Diagnostic Efficacy of Molecular Techniques for Detection and Identification of Leishmania Species in Human Whole Blood and Skin Samples from Ecuador

Muñoz, Érika; Santander, Stephanie; Rojas-Silva, Patricio; Cárdenas, Paúl; Fornasini, Marco; Cifuentes, Sara C; Salvador, Daniela; Baldeón, Manuel E.

doi:10.4269/ajtmh.16-0385

Cited by 4 publications

(4 citation statements)

References 19 publications

(28 reference statements)

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“…Therefore, more sensitive methods such as the polymerase chain reaction (PCR) have been developed as an alternative for the diagnosis and identification of Leishmania species. PCR platforms show sensitivity values between 92 and 100% and specificity of 100% ( Reithinger and Dujardin, 2007 ; Shahbazi et al, 2008 ; Mohammadiha et al, 2013 ; Adams et al, 2014 ; Munoz et al, 2016 ). For the amplification of DNA fragments of Leishmania species, the use of genetic targets such as kinetoplast DNA (kDNA), which has a sensitivity of 97% and a specificity of 87%, has been reported in several studies ( Marques et al, 2001 ; Rodriguez et al, 2002 ; Jara et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

et al. 2017

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Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

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Section: Introductionmentioning

confidence: 99%

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

et al. 2017

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“…Blood samples associated with molecular methods have already been widely used for visceral leishmaniasis diagnosis and showed sensitivity equal to or better than bone marrow or splenic aspirates [ 81 , 82 ]. For CL diagnosis, as expected, the blood samples even with leukocyte enrichment showed reduced sensitivity compared to samples obtained directly from the lesion [ 38 , 69 ], but they also showed that the dissemination of the parasite with the detection of DNA in the blood occurs even before the appearance of the mucosal lesion and is persistent after treatment [ 83 , 84 , 85 ]. Previous research has reported the isolation of the parasite in direct culture of blood samples from patients with ML [ 86 , 87 , 88 ].…”

Section: Discussionmentioning

confidence: 83%

“…Although in this study, standardized protocol was employed for sample collection and slide preparation, and the same professional examined all the slides, 206 patients would not have been diagnosed if only DP was performed. Molecular tests are more sensitive for TL than for DP, with sensitivity for TL of up to 100% [ 35 , 68 , 69 , 70 ]. As expected, higher sensitivity was observed for molecular tests (PCR) employed in this work, and only 10 samples positive for DP were negative for both molecular tests.…”

Section: Discussionmentioning

confidence: 99%

Overcoming the Negligence in Laboratory Diagnosis of Mucosal Leishmaniasis

et al. 2021

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The northern region of Brazil, which has the largest number of cases of tegumentary leishmaniasis (TL) in the country, is also the region that has the highest diversity of species of vectors and Leishmania parasites. In this region, cases of mucosal leishmaniasis (ML), a clinical form of TL, exceed the national average of cases, reaching up to 12% of the total annual TL notifications. ML is associated with multiple factors, such as the parasite species and the viral endosymbiont Leishmania RNA virus 1 (LRV1). Being a chronic parasitological disease, laboratory diagnosis of ML poses a challenge for health services. Here, we evaluated more than 700 clinical samples from patients with clinical suspicion of TL, including patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis, comparing the results of parasitological tests—direct parasitological examination by microscopy (DP) and conventional PCR (cPCR) targeting of both kDNA and hsp70. The DP was performed by collecting material from lesions through biopsies (mucosal lesions) or scarification (cutaneous lesions); for PCR, a cervical brush was used for sample collection. Blood samples were tested employing standardized real-time PCR (qPCR) protocol targeting the HSP70 gene. PCR tests showed higher sensitivity than DP for both CL and ML samples. Considering ML samples only (N = 89), DP showed a sensitivity of 49.4% (N = 44) against 98.8% (N = 88) for kDNA PCR. The qPCR hsp70 for blood samples from patients with ML (N = 14) resulted in superior sensitivity (50%; N = 7) compared to DP (21.4%; N = 3) for samples from the same patients. Our results reinforced the need to implement a molecular test for the diagnosis of ML, in addition to proposing methods less invasive for collecting material from TL patients. Sample collection using a cervical brush in lesions observed in CL and ML patients is easy to perform and less invasive, compared to scarification and biopsies. Blood samples could be a good source for qPCR diagnosis for ML patients. Thus, we propose here a standardized method for collection and for performing of molecular diagnosis of clinical samples from suspicious ML patients that can be applied in reference services for improving ML diagnosis.

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“…2,4,5 Molecular tools have been used for the diagnosis of leishmaniasis, owing to their high levels of sensitivity (40-92%) and specificity (57.1-100%). 6,7 However, the requirement for complex equipment and infrastructure means that molecular diagnostic techniques are not suitable for use in remote areas or in the search for active cases (field work). In 2000, Notomi et al 8 designed a new technique known as loop-mediated isothermal amplification (LAMP).…”

Section: Introductionmentioning

confidence: 99%

Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis

León

Muñoz

Tabares

et al. 2018

The American Journal of Tropical Medicine and Hygiene

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Loop-mediated isothermal amplification (LAMP) is ideal for the detection of DNA as it is a quick and easy-to-perform test that does not require complex or sophisticated equipment or infrastructure. However, the application of this technique in the detection of DNA has not been comprehensively analyzed to date (analytical validation). Our objective was to evaluate the sensitivity and analytical specificity (anticipated reportable range [ARR], the limit of detection [LoD], and accuracy) of LAMP targeting the 18S rRNA gene in the diagnosis of six New World species. We then applied the validated LAMP assay across 50 samples of sandflies and 50 direct smears from a recent outbreak of cutaneous leishmaniasis in Colombia to determine its diagnostic performance. The LAMP assay exclusively amplified the DNA of spp., and an ARR of between 1 × 10 and 1 × 10 equivalent parasites/mL was determined. An LoD of 1 × 10 equivalent parasites/mL was established and there was no statistically significant variation in terms of accuracy. Finally, a sensitivity of 100% in direct smears and sandflies samples was calculated and a specificity of 90.9% for direct smears using microscopy as reference and 96.8% for sandflies using real-time polymerase chain reaction as reference were determined. To our knowledge, this is the first attempt to analytically validate a LAMP test to detect DNA, which showed good diagnostic potential from sandflies and direct smear samples.

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Diagnostic Efficacy of Molecular Techniques for Detection and Identification of Leishmania Species in Human Whole Blood and Skin Samples from Ecuador

Cited by 4 publications

References 19 publications

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

Overcoming the Negligence in Laboratory Diagnosis of Mucosal Leishmaniasis

Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis

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