Diagnosis of human toxoplasmosis by a dot enzyme-linked immunosorbent assay

A Dot enzyme-linked immunosorbent assay (Dot-ELISA) was standardized and evaluated for the serodiagnosis of human toxoplasmosis. Out of 538 serum samples tested by the immunofluorescence test for toxoplasmosis (IFAT-IgG) as reference test, 183 (34%) were positive at cut off 1:16 and 192 (36%) were positive for Dot-ELISA-IgG at cut-off 1:256. For Dot-ELISA, co-positivity was 0.94, co-negativity 0.94 and concordance 0.88 in relation to IFAT-IgG. These results suggest the usefulness of Dot-ELISA (cut-off titer of 1:256) for the serodiagnosis of human toxoplasmosis. The main advantage of this technique is simplicity, positive test can be visually identified (colored precipitate). It does not require a special equipment and it can be used as a qualitative test to screen large numbers of samples or as a quantitative assay to determine end-point titration of individual sera.

The relevance of laboratory diagnosis of human cryptosporidiosis and other coccidia

Human infection by Cryptosporidium spp and other coccidia are due to opportunist non-host specific microorganisms. In HIV seropositive patients, the gastrointestinal symptoms accompanying such infections may be serious and prolonged and may include nausea, low-grade fever, abdominal cramps, anorexia and watery diarrhoea. We studied 188 stool samples from 111 patients (84 men and 27 women) with diarrhoea. A modified Ziehl-Nielsen technique for the detection of Cryptosporidium spp and Isospora belli was employed. The mean age of the patients was 31 years. Cryptosporidium spp was seen in 18% (n=20) of the patients, 90% (n=18) of whom were HIV seropositive. Isospora belli was recorded only from HIV seropositive patients (5.4% of all the patients studied and 6.5% of those who were HIV seropositive). These data confirm the good results obtained with this technique for the identification of Cryptosporidium spp and other coccidia and also reaffirm the clinical importance of correctly diagnosing the cause of diarrhoea, particularly in HIV seropositive patients.

Association of human herpesvirus 6 infection with exanthem subitum in Belem, Brazil

Recent human herpesvirus 6 (HHV-6) infection was detected in cases of exanthem subitum (ES) involving four children, aged 10 to 24 months, between April and August 1994, in Belém, Brazil. By using the indirect immunofluorescence antibody assay (IFA), significant increases (at least eight times) in antibody concentrations were noted from the acute to the convalescent serum samples, with titers ranging from <1:10/1:80 to <1:10/1:640 (patients 3 and 2, respectively). All children had high fever (over 39ºC) for three days, followed by generalized, maculo-papular skin rash. A physical examination of the children also revealed concomitant, cervical lymph node swelling and tonsillar pharyngitis in two of them.

Etiological drug treatment of human infection by Trypanosoma cruzi

Forty-nine American Trypanosomiasis (Chagas' disease) patients, with xenodiagnosis proven parasitemia were treated by the authors. Forty-one of these patients were given benznidazole, at dosages ranging from 5mg/kg/day to 8mg/kg/day, during a pre-established period of 60 days. In this group, 17 patients had an undetermined form of the disease, whereas 22 had cardiologic disease and 4 had digestive disease (two patients had a mixed form of the disease). Side effects were frequent, and led to the discontinuation of treatment in 17 patients. The follow-up period ranged from 1 to 20 years (mean follow-up period of 6 yrs. 7 mo). 26 (63.4%) of the patients became parasitemia-negative. The other eight patients were treated with nifurtimox, during 120 days, following a variable dose regime of 5mg/kg/day (initial dose) to 17 mg/kg/day (final dose). Six of them had severe side effects, and only one patient remained parasitemia-negative throughout the observation period (ranging from 1 to 18 years). Benznidazole proved to be better tolerated and more effective in the management of parasitemia when compared to nifurtimox, although more effective and less toxic drugs are still desirable.

Seroepidemiology of human toxoplasmosis in Chile

A series of already published and unpublished seroepidemiological surveys for toxoplasmosis, carried out in Chile in 1982-1994, is reviewed, expanded and analyzed. The surveys included 76,317 apparently healthy individuals of different ages (0.57% of the country's total population), from 309 urban and rural-periurban localities. Urban groups were integrated by blood donors, delivering mothers and middle grade schoolchildren, while rural-periurban individuals corresponded to unselected family groups. Blood samples were collected in filter paper. The presence of antibodies to Toxoplasma gondii was determined by the indirect hemagglutination test (IHAT), titers > 16 were considered positive. The test resulted positive in 28,124 (36.9%) of the surveyed people. Two hundred and six (0.3%) individuals presented IHAT titers > 1000, probably corresponding to acute or reactivated infections. A progressive increase of positive IHAT from northern to southern regions of the country was noted, phenomenom probably related to geographical conditions and to a higher production and consumption of different types of meat in the latter regions. It is postulated that ingestion of T. gondii cysts by humans is epidemiologically as important as ingestion of oocysts. The results presented stress the epidemiological importance of toxoplasmosis in humans, and warn about eventual implications in immunocompromised patients and in transplacental transmission, organ transplants and transfusions.

HEMAGGLUTINATION TEST FOR THE DIAGNOSIS OF HUMAN NEUROCYSTICERCOSIS: DEVELOPMENT OF A STABLE REAGENT USING HOMOLOGOUS AND HETEROLOGOUS ANTIGENS

A hemagglutination (HA) test was standardized using formalin- and tannin-treated gander red blood cells sensitized with a total salt extract of C. cellulosae (HA-Cc) and an antigenic extract of Cysticercus longicollis (HA-Cl) vesicular fluid. A total of 61 cerebrospinal fluid (CSF) samples were assayed, 41 from patients with neurocysticercosis and 20 from a control group, which were, respectively, reactive and non-reactive to ELISA using C. cellulosae. The CSF samples from the control group did not react and 35 (85.4%) and 34 (82.9%) CSF samples from patients were reactive to the HA-Cc and HA-Cl tests, respectively. The reagents ready for use were stable up to 6 months when stored at 4°C in 50% glycerol. The present results confirm that the reagent using Cysticercus longicollis stabilized with glycerol can be used as an alternative in the immunological diagnosis of neurocysticercosis

POSSIBLE ROLE OF LUTZOMYIA MARANONENSIS AND LUTZOMYIA ROBUSTA (DIPTERA: PSYCHODIDAE) AS VECTORS OF HUMAN BARTONELLOSIS IN THREE PROVINCES OF REGION NOR ORIENTAL DEL MARAÑON, PERU

Human bartonellosis is found predominantly in Perú2, 6, 8, 12, 15, as well as in Ecuador3, 7, 10 and Colombia13, 15. In Peru, the disease is restricted to the valleys of the western-side and a few inter-andean and eastern-slopes of the andean valleys6, 15, 18 at altitudes between 1000 and 3200 masl. Most human cases are reported from the regions of Chavin, Nor Oriental del Marañon and Lima16. Lutzomyia verrucarum is presumed to be the only vector of human bartonellosis in the valleys of Peru1, 2, 8, 11, 17, 19/ Our research objetive was to detect the presence of Lu. verrucarum in various localities known to be endemic for human bartonellosis in three provinces of Region Nor Oriental del Marañon. Sandfly collections were made between 1987 and 1992 during four visits to bartonellosis-endemic provinces: San Ignacio (districts of San José de Lourdes: 1020-1260 m and La Coipa: 1200-1560 m), Jaén (districts of Santa Rosa: 1300-1680 m and Jaén: 1220-1680 m) and Utcubamba (districts of Lonya Grande: 1200 m and El Milagro: 1200-1540 m)

A case of human rabies in the Urban Area of Ribeirão Preto, SP, Brazil

A 39-year old male patient was admitted to the University Hospital of the Faculty of Medicine of Ribeirão Preto with signs and symptoms of sudden dyspnea, generalized myalgia and behavioral disorders. The initial suspicion was alcohol abstinence syndrome and the patient was referred for psychiatric and neurologic care. The evolution of the patient with a worsening of signs and symptoms, presence of crises of tachypnea, agitation, difficulty to swallow, irritability and hydrophobia, and his report of having been bitten by a suspected dog raised the hypothesis of rabies. The diagnosis was confirmed by examination of a corneal impression, biological tests in the cerebrospinal fluid (CSF) and saliva and visualization of Negri bodies in nervous tissue (direct immunofluorescence). The patient evolved with agitation, aggressiveness, and worsening tachypnea intercalating with apnea, and died on the 4th day after admission

Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

Neutralizing antibodies in Brazilian sera against three strains of human immunodeficiency virus type 1 (HIV-1)

The MN strain of HIV-1 is known to be more prevalent in Brazil, the BRU strain is more prevalent in Europe, and the NDK strain in Africa. It has been suggested in the literature to include different strains in the same vaccine against HIV-1. To contribute to the studies for the development of a universal vaccine, the occurrence of antibodies (Ab) against three HIV-1 strains (MN, BRU and NDK) was determined in serum samples from 85 HIV-1-positive patients, adult volunteers seen at the University Hospital of the Faculty of Medicine of Ribeirão Preto-USP. One-hundred tissue culture infective unit (TCIU) of the viruses reacted with serial dilutions of the sera (2x) and with MT4 cells added at a final concentration of 0.3 × 106 cells/ml, and a cytopathic effect was observed on the 7th and 11th days of incubation. Titres of less than 1/50 were considered to be negative. In 129 tests, the sera were negative for one of the three strains: 40 for MN, 29 for BRU and 60 for NDK. There was a predominance of strains MN and BRU, most of them presenting titres from 1/50 to 1/200. Titres for NDK were detected in 25 sera. We conclude that there seems to be a predominance of strains MN and BRU among the individuals from the region tested; however, the detection of sera with positive NKD titres indicates the need for further studies of this strain in other populations and regions of Brazil

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

EPIDEMIOLOGICAL SURVEY ON CANINE POPULATION WITH THE USE OF IMMUNOLEISH SKIN TEST IN ENDEMIC AREAS OF HUMAN AMERICAN CUTANEOUS LEISHMANIASIS IN THE STATE OF RIO DE JANEIRO, BRAZIL

A survey for canine tegumentary leishmaniasis (CTL) has been carried out between 1986 and 1993 in seven endemic localities for American cutaneous leishmaniasis in the State of Rio de Janeiro. 270 dogs have been examined for their clinical aspects, the development of delayed hypersensitivity (DHS) with Immunoleish antigen and with immunofluorescent antibody research of IgG (IF). 28.2% of them had ulcer lesions and 3.3% had scars. The lesions consisted of single (39.5%) and mucocutaneous lesions (31.6%), multiple cutaneous (25.0%) and mucocutaneous lesions associated with cutaneous ulcers (4.0%). Twelve (15.8%) isolates from biopsies were analyzed by zimodeme and schizodeme and identified as L. (V.) braziliensis. The overall prevalence of canine infection that was evaluated with the skin test was of 40.5% and with IF it was of 25.5%. Both tests showed a high positive rate with relation to the animals with mucosal lesions, as in the case of human mucocutaneous leishmaniasis. The comparison of the two tests showed the skin test to have a better performance although there was no statistical difference (p>0.05) between them. The proportional sensitivity and specificity was of 84.0% and 74.0%, respectively. The Immunoleish skin test and IF are useful tools to be employed in CTL field epidemiological surveys.

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.