A Simplified and Standardized Polymerase Chain Reaction Format for the Diagnosis of Leishmaniasis

Deborggraeve, Stijn; Laurent, Thierry; Espinosa, Diego A.; Auwera, Gert Van der; Mbuchi, Margaret; Wasunna, Monique; El-Safi, Sayda; Al-Basheer, Ahmed Almustafa; Arévalo, Jorge; Miranda‐Verástegui, César; Leclipteux, T.; Mertens, Pascal; Herdewijn, Piet; Büscher, Philippe

doi:10.1086/592509

Cited by 76 publications

(75 citation statements)

References 34 publications

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“…Furthermore, there is a lack of standardization in the selection of target Leishmania DNA sequences and experimental PCR protocols used in laboratories worldwide, which complicates objective comparisons of test sensitivity and specificity. In order to address some of these issues, a commercially available PCR test kit (Leishmania OligoC-TesT) has been developed and validated for detection of Leishmania parasite DNA in human specimens (3,6). Sensitivities of the test ranged from 77.8% to 100% for clinical samples from patients with visceral leishmaniasis in Sudan and Kenya, with a limit of detection of 1 parasite in 180 l blood (3).…”

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confidence: 99%

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

et al. 2010

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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 < 0.05). Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries.

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confidence: 99%

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

et al. 2010

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“…Recently, the Leishmania OligoC-TesT was introduced as an innovative PCR format for simplified and standardized molecular diagnosis of Leishmania infections (7). We have demonstrated that the Leishmania OligoC-TesT offers performance comparable to that of conventional kDNA PCR for lesion scrapings in cases of suspected and confirmed CL in Peru.…”

Section: Discussionmentioning

confidence: 98%

“…The Leishmania OligoC-TesT was performed as described by Deborggraeve et al (7). Briefly, Leishmania DNA was amplified by PCR and mixed with an equal volume of migration buffer preheated at 55°C.…”

Section: Patientsmentioning

confidence: 99%

“…Access to sophisticated equipment such as real-time PCR machines is often limited in Peru. Recently, a simple and rapid dipstick format for detection of amplified Leishmania DNA was developed (Leishmania OligoC-TesT) (7). The test is based on PCR amplification of a small sequence of the 18S rRNA gene followed by visualization of the PCR products on a dipstick by hybridization with a gold-conjugated probe.…”

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Leishmania OligoC-TesT as a Simple, Rapid, and Standardized Tool for Molecular Diagnosis of Cutaneous Leishmaniasis in Peru

et al. 2009

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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 LeishmaniaOligoC-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.

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“…The implementation of more simple assays such as PCR-oligochromatography might constitute a promising alternative in the future. 65 Serologic diagnosis is not used in CL diagnosis in Bolivia. Sensitivity and specificity of tests can be variable, with tests suffering from cross-reaction with Trypanosoma cruzi , which is sympatric in many leishmaniases-endemic areas.…”

Section: Disease Distribution Notification and Incidencementioning

confidence: 99%

Leishmaniases in Bolivia: Comprehensive Review and Current Status

García

Parrado²,

Rojas³

et al. 2009

The American Journal of Tropical Medicine and Hygiene

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Abstract. The leishmaniases are protozoan, zoonotic diseases transmitted to human and other mammal hosts by the bite of phlebotomine sandflies. Bolivia has the highest incidence of cutaneous leishmaniasis (CL) in Latin America (LA), with 33 cases per 100,000 population reported in 2006. CL is endemic in seven of the country's nine administrative departments. Visceral leishmaniasis (VL) is comparatively rare and is restricted to one single focus. Most CL cases are caused by Leishmania ( Viannia ) braziliensis (85% cases); VL is caused by L. (L.) infantum. Seven sandfly species are incriminated as vectors and Leishmania infections have been detected in several non-human mammal hosts. Transmission is associated with forest-related activities, but recently, cases of autochthonous, urban transmission were reported. Because most cases are caused by L. (V.) braziliensis, Bolivia reports the greatest ratio (i.e., up to 20% of all cases) of mucosal leishmaniasis to localized CL cases in LA. Per national guidelines, both CL and VL cases are microscopically diagnosed and treated with pentavalent antimony.

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A Simplified and Standardized Polymerase Chain Reaction Format for the Diagnosis of Leishmaniasis

Cited by 76 publications

References 34 publications

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

Leishmania OligoC-TesT as a Simple, Rapid, and Standardized Tool for Molecular Diagnosis of Cutaneous Leishmaniasis in Peru

Leishmaniases in Bolivia: Comprehensive Review and Current Status

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