Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp.
ABSTRACT Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), an...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-06-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03354-22 |
| _version_ | 1827925135980494848 |
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| author | Carmen Chicharro Javier Nieto Silvia Miguelañez Emilia Garcia Sheila Ortega Ana Peña Jose Miguel Rubio Maria Flores-Chavez |
| author_facet | Carmen Chicharro Javier Nieto Silvia Miguelañez Emilia Garcia Sheila Ortega Ana Peña Jose Miguel Rubio Maria Flores-Chavez |
| author_sort | Carmen Chicharro |
| collection | DOAJ |
| description | ABSTRACT Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis. |
| first_indexed | 2024-03-13T05:20:52Z |
| format | Article |
| id | doaj.art-0ff598ab782e4bbfbaeeedf73d5c50f9 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-13T05:20:52Z |
| publishDate | 2023-06-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-0ff598ab782e4bbfbaeeedf73d5c50f92023-06-15T13:18:30ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-06-0111310.1128/spectrum.03354-22Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp.Carmen Chicharro0Javier Nieto1Silvia Miguelañez2Emilia Garcia3Sheila Ortega4Ana Peña5Jose Miguel Rubio6Maria Flores-Chavez7Reference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainReference and Research Laboratory for Parasitology, National Centre for Microbiology, Instituto de Salud Carlos III (ISCIII), Madrid, SpainABSTRACT Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.https://journals.asm.org/doi/10.1128/spectrum.03354-22leishmaniasismolecular diagnosissensitivityspecificitynested PCRqPCR |
| spellingShingle | Carmen Chicharro Javier Nieto Silvia Miguelañez Emilia Garcia Sheila Ortega Ana Peña Jose Miguel Rubio Maria Flores-Chavez Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp. Microbiology Spectrum leishmaniasis molecular diagnosis sensitivity specificity nested PCR qPCR |
| title | Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp. |
| title_full | Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp. |
| title_fullStr | Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp. |
| title_full_unstemmed | Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp. |
| title_short | Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp. |
| title_sort | molecular diagnosis of leishmaniasis in spain development and validation of ready to use gel form nested and real time pcrs to detect leishmania spp |
| topic | leishmaniasis molecular diagnosis sensitivity specificity nested PCR qPCR |
| url | https://journals.asm.org/doi/10.1128/spectrum.03354-22 |
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