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Research Article
Comparison of Three PCR-Based Methods to Detect Loa loa and Mansonella perstans in Long-Term Frozen Storage Dried Blood Spots
Thuy-Huong Ta-Tang
Instituto de Salud Carlos III
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7058-4839
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Loa loa and Mansonella perstans are two filarial species very common in Africa, with overlapping geographic distribution in some areas. Microscopy is the traditional diagnostic method for human loiasis and mansonellosis, but is a time-consuming, labor intensive and tedious. Polymerase chain reaction (PCR) methods have emerged as an alternative approach for identification of filarial parasites. Dried blood spot (DBS) has been reported as a convenient way to keep DNA for epidemiological investigations and diagnosis of infectious diseases, and does not require venipuncture. The finding of a highly sensitive DNA extraction method for filarial nematodes is also required for a good molecular performance. The aim of this study was to compare three different molecular methods to diagnose human loiasis and mansonellosis using DBS as a medium of sample collection and storage. The saponin/Chelex method for extracting filarial DNA was also applied.
Methods: A total of 100 clinical samples were randomly selected for this study. Microscopy was used as the reference method for diagnosing and calculating the microfilaraemia. Filarial DNA was extracted using saponin/Chelex method from DBS. DNA isolated was assayed by three different molecular methods: qPCR, Filaria-Nested PCR, and cytochrome oxidase I PCR. All PCR-products were subsequently purified and sequenced. Statistical values for each molecular test were computed and compared.
Results: Overall, 64 samples were identified as negative by all the tests and 36 samples were positive by at least one of the methods tested. Microscopy detected 27 positive samples, meanwhile qPCR, Filaria-Nested PCR and COI PCR detected 30, 31 and 33 positive samples, respectively. The best overall results were obtained with COI PCR protocol (sensitivity 92.6%; specificity 89.0%; kappa index 76.3%).
Conclusions: Despite the good statistical values obtained for COI PCR, this method needs the sequencing of the fragment obtained to identify the filarial species; thus the optimal technique to diagnose filarial infection was qPCR, it was very similar in terms of sensitivity and specificity compared to microscopy and for its capacity to detect a wide range of human filariae. It is an appropriate method for filarial diagnosis in non-endemic settings.
Keywords
Filariae, Loa loa, Mansonella perstans, dried blood spots, saponin/Chelex, microscopy, molecular diagnosis, Real-time PCR, Nested-PCR
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Due to technical limitations, table 1 to 4 xlsx are only available as a download in the Supplemental Files section.
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This is a preprint. It has not completed peer review.
Research Article
Comparison of Three PCR-Based Methods to Detect Loa loa and Mansonella perstans in Long-Term Frozen Storage Dried Blood Spots
Thuy-Huong Ta-Tang
Instituto de Salud Carlos III
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7058-4839
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 18 Sep, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Infectious Diseases
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Loa loa and Mansonella perstans are two filarial species very common in Africa, with overlapping geographic distribution in some areas. Microscopy is the traditional diagnostic method for human loiasis and mansonellosis, but is a time-consuming, labor intensive and tedious. Polymerase chain reaction (PCR) methods have emerged as an alternative approach for identification of filarial parasites. Dried blood spot (DBS) has been reported as a convenient way to keep DNA for epidemiological investigations and diagnosis of infectious diseases, and does not require venipuncture. The finding of a highly sensitive DNA extraction method for filarial nematodes is also required for a good molecular performance. The aim of this study was to compare three different molecular methods to diagnose human loiasis and mansonellosis using DBS as a medium of sample collection and storage. The saponin/Chelex method for extracting filarial DNA was also applied.
Methods: A total of 100 clinical samples were randomly selected for this study. Microscopy was used as the reference method for diagnosing and calculating the microfilaraemia. Filarial DNA was extracted using saponin/Chelex method from DBS. DNA isolated was assayed by three different molecular methods: qPCR, Filaria-Nested PCR, and cytochrome oxidase I PCR. All PCR-products were subsequently purified and sequenced. Statistical values for each molecular test were computed and compared.
Results: Overall, 64 samples were identified as negative by all the tests and 36 samples were positive by at least one of the methods tested. Microscopy detected 27 positive samples, meanwhile qPCR, Filaria-Nested PCR and COI PCR detected 30, 31 and 33 positive samples, respectively. The best overall results were obtained with COI PCR protocol (sensitivity 92.6%; specificity 89.0%; kappa index 76.3%).
Conclusions: Despite the good statistical values obtained for COI PCR, this method needs the sequencing of the fragment obtained to identify the filarial species; thus the optimal technique to diagnose filarial infection was qPCR, it was very similar in terms of sensitivity and specificity compared to microscopy and for its capacity to detect a wide range of human filariae. It is an appropriate method for filarial diagnosis in non-endemic settings.
Figure 1
Due to technical limitations, table 1 to 4 xlsx are only available as a download in the Supplemental Files section.