Diagnosis of visceral leishmaniasis
Parasitological diagnosis
Diagnosis of VL is usually based on the demonstration of amastigotes in Giemsa stained bone-marrow aspirates. However, in our laboratory, bone-marrow smears are mainly carried out and read in doubtful cases, because they usually are performed and interpreted by the haematologists and sometimes the pediatricians themselves and the results of the examination are later sent to us together with sera specimens.
For the purpose of parasitological diagnosis of VL, since 1996, we have been using the cytoconcentration of peripheral blood according to the technique of Petithory et al, 1997. Cytoconcentration is usually practiced first, and when positive the painful bone marrow aspiration is no more needed and the diagnosis of VL is established.
Serodiagnosis of leishmaniasis
All sera from suspected or confirmed VL cases are sent to our laboratory for serodiagnosis. Our reference technique is the fluorescent antibody test (FAT). It is carried out by using Leishmania-spot IF® slides (bioMerieux, France) on sera serially diluted starting from 1/100. Sera showing fluorescence at > 1/200 dilution are considered positive.
In addition to FAT, we have been using, since 2003, the rK39 dipstick test (DST). The first one we used was from Inbios International, USA (kalazar detect®). Then, since 2007 and up to date, we have been using the Dia. Med IT Leish® (Dia Med, Switzerland), according to Saghrouni et al., 2009.
Culture of Leishmania
Our laboratory has mainly been involved in the culture of dermotropic Leishmania, even though isolates from VL bone-marrow aspirates were occasionally cultured. Culture was mainly carried out for the purpose of typing strains by isoenzyme electrophoresis (IEE) technique. Media we have been using are the NNN (Nicolle Novy Mc Neal), the BHI (brain heart infusion agar) and the CRS (coagulated rabbit serum), the last being best for cutaneotropic L. infantum strains. Leishmania isolates were typed either in the IEE unit of the LEEP or in the laboratory of parasitology of the Faculty of Pharmacy, Monastir, where an IEE unit was set up in 2001.
Molecular biology
Up to 2008, our laboratory was not equipped for molecular biology techniques. In 2007, as part of a collaborative project on "molecular tools for accurate diagnosis and better assessment of leishmaniases", codirected by the LEEP and our own laboratory and financed by the IAEA, a molecular biology unit was set up in our department and has just started working. In the meanwhile we have been collaborating with the molecular biology unit of the LEEP, mainly for identifying Leishmania strains. The first study, conducted in 2005, aimed at identifying strains isolated from patients suspected for having SCL, by using K-DNA-PCR according to Smyth et al., 1992 and PCR-RFLP according to Guerbouj et al., 2001 (Ben Said et al., 2006).
Later, additional strains isolated from CL patients were typed by a novel Multiplex PCR (Saadi et al., in preparation) as part of the IAEA project that is still ongoing.
In addition, specimens from patients with lesions highly evocative of CL but found negative in parasitological examination were addressed for PCR to the laboratory of parasitology of the Faculty of Pharmacy, Monastir, where a molecular biology unit has just been set up. PCR was performed according to Chargui et al., 2005.
Results
Cutaneous leishmaniasis
Over the 25 year period study, 4329 patients were investigated for CL. Most of them were referred to our laboratory by the service of Dermatology of Farhat Hached hospital, Sousse. Leishmania parasites were demonstrated in 2087 cases (48.2%). In addition, out of 86 PCR performed on samples found negative in direct examination of dermal Giemsa stained smears, 17 were positive. So, the total number of CL cases diagnosed during the study period was 2104. Most of them were diagnosed during the last decade.
Out of the 2104 diagnosed cases 50 were confirmed or very likely SCL form. Fourteen came from areas known to be endemic for SCL (Le Kef, Jendouba, Siliana, Zaghouan and Bizerte governorates). The remaining 36 cases were from areas located in central Tunisian governorates where SCL has never been described: 13 were from Monastir, 12 from Sousse, 6 from Mahdia and 5 from Kairouan. Out of the 36 cases, 13 originated from areas known to be endemic for ZCL, in Kairouan, Mahdia and Sousse governorates. The age of patients ranged from to 5.5 to 63 years (median = 28.5 years). Twenty six were males and 24 were females (sex ratio M/F = 1.08).
In three patients, the isolate proved to be L. killicki. The first patient was a 5 year old child from Meknassi in Sidi Bouzid governorate where CCL was unknown. The second was a 30 year old woman from Ghomrassen, known to be endemic for CCL. The third patient was a 21 year old woman who came from Nasrallah, one of the most active ZCL foci in Kairouan governorate and from where no CCL cases were reported before.
All the remaining 2051 patients were suffering from ZCL. 1182 were females and 869 were males (sex ratio F/M = 1.36). Their age ranged from 1 month to 90 years (median = 28 years). The annual distribution of ZCL cases diagnosed over the 25 year period is shown in figure 9. The number of cases ranged from 5 in 1997 to 443 in 2004.
The place of contamination could be ascertained in 1873 out of the 2051 patients. In the 178 remaining cases, the geographical origin of contamination could not be determined with certainty because of multiple displacements of patients across two, three or more endemic areas. In addition, some patients were originating from Libya and Algeria and were not included in the analysis of the spatio-temporal distribution of ZCL cases. The distribution of the 1873 ZCL cases according to the area where the contamination took place is given in table 3. Most of the patients came from Sidi Bouzid (610 = 32.6%), Mahdia (494 = 26.4%), Kairouan (369 = 19.7%), and Sousse (306 = 16.3%) governorates. The distribution of cases according to delegations inside the four governorates mentioned above is shown in figure 10, and the annual distribution of diagnosed cases in figure 11. According to seasonal distribution 1745 (85.1 %) cases were diagnosed between October and February. All confirmed CL patients were treated with local or parenteral N-methylglucamine antimoniate (glucantime®) together with cryotherapy in those with few lesions. Most of treated patients responded well to antimonial treatment, and scarring of lesions was obtained in a few weeks (one to three) after the treatment was initiated.
Fig. 9. Annual distribution of 2051 zoonotic cutaneous leishmaniasis cases (1986-2010).
|
Governorate |
Delegation |
Nb/delegation |
Nb (% / governorate) |
|
Sidi Bouzid |
Ouled Haffouz |
453 |
610 (32.6 %) |
|
Jelma |
41 |
||
|
Sidi Bouzid city |
33 |
||
|
Others |
88 |
||
|
Mahdia |
Chorbène |
321 |
494 (26.4 %) |
|
Souassi |
54 |
||
|
Hbira |
54 |
||
|
Ouled Chamekh |
51 |
||
|
Others |
14 |
||
|
Kairouan |
Nasrallah |
127 |
369 (19.7 %) |
|
Bouhajla |
56 |
||
|
Kairouan south |
45 |
||
|
Hadjeb layoun |
42 |
||
|
Others |
99 |
||
|
Sousse |
Sidi El Heni |
241 |
306 (16.3 %) |
|
Msaken |
33 |
||
|
Others |
32 |
||
|
Others : Monastir, Gafsa, Kasserine, Sfax, Tataouine, Tozeur, Gabes, Kébili TOTAL |
- |
- |
94 (5%) |
|
- |
- |
1873 |
Table 3. Distribution of 1873 zoonotic cutaneous leishmaniasis cases according to governorate and delegation.
Fig. 10. Distribution of 1779 zoonotic cutaneous leishmaniasis cases diagnosed over the 25 year period originating from Sidi Bouzid, Mahdia, Kairouan and Sousse. The dots represent the number of cases. The limits of the governorates are shown in bold lines. The administrative district subdivisions are illustrated by different colours in each governorate.
However, in some patients, the outcome was unexpectedly atypical in that the lesions took much more delay to heal as demonstrated by the persistence of Leishmania in direct examination. In some adequately treated patients, the lesions persisted longer than one year and up to 4.5 years in one of them. In another patient, nearly 100 glucantime® injections were needed before the lesions resolved. In some additional cases, new lesions appeared while the patient was under specific treatment for previous ulcers. On the other hand, in many patients treated with in situ antimonial infiltrations, sporotrichoid nodules developed a few days or weeks later, next to the treated lesion.
Fig. 11. Annual distribution of 1779 zoonotic cutaneous leishmaniasis cases in Sidi Bouzid (A), Mahdia (B), Kairouan (C) and Sousse (D) governorates (1986 to 2010).
