Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays

Thiam, Laty G.; Nyarko, Prince B.; Kusi, Kwadwo Asamoah; Niang, Makhtar; Aniweh, Yaw; Awandare, Gordon A.

doi:10.1038/s41598-021-86438-1

Cited by 8 publications

(8 citation statements)

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“…We used 1 mg/mL of chymotrypsin in the present study based on previous knowledge that higher concentrations yielded similar levels of efficiency. Moreover, we recently showed that the efficiency of enzyme treatment could be influenced by erythrocytes' surface receptor densities (31). Notwithstanding, the current observation about Rh2b, together with previous data, highlight the contributory role of Rh2b to erythrocyte invasion and its likely involvement in dictating phenotypic variation.…”

Section: B Asupporting

confidence: 50%

Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population

et al. 2022

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BackgroundErythrocyte invasion by P. falciparum involves functionally overlapping interactions between the parasite’s ligands and the erythrocyte surface receptors. While some P. falciparum isolates necessarily engage the sialic acid (SA) moieties of the erythrocytes during the invasion, others use ligands whose binding is independent of SA for successful invasion. Deciphering the major pathway used by P. falciparum clinical isolates represent a key step toward developing an efficient blood stage malaria vaccine.MethodsWe collected a total of 156 malaria-infected samples from Ghanaian children aged 2 to 14 years and used a two-color flow cytometry-based invasion assay to assess the invasion phenotype diversity of Ghanaian P. falciparum clinical isolates. Anti-human CR1 antibodies were used to determine the relative contribution of the PfRh4-CR1 interaction in the parasites invasion phenotype and RT-qPCR was used to assess the expression levels of key invasion-related ligands.ResultsOur findings show no clear association between demographic or clinical data and existing reports on the malaria transmission intensity. The complete invasion data obtained for 156 isolates, showed the predominance of SA-independent pathways in Ghanaian clinical isolates. Isolates from Hohoe and Navrongo had the highest diversity in invasion profile. Our data also confirmed that the PfRh4-CR1 mediated alternative pathway is important in Ghanaian clinical isolates. Furthermore, the transcript levels of ten invasion-related genes obtained in the study showed little variations in gene expression profiles within and between parasite populations across sites.ConclusionOur data suggest a low level of phenotypic diversity in Ghanaian clinical isolates across areas of varying endemicity and further highlight its importance in the quest for new intervention strategies, such as the investigation of blood-stage vaccine targets, particularly those targeting specific pathways and able to trigger the stimulation of broadly neutralizing invasion antibodies.

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Section: B Asupporting

confidence: 50%

Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population

et al. 2022

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“…Samples used in this study were collected from Ghanaian children with uncomplicated malaria visiting the LEKMA hospital in Accra. To monitor the in vitro growth patterns of freshly collected P. falciparum clinical isolates, parasites were initially allowed to grow in the patient-derived erythrocytes for a minimum period of 96 hours after which freshly washed O + erythrocytes (from a single donor) ( Thiam et al., 2021 ) were added and growth tests were performed for isolates that successfully grew. Of the 25 isolates used in this study, 19 (76%) yielded detectable parasitemia 48 hours following in vitro adaptation.…”

Section: Resultsmentioning

confidence: 99%

“…Target erythrocytes were treated with either 250 mU/mL of neuraminidase, 1 mg/mL of trypsin or 1 mg/mL of chymotrypsin for 1 hour at 37° C with gentle shaking and washed thrice with RPMI1640 medium. The efficiency of enzyme treatment was assessed as previously described ( Thiam et al., 2021 ). Treated erythrocytes were then labelled with 20 µM of carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (Thermo Fisher Scientific) for two hours at 37° C with gentle shaking and protected from light exposure.…”

Section: Methodsmentioning

confidence: 99%

Short-term cryopreservation and thawing have minimal effects on Plasmodium falciparum ex vivo invasion profile

Thiam

Ansah

Niang

et al. 2022

Front. Cell. Infect. Microbiol.

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Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

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“…Target erythrocytes were treated with either 250 mU/mL of neuraminidase, 1 mg/mL of trypsin or 1 mg/mL of chymotrypsin for 1 hour at 37º C with gentle shaking and washed thrice with RPMI1640 medium. The efficiency of enzyme treatment was assessed as previously described 18 . Treated erythrocytes were then labelled with 20 µM of carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (Thermo Fisher Scientific) for two hours at 37º C with gentle shaking and protected from light exposure.…”

Section: Enzyme Treatment and Erythrocyte Invasion Phenotyping Assaysmentioning

confidence: 99%

“…Samples used in this study were collected from Ghanaian children with uncomplicated malaria visiting the LEKMA hospital in Accra. To monitor the in vitro growth patterns of freshly collected P. falciparum clinical isolates, parasites were initially allowed to grow in the patient-derived erythrocytes for a minimum period of 96 hours after which freshly washed O + erythrocytes (from a single donor) 18 were added and growth tests were performed for isolates that successfully grew. Of the 25 isolates used in this study, 19 (76%) yielded detectable parasitemia 48 hours following in vitro adaptation.…”

Section: P Falciparum Clinical Isolates Show Different Growth Patterns During Early In Vitro Culture Adaptationmentioning

confidence: 99%

Short-Term Cryopreservation and Thawing have Minimal Effects on Plasmodium Falciparum Ex Vivo Invasion Profile

Thiam¹,

Ansah²,

Niang³

et al. 2021

Preprint

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Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. Our findings indicate that natural P. falciparum infections mostly occur as polyclonal infections. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

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Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays

Cited by 8 publications

References 42 publications

Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population

Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population

Short-term cryopreservation and thawing have minimal effects on Plasmodium falciparum ex vivo invasion profile

Short-Term Cryopreservation and Thawing have Minimal Effects on Plasmodium Falciparum Ex Vivo Invasion Profile

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