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Methodology
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Background
The ability to culture P. falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture adapted transmissible P. falciparum isolates, originating from distinct geographical locations.
Results
Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to A. stephensi or A. coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3).
Conclusions
These new P. falciparum culture adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.
Keywords
Malaria, transmission, parasite culture, culture adaptation, cloning, clinical isolates, Anopheles, gametocyte, oocysts
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1supplementaryfigure1.tif
Supplementary figure 1: Isolate infection intensity of cryopreserved vials The infection intensity in oocysts per mosquito of three identical cryopreserved vials of NF54 (panel a, b and c) and NF175 (panel d, e and f). After culture initiation (zero), a subculture number was added each time fresh erythrocytes were added to the culture. Between isolates SMFAs were not always performed on the same subcultures. Bars present the mean infection intensity.
- Supplementarytable1.docx
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Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 01 Jun, 2021
No community comments so far
Review #2 received
Received 26 Jul, 2021
Editorial decision: Minor Revision
On 26 Jul, 2021
Review #1 received
Received 22 Jun, 2021
Reviewer #2 agreed
On 10 Jun, 2021
Reviews received
Received 03 Jun, 2021
Reviewer #1 agreed
On 02 Jun, 2021
Reviewers invited
Invitations sent on 31 May, 2021
Editor invited
On 28 May, 2021
Editor assigned
On 28 May, 2021
Submission checks complete
On 27 May, 2021
First submitted
On 26 May, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Infectious Diseases
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
This preprint is under consideration at Malaria Journal. A preprint is 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
Methodology
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: Under Review
Version 1
Posted 01 Jun, 2021
No community comments so far
Review #2 received
Received 26 Jul, 2021
Editorial decision: Minor Revision
On 26 Jul, 2021
Review #1 received
Received 22 Jun, 2021
Reviewer #2 agreed
On 10 Jun, 2021
Reviews received
Received 03 Jun, 2021
Reviewer #1 agreed
On 02 Jun, 2021
Reviewers invited
Invitations sent on 31 May, 2021
Editor invited
On 28 May, 2021
Editor assigned
On 28 May, 2021
Submission checks complete
On 27 May, 2021
First submitted
On 26 May, 2021
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
The ability to culture P. falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture adapted transmissible P. falciparum isolates, originating from distinct geographical locations.
Results
Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to A. stephensi or A. coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3).
Conclusions
These new P. falciparum culture adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1supplementaryfigure1.tif
Supplementary figure 1: Isolate infection intensity of cryopreserved vials The infection intensity in oocysts per mosquito of three identical cryopreserved vials of NF54 (panel a, b and c) and NF175 (panel d, e and f). After culture initiation (zero), a subculture number was added each time fresh erythrocytes were added to the culture. Between isolates SMFAs were not always performed on the same subcultures. Bars present the mean infection intensity.
- Supplementarytable1.docx
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