Mario E. Camargo

Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma gondii

A caracterizacao de infeccao primaria recente pelo Toxoplasma gondii se apoia principalmente na presenca, no soro, de anticorpos especificos IgM. Para fins diagnosticos de toxoplasmose aguda, ou de contagio recente, a possibilidade de outros marcadores e altamente desejavel. Um marcador de infeccao recente atualmente referido e a baixa afinidade ou avidez de anticorpos especificos IgG. Para avaliacao do novo marcador, titularam-se os soros contra poliantigenos do T. gondii pelo teste imunoenzimatico (ELISA), antes e apos tratamento dos complexos antigeno-anticorpo formados, com solucao de ureia 6 M como agente dissociante. O deslocamento de anticorpos de baixa avidez foi indicado por uma queda de titulos, calculada em porcentagem em relacao aos titulos iniciais. Foram estudados 69 soros, 23 de cada um dos 3 perfis sorologicos sucessivos, observados na infeccao, e que a caracterizam respectivamente como recente, em fase de transicao e cronica. Os perfis foram determinados segundo os resultados de uma bateria de testes, incluindo os de imunofluorescencia IgG e IgM, de captura de anticorpos IgM e de hemaglutinacao. Para os soros de infeccao cronica a queda observada foi de 3% ± 3%, de 34% ± 12% para toxoplasmose recente e de 12% ± 9% para a fase de transicao. Conclue-se que a determinacao da avidez de anticorpos IgG pode ser utilizada como marcador de infeccao primaria recente pelo T. gondii.

Detection of antibodies in sera from Chagas' disease patients using a Trypanosoma cruzi immunodominant recombinant antigen

A Trypanosoma cruzi DNA fragment encoding an immunodominant repetitive antigen (H49) was subcloned into a protein purification and expressed system. Purified H49 peptide reacted specifically in an enzyme‐linked immunosorbent assay (ELISA) with sera from T. cruzi‐infected patients, but not with sera from patients with other parasitic diseases such as leishmaniasis and T. rangeli‐infection. The H49 recombinant ELISA was able to detect specific antibodies in 84% of chronic chagasic serum samples tested. One of the major advantage of the recombinant ELISA for serodiagnosis of chronic Chagas’disease resides in its high specificity (100%). Our data suggest that recombinant peptides could provide a practical basis for specific diagnosis tests for Chagas’disease.

Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi

We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.

Investigação epidemiológica de um caso de leishmaniose visceral autóctone da Grande São Paulo, Brasil

Description of an epidemiological survey carried out in the urban area of Diadema, a district of Great S. Paulo (Brazil), with the aim of elucidating the source of infection and the mechanism of transmission of one case of visceral leishmaniasis in the area. Serological surveys were carried out (imunofluorescent test) in the human population (591 serums) and in the canine population (55 serums). Also an entomological survey was done in the neighbourhood of the patients house and in a florest reserve 500m from the house.Foi descrita uma investigacao epidemiologica realizada na zona urbana de Diadema, municipio da Grande Sao Paulo (Brasil), com a finalidade de elucidar a fonte de infeccao e o mecanismo de transmissao de um caso de leishmaniose visceral autoctone da area. Foram realizados inqueritos sorologicos atraves da reacao de imunofluorescencia indireta na populacao humana (591 soros) e na populacao canina (55 soros), e levada a efeito pesquisa entomologica no local da residencia do doente e em uma area de reserva florestal situada a 500m desta residencia.

Immunoglobulin M (IgM)-Glycoinositolphospholipid Enzyme-Linked Immunosorbent Assay: an Immunoenzymatic Assay for Discrimination between Patients with Acute Toxoplasmosis and Those with Persistent Parasite-Specific IgM Antibodies

ABSTRACT In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin M (IgM) specific for glycoinositolphospholipids (GIPL) derived from tachyzoite membrane (IgM-GIPL ELISA). The sensitivity and specificity of the assay were compared with those of commercially available Toxoplasma-specific IgM serological tests, namely, immunofluorescence assay (IFA) with fixed tachyzoites and capture ELISA employing tachyzoite extracts. Our results show that all patients with acute toxoplasmosis, as determined by clinical data and conventional serological tests, were also positive by the IgM-GIPL ELISA. Interestingly, many patients that were classified as indeterminate, who had IgG with high avidity but positive results in the IgM-specific IFA and capture ELISA, were negative by the IgM-GIPL ELISA. Finally, we tested the sera from patients with rheumatoid arthritis and various parasitic infections and found no evidence of false positives in the IgM-GIPL ELISA.

LECTIN USED IN THE PURIFICATION PROCESS OF TOXOPLASMA GONDII TACHYZOITES

Toxoplasma gondii tachyzoites were purified from mouse peritoneal exudates using lectins to remove the host cells. Best results were obtained with phytohemagglutinin P at a concentration of 0.01% (W/V), which provided a 99.7% pure Toxoplasma suspension. The process was reproducible and easy to perform. Toxoplasmas so obtained were infective and served as sources of high quality antigens for immunofluorescence, hemagglutination, and complement fixation tests.

Toxoplasmosis and the laboratory: diagnosis and a constant striving for improvement

Indeed, continuous scientific and technical evolution has led to a decisive progress in diagnosis through the search of “gold standard” tests that will provide safe information about the real health status of the patient. However, in our opinion, faults are often observed in the training of clinicians or laboratory technicians concerning the selection of tests and mainly their use at an opportune time and the correct interpretation of their results with respect to the clinical signs and symptoms presented by the patients. Fortunately, great efforts are being made in this respect both by Health Offices and by medical societies and entities in various States in Brazil. The serological profiles observed during the course of Toxoplasmosis are classical, respectively representing recent infection, previous infection or a transition phase, and have been defined by experienced investigators such as G. DESMONT in France, J.S. REMINGTON in the United States, and others, including Brazilian researchers, such as M.E. CAMARGO from this Tropical Medicine Institute. Successively available immunological resources, such as the Complement Fixation test, Indirect Hemagglutination and detection of anti-Toxoplasma gondii IgG and IgM antibodies especially by ELISA and Immunofluorescence, have been thoroughly investigated. For years these serological profiles of toxoplasmosis have been exhaustively taught at medical and paramedical schools and discussed at Congresses and Scientific Meetings, being unanimously accepted in laboratory diagnostic routine. However, technology has evolved, especially with the development of more sensitive automated equipment. The Universities, historical seats of research, have been rapidly superseded by large industries which have heavily invested in the development of new products and in equipment of high sensitivity. However, some errors have started to arise especially in the search for IgM antibodies by attributing excessive value to their residual levels, which are nonsignificant and erroneously interpreted as positive results indicating current infection, with serious consequences especially for women in the early phase of pregnancy, who are submitted to unnecessary treatment or even to interruption of pregnancy. Fortunately, research and development have continued to advance and new parameters have been developed for the diagnosis of toxoplasmosis in its different phases, such as the avidity test of IgG antibodies and detection of the parasite by a molecular method such as the polymerase chain reaction (PCR). IgG antibodies of low avidity characterize the beginning of infection, being present up to the third or fourth month, whereas the PCR method permits to confirm fetal infection by detection of the parasite DNA in amniotic fluid.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

DOT-ELISA for detection of anti-Cysticercus cellulosae antibodies in human cerebrospinal fluid using a new solid-phase (resin-treated polyester fabrics). Preliminary report

A dot enzyme-linked immunosorbent assay (DOT-ELISA) was developed to detect specific antibodies in cerebrospinal fluid (CSF) for human neurocysticercosis immunodiagnosis, with Cysticercus cellulosae antigen dotted on a new solid-phase. This was represented by sheets of a synthetic polyester fabric impregnated with a polymerized resin (N-methylol-acrylamide). A very stable preparation was thus obtained, the antigen being covalently bound by cross-linking with free N-methylol groups on the resin. Since robust, no special care was necessary for handling the solid-phase. The test could be performed at room-temperature. From 30 CSF samples assayed, 14 were positive, from a group of 15 cases of neurocysticercosis, with titers from 1 to 128; 15 other samples, from normals or other neurological diseases, were all negative. Test characteristics seem to indicate it as adequate for epidemiological surveys. A more detailed study on sensitivity, specificity, reproducibility and the use in serum samples is being conducted.

Prevalência de esofagopatia chagásica no Município de Mambaí, Goiás - Brasil

In Mambai, an endemic area for Chagas’ disease, contrast radiography of the oesophagus using 70 mm film was done in 1,145 male and I,184 female individuals. Ages ranged from 4 to 87 years. The patients were positioned in the right anterior oblique position and two films taken, one immediately after ingestion of 75 ml of barium contrast and another 60 seconds later. Seventy six (3.2%) individuals had oesophageal abnormalities of whom 71(7%) were among 1,006 seropositives and 5(0.37%) were among 1,323 seronegatives. Of the 76 patients, 47 (61.8%) were male and 29(38.1%) female. Following the classification of Rezende and colleagues, 48(63.1%), belonged to group 1,18(23.7%), to group II, 5(6.6%) and 5(6.6%) to group IV. The prevalence of oesophageal disease increased with age especially after 30 years, reaching 21.5% in the group over 59 years of age.