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Ingrid Felger
Swiss Tropical and Public Health Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1255-2606
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Background Molecular detection of low-density Plasmodium falciparum infections is essential for surveillance studies conducted to inform malaria control strategies in close-to-elimination settings. Molecular monitoring of residual malaria infections usually requires a large study size, therefore sampling and diagnostic processes need to be economical and optimized for high-throughput. A method comparison was undertaken to identify the most efficient diagnostic procedure for processing large collections of community samples with optimal test sensitivity, simplicity, and minimal costs. Methods In a reactive case detection study conducted on Zanzibar, parasitaemia of 4590 individuals of all ages was investigated by a highly sensitive quantitative (q) PCR that targets multiple var gene copies per parasite genome. To reduce cost, a first round of positivity screening was performed on pools of dried blood spots from five individuals. Ten cycles of a pre-PCR were performed directly on the filter paper punches, followed by qPCR. In a second round, samples of positive pools were individually analysed by pre-PCR and qPCR. Results Prevalence in household members and neighbors of index cases was 1.7% (78/4590) with a geometric mean parasite density of 58 parasites/µl blood. Using qPCR as gold standard, diagnostic sensitivity of rapid diagnostic tests (RDTs) was 37% (29/78). Infections positive by qPCR but negative by RDT had mean densities of 15 parasites/µl blood. Conclusion The approach of pre-screening reactive case detection samples in pools of five was ideal for a low prevalence setting such as in Zanzibar. Performing direct PCR on filter paper punches saves substantial time and justifies the higher cost for a polymerase suitable for amplifying DNA directly from whole blood. Molecular monitoring in community samples provided a more accurate picture of infection prevalence, as it identified a potential reservoir of infection that was largely missed by RDT. The developed qPCR-based methodology for screening large sample sets represents primarily a research tool that should inform the design of malaria elimination strategies. It may also prove beneficial for diagnostic tasks in surveillance-response activities.
Keywords
qPCR, pooling, community-wide molecular diagnostics, Plasmodium falciparum surveillance, malaria elimination program.
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View the published article at Malaria Journal.
Version 2
Posted 15 Jan, 2020
No community comments so far
Editorial decision: Accept
On 14 Jan, 2020
Editor assigned
On 13 Jan, 2020
Submission checks complete
On 12 Jan, 2020
Editor invited
On 12 Jan, 2020
No comments provided
Review #2 received
Received 13 Dec, 2019
Editorial decision: Minor Revision
On 13 Dec, 2019
Reviewer #4 agreed
On 04 Dec, 2019
Review #1 received
Received 02 Dec, 2019
Reviewer #3 agreed
On 30 Nov, 2019
Reviewer #1 agreed
On 29 Nov, 2019
Reviewer #2 agreed
On 29 Nov, 2019
Reviewers invited
Invitations sent on 28 Nov, 2019
Editor assigned
On 21 Nov, 2019
First submitted
On 20 Nov, 2019
Submission checks complete
On 20 Nov, 2019
Editor invited
On 20 Nov, 2019
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Subject Areas
Infectious Diseases
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See the published version of this preprint at Malaria Journal.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Ingrid Felger
Swiss Tropical and Public Health Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1255-2606
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Malaria Journal.
Version 2
Posted 15 Jan, 2020
No community comments so far
Editorial decision: Accept
On 14 Jan, 2020
Editor assigned
On 13 Jan, 2020
Submission checks complete
On 12 Jan, 2020
Editor invited
On 12 Jan, 2020
No comments provided
Review #2 received
Received 13 Dec, 2019
Editorial decision: Minor Revision
On 13 Dec, 2019
Reviewer #4 agreed
On 04 Dec, 2019
Review #1 received
Received 02 Dec, 2019
Reviewer #3 agreed
On 30 Nov, 2019
Reviewer #1 agreed
On 29 Nov, 2019
Reviewer #2 agreed
On 29 Nov, 2019
Reviewers invited
Invitations sent on 28 Nov, 2019
Editor assigned
On 21 Nov, 2019
First submitted
On 20 Nov, 2019
Submission checks complete
On 20 Nov, 2019
Editor invited
On 20 Nov, 2019
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
View the published article at Malaria Journal.
Background Molecular detection of low-density Plasmodium falciparum infections is essential for surveillance studies conducted to inform malaria control strategies in close-to-elimination settings. Molecular monitoring of residual malaria infections usually requires a large study size, therefore sampling and diagnostic processes need to be economical and optimized for high-throughput. A method comparison was undertaken to identify the most efficient diagnostic procedure for processing large collections of community samples with optimal test sensitivity, simplicity, and minimal costs. Methods In a reactive case detection study conducted on Zanzibar, parasitaemia of 4590 individuals of all ages was investigated by a highly sensitive quantitative (q) PCR that targets multiple var gene copies per parasite genome. To reduce cost, a first round of positivity screening was performed on pools of dried blood spots from five individuals. Ten cycles of a pre-PCR were performed directly on the filter paper punches, followed by qPCR. In a second round, samples of positive pools were individually analysed by pre-PCR and qPCR. Results Prevalence in household members and neighbors of index cases was 1.7% (78/4590) with a geometric mean parasite density of 58 parasites/µl blood. Using qPCR as gold standard, diagnostic sensitivity of rapid diagnostic tests (RDTs) was 37% (29/78). Infections positive by qPCR but negative by RDT had mean densities of 15 parasites/µl blood. Conclusion The approach of pre-screening reactive case detection samples in pools of five was ideal for a low prevalence setting such as in Zanzibar. Performing direct PCR on filter paper punches saves substantial time and justifies the higher cost for a polymerase suitable for amplifying DNA directly from whole blood. Molecular monitoring in community samples provided a more accurate picture of infection prevalence, as it identified a potential reservoir of infection that was largely missed by RDT. The developed qPCR-based methodology for screening large sample sets represents primarily a research tool that should inform the design of malaria elimination strategies. It may also prove beneficial for diagnostic tasks in surveillance-response activities.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
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