

Filarial infections induce a spectrum of disease in their natural hosts, and by correlating immunity found in individuals with their disease pattern, one may delineate non-pathogenic, protective mechanisms. Loa loa is causal of mild to moderate pathology, and it is unique among the human filaria in that adult worms are occasionally visible during subconjunctival migration. To study immune mechanisms controlling microfilaremia, sera from 15 subjects with amicrofilaremic occult loiasis (OL) were compared with sera from 10 subjects with microfilaremic loiasis (ML) microfilaremia, (greater than 4000/ml) for their reactions with living microfilariae (mf). An IFA was first used to detect antibodies able to bind to the surface of living L. loa mf. ML subjects either did not react (7/10) or reacted only very weakly (3/10). Highly reactive sera were found only in OL subjects; 7/15 gave very bright fluorescence, 5/15 gave moderate reactions, and 3/15 were negative. Most of these antibodies were of the IgG class. Sera from all subjects were also reacted with living mf in an antibody-dependent cellular adherence test using normal leukocytes. Sera that were strongly positive in IFA showed strong adherence and IFA-negative sera were non-reactive. To identify the Ag involved, mf were surface iodinated, detergent-extracted Ag were immunoprecipitated, and Mr was determined on SDS-PAGE. Several OL sera, all highly reactive in the above tests, precipitated a 23-kDa molecule with which all ML sea failed to react. Sera from a mandrill experimentally infected with L. loa also precipitated the 23-kDa Ag when taken post-patency. In conclusion, it appears that certain people who control L. loa microfilaremia have high levels of IgG antibodies that bind to a surface Ag of 23 kDa and are able to mediate cellular adherence.

Adhesiveness

High levels of parasite-specific IgG4 in the absence of microfilaremia in Loa loa infection.

The specificity of IgG4 as marker of infection has been investigated. The study was based on two well defined clinical groups: amicrofilaremic individuals with verified ocular passage of adult worms of Loa loa, and microfilaremic patients. Both groups were compared to Africans not exposed to loiasis infection and to Europeans. The study revealed that there is no significant difference in the level of parasite-specific IgG4 between individuals with occult infection (i.e. amicrofilaraemic, but infected) and microfilaremic individuals, but there is a significant difference between L. loa infected individuals and Mansonella perstans exposed people. IgG4 of individuals exposed to L. loa infection recognised specific antigens in the molecular weight range 12-30 kDa. We conclude that the elevated level of filarial-specific IgG4 is therefore not dependent upon the presence of circulating microfilariae and that serology using homologous L. loa low molecular weight antigens, can facilitate specific diagnosis of occult loiasis in an endemic area with mixed filarial infections.

Animals

Electron microscope study of the body wall and the gut of adult Loa loa.

The morphology of the body wall and the gut in the midbody region of adult male and female Loa loa originating from patients in Gabon was studied by transmission and scanning electron microscopy. The cuticle of the dorsal and ventral regions consists of ten layers. In the lateral regions the cuticle is thicker and includes two additional layers. The thin hypodermis contains numerous transhypodermal fibres. A row of median cells is situated between the syncytia in each lateral chord. No intracellular bacteria were observed. The cross-sections of each of the four muscle sectors are comprised of approximately 12 muscle cells of the coelomyarian type. The plasm of the gut cells contains large vacuoles and several mitochondria. The intestinal wall surrounds a wide lumen filled with material which occasionally contains cellular structures. The morphology of L. loa is compared with that of adult Onchocerca volvulus and Brugia malayi. The gut of the adult L. loa has the typical nematode morphology, which might be an indication of its normal function in nutrition. The multilayered cuticle with the rather smooth surface, and the prominent muscles correspond to the migratory activity of this filaria.

Animals

Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis.

Possible endocrinological repercussions of infection with Loa loa and Mansonella perstans filariae were studied in Gabonese subjects. Microfilaremic males were compared with amicrofilaremic controls. In the infected group 13/105 subjects (12%) presented only abnormally low serum levels of testosterone (less than 4 ng/ml), 25/105 (24%) only abnormally high serum levels of gonadotrophins, FSH (greater than 15 mIU/ml) and LH (greater than 20 mIU/ml), and 22/105 (21%) presented anomalies in both testosterone and gonadotrophin levels. One out of 68 control subjects had 3.6 ng/ml seric testosterone and all had normal levels of gonadotrophins. Ecdysteroids were detected (greater than 0.025 ng/ml) in the serum of 87/97 (90%) microfilaremic subjects (GM 0.123 ng/ml) compared to 12/64 (19%) controls (GM 0.030 ng/ml). Ecdysteroids were detected in the urine of all subjects, infected (GM 8.468 ng/ml) as well as control (GM 1.245 ng/ml). The hormonal perturbations were correlated with the levels of Loa loa microfilaremia but not with those of serum and urinary ecdysteroids. These results demonstrate that microfilaremic subjects often show endocrinal signs of hypogonadism and present appreciable levels of ecdysteroids in serum and urine. A direct role for parasitic ecdysteroids in hypogonadism remains to be demonstrated.

Adult

The identification and partial characterization of an immunodominant 29-31 kilodalton surface antigen expressed by adult worms of the human filaria Loa loa.

Detergent solubilized extracts of 125Iodogen surface labelled adult Loa loa revealed a relatively simple profile consisting of a strongly labelled molecule at 29-31 kDa and weakly labelled molecules at 14.5, 17, 21, 23, 34, 58, and 86 kDa. Residents of a L. loa endemic zone were assessed clinically and parasitologically and classified as microfilaremic, amicrofilaremic with documented ocular passage of adult worms, or 'resistant' subjects without any signs of infection. Sera from these subjects were used to identify L. loa adult surface antigens. All 'resistant' sera immunoprecipitated the 29-31 kDa antigen although some were more strongly reactive than others. The amicrofilaremic sera strongly immunoprecipitated the 29-31 kDa antigen, whereas microfilaremic sera reacted weakly or not at all with this antigen. Longer exposures of immunoprecipitates of strongly reactive sera revealed the recognition of additional antigens of 86, 44, 34, 23, 21, 17 and 14.5 kDa. Studies with heterologous sera demonstrated that these antigens contain cross-reactive epitopes which are restricted to filarial parasites. Biochemical characterization of the predominant 29-31 kDa antigen showed that it bound concanavalin A, was sensitive to proteases, and its antigenicity was resistant to heat but sensitive to periodate and endo-beta-N-acetylglucosaminidase H. These observations suggest that it is a glycoprotein containing mannose and N-acetylglucosamine residues and that the carbohydrate moiety is important for antibody binding. The importance of the 29-31 kDa glycoprotein in the immunobiology of loaiasis is suggested by the finding that resistant and infected amicrofilaremic individuals have strongly reactive IgG antibodies to this antigen.

Animals

Effect of attraction factors on the sampling of Chrysops silacea and C. dimidiata (Diptera: Tabanidae), vectors of Loa loa (Filaroidea: Onchocercidae) filariasis.

The effects of fire and human host density on Chrysops silacea and C. dimidiata abundance and age structure was evaluated at sites of Loa loa filariasis transmission in the Congo rain forest. Fire increased the catch of C. silacea 8.5-fold at ground level and 5-fold in the canopy, but did not modify the catch size of C. dimidiata. Catch size increased 2-fold when a pygmy camp located near the collecting station was inhabited. The presence of fire did not alter the parity and Loa loa infection rates in the populations.

Animals