Validity of serology for American Trypanosomiasis with eluates from filter paper

This study evaluates whether blood collected on filter paper kept at 4 degrees C and tested at different intervals of time (1, 7, 15, 30 and 60 days after collection) would present similar results when compared to the serum samples and whether the type of filter paper influences the results. Eluates from filter paper samples were tested for Trypanosoma cruzi antibodies using indirect immunofluorescence antibody test (IFAT), indirect haemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA) as reference, the antibody titer in sera. Analysis of data showed that results obtained with IFAT, IHA (cut off point = 1:40) and ELISA in sera had similar sensitivity and good concordance among reactions. The use of a multiple linear regression model indicated that titer fall in eluates occurs up to the 7th day after the collection, and it is more marked for samples with lower antibodies titers. However, no significant differences were observed by IFAT, IHA (cut off point = 1:20) and ELISA in the proportion of positive reactions between sera and eluates. The results also showed that Melitta, Klabin or Whatman (reference) filter papers could be indicated for surveys, since they have shown similar capacity of maintenance of anti-T. cruzi immunoglobulins.

Antibodies anti Bloodstream and Circumsporozoite Antigens (Plasmodium vivax and Plasmodium malariae/P. brasilianum) in Areas of Very Low Malaria Endemicity in Brazil

During 1992-1994, 33 malaria cases were reported in two regions in Brazil where few sporadic atypical cases occur, most of them in home owners, who are weekenders, while home caretakers live there permanently. Indirect Fluorescent Antibody Test (IFAT), with Plasmodium vivax, and Enzime Linked Immunosorbent Assay (ELISA) with repeat peptides of the circumsporozoite (CS) proteins of the 3 known P. vivax variants and P. malarie/P. brasilianum, were performed on 277 sera, obtained within a 5 to 10 km range of malaria cases. Very rarely did any of these donors recall typical malaria episodes. Blood smears of all but 5 were negative. One of the 5 malaria cases included in our serology was of a home owner, 1 of a permanent resident, 3 from Superintendência de Controle de Endemias employees who went there to capture mosquitoes. In Region 1 the prevalence of IFAT positive sera was 73% and 28% among caretakers, 18% and 9.6% among home owners. In Region 2 (3 localities) no distinction was possible between caretakers and home owners, IFAT positivity being 38%, 28% and 7%. The relative percentage of positive anti-CS repeats ELISA, differed for each of the peptides among localities. Dwellings are in the vicinity of woods, where monkeys are frequently seen. The origin of these malaria cases, geographical differences and high seropositivity is discussed

Toxoplasma gondii Antigenuria in Patients with Acquired Immune Deficiency Syndrome

A longitudinal study was performed with sera and urine of patients with acquired immune deficiency syndrome (AIDS), taken before, during and after clinically Toxoplasma infection. The tested patients were followed for an average of two years. The titres of the specific IgG and IgM antibodies were measured by an indirect fluorescent antibody test (IFAT), and the appearance of circulating antigens of T. gondii was determined in 36 urine samples of 13 patients with neurotoxoplasmosis by means of the coagglutination test. The presence of T. gondii antigens in the urine of AIDS patients by this test was correlated with the immunoblot technique, with clinical symptoms and also with pathological findings. Our results indicate that the detection of T. gondii antigens in the urine of AIDS patients can be regarded as a rapid and efficient method for the diagnosis of acute toxoplasmosis

Clinical, Parasitological and Immunological Aspects of Experimental Infection with Trypanosoma evansi in Dogs

This research investigated the pattern of antibody response by means of enzyme linked immunosorbent assay (Elisa) and indirect fluorescent antibody test (IFAT) through the course of experimental Trypanosoma evansi infection in dogs. Clinical and parasitological features were also studied. The average prepatent period was 11.2 days and parasitaemia showed an undulating course. Biometrical study of parasites revealed a mean total length of 21.68mm. The disease was characterized by intermittent fever closely related to the degree of parasitaemia and main clinical signs consisted of pallor of mucous membrane, edema, progressive emaciation and enlargement of palpable lymph nodes. Diagnostic antibody was detected within 12 to 15 days and 15 to 19 days of infection by IFAT and Elisa, respectively. High and persistent antibody levels were detected by both tests and appeared not to correlate with control of parasitaemia

Visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, State of Minas Gerais, Brazil

In the last few years the number of human cases of American visceral leishmaniasis in the Metropolitan Region of Belo Horizonte (MRBH), Minas Gerais, Brazil has increased, indicating an elevation in the transmission rate of the disease. The total number of notified human cases in the MRBH since 1994, when the first case was identified, up to 1999 was 345 of which 223 (65%) were from the city itself, indicating an urbanization of the disease in this region of Minas Gerais. The age distribution of visceral leishmaniasis cases in the MRBH shows a higher prevalence in children from 0-4 years old, responsible for 28.9% of the notifications. Clinical and immunological findings from dogs infected with Leishmania chagasi are described. The majority of these animals showed no sign of the disease. Sera from all infected dogs showed detectable Leishmania-induced high titles of antibodies based on the results of an indirect fluorescent antibody test. Samples of isolated Leishmania from human and dogs were characterized as L. (L.) chagasi by biochemical and molecular techniques.

Antibodies anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae in patients with negative serology for hantavirus. Retrospective study

The seroprevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae in hantavirus seronegative patients, who had symptoms and signs compatible with pneumonia was established. For this purpose we used the indirect fluorescent antibody test. Titers ³ 1:16 for C. pneumoniae and M. pneumoniae were found in 8.6% and 17.1% of the serum, respectively, showing evidence of recent or current infection.

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

A total of 206 serum samples from children (3-14 years old) living in the Amador County (La Chorrera District, Province of Panama) were screened by indirect immunofluorescence antibody test (IFAT) for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR) followed by dot blot hybridization. The results indicated a prevalence of 9.7% for trypanosome infections, a seroprevalence of 2.9% against T. cruzi and a predominance of T. rangeli infection (6.8%). The immunological and clinical implications of these findings are discussed.

Isolation of respiratory syncytial virus from nasopharyngeal aspirates stored at 20ºC from one to fifteen months after collection

Cell culture isolation is used for recovering respiratory syncytial virus (RSV) from respiratory specimens. As RSV is a thermolabile virus, specimens destined for inoculation into cell culture require special transport, handling, and storage. The isolation rate of RSV from nasopharyngeal aspirates (NPA) stored at 20ºC for one to 15 months after collection was investigated. A total of 126 samples considered positive for RSV by indirect fluorescence-antibody were tested by virus isolation in HEp-2 cell culture. RSV was isolated from 47/126 specimens (37.3%). These results show that RSV may be recovered from NPA stored at 20ºC by cell culture.

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100%. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72%), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40%. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

Serodiagnosis of Babesia equi in horses submitted to exercise stress

A complement fixation test (CFT), performed in microtitre plates, based upon the use of crude antigenic preparation of Babesia equi was adapted for the detection of antibodies in serum of infected horses. The indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were also used for the immunodiagnosis of B. equi. Serum samples from 15 apparently healthy horses, previously conditioned to a high-speed equine treadmill, were taken before and after exercise. All the samples analyzed were positive for B. equi infection. There were no significant differences (P<0.01) between these 3 tests, or the condition of rest or stress. The combined use of CFT and IFAT or ELISA should be recommended in order to enable veterinary services to more efficiently prevent introduction of infected horses into disease-free areas.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT>50) and 36 (32.7%) for T. gondii (IFAT>16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

IgM and IgA Antibody Responses in 12 Cases of Human Acquired Toxoplasmosis

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels £ 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis

Use of an indirect haemagglutination test, for the detection of Clostridium perfringens type A enterotoxin

An indirect haemagglutination (IH) test is described for the detection of Clostridium perfringens type A enterotoxin, produced by strains isolated from human cases of food poisoning and from contaminated food. Though no strict relationship could be observed between titers in the IH test and the time it took mice to die from the intravenous inoculation of mice (IIM), results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the ILM one. No unspecific reaction was obtanined int he IH wirh a negative control and the inhibitions of the IH and IIM tests by specific antiserum against C. perfringens enterotoxin showed that the IH test is very spcific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data.