PALEOPARASITOLOGY: PERSPECTIVES WITH NEW TECHNIQUES

Paleoparasitology is the study of parasites found in archaeological material. The development of this field of research began with histological identification of helminth eggs in mummy tissues, analysis of coprolites, and recently through molecular biology. An approach to the history of paleoparasitology is reviewed in this paper, with special reference to the studies of ancient DNA identified in archaeological material.

TOXOCARIASIS: SEROLOGICAL DIAGNOSIS BY INDIRECT ANTIBODY COMPETITION ELISA

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB). We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0%) had history of sexually transmitted diseases; 18/84 (21.4%) informed receiving or paying for sex; 3/84 (3.6%) had homosexual contact; 2/26 women (7.6%) had past history of multiple illegal abortions and 3/84 (3.6%) had been previously transfused. Four out of 62 donors (6.5%) had positive anti-nuclear factor (Hep2), with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4%) donors and other parasites in 8/62 (12.9%). Five (5.9%) of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2%) had negative results on the last visit, while 36/84 (42.9%) remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

Cyclospora cayetanensis in sputum and stool samples

We report the observation of acid-fast Cyclospora cayetanensis oocysts in a sputum sample. The patient, a 60 year-old, HIV negative man, was successfully treated for pulmonary tuberculosis during 1997. On February 1998, he was admitted to our center due to loss of weight, cough with purulent expectoration, dysphonia and a radiological picture of pulmonary fibrosis. Bacilloscopic study of sputum (negative for acid-fast bacilli) stained with Ziehl-Neelsen technique showed large (8-10 µm) spherical, acid-fast Cyclospora cayetanensis oocysts. No other pathogens were isolated on cultures from this sample or from laryngeal biopsy. Serial parasitologic studies showed C. cayetanensis and also eggs of Trichuris trichiura, Ascaris lumbricoides and Hymenolepis nana and of Entamoeba coli cysts. The patient lives in the outskirts of Buenos Aires in a brick-made house with potable water and works as builder of sewers. He travelled in several occasions to the rural area of province of Tucumán which has poor sanitary conditions. C. cayetanensis is an emergent agent of diarrhea and as far as we know this is the first time the parasite is observed in respiratory samples.

Paragonimosis in the Cajabamba and Condebamba districts, Cajamarca, Peru

Stool samples from 409 pre-school and school students, living in six villages of the Cajabamba and Condebamba districts, Cajamarca, Perú, were examined using wet preparations and Lumbreras' method, looking for Paragonimus eggs. Fecal and sputum samples from two children (0.5%) of 6 and 8 year-old showed eggs of Paragonimus. One hundred and twenty freshwater crabs, Hypolobocera chilensis eigenmanni, were collected from the Condebamba valley and 21 (17.5%) of them were infected with P. mexicanus (syn. P. peruvianus) metacercariae. Our results show the persistence of Paragonimus in human beings and in the main source of infection, the crabs.

Liver abscess caused by Ascaris lumbricoides: case report

A case is reported of a woman who lived in a rural area with a chronic illness that consisted of weight loss and abdominal pain in the epigastrium and upper right quadrant. The initial diagnosis was a mass in the liver, which was later, demonstrated, both by direct and histological examination, to be an abscess caused by Ascaris lumbricoides. Eggs of Ascaris lumbricoides and abundant Charcot-Leyden Crystals were found.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Human toxocariasis: a seroepidemiological survey in the municipality of Campinas (SP), Brazil

The occurrence of human Toxocara infection was evaluated in three neighborhoods of the periphery of the Campinas municipality (Jardim Santa Mônica, Jardim São Marcos and Jardim Campineiro) in 1999. Forty residences and 138 residents were randomly selected by drawing lots and were submitted to a seroepidemiological survey, which included blood collection for the immunoenzymatic detection (ELISA) of anti-Toxocara antibodies and a blood count, and the application of a semi-structured questionnaire for the evaluation of epidemiological data. Significant levels of anti-Toxocara antibodies were detected in 23.9% of the 1999 samples. No significant difference in the frequency of infection according to age was observed. Environmental contamination with Toxocara eggs was observed in 12.3 and 14.0% of 57 soil samples collected in the same region in December 1998 and July 1999, respectively. Univariate analysis and multiple logistic regression of the data obtained from the questionnaires and of the results of the serological tests, suggest a significant influence of socioeconomic variables on the frequency of human infection with Toxocara under the conditions prevalent in the study area.

Human bertiellosis in Goiás, Brazil: a case report on human infection by Bertiella sp. (Cestoda: Anoplocephalidae)

The authors report on a new case of human Bertiellosis in a 2-year old female patient who was born in Goiânia-Goiás (Brazil) and has had history of permanent dwelling in an area frequently visited by simians in Mato Grosso (Brazil). At the time of diagnosis the patient showed inappetence, abdominal pain, and loss of weight. Eggs and proglottids were found in her stool and were identified as Bertiella sp. The objective of this report is to register the third case of human Bertiellosis in Brazil, characterizing one more case of helminthic zoonosis.

A preliminary investigation on the gastrointestinal helminths of the Barbados green monkey, Cercopithecus aethiops sabaeus

Faecal samples were collected from fifty three freshly captured monkeys which were kept at the Barbados Primate Research Centre and Wildlife Reserve (BPRCWR). Examination of these samples for gastrointestinal helminths using the zinc sulphate floatation method revealed an overall infection rate of 88.7%.The parasites observed included Strongyloides (62.4%), Physaloptera (58.5%), Trichuris (52.8%), Hookworm (34.0%), Oesophagostomum (30.2%), Trichostrongylus (3.8%) and Ascaris (5.7%). No significant differences in overall prevalence were observed according to sex or age. Polyparasitism appeared to be common as it was observed in 92.5% of all monkeys examined. It is concluded that these monkeys could act as reservoirs of some of the parasites which can infect man.

Study of enteroparasites infection frequency and chemotherapeutic agents used in pediatric patients in a community living in Porto Alegre, RS, Brazil

Parasitic infections caused by intestinal protozoan and helminths affect more than two billion people worldwide and chemotherapy is the most commonly used therapeutic procedure. Considering the problems created by parasitic infections and the incorrect use of drugs, the aim of this work was to detect the frequency of enteroparasites infection and to estimate the use of chemotherapeutic agents in children living in the periphery of the city of Porto Alegre, RS, Brazil. Ninety-six preschool age children, who had parasitological exams and who used antiparasitic drugs, were analyzed. The efficacy of treatment was evaluated by stool examination repeated six months after treatment. The same diagnostic test was used to evaluate parasitological cure, which was defined as absence of eggs and cysts in the stool. From these children, 79 (82.3%) were contaminated by some species of parasite, the most prevalent were Ascaris lumbricoides, Trichuris trichiura and Giardia lamblia. The most commonly used drugs were mebendazole (86% of prescriptions) and metronidazole (30.3%). The cure rate in the 79 children, examined 6 months after treatment, was 65.3% for A. lumbricoides and 66.1% for T. trichiura. This study suggests that a continuous education program regarding the prevention and treatment of parasitic infections is an essential tool for their eradication.

The frequency rate of Toxocara species contamination in soil samples from public yards in a urban area "Payathai", Bangkok, Thailand

Toxocara species are most common roundworms of Canidae and Felidae. Human toxocariasis develops by ingesting of embryonated eggs in contaminated soil. There is no previous report of Toxocara contamination in the soil samples from the public areas in Bangkok. For this reason our study have been carried out to examine the frequency of Toxocara eggs in public yards in Bangkok, Thailand. A total of 175 sand and clay samples were collected and examined for parasite eggs. According to this study, Toxocara eggs were detected from 10 (5.71%) of 175 soil samples. The high rate of contamination in this study implies the importance of the control of this possible zoonotic disease: control of abandon of dogs and cats, is still necessary.

Comparative study of the fecundity and fertility of Biomphalaria glabrata (Say, 1818) and Biomphalaria straminea (Dunker, 1848) in a laboratory through self-fertilization and cross-fertilization

The aim of this study was to compare the fecundity and fertility of B. glabrata and B. straminea by cross- and self-fertilization. To attain this objective, laboratory-raised strains of B. glabrata and B. straminea were used. The former originated from natural breeding grounds in the municipality Paulista, state of Pernambuco, Brazil. The latter originated from irrigation ditches in the municipality of Petrolândia, in the same state. Snail populations of B. glabrata and B. straminea were maintained for 240 days in laboratory. Their fecundity was evaluated by noting the number of egg-masses, eggs and eggs per mass. Their fertility was evaluated by the number of viable eggs and the hatching rate. B. straminea was markedly more fecund than B. glabrata through cross- and self-fertilization, namely: greater egg-mass; higher egg production and more eggs per mass. Regarding fertility, there seemed to be no preferential period for occlusion to occur or a trend in the rhythm of producing viable eggs.

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

Neuroschistosomiasis due to Schistosoma mansoni: a review of pathogenesis, clinical syndromes and diagnostic approaches

Neuroschistosomiasis (NS) is the second most common form of presentation of infection by the trematode, Schistosoma mansoni. Granulomatous inflammatory reaction occurs as a result of schistosome eggs being transmitted to spinal cord or brain via the vascular system, or by inadvertent adult worm migration to these organs. The two main clinical syndromes are spinal cord neuroschistosomiasis (acute or subacute myelopathy) and localized cerebral or cerebellar neuroschistosomiasis (focal CNS impairment, seizures, increased intracranial pressure). Presumptive diagnosis of NS requires confirming the presence of S. mansoni infection by stool microscopy or rectal biopsy for trematode eggs, and serologic testing of blood and spinal fluid. The localized lesions are identified by signs and symptoms, and confirmed by imaging techniques (contrast myelography, CT and MRI). Algorithms are presented to allow a stepwise approach to diagnosis.