Browsing by Author "Ayanful-Torgby, R."
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Item A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs(2013-12-17) Quashie, N.B.; Duah, N.O.; Abuaku, B.; Quaye, L.; Ayanful-Torgby, R.; Akwoviah, G.A.; Kweku, M.; Johnson, J.D.; Lucchi, N.W.; Udhayakumar, V.; Duplessis, C.; Kronmann, K.C.; Koram, K.A.Abstract Background Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. Methods A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator. Results Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC50 value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs. Conclusion Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC50 value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended.Item An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs(2015-12-01) Dery, V.; Duah, N.O.; Ayanful-Torgby, R.; Matrevi, S.A.; Anto, F.; Quashie, N.B.Abstract Background The recently introduced SYBR Green1 (SG) assay for testing parasites susceptibility to anti-malarial drugs needs further improvement. This has been necessitated by various setbacks, the major one being the low fluorescence intensity associated with it use. This shortcoming diminishes the anticipated hope that this novel method was going to replace the more traditional ones, such as the isotopic and microscopy. In order to restore confidence in its use, series of experiments to determine conditions that give the best fluorescence intensity were conducted. Methods Conditions that yield the maximum fluorescent signal were ascertained by measuring the fluorescence after incubation of Plasmodium falciparum culture at different parasites concentration with lysis buffer containing SYBR Green (LBS) at different time period. In order to ascertain the effect of freeze–thaw on fluorescence intensity, P. falciparum culture was frozen for 1 h, thawed, incubated with LBS and the fluorescence measured. The optimized conditions determined in this study were then used to assess the susceptibility of clinical isolates of P. falciparum to artesunate, chloroquine and mefloquine. The concentration of anti-malarial drug inhibiting parasite growth by 50 % (IC50) for each drug was estimated using the online ICEstimator. The IC50 generated using the optimized SG method determined in this study was compared with that obtained using microscopic method and the previously reported standard SG method. Results Over all, the SG method was found to be easy to perform and sensitive. Freeze–thaw of parasite culture followed by incubation with lysis buffer containing the dye for 3 h was consistently observed to give the highest fluorescence signal. The IC50 values for chloroquine, mefloquine and artesunate determined were consistent and comparable with that determined with the previously reported standard SG method and the microscopic method. Conclusion The authors conclude that freezing and thawing of parasite culture, followed by incubation with LBS in the dark for 3 h provided a significant improvement in fluorescence signal. The IC50 generated using the improved SG method is comparable with that from microscopy and the standard method.Item Dynamics of anti-MSP3 and Pfs230 antibody responses and multiplicity of infection in asymptomatic children from southern Ghana(Parasites & vectors, 2018-01) Amoah, L.E.; Acquah, F.K.; Ayanful-Torgby, R.; Oppong, A.; Abankwa, J.; Obboh, E.K.; Singh, S.K.; Theisen, M.Background During a Plasmodium infection, exposure of human host immune cells to both the asexual and the sexual stages of the parasite elicit immune responses. These responses may be protective and prevent the development of high parasitaemia and its associated clinical symptoms, or block the transmission of malaria to an uninfected person. This study aimed at examining the dynamics of naturally acquired immune responses against the asexual and sexual forms of Plasmodium falciparum as well as assessing differences in the multiplicity of infection (MOI) in asymptomatic Ghanaian children living in two communities with varying malaria transmission intensities. Methods School children aged between 6 and 12 years were recruited from Obom, a high malaria prevalence setting and Abura, a low malaria prevalence setting and enrolled in monthly multiple cross sectional surveys between February and May 2015. Filter paper blood blots (DBS) as well as thick and thin blood smears were made from finger-pricked blood at each visit. Plasmodium falciparum parasite prevalence was determined by microscopy and PCR. Serum eluted from the DBS were used to assess anti-Pfs230 (sexual stage) and anti-MSP3 (asexual stage) antibody levels using indirect ELISA and DNA extracted from the DBS used to assess MOI. Results Malaria parasite point prevalence and MOI throughout the study was higher in Obom than Abura. The trend of parasite prevalence estimated by microscopy was similar to that determined by PCR in Obom but not in Abura. The trend of MSP3 antibody seroprevalence followed that of PCR-estimated parasite prevalence in Obom, while in Abura the trend of Pfs230 antibody seroprevalence followed that of PCR-estimated parasite prevalence. Conclusions Microscopy can more accurately predict changes in parasite prevalence in high transmission settings than low transmission settings. In high transmission settings, P. falciparum parasite prevalence can predict antibody seroprevalence to MSP3, whilst in low transmission settings, seroprevalence against Pfs230 may be a useful predictor of parasite prevalence.Item High Prevalence Of Impaired Glucose Metabolism Among Children And Adolescents Living With HIV In Ghana(HIV Medicine, 2023) Ayanful-Torgby, R.; Shabanova, V.; Essuman, A.A.; et al.Background: Antiretroviral therapy (ART)-associated metabolic abnormalities, including impairment of glucose metabolism, are prevalent in adults living with HIV. However, the prevalence and pathogenesis of impaired glucose The metabolism of children and adolescents living with HIV, particularly in sub-Saharan Africa, is not well characterized. We investigated the prevalence of impaired glucose metabolism among children and adolescents living with perinatally infected HIV in Ghana. Methods: In this multicenter, cross-sectional study, we recruited participants from 10 paediatric antiretroviral treatment clinics from January to June 2022 in 10 facilities in the Greater Accra and Eastern regions of Ghana. We determined impaired glucose metabolism in the study sample by assessing fasting blood sugar (FBS), insulin resistance as defined by the homeostatic model assessment for insulin resistance (HOMA-IR) index and glycated haemoglobin (HbA1c) levels. The prevalence of impaired glucose metabolism using each criterion was stratified by age and sex. The phenotypic correlates of glucose metabolism Markers were also assessed among age, sex, body mass index (BMI), and waist-to-hip ratio (WHR). Results: We analysed data from 393 children and adolescents living with HIV aged 6–18 years. A little over half (205/393 or 52.25%) of the children were female. The mean age of the participants was 11.60 years (SD = 3.50), with 122/393 (31.00%) aged 6–9 years, 207/393 (52.67%) aged 10–15 years, and 62/393 (15.78%) were aged 16–18 years. The prevalence rates of glucose impairment in the study population, 15.52% [95% confidence interval (CI): 12.26– 19.45], 22.39% (95% CI: 18.54–26.78), and 26.21% (95% CI: 22.10–30.78) using HbA1c, HOMA-IR, and FBS criteria, respectively. Impaired glucose metabolism detected by FBS and HOMA-IR was higher in the older age group, whereas the prevalence of abnormal HbA1c levels was highest among the youngest age group. Age and BMI were positively associated with FBS and HOMA-IR (p < 0.001). However, there was negative correlation of WHR and HOMA-IR (p < 0.01) and HbA1c (p = 0.01).Conclusion: The high prevalence of impaired glucose metabolism observed among the children and adolescents living with HIV in sub-Saharan Africa is of concern as this could contribute to the development of metabolic syndrome in adulthood.Item An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs(Malaria Journal, 2015-12) Dery, V.; Duah, N.O.; Ayanful-Torgby, R.; Matrevi, S.A.; Anto, F.; Quashie, N.B.Background The recently introduced SYBR Green1 (SG) assay for testing parasites susceptibility to anti-malarial drugs needs further improvement. This has been necessitated by various setbacks, the major one being the low fluorescence intensity associated with it use. This shortcoming diminishes the anticipated hope that this novel method was going to replace the more traditional ones, such as the isotopic and microscopy. In order to restore confidence in its use, series of experiments to determine conditions that give the best fluorescence intensity were conducted. Go to: Methods Conditions that yield the maximum fluorescent signal were ascertained by measuring the fluorescence after incubation of Plasmodium falciparum culture at different parasites concentration with lysis buffer containing SYBR Green (LBS) at different time period. In order to ascertain the effect of freeze–thaw on fluorescence intensity, P. falciparum culture was frozen for 1 h, thawed, incubated with LBS and the fluorescence measured. The optimized conditions determined in this study were then used to assess the susceptibility of clinical isolates of P. falciparum to artesunate, chloroquine and mefloquine. The concentration of anti-malarial drug inhibiting parasite growth by 50 % (IC50) for each drug was estimated using the online ICEstimator. The IC50 generated using the optimized SG method determined in this study was compared with that obtained using microscopic method and the previously reported standard SG method. Go to: Results Over all, the SG method was found to be easy to perform and sensitive. Freeze–thaw of parasite culture followed by incubation with lysis buffer containing the dye for 3 h was consistently observed to give the highest fluorescence signal. The IC50 values for chloroquine, mefloquine and artesunate determined were consistent and comparable with that determined with the previously reported standard SG method and the microscopic method. Go to: Conclusion The authors conclude that freezing and thawing of parasite culture, followed by incubation with LBS in the dark for 3 h provided a significant improvement in fluorescence signal. The IC50 generated using the improved SG method is comparable with that from microscopy and the standard method.Item An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs(Malaria Journal, 2015-12) Dery, V.; Duah, N.O.; Ayanful-Torgby, R.; Matrevi, S.A.; Anto, F.; Quashie, N.B.Background The recently introduced SYBR Green1 (SG) assay for testing parasites susceptibility to anti-malarial drugs needs further improvement. This has been necessitated by various setbacks, the major one being the low fluorescence intensity associated with it use. This shortcoming diminishes the anticipated hope that this novel method was going to replace the more traditional ones, such as the isotopic and microscopy. In order to restore confidence in its use, series of experiments to determine conditions that give the best fluorescence intensity were conducted. Go to: Methods Conditions that yield the maximum fluorescent signal were ascertained by measuring the fluorescence after incubation of Plasmodium falciparum culture at different parasites concentration with lysis buffer containing SYBR Green (LBS) at different time period. In order to ascertain the effect of freeze–thaw on fluorescence intensity, P. falciparum culture was frozen for 1 h, thawed, incubated with LBS and the fluorescence measured. The optimized conditions determined in this study were then used to assess the susceptibility of clinical isolates of P. falciparum to artesunate, chloroquine and mefloquine. The concentration of anti-malarial drug inhibiting parasite growth by 50 % (IC50) for each drug was estimated using the online ICEstimator. The IC50 generated using the optimized SG method determined in this study was compared with that obtained using microscopic method and the previously reported standard SG method. Go to: Results Over all, the SG method was found to be easy to perform and sensitive. Freeze–thaw of parasite culture followed by incubation with lysis buffer containing the dye for 3 h was consistently observed to give the highest fluorescence signal. The IC50 values for chloroquine, mefloquine and artesunate determined were consistent and comparable with that determined with the previously reported standard SG method and the microscopic method. Go to: Conclusion The authors conclude that freezing and thawing of parasite culture, followed by incubation with LBS in the dark for 3 h provided a significant improvement in fluorescence signal. The IC50 generated using the improved SG method is comparable with that from microscopy and the standard method.Item Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes(Nature Communications, 2019-05) Usui, M.; Prajapati, S.K.; Ayanful-Torgby, R.; Acquah, F.K.; Cudjoe, E.; Kakaney, C.; Amponsah, J.A.; Obboh, E.K.; Reddy, D.K.; Barbeau, M.C.et.al.Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0–78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo.Item Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes(Nature Communications, 2019) Usui, M.; Prajapati, S.K.; Ayanful-Torgby, R.; Acquah, F.K.; Cudjoe, E.; Kakaney, C.; Amponsah, J.A.; Obboh, E.K.; Reddy, D.K.; Barbeau, M.C.; Simons, L.M.; Czesny, B.; Raiciulescu, S.; Olsen, C.; Abuaku, B.K.; Amoah, L.E.; Williamson, K.C.Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0–78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo. © 2019, The Author(s).Item Publisher Correction: Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes(Nature Communications, 2019) Usui, M.; Prajapati, S.K.; Ayanful-Torgby, R.; Acquah, F.K.; Cudjoe, E.; Kakaney, C.; Amponsah, J.A.; Obboh, E.K.; Reddy, D.K.; Barbeau, M.C.; Simons, L.M.; Czesny, B.; Raiciulescu, S.; Olsen, C.; Abuaku, B.K.; Amoah, L.E.; Williamson, K.C.The original version of this Article contained an error in Fig. 6, in which the right panel showing gametocytemia in the 6th cycle was flipped horizontally. This has been corrected in both the PDF and HTML versions of the Article. © 2019, The Author(s).Item Seasonal variations in Plasmodium falciparum genetic diversity and multiplicity of infection in asymptomatic children living in southern Ghana(BMC Infectious Diseases, 2018-08) Adjah, J.; Fiadzoe, B.; Ayanful-Torgby, R.; Amoah, L.E.Background Genetic diversity in Plasmodium falciparum (P. falciparum) parasites is a major hurdle to the control of malaria. This study monitored changes in the genetic diversity and the multiplicity of P. falciparum parasite infection in asymptomatic children living in southern Ghana at 3 month intervals between April 2015 and January 2016. Methods Filter paper blood spots (DBS) were collected quarterly from children living in Obom, a community with perennial malaria transmission and Abura, a community with seasonal malaria transmission. Genomic DNA was extracted from the DBS and used in polymerase chain reaction (PCR)-based genotyping of the merozoite surface protein 1 (msp 1) and merozoite surface protein 2 (msp 2) genes. Results Out of a total of 787 samples that were collected from the two study sites, 59.2% (466/787) tested positive for P. falciparum. The msp 1 and msp 2 genes were successfully amplified from 73.8% (344/466) and 82.5% (385/466) of the P. falciparum positive samples respectively. The geometric mean MOI in Abura ranged between 1.17 (95% CI: 1.08–1.28) and 1.48 (95% CI: 1.36–1.60) and was significantly lower (p < 0.01, Dunn’s multiple comparison test) than that determined in Obom, where the geometric mean MOI ranged between 1.82 (95% CI: 1.58–2.08) and 2.50 (95% CI: 2.33–2.678) over the study period. Whilst the msp 1 R033:MAD20:KI allelic family ratio was dynamic, the msp 2 3D7:FC27 allelic family ratio remained relatively stable across the changing seasons in both sites. Conclusions This study shows that seasonal variations in parasite diversity in these communities can be better estimated by msp 1 rather than msp 2 due to the constantly changing relative intra allelic frequencies observed in msp 1 and the fact that the dominance of any msp 2 allele was dependent on the transmission setting but not on the season as opposed to the dominance of any msp 1 allele, which was dependent on both the season and the transmission setting.Item Seasonal variations in Plasmodium falciparum parasite prevalence assessed by varying diagnostic tests in asymptomatic children in southern Ghana(PLoS ONE, 2018-06) Ayanful-Torgby, R.; Quashie, N.B.; Boampong, J.N.; Williamson, K.C.; Amoah, L.E.Plasmodium falciparum infections presenting either as symptomatic or asymptomatic may contain sexual stage parasites (gametocytes) that are crucial to malaria transmission. In this study, the prevalence of microscopic and submicroscopic asexual and gametocyte parasite stages were assessed in asymptomatic children from two communities in southern Ghana. Eighty children aged twelve years and below, none of whom exhibited signs of clinical malaria living in Obom and Cape Coast were sampled twice, one during the rainy (July 2015) and subsequently during the dry (January 2016) season. Venous blood was used to prepare thick and thin blood smears, spot a rapid malaria diagnostic test (PfHRP2 RDT) as well as prepare filter paper blood spots. Blood cell pellets were preserved in Trizol for RNA extraction. Polymerase chain reaction (PCR) and semi-quantitative real time reverse transcriptase PCR (qRT-PCR) were used to determine submicroscopic parasite prevalence. In both sites 87% (95% CI: 78–96) of the asymptomatic individuals surveyed were parasites positive during the 6 month study period. The prevalence of asexual and gametocyte stage parasites in the rainy season were both significantly higher in Obom than in Cape Coast (P < 0.001). Submicroscopic gametocyte prevalence was highest in the rainy season in Obom but in the dry season in Cape Coast. Parasite prevalence determined by PCR was similar to that determined by qRT-PCR in Obom but significantly lower than that determined by qRT-PCR in Cape Coast. Communities with varying parasite prevalence exhibit seasonal variations in the prevalence of gametocyte carriers. Submicroscopic asymptomatic parasite and gametocyte carriage is very high in southern Ghana, even during the dry season in communities with low microscopic parasite prevalence and likely to be missed during national surveillance exercises.Item A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs(Malaria Journal, 2013-12) Quashie, N.B.; Duah, N.O.; Abuaku, B.; Quaye, L.; Ayanful-Torgby, R.; Akwoviah, G.A.; Kweku, M.; Johnson, J.D.; Lucchi, N.W.; Udhayakumar, V.; Duplessis, C.; Kronmann, K.C.; Koram, K.A.Background: Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. Methods. A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC§ssub§50§esub§) for each drug was estimated using the online program, ICEstimator. Results: Pooled results from all the sentinel sites indicated geometric mean IC§ssub§50§esub§ values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC§ssub§ 50§esub§ value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC§ssub§50§esub§ value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs. Conclusion: Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC§ssub§50§esub§ value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended. © 2013 Quashie et al.; licensee BioMed Central Ltd.