Comparison of PCR and antigen detection methods for diagnosis of Entamoeba histolytica infection.

AIMS: To assess different laboratory methods for the identification of Entamoeba histolytica in clinical samples. METHODS: Antigen detection enzyme linked immunosorbent assay, polymerase chain reaction solution hybridisation enzyme linked immunoassay (PCR-SHELA), and a commercial Lightcycler PCR were compared using 101 stool and pus samples. RESULTS: Fifteen of the 101 samples were positive for E histolytica by one or more method. There were discrepancies between the results in five of these 15 samples when the assays were compared. CONCLUSIONS: All three methods performed adequately, so that the choice of assay will depend on each individual laboratory's budget and projected turnaround time.

Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present.

A new, rapid assay, based on a single-round, multiplex PCR, can be used to detect Plasmodium falciparum, P. vivax, P. malariae or P. ovale in human blood. The PCR, which targets the conserved 18S small-subunit RNA genes of the parasites, not only permits a malarial infection to be detected but also allows each Plasmodium species present to be identified, even in cases of mixed infection.

Non-microscopic method for malaria diagnosis using OptiMAL IT, a second-generation dipstick for malaria pLDH antigen detection.

Rapid diagnostic tests for malaria are now a commonly used procedure for malaria diagnosis. New or improved devices need to be evaluated against a recognised gold-standard procedure and subjected to conditions of temperature and humidity that may affect their performance. The OptiMAL 48 RDT has now been available commercially for several years and a second-generation OptiMAL IT test is now coming onto the market. In this study the problems associated with the routine use of OptiMAL 48 is investigated and its performance compared with a second-generation individual test, OptiMAL IT. Sensitivity and specificity for detection of all malaria species for both tests were comparable but loss of sensitivity of the test strips due to humidity or temperature found with the routine use of OptiMAL 48 was not seen with the individual OptiMAL IT. False-positive results for Plasmodium falciparum, seen in two negative blood samples, were attributed to the presence of high levels of heterophile antibodies.

Comparison of blood-film microscopy, the OptiMAL dipstick, Rhodamine-123 fluorescence staining and PCR, for monitoring antimalarial treatment.

In an attempt to see if the OptiMAL dipstick (Flow Inc., Portland, OR) can be used to monitor antimalarial treatment, a pilot study of 17 patients with Plasmodium falciparum malaria, admitted to the Hospital for Tropical Diseases in London, U.K., was conducted. Sequential, follow-up, blood specimens were obtained from day 1 to day 3, 4 or 5 post-admission. Thin and thick films prepared from these samples were examined for the presence of malarial parasites, and the intensities of parasitaemia were estimated. In addition, each specimen was tested with the OptiMAL dipstick, Rhodamine-123 fluorescence staining and, on specimens collected on day 1 and the last follow-up before discharge, by a PCR-based test. The results showed that OptiMAL has good sensitivity for the initial diagnosis of P. falciparum malaria and also mirrors the decline in viability of the parasites on treatment, giving the potential to follow the efficacy of drug treatment. The results of the PCR-based tests were still positive when the blood film and OptiMAL result were negative. The OptiMAL dipstick compared well with blood-film microscopy for monitoring antimalarial treatment and could be a useful replacement for microscopy to monitor treatment in places where facilities for microscopy are either lacking or inadequate. In developed countries it could be a useful adjunct to blood-film microscopy, and it might permit a reduction in the duration of hospitalization and give an early warning of treatment failure.

Comparison of a parasite lactate dehydrogenase-based immunochromatographic antigen detection assay (OptiMAL) with microscopy for the detection of malaria parasites in human blood samples.

Microscopic examination of blood smears remains the gold standard for malaria diagnosis, but is labor-intensive and requires skilled operators. Rapid dipstick technology provides a potential alternative. A study was conducted in The Gambia to compare the performance of OptiMAL, an immunochromatographic antigen detection assay for the diagnosis of malaria using parasite lactate dehydrogenase, against standard microscopy in patients with suspected malaria. For initial diagnosis of Plasmodium falciparum, irrespective of stage, this assay had a sensitivity of 91.3%, a specificity of 92%, a positive predictive value of 87.2%, and a negative predictive value of 94.7%. The sensitivity of the test decreased markedly at parasitemias < 0.01%. This assay can be used for the diagnosis of malaria in areas where microscopy is not available and for urgent malaria diagnosis at night and at weekends, when routine laboratories are closed and when relatively inexperienced microscopists may be on duty.

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.

Detection and identification of gastrointestinal microsporidia using non-invasive techniques.

AIMS: To detect enteric microsporidia in faecal specimens from patients with the acquired immunodeficiency syndrome (AIDS), and to identify the spores to species level without using invasive procedures. METHODS: Formalised faecal preparations were examined using a modification of the strong trichrome staining method to demonstrate microsporidian spores. Six positive specimens were prepared for electron microscopy by emulsification and separation using a 9% Ficoll gradient. RESULTS: The modified staining technique readily identified microsporidian spores. Spores of different species showed variation in size. Identification using electron microscopy was successful for five of the six positive specimens examined. It was unsuccessful for one specimen in which spores were less abundant on initial staining. CONCLUSIONS: The modified strong trichrome staining method is a useful way of detecting spores of intestinal microsporidia in faecal specimens. Variation in spore size may permit provisional identification by light microscopy. Electron microscopic examination of faecal preparations is useful for identifying spores to species level.

Rapid latex agglutination test for extraluminal amoebiasis.

AIMS: To develop a rapid latex agglutination screening test for invasive amoebiasis. METHODS: The performance of an in-house latex agglutination test was compared with three standard serological techniques--the immunofluorescent antibody test (IFAT), the indirect haemagglutination test (IHA), and the cellulose acetate precipitin (CAP) test. Forty six sera were screened; 12 from negative controls; 10 sera from infections other than amoebiasis, and 24 sera from patients with luminal or extraluminal infection with Entamoeba histolytica. RESULTS: Strong positive latex agglutination reactions were observed, with 12 of 12 sera giving combined CAP positive, IFAT positive, and IHA positive results. These results are indicative of invasive amoebiasis. Twelve CAP negative, IFAT positive sera, and 10 of 12 IHA negative gave weak or negative agglutination reactions. One of 12 CAP negative, IFAT positive, and IHA positive sera gave a strong positive latex agglutination result; one with CAP negative, IFAT positive, and IHA positive sera gave a weak latex agglutination reaction. These results correlate with either treated amoebiasis or with the early stages of invasive amoebiasis for which the CAP test is known to have a lower sensitivity than the IFAT, but a higher specificity. No reactions were observed with 12 out of 12 CAP negative, IFAT negative, and IHA negative control sera and all 10 sera from other infections (two giardiasis, three schistosomiasis, three malaria, one filariasis). CONCLUSIONS: The latex agglutination test was a useful indicator test, paralleling the results obtained with standard serological techniques. It could also be a useful screening tool in the field.

Treatment of 'Old World' cutaneous leishmaniasis with aminosidine ointment: results of an open study in London.

Five preparations of ointment containing aminosidine were used to treat lesions of 'Old World' cutaneous leishmaniasis. A preparation containing 12-15% aminosidine with 10% urea in white soft paraffin was nontoxic. 23 of 27 patients treated healed in a mean of 6.7 weeks, the ointment being applied daily for up to 12 weeks.

Comparison of techniques for detecting antigens of Giardia lamblia and Cryptosporidium parvum in faeces.

AIM: To compare the use of commercial monoclonal antibody test systems--the Giardia CEL IF test and the Crypto CEL IF test--for the detection of Giardia lamblia and Cryptosporidium parvum antigens in faeces with conventional techniques. METHODS: Sensitivity and specificity were evaluated using preparations of cysts of G lamblia and purified oocysts of C parvum. Evaluation of 59 random faecal samples passing through the Department of Clinical Parasitology, Hospital for Tropical Diseases, London, was carried out for both organisms. RESULTS: The fluorescence staining techniques proved more sensitive than other tests routinely used for diagnosis.

Parasitology: United Kingdom National Quality Assessment Scheme.

AIMS: To assess the results from parasitology laboratories taking part in a quality assessment scheme between 1986 and 1991; and to compare performance with repeat specimens. METHODS: Quality assessment of blood parasitology, including tissue parasites (n = 444; 358 UK, 86 overseas), and faecal parasitology, including extra-intestinal parasites (n = 205; 141 UK, 64 overseas), was performed. RESULTS: Overall, the standard of performance was poor. A questionnaire distributed to participants showed that a wide range of methods was used, some of which were considered inadequate to achieve reliable results. Teaching material was distributed to participants from time to time in an attempt to improve standards. CONCLUSIONS: Since the closure of the IMLS fellowship course in 1972, fewer opportunities for specialised training in parasitology are available: more training is needed. Poor performance in the detection of malarial parasites is mainly attributable to incorrect speciation, misidentification, and lack of equipment such as an eyepiece graticule.