Asymptomatic parasite carriers represent a “silent” infective reservoir for malaria transmission and contributes to malaria persistence. However, limited data are available on asymptomatic malaria in Vietnam. Between November 2018 and March 2019, we conducted a malaria epidemiological survey of asymptomatic people  residing in three communes in Tuy Duc district, Dak Nong province in the Central Highlands of Vietnam. Based on the national stratification of malaria risk, Dak Buk So, Dak Ngo and Quang Truc communes were classified by the National Malaria Control Programme as low, moderate and high malaria endemic areas, respectively. Using participants’ finger prick blood samples, malaria parasites were detected by one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The median age (Interquartile Range) for adults and children were 35 years (26–50) and 12 years (11–14), respectively. The prevalence of asymptomatic malaria was 1.7% , 3.5% (31/890) and 12.2% (72/591) for participants from Dak Buk So, Dak Ngo and Quang Truc, respectively. The prevalence of asymptomatic malaria was lower in children compared to adults: 2.6% (9/352) versus 4.7%  . Ownership of long-lasting insecticide-treated bed nets and hammocks was 97.1%, 99.0% and 94.7% for participants in Dak Buk So, Dak Ngo and Quang Truc, respectively, however, only 66.0%, 57.3% and 42.8% of the participants reported using bed nets every night. Of the several risk factors examined, going to the forest two weeks prior to enrolment into the study and sleeping in the forest had a significant association with participants being infected with asymptomatic malaria in Quang Truc, but not in the other two communes. Knowledge of the prevalence and distribution of asymptomatic malaria will help design and evaluate future intervention strategies for malaria elimination in Vietnam. Nations within the Greater Mekong Sub-region (GMS), including Vietnam, have adopted the goal to eliminate malaria by 2030 . In VietThe global prevalence of asymptomatic malaria varies widely from 0.4% to 90.6% in different endemic settings with the median parasite density correlating with the prevalence of asymptomatic infections and ranging from 1 to 1,336 parasites per μL . A recenPlasmodium falciparum and P. vivax infections persist frequently for months or years [In the absence of a curative drug treatment, asymptomatic or years , 20, 21,or years ). Anemiaor years . TargetiPlasmodium spp. prevalence rate for the four villages ranged from 7.4% to 14.7%. The uPCR method was also able to determine that of the asymptomatic participants, 2.3% , 4.0% , 1.6%  and 4.2%  were infected with P. falciparum, P. vivax, mixed P. falciparum and P. vivax and other than P. falciparum and P. vivax malaria, respectively [Gaining an understanding of the prevalence and distribution of asymptomatic malaria in Vietnam will help design intervention strategies to achieve malaria elimination. Surveys of submicroscopic malaria infections, detectable only by polymerase chain reaction (PCR) have revealed asymptomatic malaria to be common near and in forested areas of south-central , 23, cenectively .Plasmodium infections in 19.7% (175/888) of the study population [In other Southeast Asian countries where uPCR has been used, high asymptomatic malaria prevalence rates were detected in three western Cambodian villages  and in four villages on the Thailand-Myanmar border  . In 2015Other than the studies conducted in participants from Binh Phuoc, Quang Nam and Ninh Thuan provinces , 25, 26,Fig 1). The district has 17 ethnic minority groups: the two major ethnic groups are the M’Nong and H’Mong people. The main income for the people living in the district is generated from agricultural production, including rice farming near their homes or in forested areas, as well as from coffee, pepper and potato plantations.Tuy Duc district, Dak Nong province borders with Cambodia and Binh Phuoc province in south-western Vietnam  were surveyed at the end of the wet season (November-December 2018) and during the dry season (February-March 2019), respectively. Individuals, who lived near the communal health stations and preferably worked in rural areas, were invited to participate in the study and different participants were recruited for each cohort.The cross-sectional survey was carried out in people from three communes, Dak Buk So, Dak Ngo and Quang Truc, which were stratified by NMCP as low, moderate and high malaria risk areas, respectively . The stuThe inclusion criteria for participants were: absence of malaria symptoms, age ≥10 years and willingness to provide a finger prick capillary blood sample. Participants with tympanic temperatures <38.0°C were considered to be afebrile. People who were recruited into the study were invited to complete a questionnaire consisting of questions of demographic characteristics, recent medical history, travel in the past two weeks and malaria prevention measures, such as sleeping under a bed net and using a topical repellent.2 p (1-p) /d2, where N is the sample size, Z- statistics equals 1.96 at a confidence level of 95% and applying p  values of 0.05, 0.10 and 0.15 with respective d (precision) values of 0.012, 0.02 and 0.03, the minimum numbers of people to be recruited were 1,267 in Dak Buk So, 864 in Dak Ngo and 544 in Quang Truc. These numbers were increased by 5% for unexpected withdrawals with a total of 2,809 participants to be recruited.Based on the asymptomatic prevalence rates seen in Vietnamese villages in Binh Phuoc and Ninh Thuan provinces , it was ® LoBind microcentrifuge tube containing 250 μL of RNAprotect reagent  and stored at -20°C until shipment on dry ice to the Institute of Malariology, Parasitology and Entomology, Quy Nhon (IMPE-QN) for laboratory analysis.Participating adults and children were examined by a study doctor. Their vital signs  and clinical information, such as body weight, body temperature and recent medical history were collected. A finger prick capillary blood sample (~130 μL) was collected from each participant and transferred to a Becton-Dickinson (BD) microtube (500 μL) containing EDTA as an anticoagulant and gently mixed. Fifty-μL was transferred to an EppendorfP. falciparum and lactate dehydrogenase (pLDH) of the four Plasmodium species; P. falciparum, P. vivax, P. malariae and P. ovale. Testing was carried out according to the manufacturer’s instructions. Thick and thin blood films (quantity two) were prepared for malaria diagnosis. The dried thin films were fixed with methanol. The blood films were stained with 4% Giemsa for 45 min and examined at a magnification of 1000× by two trained independent microscopists. Parasites were counted per 200 white blood cells (WBCs) and parasite density was calculated by assuming a WBC count of 8,000/μL in accordance with the WHO guidelines [Malaria RDT  is based on the detection of histidine-rich protein 2 (HRP-2) produced by idelines . A bloodTo determine the participant’s Hb concentration, 7 μL of mixed EDTA blood from the BD microtube was transferred to a CareSTART Hemoglobin Monitoring System  and the value was recorded.Blood samples preserved in RNAprotect reagent were used for RNA/DNA extraction using an RNeasy kit . The RNA/DNA extraction procedure was tested and modified to accommodate the extraction using 300 μL sample volume as follows: 1.1 mL of RLT-β buffer containing β-mercaptoethanol was added to the 300 μL sample followed by the addition of 1.4 mL of 70% ethanol. The procedure was then conducted in accordance with manufacturer’s instructions with no DNA elimination step carried out. Parasite RNA and DNA were eluted in 50 μL RNAase-free water .Plasmodium species  using primers QMAL_Fw- TTAGATTGCTTCCTTCAGT(A/G)CCTTATG and QMAL_Rev-GTTGAGTCAAATTAAGCCGCAA as previously described [TATTGCTTTTGAGAG GTTTTGTTACTTTG and Pfal_Rev-TATTCCATGCTGTAGTATTCAAACACAA for P. falciparum or Pv-Fw- GCTTTG TAATTGGAATGATGGGAA T and Pv_Rev- ATGCGCACAAAGTCGATACGAAG for P. vivax were used in respective RT-qPCR reactions as described above.Malaria positive samples were determined in a one-step RT-qPCR analysis, targeting 18S small sub-unit ribosomal RNA (SSU rRNA) gene transcripts for a simultaneous detection of the four escribed , with thP. falciparum D6 laboratory line  containing predominantly young rings (>95%). Aliquots (50 μL) of cultured parasites at 5.5% parasitemia (quantified by microscopy) and 46% hematocrit in human plasma were prepared, to which RNAprotect reagent (250 μL) was added and the standards were stored at -80°C. Ten-fold serial dilutions with blood/RNAprotect mixture (up to 4 x 10−9 fold) were made to test the sensitivity of detection. The detection was reproducibly achieved with a threshold cycle (Ct) of 32 in at least 10 independent experiments, using 2 μL (out of 50 μL) of RNA/DNA template (in water) containing equivalent of 0.001012 parasites, corresponding to 0.0253 parasites/μL (or 25 parasites/mL) in a positive control dilution. For lower dilutions, the detection of positive samples became stochastic. It follows that if at least one parasite is present in the initial volume of finger prick capillary blood sample (50 μL) the assay would be sufficiently sensitive to detect positive samples by RT-qPCR. The efficiency and sensitivity of detection for the other three Plasmodium species was not tested.To test the sensitivity of the assay, standards were prepared from highly synchronous parasite cultures of the Plasmodium speciation was carried out as described in Wampfler et al. [P. falciparum standard dilution containing approximately 250 parasites, with an expected Cts of 19–20. The RNA/DNA extracted from a P. falciparum standard dilution containing 25 parasites was aliquoted and used as a RT-qPCR positive control (Ct of 23–24). The positive samples were identified by analysis of the melting curve profiles of the RT-qPCR products and compared to the melting temperatures (Tm) of the RT-qPCR products and those obtained from the positive controls. The samples with 78°C <Tm<80°C were selected as possibly positive and subsequently confirmed as positive by at least one more RT-qPCR.Following detection of malaria positive samples, the r et al.  by speciP<0.05.Statistical analysis of the demographic and clinical data was performed using SigmaStat . Descriptive statistics were used to summarize baseline values and demographic data. Variable comparisons were analysed using the Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks and Mann-Whitney rank sum test. Values of normally distributed data were expressed as means with standard deviations (SD) and non-normally distributed data as medians with interquartile ranges (IQR). Comparison between blood Hb concentrations in RT-qPCR-positive and -negative participants was carried out using the Fisher’s exact test of contingency tables for Hb values of <11 g/dL and ≥11 g/dL using GraphPad Prizm Version 9.0.1 software package . Risk analysis using contingency tables was also used to evaluate association of asymptomatic malaria with factors, such as occupation , time spent in the forest and use of LLINs and repellents. Multiple logistic regression analysis was used to analyse the association of physical characteristics  with RT-qPCR positivity (dependent variable) using GraphPad Prizm software. Differences were considered statistically significant when Ethics approval for the study was obtained from the Institutional Review Board of the Institute of Malariology, Parasitology and Entomology Quy Nhon (Ref. No. 533/VSR-LSDT) and mutual recognition by the Australian Government Departments of Defence and Veterans’ Affairs Human Research Ethics Committee (Ref. No. 2018/R32250633). Extramural research review was also conducted in accordance with the U.S. Navy Human Research Protection Program (HRPO.NMRCA 2018.0012) in compliance with all applicable federal regulations governing the protection of human subjects. All adults gave written informed consent and children (≥10 to < 18 years old) gave written assent with permission from their parent or guardian to be in the study.Table 1. Of these participants, 37.9%  were males and 87.5%  were adults. The median (IQR) ages for adults and children were 35 years (26–50) and 12 years (11–14), respectively. All children were greater than 10 years old. The median (IQR) body weights were 53 kg (49–59) for adults and 39 kg (34–41) for children. The participants were afebrile with a median (IQR) tympanic body temperature of 36.9°C (36.7–37.0). Multiple comparisons of the physical characteristics of the participants revealed statistical differences (P<0.05) in the age and body weight of adults and children between the three communes, whereas there were no differences in gender and body temperature in adults and children.The demographics of the 2,809 participants from the three communes are shown in P. falciparum—RDT negative), QT554 (P. vivax—RDT positive) and one participant from Dak Buk So, DBS1026 . Despite microscopically detected parasitemia, these three participants did not report any malaria symptoms and had body temperatures <37.5°C at enrolment, but two participants with P. falciparum infections developed malaria symptoms soon after and were treated for their malaria infections.None of the 2,809 participants in the cross-sectional asymptomatic malaria prevalence survey reported malaria symptoms or had taken antimalarial drugs for 28 days prior to enrolment. All but one participant (QT554) were RDT negative and three were blood film microscopy positive for malaria infections: two participants from Quang Truc, QT528  for the 2nd cohort, with an overall prevalence of 4.4%  (Table 2), with no statistical difference in prevalence between the two cohorts (P>0.05). The point prevalence of asymptomatic malaria determined by RT-qPCR were 1.7% , 3.5% (31/890) and 12.2% (72/591) for participants from Dak Buk So, Dak Ngo, and Quang Truc, respectively . This prevalence of asymptomatic malaria was consistent with the stratification of malaria risk and the incidence of symptomatic malaria infections reported by the NMCP [Table 2).The point prevalence of participants infected with asymptomatic malaria determined by RT-qPCR were 4.4%  for the 1the NMCP  for 2018Plasmodium positive cases, only 7.2% (9/125) were detected in children  and 92.8% (116/125) in adults , of which 60.8% (76/125) were females. The overall prevalence of participants infected with asymptomatic malaria between the three communes was statistically different (P<0.05).Across the three communes, of the  In children (≥10 to < 18 years old) there were no asymptomatic malaria cases detected in Dak Buk So (0/152) and only 1.1% (1/93) detected in Dak Ngo, whereas in Quang Truc 7.5% (8/107) of surveyed children were positive for asymptomatic malaria compared to an overall prevalence in adults of 13.2%  (S1 Appendix). Furthermore, in Quang Truc young adults (18–25 years old) had the highest prevalence of asymptomatic malaria of 16.7%  amongst all age groups, followed by 16.0%  amongst 56–65 years old and 13.8%  in 26–35 years old (S1 Table in S1 Appendix). Because of the low number of positive cases for all age groups in the three communes, the 95% CI intervals were wide, with none of the ORs ratios reaching statistical significance (P<0.05) (S1 Table in S1 Appendix). Note that in low and moderate risk areas the highest prevalence of asymptomatic malaria was observed in the older age groups (S1 Appendix), in contrast to younger adults in the high risk area. These data might be useful for the identification of populations with the highest risk of asymptomatic malaria.The prevalence of RT-qPCR positive samples amongst different age groups in the three communes is shown in Plasmodium RT-qPCR positive samples had high Ct values, with mean (range) values of 34 (8–38), 33 (16–38) and 31 (7–38) for Dak Buk So, Dak Ngo and Quang Truc, respectively. The lowest Ct values were recorded for the three blood microscopy positive samples: DBS1026 (Ct = 8), QT528 (Ct = 7) and QT554 (Ct = 11) . Based on the standards derived from the cultured laboratory P. falciparum D6 line, the RT-qPCR assay limit of quantification was 0.025 parasites/μL (Ct~32 cycles), after which the detection became stochastic. The participants’ mean parasite density in Quang Truc was the highest at approximately 0.050 parasites/μL or ~50 parasites/mL, whereas participants from Dak Ngo and Dak Buk So had lower mean parasite densities of less than 12 and 6 parasites/mL, respectively. The majority of the positive samples had parasite densities either equal to or below the limit of quantification: 90.9% (20/22), 77.4% (24/31) and 75.0% (54/72) in Dak Buk So, Dak Ngo and Quang Truc, respectively. Furthermore, P. falciparum parasites were detected by RT-qPCR in 32.0% (40/125) of positive cases: 27.4% (17/62) of participants from the 1st cohort and 36.5% (23/63) from the 2nd cohort. There were only three P. vivax infections detected amongst the positive cases by RT-qPCR, and all were from Quang Truc. For the remaining 82 malaria RT-qPCR-positive samples, the Plasmodium species could not be determined, presumably due to the very low parasite densities in the finger prick capillary blood samples.Most of the Data on participants’ occupation, visiting forest, sleeping in the forest and LLINs and repellent use collected from the study participants were analysed to appreciate adherence to the malaria protective measures in the communities and to identify risk factors associated with asymptomatic malaria.S3 Table in S1 Appendix). However, of the study population, 66.0% , 57.3% (510/890) and 42.8% (253/591) reported using a bed net every night at home in Dak Buk So, Dak Ngo and Quang Truc, respectively. In these communes, 40.7% , 33.7% (300/890) and 29.3% (173/591) reported going and sleeping in the forest two weeks prior to enrolment in the study, however, of these participants only 30.6% , 8.3% (74/890) and 10.2% (60/591) reported sleeping under LLINs or hammocks in the forest (S3 Table in S1 Appendix), which encompasses 75.2%, 24.7% and 34.7% of LLINs owners in Dak Buk So, Dak Ngo and Quang Truc, respectively. Only 1.7% (10/591) of participants from Quang Truc and none in other communes acknowledged using a topical repellent.In Dak Buk So, Dak Ngo and Quang Truc 97.1%, 99.0% and 94.7% of participants, respectively, reported having LLINs or long lasting insecticide-treated hammocks at home in the commune or in the forest (P = 0.41) in blood Hb concentration in participants with or without asymptomatic malaria, with mean concentrations of 11.9 g/dL (95% CI 11.3–12.3) and 12.0 g/dL (95% CI 12.1–12.4), respectively, nor for participants at each of the three communes (S2 Table in S1 Appendix). There were also no significant differences (P>0.05) in prevalence of individuals with lower blood Hb concentrations (<11 g/dL) for RT-qPCR-positive compared to RT-qPCR-negative participants from Dak Buk So, Dak Ngo and Quang Truc, as well as for combined data for all three communes (S2 Table in S1 Appendix).Multiple logistic regression analysis did not reveal any significant associations of asymptomatic malaria prevalence with gender, body temperature, and pulse and respiratory function (data not shown). There was no significant difference (S3 Table in S1 Appendix). The majority of children  and adults  were students and farmers, respectively. Amongst the positive cases, 100% (9/9) of children were students and 87.1% (101/116) of adults were farmers. No significant associations between asymptomatic malaria and being a farmer or a student were found in the study population, whereas in Quang Truc the prevalence of asymptomatic malaria was significantly higher (P = 0.026) amongst forest rangers  (S3 Table in S1 Appendix).Participants’ responses to the questionnaire were analysed to determine whether an associations exist between the presence or absence of asymptomatic malaria (RT-qPCR-positivity or negativity) and occupation, living in the forest and adherence to malaria protection measures using contingency table analyses (P = 0.048 95% CI 1.15 to 5.23) (S3 Table in S1 Appendix), whereas in Quang Truc asymptomatic malaria was linked to going to the forest  and sleeping in the forest under a bed net OR = 2.48  (S3 Table in S1 Appendix).Only in Dak Ngo there was a significant association between the number of participants reporting using bed nets rarely and asymptomatic malaria cases  in Dak Buk So, 3.5% (31/890) in Dak Ngo and 12.2% (72/591) in Quang Truc. The RT-qPCR findings corroborate the malaria risk stratification by the NMCP of Dak Buk So, Dak Ngo, and Quang Truc being areas of low, moderate and high malaria risk areas, respectively. The prevalence of asymptomatic malaria in Quang Truc was similar to that reported in Binh Phuoc and Ninh Thuan provinces of Vietnam and western Cambodia, but lower than that in Lao-PDR [As Vietnam heads towards malaria elimination it is important to develop strategies to deal with the asymptomatic malaria, a “silent reservoir” of malaria infections. Understanding the extent and the local context of the problem is a necessary step towards the goal of malaria elimination. This is the first study to report on the prevalence of asymptomatic malaria in the Central Highlands of Vietnam. The overall point prevalence of  Lao-PDR . By cont Lao-PDR .In this study, only children ≥10 to < 18 years of age were included, with 96.3% of children attending school across the three communes, whereas the majority of adults, (86.7%) were farmers, often working in or near forested areas. Absence of asymptomatic malaria in children in Dak Buk So and with only one child  of surveyed children with asymptomatic malaria in Dak Ngo suggests that children are at lower malaria risk in these areas. However, in Quang Truc 7.5% (8/107) of children had asymptomatic malaria compared to 13.2% (64/484) for adults. The higher prevalence of asymptomatic malaria in adults is likely to be due to different behavioural and occupational factors resulting in higher carriage of asymptomatic parasites in adults, particularly in the high risk area of Quang Truc.Specific local factors appear to influence the prevalence of asymptomatic malaria, even in the same geographical region. In 2014, the prevalence of asymptomatic malaria in two villages in Myanmar along the China border was vastly different . In the (S4 Table in S1 Appendix) showed that there were no recorded malaria cases in Dak Buk So during the second half of 2018, yet 1.7% of the surveyed population had asymptomatic malaria parasites detected from the same commune. Interestingly, in February to March 2019 there were 6 symptomatic P. falciparum malaria cases detected in the low risk area of Dak Buk So (S4 Table in S1 Appendix). Of these cases, one was found to have asymptomatic malaria in our study prior to developing symptomatic malaria. In Dak Ngo and Quang Truc, the moderate and high risk areas, respectively, the prevalence of asymptomatic malaria cases also exceeded that of symptomatic malaria cases. Our data are consistent with previous observations that in low transmission areas the majority of malaria infections are submicroscopic and asymptomatic, but are responsible for 20–50% of the malaria transmission [There were no significant differences in the prevalence of asymptomatic malaria cases between the two cohorts for each commune covering the end of the wet season and during the dry season, which was most likely due to only a 6-week interval between the two collection periods. Note that the data on symptomatic malaria cases provided by NMCP   asymptomatic P. falciparum cases. Note that according to NMCP data in 2018, the proportion of symptomatic P. vivax infections was 42.9% (6/14) for Dak Ngo and 47.4% (27/57) for Quang Truc (Table 2) in subjects presenting to the communal health stations and district hospital. However, the distribution of species in asymptomatic malaria may not resemble that of symptomatic cases, with P. vivax having a greater propensity (64% to 100% of infections) to remain asymptomatic compared with P. falciparum [A distinct advantage of using the RNA-based 18S SSU rRNA RT-qPCR assay is its greater sensitivity compared to DNA-based standard qPCR in detecting submicroscopic parasitemia, with an LOD of 0.02–0.002 parasite/μL of blood , 42, 43.g et al. . The estlciparum , 46.In contrast to symptomatic malaria, there was no associations between asymptomatic malaria and participants’ characteristics, such as body temperature, pulse,respiratory rate and blood Hb concentrations. In other studies anemia has been linked not only to symptomatic malaria with high parasitemia, but is also associated with asymptomatic or “chronic” malaria , 47. TheP = 0.026) or going to the forest two weeks prior to enrolment  and sleeping in the forest under a bed net . These findings further support the notion that forest goers are at the highest risk to malaria exposure and interventions need to be prioritized for this group, consistent with previous recommendations [The majority of the 125 RT-qPCR positive asymptomatic participants were adult farmers living or working in or near forested areas of the communes and thus were most likely to be exposed to malaria, since malaria transmission mainly occurs in remote forested areas of the Central Highlands of Vietnam and along the international borders with Cambodia and Lao-PDR , 48. In ndations . As no sndations , 50.Anopheles mosquito vectors in forested areas and on forest fringes, with primary malaria vectors Anopheles dirus and An. minimus predominantly biting outdoors early at dusk, as well as secondary vectors that bite from dusk to down [In Central Vietnam malaria transmission is sustained by a variety of  to down , 52. LLI to down . Importa to down . In the S3 Table in S1 Appendix). In the moderate risk area (Dak Ngo), infrequent sleeping under LLINs was significantly associated with asymptomatic malaria prevalence. In Quang Truc the asymptomatic malaria prevalence was associated with going to the forest and sleeping in the forest under a bed net (S3 Table in S1 Appendix), likely indicating that spending a significant amount of time and sleeping in the forest is associated with the risk of having asymptomatic malaria. The relatively low utilization of LLINs and regular retreatment or replacement of LLINs in all three communes needs to be addressed in order to further reduce both, symptomatic and asymptomatic malaria in the area. Furthermore, our results confirmed that only 1.7% of participants used a topical repellent in Quang Truc, with none reported by participants in the other two communes, which was also observed in other areas of Central Vietnam and GMS [The highest adherence to protective measures was in the low risk area (Dak Buk So), whereas in the high risk area (Quang Truc) the lowest usage of LLINs and hammocks was reported Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.S3 Appendix(XLSX)Click here for additional data file.


Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose. Colletotrichum higginsianum is an important pathogen that causes anthracnose on various cruciferous plants and leads to serious economic losses worldwide [C. higginsianum attach onto the host surfaces, germinate and produce dark appressoria. Then, the fungus penetrates epidermal cells and generates large bulbous biotrophic hyphae in the first infected living cell. Finally, thin necrotrophic hyphae are produced which grow to kill host tissues, resulting in formation of necrotic leaf lesions [The hemibiotrophic ascomycete fungus orldwide . In the  lesions .Saccharomyces cerevisiae [Uromyces fabae  [Ustilago maydis  [Puccinia striiformis  [Geosiphon pyriformis [Colletotrichum graminicola [Magnaporthe oryzae [U. fabae (UfHXT1) and G. pyriformis (GpMST1) were monosaccharide transporters that restored the growth of hexose uptake-deficient yeast strains on mannose and glucose, and the lost transport activities in yeast [U. maydis transporter UmSRT1 is specific for sucrose and the UmSRT1 deletion mutants of U. maydis showed strongly reduced virulence on maize plants [U. maydis was interpreted as a mechanism to avoid apoplastic signals (glucose) potentially recognized by the host [Hxt1 identified in U. maydis encodes a high-affinity transporter for glucose, fructose, and mannose, and disruption of Hxt1 resulted in reduced growth on these substrates and showed decreased symptom development on maize [P. striiformis, an invertase PsINV is unique in fungi and important for sugar uptake during infection. PsINV is abundantly secreted into the host to secure sugar absorption for P. striiformis, and silencing PsINV inhibits growth and development as well as sporulation [P. striiformis PsHXT1 is required for pathogenicity through sugar uptake. PsHXT1 is a glucose–proton symporter, silencing PsHXT1 restricts normal growth and development of P. striiformis during infection [C. graminicola, hexose transporter genes CgHXT1-CgHXT5 exhibit various expression profiles during infection of maize and in hexose uptake [M. oryzae plays a specific role for conidiation and mycelial melanization [In fungi, the system for uptake and utilization of carbon sources is highly conserved and enables fungal cells to sense and respond to extracellular conditions ,4. The hrevisiae ,6. So faan rust) ,8, Ustilrn smut) ,10, and pe rust) ,12, the riformis , as wellminicola , and thee oryzae . The proin yeast . The U. e plants . Direct the host . The genrulation . Hexose e uptake . MoST1 inization . HoweverChHxt6 from a T-DNA insertion pathogenicity-deficient mutant, G256, in C. higginsianum. Deletion of ChHxt6 showed reduced fungal virulence and caused less necrotrophic hyphae formation. Furthermore, ChHxt6 was found to be important for uptake of multiple hexoses. Site-directed mutants of Thr169 and Pro221 positions on ChHxt6 also revealed deficiencies in virulence and hexose transportation, suggesting that ChHxt6 is essential for virulence possibly mediated by affecting hexose uptake in the switch from biotrophy to necrotrophy.In this study, we identified and characterized a new HXT gene named C. higginsianum T-DNA insertion mutant G256 was not noticeably different from the wild-type on PDA , we selected homologues of these five Hxt proteins  in C. higginsianum as well as ChHxt6 identified in this study for further function analysis.Since previous study revealed that five CgHXT proteins in C. graminicola. In addition, the ChHxt6 gene identified from G256 T-DNA mutant was separated as outgroup, indicating that the ChHxt6 is a novel hexose transporter in C. higginsianum.Phylogenetic analysis was performed with putative Hxts of functionally characterized proteins from some phytopathogenic fungi and functionally characterized HXT-type transporters A. The reChHxt genes during infection stages were analyzed by RT-qPCR . ChHxt6 expression levels were significantly up-regulated at both 48 and 72 hpi. Expression profiling analysis revealed that ChHxt1 to ChHxt6 exhibited specific expression profiles in different infection phases of C. higginsianum, suggesting differences in functional characteristics for these six ChHxt proteins.Expression of the six  RT-qPCR B. ChHxt5ChHxt1 to ChHxt6, were separately knocked out using homologous recombination   C. In conChHxt1, ΔChHxt2, ΔChHxt3 and ΔChHxt5 penetrated to form both bulbous biotrophic hyphae and thin filamentous necrotrophic hyphae . To explore functions of hexose transport structure of ChHxt6, mChHxt6(T169S), mChHxt6(P221L) and mChHxt6 point mutants were generated by PCR-based site-directed mutagenesis. All ChHxt6 point mutants were verified by PCR with ChHxt6-KF/ChHxt6-KR primers  A. There  primers , followiin planta inoculation assays at 4 dpi, virulence of the point mutants m-5ChHxt6(T169S) and m-2ChHxt6 was decreased significantly compared to Ch-1 (m-27ChHxt6(P221L)  was similar to that of the wild-type  m-2ChHxt6(T169S)  were lower than that of the wild-type but higher than ΔChHxt6-2 , mChHxt6(P221L) and mChHxt6 expressing yeast cells grew weaker than ChHxt6-expressing cells on all hexoses , ChHxt6(P221L), and ChHxt6 in ΔChHxt6 resulted in reduced virulence. In previous works, the mutation analogous to T169S was known to induce constitutive active glucose signaling in the hexose sensors of several yeasts [Expression of l yeasts ,5, probaChHxt6 seemed to be unique among hexose transporter family genes in C. higginsianum. We demonstrated that ChHxt6 is needed for hexose uptake and that hexose uptake might be involved in the necrotrophic stage of C. higginsianum during the infection process. This study demonstrated that ChHxt6 is a novel hexose transporter in hemibiotrophic fungi, and it affects the virulence of C. higginsianum through influencing the uptake of hexose in the necrotrophic stage.Overall, functions of C. higginsianum strain Ch-1, isolated from diseased tissues of Brassica campestris, was kindly provided by Prof. Yangdou Wei, University of Saskatchewan, Canada. Mycelia were placed on potato dextrose agar (PDA) plates and cultured in darkness for 7 days at 25 °C for conidial production. The Escherichia coli strain DH5α  was used for cloning. Vectors pMD18T-HYG and pNeoP3300III were used for gene disruption and complementation vector construction. The Agrobacterium tumefaciens strain EHA105 was used for transformation and stored at −80 °C in 20% glycerol (v/v) as a bacterial suspension [The spension .Saccharomyces cerevisiae mutant strain EBY.VW4000, which lacks all 20 identified hexose transporters was grown in a YNB medium (0.67% yeast nitrogen base supplied with tryptophan histidine and leucine) with 2% maltose at 29 °C [The at 29 °C . ComplemArabidopsis thaliana ecotype Col-0 which is susceptible to C. higginsianum was used in virulence assays. Arabidopsis seeds were sown on the surface of a peat-based compost and placed in a growth chamber at a 16 h/8 h (day/night) photoperiod with temperatures of 22 and 18 °C, respectively.The A. thaliana Col-0, a virulence-reduced mutant G256 was screened from a C. higginsianum T-DNA insertion library. Flanking sequences of the insertion site were isolated with the inverse PCR strategy [C. higginsianum genome database to obtain the full sequence of the target gene.After inoculation on strategy . PCR proChHxt genes was achieved through homologous recombination. The total DNA of the C. higginsianum wild-type strain Ch-1 was isolated using a CTAB method [ChHxt_F1FP/ChHxt_F1RP and ChHxt_F2FP/ChHxt_F2RP  [ChHxt knockout mutants, the transformants were grown on PDA supplied with 50 μg/mL of hygromycin  and 500 μg/mL of cephalosporin , and then sub-cultured on PDA supplemented with 150 μg/mL antibiotic G418  and 500 μg/mL of cephalosporin . Transformants which grew on PDA plates supplemented with hygromycin but did not grow on PDA supplemented with G418 were selected as candidate knockout mutants. These were further verified by PCR amplification with ChHxt-KF/ChHxt-KR and HphSP/HphAP primers (The vector p3300neo-n (ATMT) . To scre primers .ChHxt knockout mutants were due to the targeted gene deletion, the knockout mutant ΔChHxt4-5 or ΔChHxt6-2 was complemented with a DNA fragment containing the complete coding region of ChHxt4 or ChHxt6 gene. The DNA fragment including the whole coding region of ChHxt6 and 1.5kb upstream sequence, which is considered as promoter, were amplified with ChHxt-comFP/ChHxt-comRP primers . The complement transformants were screened on PDA containing 150 μg/mL G418, and further confirmed using PCR with gene-specific primers, ChHxt-KF/ChHxt-KR  were also spotted onto microscope plastic coverslips in 9-cm-diameter petri dishes, lids replaced, and incubated at 25 °C. After 12 h, the conidial germination and appressorial formation were examined by light microscopy .6 spores/mL) were sprayed onto 4 to 5-week-old Arabidopsis plants. The plants treated with sterile deionized water served as control. The inoculated plants were incubated at 25 °C and 100% RH in a plant growth chamber (18-h photoperiod and a flux rate of 40 μmol‧m−2‧s−1). Symptoms were recorded daily. To observe fungal infection process, inoculated leaf tissues were cleared in a solution of methanol: chloroform: glacial acetic acid  for 24 h and stained with trypan blue (0.4%) for 10 h. Inoculated leaves were collected at 24, 48 and 72 hpi for light microscopy observation and formation rate of infection structure were calculated.The strains were propagated on PDA for 7 days at 25 °C. Sterile distilled water was added to each plate and gently scraped. The liquid was collected and conidia were harvested by passing through four layers of lens paper to remove debris and mycelium. In intact plant assays, conidial suspensions (1 × 10ChHxt6 (T169S), ChHxt6 (P221L), and ChHxt6  were generated by PCR-based site-directed mutagenesis [C. higginsianum by ATMT. After expression in S. cerevisiae, sequences of ChHxt6, ChHxt6 (T169S), ChHxt6 (P221L), and ChHxt6  were amplified and cloned into plasmid pMD18-T. Primer sequences are presented in Open reading frames of ChHxt6 P2L, and ChChHxt genes and ChHxt6 site-directed mutant genes in S. cerevisiae, the coding region of ChHxt genes were amplified from the cDNA of Ch-1 and ChHxt6 site-directed mutants using primers containing SpeI and ClaI restriction sites . The sequences transformed into pDM18-T were verified by PCR using ChHxt specific primers and digested with SpeI and ClaI, and then the fragments were ligated into the yeast expression vector pEBY using T4 DNA Ligase . Plasmid DNA of pEBY-ChHxts was extracted using the Qiagen plasmid extraction kit , and correct insertion of the fragments were verified by sequencing the cDNA/vector junctions . The plasmids of pEBY-ChHxts were transformed into chemically competent cells of S. cerevisiae strain EBYVW.4000. The yeast transformants expressing the ChHxt genes were verified by PCR. Yeast transformants were selected for the ability to grow on selective YNB medium supplied with appropriate amino acids and 1% maltose [ChHxt genes were compared to the strain harboring plasmid pEBY without insertion.For heterologous expression of  maltose . Some ofGenomic DNA was extracted using a CTAB method . SoutherArabidopsis leaves inoculated with C. higginsianum at 24, 48 and 72 hpi were collected, flash frozen in liquid nitrogen, and stored at −80 °C. RNA isolation was carried out using the TRIzol® Plus RNA Purification Kit , and potential DNA contamination was removed by DNase I treatment (RNase Free)  following the manufacturer’s instructions. First-strand cDNA was synthesized with the Revert Aid first strand cDNA Synthesis kit  following the manufacturer’s instructions. RT-PCR was used for confirmation of deletion and complementation mutants following the methods in previous work [ChHxt genes at different infection stages were analyzed by Real Time-PCR, and α-tubulin  was used as the reference gene. The primers used in RT-qPCR assays are listed in The ous work . ExpressS. cerevisiae Hxts were downloaded from NCBI . The Hxt homologues in C. higginsianum were located in the C. higginsianum genome using BLASTP (v2.2.31+) [http://smart.embl.de/, accessed on 5 April 2021) [http://www.clustal.org/clustal2/, accessed on 5 April 2021), and a phylogenetic tree was constructed using the Neighbor-Joining method with the MEGA software  [http://cctop.enzim.ttk.mta.hu/?_=, accessed on 5 April 2021) [The amino acid sequences of the 2.2.31+) . Open re2.2.31+) . The sofil 2021) . Multiplil 2021) . Transmeil 2021) .p < 0.05), the means were further separated using the test of least significant difference .The statistical analysis software DPS  was used to analyze all data from the quantitative assay with ANOVA . When significant treatment effects were found  genes in phytopathogenic fungi


Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasite’s maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurer’s clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites.Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of IMPORTANCE Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we used transcriptome sequencing (RNA-seq) to measure the impact of HbAS on the blood-stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections. Our in vitro time series data reveal that, during its blood stage, P. falciparum’s gene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, our in vitro and in vivo data indicate that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralize P. falciparum. In regions across Africa where malaria is endemic, red blood cell (RBC) variants are highly prevalent and can confer significant protection against severe malaria . HeterozPlasmodium falciparum’s pathogenesis, is reduced in HbAS iRBCs. HbAS reduces both the display on the iRBC surface of P. falciparum erythrocyte membrane protein 1 (PfEMP-1) and knob density (in vitro (in vivo (Candidate protective mechanisms reported thus far are diverse and provide evidence that HbAS disrupts mediators of pathogenesis and the parasite life cycle in infected RBCs (iRBCs). Parasite maturation may be inhibited by either low oxygen tension –6 or hos– density . This is density . The attin vitro —as well  in vitro in tightly synchronized parasite populations in HbAA and HbAS iRBCs as well as in freshly collected parasites from Malian children with uncomplicated malaria with HbAA or HbAS.The natural protection against severe falciparum malaria conferred by HbAS provides an opportunity to identify parasite mechanisms that are neutralized and therefore critical to pathogenesis. In this study, we used transcriptome sequencing (RNA-seq) to perform an unbiased comparative transcriptomic survey of the Plasmodium falciparum strains that originated where the sickle allele of β-globin is common  based on transcript counts clustered samples by parasite line and developmental stage . NotablyAS iRBCs . These dP. falciparum maturation in HbAS iRBCs was described previously to be delayed compared to that in HbAA iRBCs , which comprises eight transcripts that encode the core histones and histone variants in t 48 hpi . SimilarAmong molecular function GO terms, downregulated transcripts were enriched only in that of cysteine-type peptidase activity (GO:0008234), which primarily comprises the serine repeat antigen (SERA) proteases that play a pivotal role enabling parasite egress from the iRBC. SERA2 to -7 were significantly downregulated in 3D7 and FUP parasites in HbAS iRBCs at late time points .r = 0.524; P < 0.0001), indicating a conserved impact on transcript timing across strains.To identify the transcripts that are temporally dysregulated, we analyzed our time series data using dynamic time warping (DTW) , which dWe observed in HbAS iRBCs 155 temporally dysregulated transcripts in 3D7 and 162 in FUP . In contWe focused more closely on ring-stage expression by screening with the temporal alignment Kendall-Tau (TAKT) algorithm, which measures the overall similarity and temporal shift between two curves , and theP. falciparum episodes in HbAS children matched to 16 episodes in HbAA children in Kéniéroba, Mali. We first used stage-specific ring (n = 7 transcripts), trophozoite (n = 4), and schizont (n = 6) transcripts to classify 24 infections as ring stage and 12 as trophozoite stage , a peptidyl-prolyl cis-trans-isomerase (PPIase) (cyp19B in those with HbAS was downregulated 3-fold (adjusted P = 0.023010). Quantitative PCR (qPCR) confirmed that cyp19B is reduced in ring-stage parasite transcriptomes in HbAS. Cyclophilins function in protein folding and trafficking, in RNA processing, and generally as molecular chaperones  : compareaperones  and are in vitro .2 fold change = 5.00; adjusted P = 1.08 × 10−18). Also among the 10 most upregulated transcripts in HbAS trophozoites were the gene for the 130-kDa glycophorin binding protein  and the two glycophorin binding protein homologs , each upregulated more than 8-fold in HbAS trophozoites. Additionally, one of the most significantly upregulated transcripts in HbAS trophozoites  encodes the heat shock protein 70-x , which is thought to chaperone export of PfEMP-1 via J-dots and Maurer’s clefts (P = 0.041) and MAHRP2 .We observed in trophozoite-stage patient samples differential expression of 74 transcripts , among ws clefts , similar2 fold change = −2.37; adjusted P = 1.77 × 10−6). We also observed among the top 10 downregulated transcripts the genes for putative autophagy protein 5 (ATG5) , which associates with autophagosome-like structures (P = 0.0091), which has been linked to delayed clearance following artemisinin treatment , was upregulated in children with HbAS. Five of the six transcripts that were upregulated in severe malaria were downregulated in children with HbAS . Among the transcripts with statistically significant upregulation in severe malaria but downregulation in HbAS RBCs was that of a putative acid phosphatase (PF3D7_1430300), which was the most statistically significantly downregulated transcript in our HbAS trophozoite samples compared to HbAA trophozoites.Because HbAS confers protection specifically from severe malaria, we compared our data with those from a prior study comparing parasite gene expression in severe and uncomplicated malaria , which iin vivo infections in the same direction. The sole transcript differentially expressed in in vivo ring-stage parasites, cyp19B, was also downregulated in both 3D7 and FUP in HbAS  demonstrated a pattern of elevated transcript abundance at late time points in 3D7 and FUP, although statistical significance was achieved only in FUP , a thiamine-phosphate pyrophosphorylase (PF3D7_0614000), an exported protein  and two unannotated proteins of unknown function (PF3D7_0316200 and PF3D7_1449200). The protein phosphatase (PF3D7_1127000) that was the most highly upregulated transcript in HbAS trophozoites was highly expressed in HbAS and was also highly expressed and slightly upregulated in HbAS FUP, although it did not pass the threshold for statistical significance. In 3D7, however, this transcript was expressed at low levels in all samples.Among the genes that were significantly upregulated y in FUP  and D. A in vivo  and D. Ain vivo that were similarly expressed between Hb genotypes in vitro, a set which may indicate parasite responses that are specific to physiologic conditions. These 17 in vivo-specific transcripts include upregulation of atg5 (PF3D7_1430400) and rad5 (PF3D7_1343400), suggesting that autophagy and DNA repair, as well as the differential expression of enzymes whose specific functions are currently not well understood, including the putative protein phosphatase PF3D7_112700 and the putative acid phosphatase PF3D7_1430300, are impacted by HbAS in vivo.Finally, we examined the set of candidate aberrant transcripts identified P. falciparum malaria by mechanisms that remain obscure. In this study, we investigated the transcriptional changes that occur in P. falciparum in response to HbAS, and overall, the parasite’s transcriptional program remains largely unperturbed in HbAS iRBCs. However, hundreds of transcripts demonstrated differential expression in HbAS, with the bulk of these changes occurring late in the IDC. Combining our in vitro experiments with parasite transcript expression in vivo, we observed impacts of HbAS on transcripts encoding the parasite’s protein chaperone and folding machinery, oxidative stress response, and protein export machinery.HbAS consistently protects against severe var genes and the presentation on the RBC surface of P. falciparum erythrocyte membrane protein 1 (PfEMP1)  . In thosin vitro time series. The molecular functions and cellular components that were most downregulated in HbAS include cysteine-type peptidase activity, including the SERA proteins and the core histones and histone variants  and the RBC membrane  parasites in a Southeast Asian population transcriptomic survey, in which its expression was the most positively correlated single transcript with increased parasite clearance half-life , and we observed a downregulation of a majority of the members of PROSC in HbAS in vitro at late time points in the IDC in both 3D7 and FUP, including cyp19B, hsp70-2, grp94, pdi8, and pferc, though not in vivo. Additionally, the relatively high levels of cyp19B expression in HbAA in vivo ring-stage samples were unexpected, given that both transcript expression (in our in vitro time series) and protein expression  and HbAA children (5/16)  and B anin vitro time series data in HbAS RBCs mirrored key aspects of the transcriptional changes observed in P. falciparum lines with mutations of the drug resistance transporter genes pfmdr-1 and pfcrt , where HbAS reduces the risk of uncomplicated malaria by 34%  number Pro00007816 for a by 34% . Inclusia by 34% , and stoP. falciparum parasites of the 3D7 and FUP strains were cultured in human RBCs in RPMI 1640 supplemented with Albumax II, l-glutamine, hypoxanthine, and d-glucose at a 2% hematocrit in 1% O2–5% CO2 in N2 at 37°C  were sequenced on a full flow cell on the NovaSeq 6000 S2 platform with 150-bp paired-end reads. Field study samples (n = 32) were sequenced on a full flow cell of the NovaSeq 6000 S1 platform with 50-bp paired-end reads.Total RNA was isolated using TRIzol and the RNeasy minikit (Qiagen) after DNase treatment and quantified by a Qubit high-sensitivity RNA assay (Thermo Fisher Scientific) before storage at −80°C. Libraries were prepared from total RNA with the Kapa stranded mRNA-seq library prep kit. Libraries from each of the two time series experiments . Quantification files of parasite gene expression were summarized with the tximport package in R | was >log2(1.5) in the time series and, with a false-discovery rate set to 0.1, the adjusted P value was <0.05 and |log2(HbAS/HbAA)| was >log2(1.5) in the field samples.All differential expression analyses were performed with the DESeq2  R packagftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2go.gz), and P. falciparum’s annotations were extracted with the taxonomic identifier 36329. Using the transcripts identified as differentially expressed by DESeq2 according to the conditions described above as input, gene ontology terms were accepted for Benjamini-Hochberg-adjusted P values of <0.05.Gene ontology enrichment analysis was performed with GOATOOLS . Gene IDDynamic time warping (DTW) of time series transcript expression data was computed with the tslearn toolkit . We consP value of ≤0.05 was used to identify transcripts with similar dynamics across all β-globin types within each parasite.We used the temporal alignment Kendall-Tau algorithm (TAKT)  to identin vitro time series that peaked within rings, trophozoites, or schizonts and then selected transcripts that were highly expressed at their peak, discarding those that did not exceed 1,000 TPM. We also estimated parasite stage with a previously published mixture model . All scripts, protocols, analyses, and supplemental output are available on GitHub (The RNA-seq data are available in GEO (accession no. n GitHub . See Sup10.1128/mSphere.00755-21.1TEXT S1Text S1, DOCX file, 0.04 MB.Supplemental text Copyright © 2021 Saelens et al.2021Saelens et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Plasmodium falciparum Histidine Rich Protein 2-based rapid diagnostic tests (PfHRP2-based RDTs) are widely used. However, in the last decade, the accuracy of PfHRP2-based RDTs has been challenged by the emergence of P. falciparum strains harbouring deletions of the P. falciparum histidine rich protein 2 (pfhrp2) gene, resulting in false-negative results. In the Democratic Republic of Congo (D.R. Congo), little is known about the prevalence of the pfhrp2 gene deletion among P. falciparum isolates infecting symptomatic patients, especially in low to moderate transmission areas where pfhrp2 deletion parasites are assumed to emerge and spread. Here we determine the local prevalence and factors associated with pfhrp2 gene deletions among symptomatic malaria patients in the Kwilu Province of the D.R. Congo.Malaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where PfHRP2-RDT, and spotted onto Whatman filter paper for downstream genetic analysis. Genomic DNA was extracted and used to perform PCR assays for the detection and confirmation of pfhrp2 gene deletions. Fischer’s exact and the Kruskal–Wallis tests were applied to look for associations between potential explanatory variables and the pfhrp2 gene deletion with a level of statistical significance set at P < 0.05.We used secondary data from a prospective health facility-based cross-sectional study conducted in 2018. Blood was collected for microscopy, pfhrp2 gene deletion was 9.2% (95% CI: 6.7%–12.1%). The deletion of the pfhrp2 gene was associated with health zone of origin (P = 0.012) and age (P = 0.019). Among false-negative PfHRP2-RDT results, only 9.9% were due to pfhrp2 gene deletion.Of the 684 enrolled symptomatic patients, 391 (57.7%) were female. The majority (87.7%) reported the presence of mosquito breeding sites within the household’s compound, and fever was the most reported symptom (81.6%). The overall prevalence of the P. falciparum isolates with pfhrp2 gene deletions are relatively common among symptomatic patients in Kwilu province. Further investigations are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting PfHRP2 and parasite lactate dehydrogenase (pLDH) antigens could limit the spread of deleted isolates. Malaria remains a global health issue despite progress over the last decade. In 2018, there were an estimated 228 million new malaria cases, including 405 000 deaths ).The CareStart™ Malaria P. falciparum HRP2 specific monoclonal antibody as a single line across the test strip. The reported panel detection score is 91.0% at 200 parasites/µl with a false positive rate of 0.9% [The test membrane strip is pre-coated with a  of 0.9% , 37A team of four medical technologists read the slides in the laboratories of health facilities where samples were collected. When a health facility did not have the necessary equipment to perform the examination, slides were read at the nearest laboratory possessing adequate equipment. For quality assurance, one expert microscopist randomly selected positive and negative slides to cross-check results. In the case results were not concordant, another reading was performed. About five percent of slides went through another quality control in the vector control laboratory of the Kinshasa School of Public Health.Thick and thin smears were made on the same slide. The part of the slide containing the thin smear was fixed with methanol and dried. Then the whole slide was stained with 10% Giemsa’s solution for ten minutes and finally washed off with distilled water and air-dried. Stained smears were examined under a microscope for malaria parasite identification. For positive slides, malaria parasites were counted against 200 white blood cells (WBC), and parasite density was calculated based on a total of 8000 WBC/µl using the following formula: (Number of Parasites counted × 8000)/Number of counted WBC.Parasite density calculation was immediately performed when 100 parasites were counted against 200 WBC. However, in the case that fewer than 100 parasites were counted against 200 WBC, the count continued until 500 WBC.PfHRP2-RDT cassettes and DBS using the QIAGEN QIAmp®DNA extraction kit  according to the manufacturer’s instructions. We also adapted a previously described method to recover DNA from 197 spent RDTs membranes from Bandundu Health Zone [Genomic DNA was extracted from membranes of spent lth Zone .P. falciparum infection, we designed specific primers targeting a 226 base pair region of the P. falciparum lactate dehydrogenase (pfldh) gene and performed a real-time PCR assay  , as well as negative controls containing DNA from blood spots prepared from known malaria negative individuals. We repeated the assay for all discordant duplicates and counted three consistent results as the final result.Samples were duplicated and loaded in 96-wells plates along with serially diluted positive controls  and 3D7 (pfhrp2 positive) as controls for all assays.For detection of the We repeated the nested PCR for all negative results. In the case of discordant results, we performed the amplification a third time and counted two consistent results as the final result.Reaction components for both real-time and nested PCR are summarized in Table PCR amplicons were separated by electrophoresis on a 2% agarose gel stained with Gel Red® Nucleic Acid Stain 10 000 × in water. A total of 12 µl of PCR amplicons (6 µl) and loading dye (6 µl) were loaded onto the gel, which was run for 35 min at 100 V and observed under UV light. A 500 µg/ml Gene Ruler 100 bp DNA Ladder  was loaded onto the same gel to determine the sizes of the resolved fragments.Data were entered and analyzed using STATA15 . Tables have been used to describe categorical variables. Continuous variables have been summarized using median and interquartile ranges. Proportions have been used to summarize categorical variables.pfhrp2 gene deletion). We computed the 95% Confidence Interval (95% CI) for the prevalence of pfhrp2 gene deletion. We considered a P-value less than 0.05 statistically significant.Fischer’s exact test  and the Kruskal–Wallis test  were applied to look for associations between potential explanatory variables and the main outcome  were female, and 287 (42.3%) were male. Kikwit-Nord Health Zone accounted for the majority of patients 362 (52.9%) while Bandundu and Kikwit-Sud Health Zones accounted for 197 (28.8%) and 125 (18.3%) patients, respectively.The median age was nine years, with an interquartile range from 3 to 26 years old. Children under five years old represented 36% of enrolled patients. The median size of household was six, with an interquartile range from five to six. Table The majority (87.7%) of patients/patients’ guardian reported the presence of mosquito breeding sites within the compound where the household was located. Two-thirds of household 64.2%) owned a mosquito bednet, while 49.7% of patients spent the night before the interview under a mosquito bed net. One-third (220) of patients reported prior malaria drug intake. Quinine (13%), sulfadoxine-pyrimethamine (6.1%), artemether-lumefantrine (4.8%), artesunate (2.0%), arteether (1.9%), artemether (1.8%) were the common drugs taken as illustrated in Table % owned aAt admission, the body temperature of patients ranged from 37 °C to 38 °C (median temperature of 37.5 °C). The common findings of the clinical examination were: A history of fever the last 72 h (81.6%), headaches (41.8%), vomiting (31.4%), rigor (22.8%), fatigue (22.1%) and abdominal pain (20.2%). Among the 235 patients with a microscopy positive result, malaria parasite density ranged from 64 parasites /µl to 7200 parasites /µl with a median parasite density of 880 parasites/µl. Table PfHRP2-RDT result, 63.9% were ‘false negatives’ compared to PCR. The sensitivity and specificity of RDTs with reference to PCR were 71.3% (325/456) and 42.4% (74/173), respectively. Using microscopy as the gold standard, the proportion of false-negative RDT result dropped to 26.3%. The microscopy-determined sensitivity and specificity of RDTs were 74.3% (156/210) and 36.0% (151/419), respectively . Of these, only one harboured a deletion at the PfHRP2 locus.Using PCR as the gold standard, there were 131 false-negative pfhrp2 gene deletion among PfPCR confirmed symptomatic cases was 9.2%. The highest prevalence was found in Bandundu Health Zone (15.7%), followed by Kikwit-Sud Heath Zone (9.6%) and Kikwit-Nord Health (6.2%). Table pfhrp2 gene deletion among PCR confirmed cases across Heath Zones.The overall prevalence of th Zone 1.7%, follpfhrp2 gene deletion status between males and females (8.5% vs 9.8%). Conversely, there was a statistically significant difference in pfhrp2 gene deletions status in Bandundu health zone compared to Kikwit-Nord and Kikwit-Sud health zones (P = 0.012). There was a trend towards pfhrp2 gene deletion in older compared to younger patients, but this was not statistically significant (P = 0.079). However, when analyzing age as a continuous variable, the median age among pfhrp2 gene deleted patients was higher than the median age among pfhrp2 gene non-deleted patients (18 years vs 7 years). The Kruskal Wallis test showed strong evidence suggesting that the distributions of age differed by pfhrp2 gene deletion status (P = 0.019). Similarly, there was a trend towards pfhrp2 gene deletion among negative microscopy results, but weak evidence supported this finding (P = 0.079).As shown in Table pfhrp2 gene deletion. However, the difference was not statistically significant . This finding is in keeping with results from the D.R. Congo 2013–2014 Demographic and Health Survey(DHS) which reported a sex ratio slightly in favour of females .Surprisingly, children under-five represented 36% of participants. This is in contrast with what might be expected since under-five children are known to be at higher risk of contracting malaria. Despite the burden of malaria in Kwilu Province, there is a seasonal trend in transmission, and immunity is acquired later in life, around 10 years .There were mosquito breeding sites near households of the majority of participants (87.7%). The survey was conducted during the rainy season, which is characterized by the formation of breeding sites, especially in rural and semi-urban areas with limited public facilities.. recently reported consistent data (72% ownership vs 45% use) in health zones of Kinshasa province, neighbouring Kwilu province [The use of mosquito bednets was the primary means of bite-prevention. Two-thirds of households (64.2%) owned a bednet, and only 49.7% of participants spent the night before the interview under a mosquito bednet. These results are lower than the previous report from the DHS in Kwilu Province. In 2014, 87% of households surveyed in the Kwilu province possessed a bednet, and 69.4% of participants slept under a bednet the night before the interview . Back thprovince . A low cprovince . SubsequAmong participants who reported prior malaria drug intake, 13% took quinine at least one week before the survey. This finding highlights the poor compliance with the WHO guidelines for the treatment of malaria in the study areas as well as challenges associated with the use of injectable artesunate in areas where quinine is cheaper, easy to use and available.As might be expected, 81.6% of participants reported a history of fever. In many cases, fever is suggestive of malaria but is also reported in several febrile illnesses prevailing in tropical areas. Without a reliable diagnostic tool, a syndromic approach often leads to overtreatment, especially among people living in low to moderate malaria transmission areas . A recenPfHRP2-RDT results involved parasites with pfhrp2 gene deletions. A similar result (10.6%) has been reported in Nigeria, [et al. (2016), in Ghana reported a higher proportions of 23% [Only 9.9% of false-negative Nigeria, . Converss of 23% , 5, 17.pfhrp2 gene deletion is not a major cause of false-negative PfHRP2-RDT results. Several reasons may explain a false-negative PfHRP2-RDT result such as poor quality of the test, inappropriate manipulation and interpretation, low parasite density, excess of circulating parasite antigens creating a prozone-like effect, and genetic polymorphisms in the target antigen [This weak proportion shows that  antigen , 43, 44.PfHRP2-RDT false-negative results due to pfhrp2 gene deletion has surpassed the 5% threshold set by the WHO, requiring a subnational change in malaria RDTs, the required number of P. falciparum isolates (37) to include per health facility in the sampling domain was not reached. Therefore, further investigations are needed to provide enough evidence for policy change.Even though the proportion of P. falciparum isolates with pfhrp2 gene deletion was 9.2%. This prevalence is higher than the previously reported national prevalence of 6.4% and the local prevalence of 3% [pfhrp2 gene deletion, which can survive and spread [pfhrp2 gene deletion has been shown to be higher in low to moderate transmission area in the beginning of the rainy season, which is the case for the present study [The overall prevalence of ce of 3% . The pred spread . Thirdlynt study .A similar prevalence has been reported in Eritrea (9.7%) and Kenya (9%) , 46. Howphrp2/3 deletions was released in 2014 [phrp2/3 deletions among symptomatic patients [The first protocol concerning  in 2014 . Subsequpatients . Finallypatients .pfhrp2 gene deletion prevalence in Bandundu health zone compared to Kikwit-Nord and Kikwit-Sud health zones (P = 0.012). Variations in pfhrp2 gene deletion status within regions and countries have been previously reported and depend on several factors including level of transmission and magnitude of PfHRP2-RDT use [There was a statistically significant difference in s P = 0.0. Variati-RDT use , 50. Furpfhrp2 gene deletion in older compared to younger (under-five) patients, but this was not statistically significant (P = 0.079). However, when analyzing age as a continuous variable, the Kruskal Wallis test showed strong evidence suggesting that the distributions of age differed by pfhrp2 gene deletion status (P = 0.019). This may suggest that the risk of being infected with parasites harboring pfhrp2 deletion increases with age while traditionally under-5 years old are at high risk of contracting malaria compared to the older group.There was a trend towards pfhrp2 gene deletion among negative microscopy results, but weak evidence supported this finding (P = 0.079). Microscopic examination is based on morphological aspects of the parasite and might not be influenced by genetic traits such as gene deletions. Even considering that PfHRP2 is known to be involved in the formation of hemozoin, this is not the only morphological feature allowing parasite detection by microscopic examination.Similarly, there was a trend towards pfhrp2 gene deletions. However, the difference was not statistically significant. This study could only identify 45 P. falciparum isolates with pfhrp2 gene deletions, and a larger sample size may be required to detect differences and provide evidence of association.We found more malaria signs and symptoms among patients infected with parasites not harbouring P. falciparum isolates with pfhrp2/3 gene deletion among symptomatic patients in this region. The availability of clinical, biological, and sociodemographic data allowed exploration of differences between infection by pfhrp2-deleted and wild-type P. falciparum parasites. However, the limited sample size precluded identification of predictors of pfhrp2 gene deletion and did not allow us to make solid conclusions regarding differences in pathology between pfhrp2 deleted and wild-type parasites.This study is the first to provide the local prevalence of The selection of study sites based on known burden of malaria and sociodemographic characteristics may have introduced a selection bias making the sample not representative of the whole province within which the level of transmission varies. A health facility-based design is undoubtedly the best choice to recruit symptomatic patients, but the low service utilization rate and insufficient public subsidies allocated to malaria management may have prevented some subjects with the characteristic of interest to attend health facilities and thus to be enrolled in the study.PfHRP2 based RDTs as primary diagnostic tools and co-infections with non-malaria febrile illnesses could lead to the initiation of a selective treatment favoring pfhrp2 deleted parasites. There is a need, therefore, to establish a surveillance system for pfhrp2 deleted mutants as a part of malaria control programs. Such a surveillance system should be strengthened with reliable diagnostic tools such as molecular point of care testing to ensure efficient and evidence-based allocation of resources to disease control programs.The D.R. Congo National Malaria Control Program has adopted a five year (2016–2020) strategic plan with goals including the diagnosis of at least 80% of fever cases and the treatment of all positive diagnosed cases with ACT. Increased service utilization, the significant use of In routine practice, clinicians should investigate other febrile illnesses despite a positive RDT result to minimize failure in disease management.P. falciparum isolates with a pfhrp2 gene deletion among symptomatic patients. These isolates explained only 9.9% of PfHRP2-RDT false-negative results, suggesting that factors other than pfhrp2 gene deletion are of significant importance in the false-negativity rates of PfHRP2-based RDTs. Even though the proportion of false-negative PfHRP2-RDT results due to the pfhrp2 deletion has surpassed the 5% threshold set by the WHO for a subnational change in malaria RDTs, further regional investigations with appropriate sampling are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting PfHRP2 and pLDH antigens could limit the spread of deleted isolates.We found a local prevalence of 9.2% of


Plasmodium infections significantly drive malaria transmission and impact control and elimination strategies, but are largely uncharacterized. We investigated the prevalence and risk factors of asymptomatic malaria infections to inform malaria control strategies in Ghana.Asymptomatic Five cross-sectional surveys were conducted at the end of the peak transmission season (August–September) on 4892 school children aged between 6 and 14 years in two distinct ecological settings in Ghana between 2013 and 2017. The study sites were Begoro (forest ecology) and Cape Coast . The children were screened for malaria parasites by microscopic examination of Giemsa-stained thin and thick blood films. Hemoglobin levels were measured using the Hemocue HB analyzer. In addition, height was measured and the height-for-age z-scores estimated from the reference population defined by WHO to determine children who were stunted. Proportions of categorical and means of continuous variables were compared using Chi-square test and Student’s t-test respectively, and multivariable logistic regression was done to assess risk factors associated with asymptomatic infections.p value = 0.04). The study recorded three species of Plasmodium  in both sites. Plasmodium falciparum was the predominant species, accounting for about 85% of infections in both study sites. The asymptomatic school children were more likely to be anaemic  and stunted in growth . Males carried more asymptomatic infection than females . School children aged 12–14 years had more asymptomatic infections than those aged 6–8 years .The overall prevalence of asymptomatic malaria in the school children was higher in Begoro compared to Cape Coast  vs. 24% , There is a considerable burden of asymptomatic malaria in the two regions of Ghana, which is associated with males, older children, anaemia, and stunted growth in children, and may have implications for malaria control and elimination strategies in Ghana. Malaria is still one of the leading global public health concerns. Although there was a decline in malaria morbidity and mortality worldwide in 2018, sub-Saharan Africa saw an increase of about one million cases that same year –5.Ghana is a malaria-endemic country and falls within the top 15 highest-burden countries . PregnanPlasmodium falciparum infections may result in severe, uncomplicated or asymptomatic malaria. There is a wealth of studies and data on uncomplicated and severe malaria because they are the drivers of malaria-related morbidity and mortality respectively. Comparatively, asymptomatic malaria infection is understudied and remain a challenge to malaria control due to its effect on transmission dynamics [dynamics .P. falciparum infection has been shown to persist asymptomatically in semi-immune individuals for more than 18 months especially in older children [Asymptomatic malaria, is defined as the presence of parasites in people and an absence of malaria-related symptoms such as temperature > 37.5 °C. Asymptomatic malaria occurs as a result of continuous exposure to malaria infections, leading to the acquisition of partial immunity against complications such as cerebral malaria and the accumulation of the “reservoir of infection” . P. falcchildren –15. The children . Some fichildren , 17, 18 children . Aside fchildren , 21.Like most malaria control programs, control efforts in Ghana gravitate towards a system, which identifies, treats, and reports people with malaria presenting at health care facilities giving little attention to asymptomatic cases in the community. However, through seasonal malaria chemoprevention and intermittent preventive treatments, a subset of asymptomatic infections is treated but not tracked. For successful malaria elimination, however, the full complement of the burden of infection, including the asymptomatic reservoir should be the focus. Thus, the asymptomatic carrier must be actively characterized .To better understand the asymptomatic burden in Ghana, we leveraged repeated school-based cross-sectional surveys on the impact of parasite diversity on the evolution of drug resistance. We characterized the asymptomatic burden and its associated risk factors in two sites in Ghana with varying malaria endemicities. We show that the asymptomatic burden is significant in both sites and are influenced by risk factors such as age, anaemia, and stunted growth in children.Repeated cross-sectional surveys were conducted in Cape Coast and Begoro for five consecutive years (2013–2017). Cape Coast is in the coastal savanna ecological zone of Ghana where malaria transmission is low to moderate and perennial. Begoro is situated in the forest ecological zone of Ghana, where malaria transmission is high and perennial. The detailed description of these study sites has been described elsewhere . The stuThe study primarily compared the prevalence of asymptomatic malaria in the two study sites. A minimum sample size of 1933 per study site provides 80% power to determine as low as a 4% difference in asymptomatic malaria between study sites at a 95% confidence level and 5% precision.The list of all schools in the catchment areas of this study was obtained from the municipal education office and three schools randomly selected for each study site. All the school children aged 6 to 14 years from the selected schools, whose parents gave informed consent and assented if aged 12 to 14 were screened.Blood smears were prepared for each participant in the study. Two slides per participant were obtained: one with a thick smear and the other with both thick and thin smears. The thick smear slide was stained rapidly (10% Giemsa for 10–15 min) for initial screening, while the thick and thin smear slides were retained for subsequent quantification of the parasites if the participant tested positive. The parasite density was estimated, by counting the number of asexual parasites against 200 WBC with a hand tally counter. Parasite density was calculated as follows: Parasite density (per μl) = (number of parasites counted ÷ number of leukocytes counted) × 8000. Haemoglobin levels were determined for all patients using a portable HemoCue® Hb 301 Analyzer . The body weight, height, and axillary temperature were also measured. Children who tested positive for malaria and had fever were treated with artesunate amodiaquine as recommended by the national malaria control program (NMPC).Asymptomatic malaria: the presence of parasites in peripheral thick blood smears, an axillary temperature < 37.5 °C, and an absence of malaria-related symptoms [Anaemia: Anaemia in children was defined as having haemoglobin (Hb) < 11 g/dLStunting in children is defined as height-for-age z-score (HAZ) below negative two standard deviations from the median of the reference population, using the WHO Child Growth Standardsp < 0.05 for all tests.The variables hemoglobin and height-for-age z-scores were categorized into binary variables. Height-for-age z-scores were categorized as “stunted”  and “not stunted” (z-score greater than − 2 SD), haemoglobin was categorized as anaemic (HB < 11 g/dl) and “non-anaemic” (HB ≥ 11 g/dl). All statistical analyses were performed using the statistical software STATA version 12. Continuous data were summarized into means and standard deviations (SD)  and categorical data into proportions for descriptive analysis. Parasite density which is not normally distributed was log-transformed and the geometric mean calculated. Means were compared between study sites using Student’s t-test/Mann Whitney rank test, while proportions were compared using Pearson’s Chi-square tests/Fisher exact test. Trends of asymptomatic malaria over the study period were compared using the Cochrane Armitage test of trends. Univariate analysis to determine potential risk factors of asymptomatic malaria was performed using the Student’s t-test/Mann Whitney rank test for continuous variables and Pearson Chi-square test/Fisher exact test for categorical variables. The association between asymptomatic malaria and anaemia, stunting, gender, age, and study site was explored using multivariable logistic regression and the odds ratio and confidence intervals recorded. The significance level was set at p value< 0.001). Also, the proportion of children from Cape Coast who were stunted was higher than observed in Begoro . With the exception of 2014, the prevalence of asymptomatic malaria was always higher in Begoro compared to Cape Coast. The overall prevalence of asymptomatic malaria in Begoro was higher than in Cape Coast , P. malariae , P. ovale . Cape Coast had 3% mixed infections of PF, PM, and 0.3% of PF, PO, while, Begoro had 2% of PF, PM but no mixed infection of PF, PO. . In comparison with the other age groupings, the older children (aged between 12 and 14 years) were more asymptomatically infected . The prevalence of asymptomatic malaria was higher in anaemic children compared to the non-anaemic children . Prevalence of asymptomatic malaria was higher in children who were stunted (34%) compared to those who were not . Children living in the Begoro catchment area were more likely to be asymptotic carriers than those living in Cape Coast .Table p value = 0.015). The odds of asymptomatic malaria in the 12–14 years age group was 1.28 times higher than that in the 6–8 years age group . Anaemic children were twice more likely to carry asymptomatic infections  than the non-anaemic children and stunted growth increased the odds of carrying asymptomatic parasites by 46% . In addition, the study showed an increased odd of 26% asymptomatic carriage in Begoro  compared to Cape Coast  after adjusting for age, sex, anaemia and stunted growth  , being male increased the odds of asymptomatic malaria by 18% . As expected, asymptomatic malaria was higher in Begoro, within the forest ecological niche compared to Cape Coast in the coastal region of Ghana and was associated with age, gender, anaemia and stunted growth in school children. The outcomes of this study highlight the need to expand control interventions and educational campaigns for asymptomatic malaria in Ghana with particular focus on males and older children.Our repeated cross-sectional surveys estimated the prevalence of the asymptomatic infections in catchment areas of two ecological zones in Ghana and also identified associated risk factors of asymptomatic malaria. The study revealed a high asymptomatic burden (~ 20%) in the two catchment areas studied, where most of the infections were Plasmodium falciparum, with other Plasmodium species having a low but underestimated prevalence [Plasmodium species in this study is consistent with other studies conducted in Ghana. P. falciparum is the most common malaria-causing species in Ghana, followed by P. malariae and P. ovale [The variation in asymptomatic carriage in these different ecological zones is consistent with transmission intensity in these two sites. The increased odds of asymptomatic malaria in the forest ecological zone may be due to more favorable climatic conditions such as temperature, abundant rainfall and the vegetative cover, resulting in an abundance of breeding sites for the mosquito vector in these areas. These variations observed in transmission between communities reflect the importance of micro-ecological factors and transmission in the areas studied . The bulevalence , 25. TheP. ovale .The prevalence of asymptomatic infections among children was more predominant in males than females. This disparity may come as a result of gender roles with regards to division of labour, hormonal or host genetic factors , 29, andIn this study, children aged between 12 and 14 years were 21% more asymptomatic than children aged between 6 and 8 years. This was consistent with findings from other studies conducted in Ghana –35. Age Our study recorded a doubled risk of anaemia in children who had asymptomatic infections. Malaria-associated anaemia has been well documented, however, few studies have looked at the impact of asymptomatic malaria on anaemia in healthy children in schools. In general, malaria contributes to the loss of iron from lysed cells through excretion, impairment of intestinal absorption of ingested iron, the release of storage iron from hepatocytes, and recycling of iron that is derived from phagocytosis of senescent or parasitized erythrocytes by macrophages . HoweverOur study revealed that stunting was associated with asymptomatic malaria though a similar study conducted in another forest region of Ghana did not show an association between stunting and asymptomatic malaria . The incThis study had a few limitations. The study might have under-reported the prevalence of asymptomatic malaria because microscopic detection of parasites as used in the study has low sensitivity compared to polymerase chain reaction (PCR), although it is the gold standard for diagnosing malaria in endemic countries. Evidence of under-reporting of malaria cases by microscopy has been shown , 53 compThe prevalence of asymptomatic malaria was high in both study sites in Ghana. Asymptomatic malaria among the school children was age-dependent with a higher risk in male children. A significant association was indicated between asymptomatic malaria and the risk of anaemia and stunting. Thus, malaria control programs should also focus on asymptomatic malaria as a means to monitor the impact of control interventions and to reduce malaria morbidity and mortality.