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Background: Traditional diagnostic tests, ie, smear, culture, and histopathology of a skin biopsy specimen, are not always conclusive in patients with a clinical diagnosis of cutaneous leishmaniasis (CL)., Objective: Our purpose was to find out if a polymerase chain reaction (PCR) specific for Leishmania organisms might be more sensitive than the traditional diagnostic techniques, thereby decreasing the number of false-negative diagnoses., Methods: In a prospective study, smear, culture, and histopathology of skin biopsy specimens from 46 patients with a possible diagnosis of CL were compared with PCR specific for Leishmania. In addition, the Montenegro test as a measure of cellular immunity against the Leishmania parasite was performed. Proven CL was defined as a case in which at least 1 of the 3 traditional tests showed the presence of Leishmania parasites., Results: Of our 46 patients, 22 had leishmaniasis. Of the traditional tests, culture was the most sensitive but there were no statistically significant differences between the sensitivities of the various tests. PCR results were positive in all cases of proven leishmaniasis. Moreover, 3 patients with the clinical diagnosis of CL and negative findings on traditional tests had positive PCR results. Only 1 patient with a strong clinical suggestion of CL and positive Montenegro test results had negative PCR findings; this patient also had negative smear, culture, and histopathology results., Conclusion: PCR appears to be the most sensitive single diagnostic test for CL.

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 10(8) Plasmodium falciparum parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.

An evaluation of Leishmania PCR was performed with bone marrow, lymph node, and blood samples from 492 patients, 60 positive controls, and 90 negative controls. Results were compared with microscopy results for Giemsa-stained smears. PCR and microscopy of lymph node and bone marrow aspirates from patients with microscopically confirmed visceral leishmaniasis (VL) were equally sensitive. However, in patients clinically suspected of having VL and in whom parasites could not be demonstrated by microscopy, PCR was positive for 12 of 23 (52.2%) lymph node aspirates and 8 of 12 (66.7%) bone marrow aspirates, thus confirming the clinical diagnosis of VL. With PCR on filter paper, Leishmania DNA was detected in the blood of 33 of 47 (70%) patients with confirmed VL and in 2 of 11 (19%) patients suspected of having VL. Positive PCR results were more frequently found for blood samples on filter paper than for samples stored in EDTA. In conclusion, PCR is a more sensitive method than microscopy for the detection of Leishmania in lymph node and bone marrow aspirates, being especially useful for the confirmation of cases of suspected VL. Blood from a finger prick may be used for the initial PCR screening of people suspected of having VL. If the PCR of blood is negative, one should perform PCR with lymph node and/or bone marrow material, because PCR with these materials is more often positive.

We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti-Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.

The sequence of the most variable part of the small subunit ribosomal RNA (SSU rRNA) gene, comprising 800 bases, was analysed for 9 Leishmania taxa and compared with those of Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. Considerable differences were observed between the sequence of the Leishmania taxa on the one hand and those of Crithidia and Trypanosoma on the other. Amongst the Leishmania taxa only a few point mutations were found, all located within 2 sequence blocks in the central part of the SSU rRNA gene, which are unique for Kinetoplastida. These unique sequences were used for the development of kinetoplastid-specific probes and a Leishmania-specific PCR assay of high sensitivity (less than 10 parasites could be detected). Based on the observed point-mutations an identification of the Leishmania parasites, according to complex, could be achieved by direct sequencing, restriction fragment analysis or single-stranded conformation polymorphism of the PCR-generated fragments.