Evaluation of a new Plasmodium lactate dehydrogenase assay (OptiMAL-IT) for the detection of malaria.

The new OptiMAL-IT(R) rapid diagnostic test for malaria was evaluated in 271 patients in Thailand with uncomplicated malaria between June and July 2002. The sensitivity and specificity for the diagnosis of Plasmodium falciparum parasites were 88% and 92%, respectively. For species other than P. falciparum, the sensitivity was 65% and specificity was 99%. The performance of the new test decreased markedly at low levels of parasitaemia.

A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay.

We report a double-site enzyme-linked lactate dehydrogenase immunodetection assay (DELI), a highly sensitive antigen-capture enzyme-linked immunosorbent assay, which proved to be more sensitive for the detection of Plasmodium falciparum than thick blood smears, as sensitive as the polymerase chain reaction, and probably more reliable. This technique can help to detect infra-microscopic parasitemias (one parasite in 10(6)-10(8) red blood cells) from biological samples, and being quantitative, provide a fast substitute to thick smears for epidemiologic purposes. The technique can also be used to measure the in vitro drug sensitivity of P. falciparum with greater ease, much greater speed, and simpler equipment than that required for the isotopic microtest. Results obtained with four antimalarial drugs upon 16 strains closely paralleled those obtained by the isotopic assay (R = 0.95). In contrast with the latter, much lower parasite densities could be tested in the DELI assay (as low as 0.005%), thereby extending the number of isolates that can be investigated. The ease of implementation and low cost of the DELI-microtest may contribute to a revived interest in using in vitro methods to survey resistance to antimalarial drugs, so as to better predict future in vivo drug failures and provide public health recommendations.

Early aqueous humor analysis in patients with human ocular toxoplasmosis.

To evaluate the diagnostic sensitivity of a panel of laboratory tests for ocular toxoplasmosis performed at the time of presentation, paired samples of aqueous humor and serum were collected from 49 consecutive episodes of ocular toxoplasmosis with a clinical course of less than 3 weeks. Total immunoglobulin G (IgG) and Toxoplasma gondii-specific IgG, IgM, and IgA were quantified by enzyme-linked immunosorbent assay. The avidity of T. gondii-specific IgG was determined, and DNA extracted from aqueous humor was amplified for detection of a glycoprotein B gene sequence of T. gondii. The diagnosis was confirmed for 73% (36 of 49) of the patients; this rate rose to 79.5% if data from a later analysis of aqueous humor derived from five of the negative patients were included. The analysis of serum (detection of T. gondii-specific IgM and analysis of consecutive serum samples) alone did not contribute to the diagnosis. Calculation of local antibody production lacked diagnostic sensitivity when it was determined less than 3 weeks after the manifestation of clinical symptoms (28 of 49 patients [57%]), but this rose to 70% after an analysis of a second aqueous humor sample. The antibody avidity index attained diagnostic significance in only 8 of 43 instances (19%), and T. gondii DNA was amplified from no more than 6 of 39 (16%) aqueous humor samples. However, T. gondii-specific IgA was found within the aqueous humors of 11 of 43 patients (26%); measurement of the T. gondii-specific IgA level thus contributed substantially to the diagnostic sensitivity of the laboratory tests.

[Current limits in diagnosis of ocular toxoplasmosis]. / Aktuelle Grenzen in der Diagnostik der okulären Toxoplasmose.

BACKGROUND: The diagnosis of ocular toxoplasmosis has remained a merely clinical one, because the low sensitivity of established methods does not allow clinical consequences. The underlying open prospective study was undertaken to analyse the sensitivity of a combination including the newly available tests for the diagnosis of the disease. METHODS: From 27 patients included until now, aqueous humor and serum samples were collected and sent to one of two reference laboratories according to their actual availability. From all samples, total IgG and anti-Toxoplasma IgG as well as specific IgM and IgA were quantified, and from the results, the antibody ratio was calculated according to the formula of Goldmann and Witmer. From the samples sent to laboratory 2, antibody avidity was determined and Toxoplasma DNA amplified using PCR. RESULTS: A confirmation of the clinical diagnosis was achieved in 5/9 cases (56%) from samples sent to laboratory 1, and from 14/18 samples (78%) sent to laboratory 2. Calculation of the antibody ratio was confirmed to be the most sensitive method with a confirmation rate of 41%, followed by PCR (28%), determination of specific IgA (22%) and finally antibody avidity (15%). A confirmation with two independent tests was achieved in 28% of cases. CONCLUSION: None of the methods analysed was sensitive enough to establish the diagnosis in a given case. The combination of all four methods, however, achieved a sensitivity, which is high enough to justify a clinical routine analysis of aqueous humor samples.

Accelerated detection of DNA on membranes by automated enzyme-linked immunofiltration assay.

Methods used to detect DNA after transfer to nitro-cellulose or nylon membranes are all based on slow incubation with agitation. We describe an application of the ELIFA technique (enzyme-linked immunofiltration assay) for rapid detection of DNA immobilized on a membrane by active filtration of the reagents across the membrane. The different steps (saturation, hybridization to a nonisotopically labeled probe, washing, and immunoenzymatic revelation) are automated and controlled by a microcomputer that determines the direction of flow and flow rates of the solutions through the membrane. We applied this method to the detection of Toxoplasma gondii DNA in 108 samples of amniotic fluid during antenatal tests for toxoplasmosis and compared the results with those obtained by the conventional method. In addition to a major time saving (2 h against almost 15 h), automation improves reproducibility and avoids manipulation of the membranes between the different steps, while keeping the same sensitivity and specificity as the standard method.

Investigations on a Swiss area highly endemic for Echinococcus multilocularis.

Anecdotal information suggested that a focus of hyperendemicity may be present in a small area of the Canton of Fribourg in Switzerland. Therefore, the prevalence of E. multilocularis was assessed both in the fox and the rodent population over a two-season-period. A high prevalence ranging between 47% and 56% was consistently determined in the fox population. An Arvicola terrestris population was infected at 39% in the first season of investigation and at 11% in the following season. A subsequent seroepidemiological survey in the population of inhabitants surrounding the area provided no indication of seroconversion and thus no indication of infection for humans. However, a longer-term survey will be needed to assess more precisely the risk of disease occurrence among these inhabitants.

Seroprevalence of HBV (anti-HBc, HBsAg and anti-HBs) and HDV infections among 9006 women at delivery.

Serum samples from 9006 women, who delivered in Switzerland in 1990 and 1991, were collected around the country. Of these women, 62.7% were Swiss and 37.3% originated from foreign countries. Samples were first screened for anti-HBc and those found positive were further tested for HBsAg, anti-HBs and anti-HDV. Anti-HBc was found in 640 of the 9006 women (overall prevalence, 7.1%; Swiss, 3.3%; foreigners, 13.5%). Of these 640 positive samples, 61 (9.5%) were positive for HBsAg (without anti-HBs), 467 (73.0%) positive for anti-HBs (without HBsAg) and 8 (1.3%) positive for both HBsAg and anti-HBs. The remaining 104 were thus anti-HBc positive without HBsAg or anti-HBs. These 104 specimens with the so-called "isolated anti-HBc" reactivity represented 1.2% of the whole population or 16.3% of the 640 anti-HBc positive mothers. All were HBV DNA negative (PCR). Anti-HDV antibody was found in only five women. HBsAg was seen in 38 of the cord-blood samples from the anti-HBc positive mothers. In this large sampling, we observed a relatively high seroprevalence of HBV infection. Cases with isolated anti-HBc reactivity, being HBV DNA negative by PCR, were probably non-infectious at the time of blood collection.

[Human echinococcosis in Switzerland, 1984-1992]. / Echinokokkose des Menschen in der Schweiz, 1984-1992.

In a retrospective study (1984-1992), new cases of human echinococcosis were registered in Switzerland based on information obtained from (a) questionnaires sent to 294 of the 300 acute hospitals in all parts of the country and to 17 institutes of pathology (268 answers form hospitals: 91%), and (b) from tracing back cases reported under the official notification system since 1 January 1988 by laboratories to the Federal Health Office or recorded at the Institute of Parasitology in Zurich. Cases were regarded as verified if the diagnosis was documented by unequivocal findings (by radiology, ultrasonography, pathomorphology etc. and often by additional detection of anti-Echinococcus antibodies). Patients with antibodies but without reports on further findings were classified as suspected cases. From 1984 to 1992 (9 years), 302 new cases of human echinococcosis were diagnosed in Switzerland and verified in this study, corresponding to an annual average of 34 new cases and a range between 26 and 43 cases. The total number of 302 cases included 228 (75%) of cystic echinococcosis (CE) (Echinococcus granulosus), 65 (22%) of alveolar echinococcosis (AE) (E. multilocularis), and 9 (3%) of non-specified echinococcosis (NSE) (Echinococcus sp.). Among 185 patients with CE and 60 patients with AE and known geographic origin, the ratio of Swiss nationals to foreigners was 25%:75% and 88%:12% respectively. Based on a total population (Swiss nationals and foreigners) of 6.62 million in 1988 and the case numbers of 1984-1992, the following average annual incidence rates per 100,000 inhabitants were calculated: 0.51 for all new cases, 0.38 for CE, 0.11 for AE and 0.01 for NSE. In the 37 years since 1956 there has been steady increase in new cases of CE due to the importation of such cases by foreigners, whereas the case numbers of AE have remained nearly constant with a range between 7 and 10 new cases per year. In our study 258 suspected seropositive patients were registered but not added to the total number of cases, due to the lack of further findings.

Seroprevalence of rubella among women of childbearing age in Switzerland.

A seroepidemiological study was carried out in Switzerland to define the population susceptible to rubella among women of childbearing age. IgG antibodies to rubella virus were determined in 9,046 women giving birth between 1 August 1990 and 30 September 1991 in 23 of 26 Swiss cantons. These sera represented 10-20% of the yearly total number of births in each Swiss canton. Anti-rubella IgG was measured by an automated enzyme-linked fluorescent assay for use with a commercial system (Vidas Rub IgG, bio-Mérieux, France). Before the study population was screened, the commercial system was compared to the traditional hemagglutination-inhibition (HAI) test using 500 consecutive samples from parturient women. The sensitivity was 97.7%, the specificity was 100%, and agreement between the two tests was 97.8%. The discrepancies corresponded to very low titres of antibodies as measured by HAI. The seroprevalence of rubella nationwide in women of childbearing age in Switzerland was 94.3%. The seroprevalence was higher (96.5%) in the 5,677 women of Swiss nationality than in the 3,090 women of a different nationality (90.4%) (p < 0.001). In Swiss women the seroprevalence of rubella did not increase significantly with age and was identical in primiparous and in multiparous women, thus indicating that women of childbearing age are probably not sufficiently immunised.

Toxoplasma infection among pregnant women in Switzerland: a cross-sectional evaluation of regional and age-specific lifetime average annual incidence.

A simple generalized linear model of toxoplasmosis incidence has been applied to serologic data from more than 9,000 women who delivered babies in Switzerland. This model, based on an assumption of constant incidence of toxoplasmosis throughout time and ages, yields estimates of incidence rates that show marked contrasts between subgroups of women categorized on the basis of their geographic origin and duration of residence in Switzerland. The patterns observed are in agreement with previously reported data from specific areas. When applied to Swiss resident women of different age groups, the average incidence rate estimates obtained were remarkably similar. This was not the case among the subgroup of Portuguese immigrant women, nor when the model was applied to previously published data from Sweden where toxoplasmosis incidence has clearly decreased in the last 20 years. Although models for the analysis of cross-sectional data involve major simplifying assumptions, it appears that they can yield useful epidemiologic information and that departures from the assumed simplified structure of the data can sometimes be identified.

[Parasitology and human medical preventive importance of Toxoplasma gondii]. / Parasitologie und humanmedizinisch-präventive Bedeutung von Toxoplasma gondii.

UNLABELLED: Present knowledge of the biology and distribution of Toxoplasma gondii allows to provide recommendations for primary prevention of infection with the parasite. The recommendations are chiefly designed for "seronegative" pregnant women without specific serum anti-T.-gondii-IgG and for persons with continuous or temporary immune deficiencies. Prevention should focus on 3 main sources of infection risk: Meat: meat should only be eaten when well cooked or when it has been frozen prior to preparation; do not prepare raw food in the same place and with the same utensils as for raw meat preparation; no mouth-finger-contact while handling raw meat. ENVIRONMENT: fruit and vegetables should be carefully washed prior to consumption (also including fruit and vegetables from the consumer's own garden or orchard). Cats: household cats should be preferably fed with canned food rather than with raw meat; contact with any utensil which may have been contaminated by cat's feces, as well as with the cat's litter, must be strictly avoided. If cleaning the cat's toilet is inevitable, plastic gloves must be worn. Disinfect the cat's toilet daily with boiling water. All primary prevention measures apply also to the areas of agriculture, veterinary practices, pet shops and gastronomy. Secondary prevention by means of serological monitoring of seronegative pregnant women can only be envisaged when associated with precise primary prevention recommendations.

[Epidemiology of toxoplasmosis: worldwide status]. / Epidémiologie de la toxoplasmose: situation au niveau mondial.

A literature review of toxoplasmosis seroprevalence data available over the last 40 years has been conducted both among European women of reproductive age and general populations at the global level. Summary maps are presented. In Europe, the highest seroprevalences have been reported from France, both among women of reproductive age and the general population. There is a higher seroprevalence in Central Europe than in Scandinavia or the United Kingdom. At the world level, prevalences similar to those of Central Europe have been reported in sub-Saharan Africa and Latin America. Lower prevalences are found in North America, South-East Asia, and Oceania. The limitations of such comparisons are discussed.

[The status of infection with Toxoplasma gondii in the Swiss population: contribution of a seroepidemiologic study from the Zurich canton]. / Zur Lage der Infektion mit Toxoplasma gondii bei der schweizerischen Bevölkerung: Beitrag einer seroepidemiologischen Studie aus dem Kanton Zürich.

To obtain information on the seroprevalence of specific antibodies to Toxoplasma gondii at the level of the general population, a study was performed using 4300 serum samples: 3500 samples collected at the blood donor center of Zürich (individuals between 18 and > 65 years old), 500 serum samples from children referred to the University Children's Hospital of Zürich (between 1 month and 17 years old) and 300 maternal sera, collected at delivery (from a mother-child sera collection of the University Children's Hospital of Zürich). Samples were systematically collected by age- (5 years) and sex-groups (sex ratio 1:1), until the desired number was obtained. The crude seroprevalence of specific IgG anti-T. gondii was 52.4% (confidence interval at 95% [p < 0.05]: 50.9-53.9). There were no significant differences between males and females. The seroprevalence of the group of women of childbearing age (between 20 and 40) was 40%. The comparison of this value with the results obtained from 300 maternal sera from the mother-child collection revealed no significant difference between the two groups. On the basis of these results and assuming that the information obtained at the level of the general population in the region of Zürich is representative of Switzerland, it was possible to calculate the required sample size to initiate a national study on seroprevalence of the infection with T. gondii among women at time of delivery.

[Epidemiology of toxoplasmosis in Switzerland: national study of seroprevalence monitored in pregnant women 1990-1991]. / Epidémiologie de la toxoplasmose en Suisse: étude nationale de séroprévalence menée chez les femmes enceintes en 1990-1991.

A national study was performed to assess the seroprevalence of anti-Toxoplasma gondii IgG in pregnant women at the time of delivery. The study was organized between 1990 and 1991 in 23 out of 26 Swiss cantons. 9059 women, corresponding to 11.8% of the annual total of births in those cantons, were included. The global seroprevalence of specific IgG was 46.1% (95% confidence interval: 45.0-47.1%). There was no significant difference in seroprevalence between different cantons after adjustment according to age. At the national level, the seroprevalence was 46.0% for Swiss women and 45.8% for women of other nationality (information on nationality was available only for 8382 persons). The use of a model of linear regression according to age showed that the risk of seroconversion among seronegative women during their 9 months of pregnancy was 1.21%. In addition, a certain number of data were calculated from that value to "simulate" a theoretical situation with absence of specific serological screening during pregnancy at a national level and consequently, absence of appropriate treatment. It was estimated that 548 cases of seroconversion would occur annually during pregnancy. This would lead to 183 congenital transmissions of toxoplasmosis, among which, 75% would be asymptomatic at birth. The number of expected pathologies would be: 40 cases of chorioretinitis with impared vision, 18 cases of cerebral lesions, and 2.7 cases of perinatal death. We observed positive results for specific anti-T. gondii-IgM in 1.7% of persons tested. This result can be the source of medical interpretation difficulties if the serum sample is the first done during pregnancy.

[Biological diagnosis of toxoplasmosis in the course of pregnancy: methods, interpretations and practical recommendations]. / Diagnostic biologique de la toxoplasmose au cours de la grossesse: méthodes, interprétations et recommandations pratiques.

The biological diagnosis of infection with Toxoplasma gondii during pregnancy can be performed: either (1.) with direct methods (microscopic examination, in vitro isolation on cell cultures, histology, detection of T. gondii DNA) or (2.) by indirect serological methods in order to demonstrate specific antibodies in serum. An appropriate combination of complementary techniques (antibody detection in serum and demonstration of the parasite) must lead, in the majority of cases, to a precise diagnosis of congenital toxoplasmosis. Serological examination of a woman before or at the beginning of pregnancy serves to distinguish between women with or without specific immune status. The diagnosis of a seroconversion during pregnancy causes few difficulties particularly if carefully followed up. However, it is rather difficult to date an infection during pregnancy based only on a initial positive sample with the presence of anti-T. gondii IgM. Thus, around 1 to 5% of serological examinations during pregnancy cause practical problems of interpretation. A certain number of recommendations are suggested to the laboratory supervisor: 1. Pregnant women should be systematically tested for specific anti-T. gondii IgG or total Ig and IgM. The choice of the techniques serves to reliably distinguish women without anti-T. gondii antibodies (women at risk for primary infection) from those with anti-T. gondii antibodies (immune women). 2. Interpretations of results should be well formulated to be understandable to the practitioner who must adequately inform the patient and determine the proper medical action on the basis of the serological results. 3. Sera should be preserved systematically for a minimum of 12 months (whatever the results may be). 4. Cases or situations causing problems should be referred to a specialized laboratory for expert advice.

Seroepidemiologic screening of Echinococcus multilocularis infection in a European area endemic for alveolar echinococcosis.

In a serologic survey for Echinococcus multilocularis infection, we screened sera from 7,884 subjects from the Doubs Departement in France, an area endemic for alveolar echinococcosis (AE) of the liver. An enzyme-linked immunosorbent assay (ELISA) with a highly species-specific antigen (Em2) and an E. multilocularis crude antigen (Emc) was used for screening. An evaluation of the cost/benefit relationship of this screening, followed by therapeutic management of patients, was made and compared with the actual cost of the follow-up and treatment of the disease in symptomatic cases in this endemic area. Antibody reactions to Em2 and/or Emc made possible the detection of eight asymptomatic clinical cases (seroprevalence averaging 1/1,000), with typical lesions of active AE revealed by abdominal ultrasonography and computed tomography. All were seropositive using the Emc ELISA but two were seronegative using the Em2 ELISA. In five additional seropositive cases, the radiologic investigations revealed small calcified lesions similar to the lesions of abortive AE previously found in Alaska. The cost of this serologic screening program per screened subject and per diagnosed case averaged 50.00 French Francs (FF) (U.S. $8.60) and 60,000.00 FF (U.S. $10,909.00), respectively. The cost of diagnosis, follow-up and treatment of the patients was 5,086.00 FF (U.S. $929.00) per patient per month in the case of diseases diagnosed by the screening program and 7,086.00 FF (U.S. $1,288.00) per patient per month for patients with symptomatic AE. This survey indicates a high prevalence of AE in the target area; it confirms the long latency period of the larval growth in human AE and shows that abortive AE is present in Europe. The use of both the Emc and Em2 ELISAs seems to be better than using the Em2 ELISA alone. The cost of the hospitalization and treatment of the eight screened patients would appear to be relatively high. Even though two of them were asymptomatic, they had very severe forms of the disease. In fact, the total cost was much lower than the actual cost of the disease when diagnosed from clinical symptoms.

Improved primary immunodiagnosis of alveolar echinococcosis in humans by an enzyme-linked immunosorbent assay using the Em2plus antigen.

Alveolar echinococcosis (AE) in humans is generally a fatal disease when not diagnosed early enough to provide curative treatment such as radical surgery. Immunodiagnosis for early detection of AE was improved by the isolation of an affinity-purified metacestode Em2 antigen and by the synthesis of recombinant Echinococcus multilocularis antigen II/3-10. Both antigens were individually assessed by enzyme-linked immunosorbent assay (ELISA) and demonstrated high specificities and diagnostic sensitivities, although both missed approximately 4 to 11% of diagnostic cases of AE. To provide an optimal serodiagnostic test, we investigated the two purified antigens by using a test employing a mixture of both purified antigens (designated Em2plus antigen) in one assay. For comparative purposes, crude E. multilocularis and Echinococcus granulosus metacestode antigens were investigated as well. The Em2plus ELISA proved to be the optimal diagnostic test with the highest diagnostic sensitivity, 97%, in serum samples from 140 patients with AE and an overall specificity of 99% for infections due to other Echinococcus and non-Echinococcus parasites. The new test combination (Em2plus ELISA) is suggested for the serodiagnosis of AE in patients and for seroepidemiological surveys.

Immunodiagnosis of toxocarosis in humans: evaluation of a new enzyme-linked immunosorbent assay kit.

Excretory/secretory (E/S) antigen derived from second-stage larvae of Toxocara canis maintained in defined medium in vitro has been well established worldwide for the immunodiagnosis of human toxocarosis by enzyme-linked immunosorbent assay. Such an enzyme-linked immunosorbent assay, based on the detection of human anti-T. canis (E/S antigen) serum immunoglobulin G, has recently been commercialized by Biokema-Affinity Products (Crissier-Lausanne, Switzerland). This commercial test kit was evaluated with regard to its application in a routine diagnostic laboratory and the reliability of the results. Of 78 patients with suspected clinical toxocarosis, 71 had anti-T. canis antibodies (positive serological result) corresponding to a diagnostic sensitivity of 91%; 14% of serum samples (n = 199) from patients with protozoan or with helminthic infections also showed positive reactions mainly related to infections with Trichinella, Strongyloides, and Fasciola species. An epidemiological study with 1,000 serum samples from randomly selected healthy blood donors and children in Switzerland demonstrated a seroprevalence of 2.7%. The test kit under evaluation had an overall diagnostic sensitivity of 91% and a relative specificity of 86%, the latter being related to some protozoan and helminthic infections. Because of the scarcity of such infections, potential cross-reactivity does not play a major role under the conditions found in the middle part of Europe. In conclusion, the application of the test kit provided for use in this study can be recommended for routine diagnostic use.