LEISHMANIA - Science
• Towards Point-of-Care Diagnostic and Staging Tools for Human African Trypanosomiaisis
Enock Matovu, Anne Juliet Kazibwe, Claire Mack Mugasa, Joseph Mathu Ndungu, Zablon Kithingi Njiru
Diagnosis of visceral Leishmaniasis in Sudan
• Diagnosis of Visceral leishmaniasis in Sudan:- NASBA-OligoC and PCR-OligoC-tests
Saad A, Ahmed NG, Al-Basheer AA, Hamad A, Deborggraeve S , Büscher P, Schoone GJ, Schallig H, Laurent T, Haleem A, Osman OF, Altom AM and El-Safi S.
Poster presented at the Conference of the Sudanese Association of Pathologists between 7-9 April 2009
Diagnosis of visceral Leishmaniasis in Sudan
• Diagnostic Accuracy of the Leishmania OligoC-TesT for the Diagnosis of Cutaneous Leishmaniasis in Peru.
Diego ESPINOSA, Andrea K. BOGGILD, Stijn DEBORGGRAEVE, et al.
57th ASTMH annual meeting – December 11, 2008
Diagnostic Accuracy of Leishmania OligoCTesT
• Detection of Leishmania by PCR-Oligochromatography with the "Leishmania OligoC-Test".
T. Laurent, S. Deborggraeve, D. Espinoza, T. Leclipteux, P. Büscher, J. Arevalo, P. Mertens
18th ECCMID, 19-22 April 2008
Detection of Leishmania by PCR Oligo
• Leishmania OligoC-TesT - PCR-Oligochromatography (PCR-OC) Test for Amplification and Detection of Leishamania in biopsy and blood.
Laurent T, Deborggraeve S, Espinosa D, Mertens P, Dujardin JC, Arevalo J, Buscher P and Leclipteux T
• Sensitivity and specificity of the Leishmania OligoC-TesT and NASBA-oligochromatography for diagnosis of visceral leishmaniasis in Kenya.
Basiye FL, Mbuchi M, Magiri C, Kirigi G, Deborggraeve S, Schoone GJ, Saad AA, El-Safi S, Matovu E, Wasunna MK.
Trop Med Int Health. 2010 Jul;15(7):806-10. Epub 2010 May 14.
OBJECTIVE: To estimate the sensitivity and specificity of the OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya.
METHODS: Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays.
RESULTS: The Leishmania OligoC-TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90-98.8%) and a specificity of 88.8% (95% CI: 81-93.6%), while the sensitivity and specificity of the NASBA-OC were 79.8% (95% CI: 67-87%) and 100% (95% CI: 96.3-100%), respectively.
CONCLUSION: Our findings indicate high sensitivity of the Leishmania OligoC-TesT on blood while the NASBA-OC is a better marker for active disease.
• Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania.
Mugasa CM, Deborggraeve S, Schoone GJ, Laurent T, Leeflang MM, Ekangu RA, El Safi S, Saad AF, Basiye FL, De Doncker S, Lubega GW, Kager PA, Büscher P, Schallig HD.
Trop Med Int Health. 2010 Jul;15(7):800-5. Epub 2010 May 14.
OBJECTIVE: To evaluate the repeatability and reproducibility of four simplified molecular assays for the diagnosis of Trypanosoma brucei spp. or Leishmania ssp. in a multicentre ring trial with seven participating laboratories.
METHODS: The tests are based on PCR or NASBA amplification of the parasites nucleic acids followed by rapid read-out by oligochromatographic dipstick (PCR-OC and NASBA-OC).
RESULTS: On purified nucleic acid specimens, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 91.7% and 95.5%; Tryp-NASBA-OC, 95.8% and 100%; Leish-PCR-OC, 95.9% and 98.1%; Leish-NASBA-OC, 92.3% and 98.2%. On blood specimens spiked with parasites, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 78.4% and 86.6%; Tryp-NASBA-OC, 81.5% and 89.0%; Leish-PCR-OC, 87.1% and 91.7%; Leish-NASBA-OC, 74.8% and 86.2%.
CONCLUSION: As repeatability and reproducibility of the tests were satisfactory, further phase II and III evaluations in clinical and population specimens from disease endemic countries are justified.
• Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis.
Mugasa CM, Laurent T, Schoone GJ, Basiye FL, Saad AA, El Safi S, Kager PA, Schallig HD.
Parasit Vectors. 2010 Mar 2;3(1):13.
BACKGROUND: Molecular methods to detect Leishmania parasites are considered specific and sensitive, but often not applied in endemic areas of developing countries due to technical complexity. In the present study isothermal, nucleic acid sequence based amplification (NASBA) was coupled to oligochromatography (OC) to develop a simplified detection method for the diagnosis of leishmaniasis. NASBA-OC, detecting Leishmania RNA, was evaluated using clinical samples from visceral leishmaniasis patients from East Africa (n = 30) and cutaneous leishmaniasis from South America (n = 70) and appropriate control samples.
RESULTS: Analytical sensitivity was 10 parasites/ml of spiked blood, and 1 parasite/ml of culture. Diagnostic sensitivity of NASBA-OC was 93.3% (95% CI: 76.5%-98.8%) and specificity was 100% (95% CI: 91.1%-100%) on blood samples, while sensitivity and specificity on skin biopsy samples was 98.6% (95% CI: 91.2%-99.9%) and 100% (95% CI: 46.3%-100%), respectively.
CONCLUSION: The NASBA-OC format brings implementation of molecular diagnosis of leishmaniasis in resource poor countries one step closer.
• Leishmania OligoC-TesT as a simple, rapid, and standardized tool for molecular diagnosis of cutaneous leishmaniasis in Peru.
Espinosa D, Boggild AK, Deborggraeve S, Laurent T, Valencia C, Pacheco R, Miranda-Verástegui C, Llanos-Cuentas A, Leclipteux T, Dujardin JC, Büscher P, Arévalo J.
J Clin Microbiol. 2009 Aug;47(8):2560-3. Epub 2009 Jun 24.
Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru.
• A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis.
Deborggraeve S, Laurent T, Espinosa D, Van der Auwera G, Mbuchi M, Wasunna M, El-Safi S, Al-Basheer AA, Arévalo J, Miranda-Verástegui C, Leclipteux T, Mertens P, Dujardin JC, Herdewijn P, Büscher P.
J Infect Dis. 2008 Nov 15;198(10):1565-72.
BACKGROUND: Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling.
METHODS: We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan.
RESULTS: The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 microL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%-99.7%) and 95.6% (95% CI, 85.2%-98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%-97.7%), 91.7% (95% CI, 64.6%-98.5%), and 86% (95% CI, 72.7%-93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively.
CONCLUSIONS: The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites
• Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection.
Laurent T, Van der Auwera G, Hide M, Mertens P, Quispe-Tintaya W, Deborggraeve S, De Doncker S, Leclipteux T, Bañuls AL, Büscher P, Dujardin JC.
Diagn Microbiol Infect Dis. 2009 Feb;63(2):173-81. Epub 2008 Dec 18.
We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR.
• Spread of vector-borne diseases and neglect of Leishmaniasis, Europe.
Dujardin JC, Campino L, Cañavate C, Dedet JP, Gradoni L, Soteriadou K, Mazeris A, Ozbel Y, Boelaert M
Emerg Infect Dis. 2008 Jul;14(7):1013-8.
The risk for reintroduction of some exotic vector-borne diseases in Europe has become a hot topic, while the reality of others is neglected at the public health policy level. Leishmaniasis is endemic in all southern countries of Europe, with approximately 700 autochthonous human cases reported each year (3,950 if Turkey is included). Asymptomatic cases have been estimated at 30-100/1 symptomatic case, and leishmaniasis has up to 25% seroprevalence in domestic dogs. Even though leishmaniasis is essentially associated with Leishmania infantum and visceral leishmaniasis, new species, such as L. donovani and L. tropica, might colonize European sand fly vectors. Drug-resistant L. infantum strains might be exported outside Europe through dogs. Despite this possibility, no coordinated surveillance of the disease exists at the European level. In this review of leishmaniasis importance in Europe, we would like to bridge the gap between research and surveillance and control.
• American tegumentary leishmaniasis: direct species identification of Leishmania in non-invasive clinical samples.
Garcia AL, Parrado R, De Doncker S, Bermudez H, Dujardin JC.
Trans R Soc Trop Med Hyg. 2007 Apr;101(4):368-71. Epub 2006 Sep 29.
Species identification is highly relevant for improved prognosis and adequate treatment of American tegumentary leishmaniasis (ATL). PCR-based methods are available for this purpose but should be simplified to improve accessibility. As a first step in this process, this paper describes a simplified protocol for collection of clinical samples. Using samples from 44 Bolivian patients with confirmed ATL, we demonstrated that hsp70 PCR-RFLP on skin scrapings collected with a tooth pick allowed identification of the parasite species with a sensitivity of 95% and specificity of 100%. Our method should greatly facilitate individual patient management and epidemiological surveillance of ATL
• Molecular diagnosis of leishmaniasis: current status and future applications.
Reithinger R, Dujardin JC.
J Clin Microbiol. 2007 Jan;45(1):21-5. Epub 2006 Nov 8.
• Diagnostic accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for diagnosis of leishmaniasis in Sudan.
Saad AA, Ahmed NG, Osman OS, Al-Basheer AA, Hamad A, Deborggraeve S, Büscher P, Schoone GJ, Schallig HD, Laurent T, Haleem A, Osman OF, Eltom AM, Elbashir MI, El-Safi S.
PLoS Negl Trop Dis. 2010 Aug 3;4(8):e776.
BACKGROUND: The Leishmania OligoC-TesT and NASBA-Oligochromatography (OC) were recently developed for simplified and standardised molecular detection of Leishmania parasites in clinical specimens. We here present the phase II evaluation of both tests for diagnosis of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and post kala-azar dermal leishmaniasis (PKDL) in Sudan.
METHODOLOGY: The diagnostic accuracy of the tests was evaluated on 90 confirmed and 90 suspected VL cases, 7 confirmed and 8 suspected CL cases, 2 confirmed PKDL cases and 50 healthy endemic controls from Gedarif state and Khartoum state in Sudan.
PRINCIPAL FINDINGS: The OligoC-TesT as well as the NASBA-OC showed a sensitivity of 96.8% (95% CI: 83.8%-99.4%) on lymph node aspirates and of 96.2% (95% CI: 89.4%-98.7%) on blood from the confirmed VL cases. The sensitivity on bone marrow was 96.9% (95% CI: 89.3%-99.1%) and 95.3% (95% CI: 87.1%-98.4%) for the OligoC-TesT and NASBA-OC, respectively. All confirmed CL and PKDL cases were positive with both tests. On the suspected VL cases, we observed a positive OligoC-TesT and NASBA-OC result in 37.1% (95% CI: 23.2%-53.7%) and 34.3% (95% CI: 20.8%-50.9%) on lymph, in 72.7% (95% CI: 55.8%-84.9%) and 63.6% (95% CI: 46.6%-77.8%) on bone marrow and in 76.9% (95% CI: 49.7%-91.8%) and 69.2% (95% CI: 42.4%-87.3%) on blood. Seven out of 8 CL suspected cases were positive with both tests. The specificity on the healthy endemic controls was 90% (95% CI: 78.6%-95.7%) for the OligoC-TesT and 100% (95% CI: 92.9%-100.0%) for the NASBA-OC test.
CONCLUSIONS: Both tests showed high sensitivity on lymph, blood and tissue scrapings for diagnosis of VL, CL and PKDL in Sudan, but the specificity for clinical VL was significantly higher with NASBA-OC
• Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for Diagnosis of canine Leishmania infection.
CarsonC, Quinnell RJ, Holden J, Garcez LM, Deborggraeve S, Courtenay O.
J Clin Microbiol. 2010 Sep;48(9):3325-30. Epub 2010 Jul 14.
There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P