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Evaluation comparee des outils classiques et moleculaires pour le d iagnostic et les investigations eco-epidemiologiques dans le cadre des leishmanioses

Objective

- To develop a DNA probe specific for Leishmania infantum; to measure its general efficiency for diagnosis of human and canine leishmaniasis (in particular during disease development in the dog), using the dot blot technique; to evaluate the results with those obtained by non-molecular, classical methods;
- To evaluate the comparative sensitivities and specificities of kDNA probes for the identification of L. infantum;
- To measure cytokine production by PBMC in response to parasite antigen to understand the TH1 versus TH2 balance in clinical groups as a function of age;
- To measure the intrinsic validity and extrinsic validity parameters for DNA probes specific for Leishmania major, as used in the dot blot technique for the diagnosis of zoonotic cutaneous leishmaniasis in man; and, to compare the results with the estimates of these parameters for classical techniques (direct examination, culture, inoculation of animals, serology);
- To evaluate squash blotting, ELISA and classical dissection techniques for the detection of Leishmania in the phlebotomine vector, for the pairs Phlebotomus papatasi/Leishmania major, P. perniciosus/Leishmania infantum and P. perfiliewi/L.infantum.
- In general, serological tests were the most sensitive (73% for IFAT, 94.6%% for ELISA), followed by the Balb/c inoculation (48.2%) :
- Balb/c mice (MI): the lesions appeared from 4 to 9 weeks (max. 67 days);
- NNN cultures (C): 12 were positive within the first week and 3 in the second week of subcloning. Only one of the samples giving a positive culture was negative in Balb/c mice;
- Direct smear (DS): of the 26 samples positive in Balb/c mice only 12 (46.15%) were also positive by direct smear, but 4 smears were positive when the Balb/c results were negative;
- ELISA: soluble antigens were prepared by the classical technique from a Tunisian strain of L. major (MPSA/TN/86/RON44; zymodeme MON-25/LON-1) and uses at 1/500 dilution; sera were diluted at 1/100; positive and negative controls were used on each ELISA plate; spectrophotometry reading was at 420 nm;
- IFAT : the same strain of L. major was used; sera were diluted 1/50 in PBS, and the fixed antibodies were revealed with anti-human antiglobulins labelled with fluorescein;
- Dot blots: samples from 54 patients were hybridised with the 85 bp universal probe (SU), the 450 bp TaqI probe (TaqI) and the 250 bp AvaII probe (AvaII) (see section B.2.2 of first technical report). Autoradiographic exposure was set at one week for the first two probes and two weeks for the third probe. The presence of Leishmania parasites in the samples was confirmed by at least one direct test. The variable number of dot-blot samples (3-6) made for each lesion was shown to be adequate, even though the intensity of the autoradiographic signal varied both within and among lesion samples.

- However, only some of the patients' lesions had demonstrable parasites, and so the evaluation of the various indirect techniques can be better estimated after selection of the positive patient sample. This "gold standard" was the positivity demonstrated by at least one of the three classical techniques that allow visualisation of the parasite, namely direct smear, culture and Balb/c inoculation. The gold-standard sample is constituted by 30 patients among the total of 54. For this sample the dot blot sensitivity was 46.7% (14/30) or 40.0% (12/30) depending on the probe. The 24 patients not showing any parasites in their lesions, when assessed by the three gold-standard tests, constituted the negative control sample that permitted calculation of the specificity of each of the indirect tests. The specificity was 100% for all techniques except for ELISA and IFAT.

- Probe 3E9/HaeIII-12 (= 3E9) can be considered as an excellent tool for the specific identification of L. infantum in Tunisia (and in the Mediterranean Basin in the absence of L. donovani) when 105 or fewer promastigotes are used: then, both the specificity and predictive value of the positive result were 100%. As with most diagnostic tools, there is a trade-off between specificity and sensitivity, with a loading of 105 promastigotes being optimal.
- Probe 3B8/HaeIII-2 (=3B8) was assessed to be less efficient than 3E9, its sensitivity being one order less and the predictive value of a negative result never reaching 100%.

Achievements

- Measurement of the intrinsic validity parameters for DNA probes specific for L. major, as used in the dot blot technique for the diagnosis of ZCL in man, and comparison of the results with the estimates of these parameters for classical techniques (direct examination, culture, inoculation of animals, serology).
- Using the KDNA probes previously isolated, the dot-blot DNA test was shown to be 100% specific, as were the three direct visualisation tests (direct smear, Balb/c mouse inoculation, NNN culture). The serological tests (IFAT, ELISA) showed significantly lower specificity but higher sensitivity. Inoculation of Balb/c mice proved to be the test with highest "global efficiency". The dot blot test is, therefore, a useful addition to the battery of tests now available for diagnosis of ZCL due to L. major, performing as well as all tests except inoculation of Balb/c mice and, unlike the serological tests used, having 100% predictive value of a positive result.
- Confirmation was obtained of the diagnostic value of a ribosomal IGS DNA probe for the identification in different regions of Tunisia of P. papatasi, the only known vector of L. major in north Africa. Development was started of DNA probes specific for the most abundant and widespread Tunisian vectors of L. infantum: restriction mapping, subcloning and sequencing permitted the identification of DNA fragments and internal repeat sequences that showed marked specificity for P. perniciosus and P. perfiliewi.

- Phlebotomine sandflies necessary for the various molecular tasks were successfully collected from different regions of Tunisia;
- For the evaluation of the sensitivity and specificity of probes for dot-blotted promastigotes of L. infantum 3E9/HaeIII-12 and 3B8/HaeIII-2, the two mini-circle kinetoplast (k) DNA probes were tested using 49 promastigote preparations, including 7 different species of Leishmania and Sauroleishmania tarentolae originating from several Old World countries. Promastigotes were cultured in RPMI medium with foetal calf serum. Serial dilutions of 106, 105, 104 and 103 promastigotes were applied to replicate nylon DNA transfer membranes using a vacuum blot apparatus;
- For the evaluation of the diagnostic potential of probes specific for L. infantum infecting man and dog and comparison with classical techniques' samples were collected from 32 human cases and 152 dogs;
- In a basic health care centre in the Sidi Bouzid focus, 54 patients were selected as having ZCL on a clinical basis (epidemiological context, clinical presentation and site of lesion, duration of lesion for more than 3 weeks, inefficacy of antibiotics). The patient sample constituted 25 males (43%) and 29 females (57%) varying in age from 2 to 81 years old. The number of lesions per patient varied from 1 to 11 (average = 3). 30 patients had already started a course of Glucantime treatment;


- Samples were taken from each lesion for :
- a direct dermal smear on a microscope slide was coloured by the May-Gunwald-Giemsa technique and read with an optic microscope at x1000 magnification (54 patients);
- a culture was made in NNN medium with Penicillin G (800 U/ml rabbit blood and Streptomycin (500mg/ml rabbit blood) (49 patients);
- 3 to 6 drops of dermal fluid (ca. 5ml each) were spotted on to a Genescreen Plus nylon DNA transfer membrane; after the normal denaturation and neutralisation processes, each membrane was treated with proteinase K (100 mg/ml in 0.1M Tris-HCl pH 7.5) for 1 hour at 37°C (54 patients);
- dermal fluid was inoculated in to the hind footpads of a Balb/c mouse.
A blood sample was taken for ELISA and IFAT tests.

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Coordinator

Natural History Museum
Address
Cromwell Road
SW7 5BD London
United Kingdom
 

Participants (3)

IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE
United Kingdom
Address

SW7 2AY London
 
INSTITUT PASTEUR DE TUNIS
Tunisia
Address
13,Place Pasteur 13
1002 Tunis
 
Universidade Nova de Lisboa
Portugal
Address
96,Rua De Junqueira
1300 Lisboa