Plasmodium falciparum malaria among Trypanosoma brucei rhodesiense HAT and plasma cytokine profile levels associated with HAT and/or malaria infections.Immunological Human African Trypanosomiasis (HAT) studies often exclude malaria, although both infections overlap in specific endemic areas. During this co-infection, it is not known whether this parasitic interaction induces synergistic or antagonistic cytokine response among humans. This study determined prevalence of Participants were recruited at Lwala hospital in north eastern Uganda: healthy controls (30), malaria (28), HAT (17), HAT and malaria (15) diagnosed by microscopy and PCR was carried out for parasite species identification. Plasma cytokine levels of Interferon-gamma (IFN-γ), Tumour Necrosis Factor-alpha (TNF-α), Interleukin (IL)-6, IL-10 and Transforming Growth Factor-beta (TGF-β) were measured by sandwich Enzyme-Linked Immuno Sorbent Assay and data statistically analysed using Graphpad Prism 6.0.P. falciparum malaria among T. rhodesiense HAT cases was high (46.8%). Malaria and/or HAT cases presented significant higher plasma cytokine levels of IFN-γ, TNF-α, IL-6, IL-10 and TGF-β than healthy controls (P < 0.05). Levels of IFN-γ, IL-6 and IL-10 were significantly elevated in HAT over malaria (P < 0.05) but no significant difference in TNF-α and TGF-β between HAT and malaria (P > 0.05). Co-infection expressed significantly higher plasma IFN-γ, IL-6, and IL-10 levels than malaria (P < 0.05) but no significant difference with HAT mono-infection (P > 0.05). The TNF-α level was significantly elevated in co-infection over HAT or malaria mono-infections (P < 0.05) unlike TGF-β level. Significant positive correlations were identified between IFN-γ verses TNF-α and IL-6 verses IL-10 in co-infection (Spearman’s P < 0.05).The prevalence of T. b. rhodesiense significantly induced the cytokine response more than P. falciparum infections. Co-infection led to synergistic stimulation of pro-inflammatory , and anti-inflammatory  cytokine responses relative to malaria mono-infection. Level of TNF-α partially indicates the effect induced by T. b. rhodesiense and P. falciparum mono-infections or a synergistic interaction of co-infections which may have adverse effects on pathogenesis, prognosis and resolution of the infections.The Trial registration VCD-IRC/021, 26/08/2011; HS 1089, 16/01/2012 Human African Trypanosomiasis (HAT) and/or malaria are two protozoan parasitic vector borne diseases of public health concern in endemic areas of Africa. In Uganda, malaria is prevalent throughout the country whereas HAT is endemic in specific foci. Nevertheless, malaria coincides with HAT in specific overlapping areas. Therefore plasmodium and trypanosome infection may exist within a host concurrently , 2. ThisP. falciparum infection [P. falciparum infection [T. brucei than Plasmodium berghei infected mice. Co-infected mice expressed elevated IFN-γ and TNF-α levels over P. berghei or T. brucei mono-infected group suggesting active response against secondary infection. Although IFN-γ in co-infected mice was more less than in T. brucei mono-infected group. The induction towards pro-inflammatory response  by T. brucei could account for plasmodium hepatic impairment in mice [T. b. gambiense and P. falciparum co-infection than healthy controls. Consequently, IL-10 plasma level of HAT was similar irrespective of P. falciparum infection [At immunological level, quite different immune cytokine responses are induced by different parasites. Studies in animal and human co-infections suggest significant variation in the immune cytokine response to occur in reaction to pathogen–pathogen interactions. This is essential in the pathogenesis of infections (pathogen–host interactions) and their interactions (pathogen–pathogen interactions) in the host. During co-infections, the parasites may exhibit synergistic, antagonistic, or competitive interactions with the immune system of host , 6. The nfection , 12, 15.nfection . Also idnfection , IL-10 anfection . In anim in mice . Howevernfection .T. b. rhodesiense and P. falciparum co-infection, and its relative comparison to mono-infection in naturally infected cases are not or poorly explored. Immunological HAT studies have always excluded malaria although both infections overlap in specific co-endemic areas of tropical Africa [P. falciparum malaria among T. b. rhodesiense HAT cases and plasma cytokine profile levels associated with parasitological acute sleeping sickness and/or malaria cases from north eastern Uganda. The results will provide insights that can be manipulated in future to aid clinicians, diagnostic approaches, vector or disease control policy team on how to handle HAT and malaria cases. The comparison of cytokine concentration examined how T. b. rhodesiense HAT and P. falciparum malaria co-infection modify the immunological cytokine response induced by the two parasites relative to mono-infections. This will contribute to the understanding of the immunological response of this co-infection and management of cases, emphasising the significance of immune-mediated interactions in poly-parasitism among people.The plasmodium and trypanosome infections have been extensively described separately with potential to induce cytokine production in the host. Despite epidemiological HAT and malaria co-infection studies, immunological cytokine response to l Africa , 10. It T. b. rhodesiense HAT which extended from the historical foci in the eastern part of the country [T. b. rhodesiense sleeping sickness [T. b. rhodesiense infected cattle as human reservoirs [Participants were recruited from north eastern Uganda at Lwala hospital, a sleeping sickness referral center in Kaberamaido district, providing health services especially to HAT cases. Since 2004, this area has been affected by  country . Currentsickness . The higservoirs . MalariaP. falciparum malaria among T. b. rhodesiense HAT cases and associated plasma cytokine levels of HAT and/or malaria cases from the region reporting to Lwala hospital, aged 6 years and above between October/2013 and May/2014. Patients report to hospital with common complaints of; fever (temperature > 37 °C), general body weakness, headache, joint pain, tremor, loss of appetite and sweating. It is a routine practice at Lwala hospital to microscopically check for malaria and HAT parasites. After patients had given detailed clinical descriptions to the medical physician, participants were subjected to routine laboratory diagnostic procedures at the hospital required to guide treatment. Microscopic examination of wet and thick blood films from finger prick blood stained with 10% Giemsa or Field stain for plasmodium and trypanosomes [3 was categorised as early stage HAT while late stage infection was confirmed by the presence of trypanosomes in the CSF and/or WBC count > 5 cells/mm3.This was a case control study to determine the prevalence of anosomes , or haemanosomes  were peranosomes . White banosomes . The preFollowing this diagnosis, patients were recommended for the appropriate treatment and monitoring by the clinician. For HAT, this involved administering Suramin and Melarsoprol for early and late stage HAT respectively . MalariaT. b. rhodesiense) and/or MSP-2  nested PCR was carried out. A healthy control was an individual without any detectable parasite on blood smear or other infections and SRA or MSP-2 PCR negative. Individuals diagnosed with infections of helminths (microscopic examination of filtered urine for the presence of eggs), amebiasis (detection of cysts in stool), typhoid , tuberculosis (sputum microscopy), human immunodeficiency virus (HIV) (rapid diagnostic test strip) and Urinary tract infections (UTI)  [All HAT and/or malaria cases and healthy control individuals diagnosed from the region reporting at Lwala hospital were included, after a written informed consent in their local language (Kumam) from adults or guardians in case of minors (6 < 18 years) in addition to their assent. A case was an individual with positive blood smear of trypanosome and/or plasmodium parasites in blood, or presence of trypanosomes in CSF. For parasite species identification, SRA   were excd scale) . Previoud scale)  and thos®, USA) according to manufacturer’s instructions. The eluted DNA was stored at − 20 °C for PCR analysis. Nested SRA PCR was used to detect T. b. rhodesiense using sequence primers  on test samples, positive control (Genomic DNA extracted from previously characterised strain of T. b. rhodesiense from Tororo endemic area) or negative control  with conditions as described by Maina et al. [The extraction of DNA from blood PCV was performed using QIAGEN DNeasy Blood & Tissue Kit  on test samples, positive control  or negative controls  with conditions as described by Dokomajilar et al. [Nested MSP-2 PCR was used to detect the presence of r et al.  and prodAG, Berlin) and incubated for 1 h at room temperature (RT). After washing microplates 3 times with wash buffer, 200 µl assay diluent was added, followed by 100 µl plasma sample, 100 µl standards , serially diluted in 100 µl assay diluent and 100 µl controls and incubated for 2 h at RT. After washing microplates 5 times, 100 µl working antibody detector (biotinylated detection antibody + streptavidin–horseradish peroxidase) was added and incubated for 1 h at RT. After 7 washes, 100 µl substrate solution  was added and incubated in the dark for 30 min for colour development, after which 50 µl stop solution (2 M H2SO4) was added and the plate read at 450 nm using a microplate reader . All assays were done in triplicate wells on the same plate and readings averaged. For TGF-β, the plasma sample was centrifuged for 2 h at 1800×g to release the latent TGF-β1 into plasma and then activated through acidification with 1 M HCl at 4 °C for 60 min and neutralised with 1 M NaOH. Cytokine concentrations of test samples were extrapolated from the optical density (OD)-concentration standard curve obtained from serial dilution of recombinant cytokine standards using GraphPad Prism 6.0 statistical package.The selected cytokines  have been associated with the pathogenesis, disease severity and survival in either HAT –9 or malAll numerical variables were summarized using mean and standard deviation of mean. All comparisons of categorical variables and cytokine data were analyzed using Graphpad Prism 6.0 statistical packages. Comparison of categorical variables was performed using Chi-square test at P < 0.05 significance. Deviation from normality was tested using D’Agostino and Pearson omnibus normality test of plasma cytokine data. Cytokine data was presented as medians since it did not present a normal distribution. Comparison of cytokine levels between the different groups of participants was done using nonparametric tests; Mann–Whitney U and Kruskal–Wallis tests followed by Dunn’s multiple comparisons test at a significant level . Correlation analysis between cytokines was performed using bivariate non-parametric Spearman’s correlation rank test at a significant level .P. falciparum malaria. Therefore, the prevalence of malaria among HAT cases was 46.8% (15/32)  to female (42) was approximately 1:1 with an average age of 28.8 ± 14.1 years. Participants 18 years and above were significantly more infected than those of six and below 18 years (P < 0.05). There was no significant association between sex and infection status (P > 0.05). There were 14 early and 18 late stage HAT cases in this study with no significant difference (P > 0.05) . Among the participants; malaria mono-infected cases were 31.1% (28/90), 18.9% (17/90) were HAT mono-infected, while 16.7% (15/90) had HAT and malaria co-infection and 33.3% (30/90) were healthy controls. Among the 32 HAT cases, 15 were positive for The assay was carried out for five plasma cytokines  in HAT and/or malaria cases and healthy controls. The detection limits for the cytokine assays  were 8.3, 9.1, 3.6, 4.2, and 522.2 pg/ml respectively, calculated according to Armbruster and Pry . The plaThe co-infected cases expressed a significantly higher plasma level of IFN-γ, IL-6, and IL-10  than malaria , but no significant difference with HAT   and IL-6 with IL-10 (Spearman r = 0.557) among co-infection. There was no significant correlation observed between the other cytokines amongst co-infection (P > 0.05) , which is higher than 28.9% (70/242) earlier reported in Uganda [Malaria has been reported to be prevalent among HAT cases , 34, 35.6.8% 15/3, which in Uganda .T. b. rhodesiense and/or P. falciparum infections are associated with up-regulated cytokine responses. A significantly higher level of IFN-γ, TNF-α, IL-6, and IL-10 were expressed by co-infected cases than malaria cases, except for TGF-β which did not vary significantly. Co-existence of HAT and malaria parasites in cases intensely changed their immunological cytokine response relative to malaria. Therefore, T. b. rhodesiense co-infection led to a synergistic cytokine response relative to P. falciparum infection in host. No significant difference was expressed between co-infected and HAT cases for cytokine IFN-γ, IL-6, IL-10 and TGF-β levels, apart from TNF-α levels which were significantly higher in co-infected than mono-infected HAT cases. This indicates that P. falciparum infection modified the TNF-α level synergistically in T. b. rhodesiense infected host. The HAT cases expressed significantly higher plasma IFN-γ, IL-6 and IL-10 levels than malaria cases in this study. These findings suggest that T. b. rhodesiense significantly induces the host immune cytokine network more than P. falciparum infection. No significant difference between HAT and malaria mono-infected cases was noted for TNF-α and TGF-β levels. However, no study explores the cytokine profile concentration of T. b. rhodesiense relative to P. falciparum in naturally infected humans.Cases with HAT and/or malaria parasites responded with an overall increased secretion of plasma cytokines than healthy controls. Therefore T. b. rhodesiense HAT [P. falciparum malaria [P. vivax malaria than healthy individuals. For trypanosome infection, it has been described that IFN-γ is associated with the penetration of the blood brain barrier in animal models [T. brucei infections [T. b. rhodesiense infection as compared to the P. falciparum malaria cases. The main pro-inflammatory IFN-γ response induced by T. b. rhodesiense from natural killer (NK) and T cells may partially act as a pre-regulating activation of macrophages against plasmodium infection. This establishes protective adaptive immunity through TNF-α and nitric oxide impairment of hepatic stage of plasmodium [Cases with HAT and/or malaria parasites responded with an overall increase in secretion of pro-inflammatory cytokines (IFN-γ and TNF-α), which partially suggested the effect induced by either HAT or malaria mono-infections. This is in agreement with previous studies described in separate cases of ense HAT –9 or P.  malaria , 36. But malaria , reportel models . The TNFfections , 40, simfections , 41. Thefections , 8. Prevfections  found hifections . In eastfections –45, bloofections , exo-eryfections , 47, resfections , and acufections , 48. Secfections . The curasmodium , 49.T. b. gambiense HAT and P. falciparum co-infection than healthy controls. Consequently, IL-10 plasma level of HAT was not significantly affected by P. falciparum infection [P. falciparum malaria [T. brucei infection [T. brucei [T. b. rhodesiense induces these anti-inflammatory cytokines that may regulate the pro-inflammatory cytokine response to P. falciparum during co-infection in host. The elevated TGF-β level in HAT over healthy controls was in agreement with a study by Kato et al. [The elevation of pro-inflammatory cytokine response is regulated by anti-inflammatory cytokines. Cases with HAT and/or malaria parasites responded with an overall increased secretion of anti-inflammatory cytokines . Similarly, IL-6 and IL-10 increased levels agrees with other studies in HAT , 42 or mnfection , in agrenfection . Also knnfection , IL-10 a malaria  and T. bnfection , 41. Thenfection . Both IL. brucei  showed to et al. . At higho et al. . For malo et al. , 52 as oo et al. . The supo et al. . This suo et al. .T. b. rhodesiense and/or P. falciparum infections on immune cytokine response of the host. This also limited the investigation of the cytokine levels from HAT and/or malaria infections matched by sex and age. Some pathogens for other infections might have been missed as all participants were subjected only to the routine laboratory diagnosis at the hospital associated with low sensitivity. This may explain the presence of outliers in the cytokine levels. Many HAT cases report to the hospital having been medicated or self-medicated on suspicion of malaria when it is actually HAT, since clinical symptoms are quite similar even to other disease conditions. Nevertheless, the study provides information on the immune cytokine response of T. b. rhodesiense and/or P. falciparum infections. Follow up studies on plasma and CSF cytokine profiles of HAT and malaria co-infections which are stratified would be appropriate. For example, cases with mild, severe, and cerebral malaria or symptomatic and asymptomatic malaria with either early or late stage HAT. This will also harmonise existing controversies about cytokines as potential disease biomarkers. Further studies about antibodies, chemokine and other biomarkers would be very important.The sample size was small and may not be adequate to identify the effect of T. b. rhodesiense infection significantly induced host immune cytokine response of IFN-γ, IL-6, and IL-10 more than P. falciparum infection. Malaria and HAT co-infection modified synergistically the pro-inflammatory  and anti-inflammatory  cytokine response than P. falciparum mono-infections. The TNF-α plasma level partially indicated the effect induced by mono-infections of HAT and malaria or from a synergistic effect of the co-infection which indicates a protective immunity against the parasites. The up-regulated cytokines may have adverse effects on pathogenesis, prognosis and resolution of the infections.The prevalence of malaria among HAT cases was high (46.8%). The HAT and/or malaria infection is associated with up-regulated cytokine responses. The


Malaria continued to be the major public health concern in sub-Sahara Africa, thus for better planning of control activities, periodic surveillance of both clinical and asymptomatic cases remains important. However, the usability of routinely collected malaria data in Kenyan hospitals as a predictor of the asymptomatic malaria infection in the community amidst rapid infection resurgence or reduction in different areas of disease endemicities remains widely unstudied. This study was therefore aimed to evaluate the utility of passive surveillance of malaria in health facilities as a proxy of infection transmission of the surrounding community in different transmission intensities.Prospective multiple cross-sectional surveys were done in three villages in western Kenya. Monthly asymptomatic malaria positivity among school children, number of outpatient (OPD) confirmed malaria cases and abundancy of indoor resting malaria vectors were surveyed from June 2015 to August 2016. Community surveys on antimalarial drug use among adults and children were also done. Detection of malaria parasitaemia was done using thick and thin Giemsa stained blood slide microscopy for both clinical and school participants. A questionnaire was used to collect information on self-use of antimalarial drugs from randomly selected households.p = 0.001) and at Iguhu (Moderate transmission) . The high transmission setting (Kombewa) showed no significant correlation .The overall OPD blood slide positivity from all study sites was 26.6% (95%CI 26.2–27.0) and highest being among the 5–14 years (41.2% (95% CI 40.1–42.3). Asymptomatic malaria positivity among the school children were 6.4% (95%CI 5.3–7.5) and 38.3% (95%CI 36.1–40.5) in low and high transmission settings respectively. A strong correlation between overall monthly OPD positivity and the school age children positivity was evident at Marani (low transmission)  contains supplementary material, which is available to authorized users. Malaria infection in tropical and sub-tropical countries continued to be a major public health concern . In 2015Monitoring of malaria transmission dynamics require both local and nationwide surveillance mechanisms. Malaria control programs conduct regular indicator surveys for nationwide planning and decision making , 11. ThePlasmodium falciparum is the most prevalent malaria parasite transmitted mainly by Anopheles gambiae s.s., An. arabiensis and An. Funestus [This study was performed in three villages with different altitude, topography and malaria transmission intensity Fig. . The firFunestus .Fig. 1MaThis study involved multiple cross-sectional surveys conducted monthly from June 2015 to August 2016. Surveillance of asymptomatic malaria among primary school children, OPD confirmed malaria and indoor resting malaria vectors for 15 observations. Moreover community based cross-sectional surveys on the use and source of anti-malarial were done in June and December 2016. A snap shot additional study to determine accuracy of blood slide malaria microscopy examination was conducted at Iguhu hospital (moderate transmission site).. A total of 17,231, 20,985 and 10,676 suspected patients were tested for malaria at Marani, Iguhu and Kombewa hospitals respectively.All patients attended the OPD and suspected to have malaria were examined by Giemsa stained blood slides and their results were recorded on daily basis on the specific registry as from June 2015 to August 2016. A total of 48,892 malaria suspected patients were examined by Giemsa stained blood slide in the three hospitalsSurveillance of asymptomatic infection in schools required minimum of 150 school age children per month per study site. The sample size was based on the local prevalence asymptomatic malaria infection ranging from 6.2 to 47.1% . A totalMultiple cross-sectional surveys were conducted among school children attending nine primary schools in the study areas. A finger prick blood sample was obtained from each school child, thick and thin blood smears prepared, stained with Giemsa and examined using light microscope. Sum of 1795, 1972 and 2430 blood slides were collected and examined from Kombewa, Marani and Iguhu respectively as from June 2015 to August 2016.A total of 30 randomly selected houses in each village were used to collect vectors per site per month as from June 2015 to August 2016. Pyrethrum Spray Catch (PSC) method as described elsewhere  was usedThe survey involved 756 and 1500 randomly selected participants from households who had used anti-malarial drugs prior to the interview in June 2016 and December 2016 respectively. Using a structured questionnaire, information on the use of anti-malarial drug within 2 weeks prior to the interview and type of dispensing unit were inquired.0 C. Malaria suspected found with parasitemia were termed as clinical malaria cases. Malaria parasites  and gametocytes were counted at each 200 white cell fields. Technicians who re-examined the blood slides were blinded of the hospital microscopy results and of each other’s results.About 200 Giemsa stained thin and thick smear blood sides among malaria suspected patients from a hospital located in mesoendemic transmission setting were collected and re-examined by two qualified laboratory technicians at the Kenya Medical Research Institute in Kisumu. Malaria suspected patients were those with history of chills, body malaise, joint pain and other symptoms together with fever above 37.5p-value was < 0.05. Strong correlation and modest correction were defined when the p-value was ≤0.001 and < 0.01 respectively. Graphic presentation of data, display of trend lines as well as predictor equations were computed. The correlation analyses were done by using SPSS® version 17 and determination of linearity computed using Microsoft excel® window 10.The monthly and overall prevalence of malaria parasites per site among the school children and among the symptomatic patients were computed. The mean number of female Anopheline mosquitoes identified per house per night were calculated and compared between the villages. Variations of vector abundance was compared between the study sites using one way analysis of variance (ANOVA) and Tukey-Kramer post-hoc test. The frequency of household anti-malarial drug use per age group and per site was computed. Cohen’s kappa measurement of agreement was used to determine blood slide results concordance between hospital results and those re-examined at the research centre. Relationship between monthly asymptomatic malaria positivity among school children and clinical blood slide positivity was determined by using Spearman’s correlation test. Spearman’s correlation between abundance of indoor resting female Anopheline mosquitoes collected by PSC and the different clinical age groups malaria positivity was also done. Regression analysis between the abundance of indoor vectors and the clinical blood slide positivity rate was done to determine strength of association. Spearman’s correlation coefficient (rho) and regression analysis were used to determine strength of associations. The association strength was considered significant when the The overall blood slide positivity (for 15 month observation) for all study sites was 26.6% (95%CI 26.2–27.0) and 27.4% (95%CI 26.9–27.9) for the first 12 months. The highest OPD malaria positivity rate was observed among children aged 5–14, with 45.3% (95% CI 42.8–47.8) and 44.5% (95%CI 42.8–46.2) at Kombewa and Marani hospital respectively (Table The blood slide positivity was consistently highest at the high transmission study area (Kombewa) with an overall prevalence of 38.3% (95%CI 36.1–40.5) and lowest at the low transmission setting (Marani) with 6.4% (95%CI 5.3–7.5) . Post hoc Tukey-Kramer test however revealed existence of a significant variation of indoor vectors only between Marani and Kombewa  were 0.59 ± 0.22, 0.68 ± 0.08 and 1.01 ± 0.28 female anopheline mosquitoes per house per night at Marani, Iguhu and Kombewa respectively. Moreover, for the first 12 months the mean densities were 0.52 ± 28, 0.63 ± 0.15 and 0.98 ± 0.33 female anopheline mosquitoes per house per night at Marani, Iguhu and Kombewa respectively. There was significant variation of the monthly abundance of indoor vectors between the study areas (F = 3.66p = 0.001) and moderate at Iguhu . However, the high transmission study site (Kombewa) had no significant correlation between overall OPD positivity and asymptomatic infection positivity among school children   than Iguhu  but showed no correlation at Kombewa   nor the ≥15 OPD positivity   Fig. , b, c. R76) Fig. , e, f. Fely Fig. , d. Scho16) Fig. , e. At Kely Fig. , f.Fig. p = 0.08)  and OPD malaria positivity  Fig. .Fig. 4CoAbout half of respondents from the high transmission study site (Kombewa), three quarters from the moderate transmission (Iguhu) and as low as 10% from the low transmission setting (Marani) practiced self-malaria treatment Table .Table 2Hp = 0.000). Therefore, a substantial agreement between the hospital blood slide examination results and that at the research Centre observed.A selected health facility located in the moderate malaria transmission setting revealed an agreement of 88% (175/199) of all examined blood slides between the hospital results and those by two microscopy technicians at the Research Centre  to highest at Kombewa (hyperendemic area). Kombewa registered highest prevalence of asymptomatic infection (55%) in June 2015 and lowest in May 2016 after bed nets mass distribution in December 2015. Elsewhere, increasing malaria susceptibility among the school children has been also reported , 27, 28.This study found a significant correlation between asymptomatic malaria positivity among the children and OPD positivity of all age groups at the low transmission setting. Whereas at the moderate transmission study site, the correlation was only observed with the all age groups OPD positivity and < 5 children but not with ≥15 years age group. The correlation between school children asymptomatic malaria positivity and OPD positivity rates for all age groups at the low transmission setting occurred due to fact that clinical malaria susceptibility was higher for all age groups in low transmission settings. Therefore, likelihood of seeking for medical attention is higher as compared to the high transmission settings . Indeed,In Uganda, a non-linear relationship was observed between clinical malaria attack rate and community asymptomatic malaria positivity in a defined population . In LakeImmunoepidemiology of malaria indicated that, population in high transmission setting carry high burden of asymptomatic malaria and only a proportion of the population develops clinical disease, mainly children under-five because of their high exposure to the parasites compared to population in epidemic prone areas , 16. In The population of indoor resting malaria vector collected by PSC was not correlated with the OPD malaria blood slide positivity. This could be due to the reported increasing outdoor resting behaviour, outdoor biting behaviour and changing host preference of previously known anthropophilic vectors –34. StudThis study however had some limitation including lack of a defined catchment area of the selected hospitals. Possibly some patients came from different areas with different infection transmission intensity and thus might have affected the outcome of interest especially in relatively less populated areas. The correction of variables could have been affected by accuracy of microscopic examinations. However, the accuracy of malaria diagnosis has greatly improved over the recent years compared to the past . This haThe hospital based malaria surveillance in low transmission settings strongly correlated with the asymptomatic infection transmission dynamics among school children. Only the ≤5 children OPD positivity correlated significantly with the asymptomatic infection among school age living in mesoendemic study area. No correlation observed between hospital OPD malaria positivity and asymptomatic malaria positivity at the endemic high transmission study site. The use of hospital malaria data in endemic setting as a reflection of the surrounding catchment area may be unreliable despite provision of free anti-malarial drugs at health facilities. Passive and active surveillance of clinical and asymptomatic malaria should be emphasized in endemic settings of Kenya especially in these moments when there are reports of changing infection transmission risks.Additional file 1:Monthly malaria outpatient positivity rates of the three study sites that are located in areas with different malaria infection transmission intensity in western Kenya from June 2015 to August 2018 (PPTX 306 kb).Additional file 2:Dynamics of asymptomatic malaria positivity and abundance of indoor resting malaria vectors from three study sites that are located in areas with different malaria infection transmission intensity in western Kenya from June 2015 to August 2016 (PPTX 243 kb).


Plasmodium falciparum cases imported from Southeast Asia has frequently reported especially in the China-Myanmar border (CMB) area. Though, information is scant on P. falciparum genetic variability in this area.China has made progress in malaria control and aims to eliminate malaria nationwide, but implementing effective interventions along the border regions remain a huge task. The P. falciparum isolates genome sequence of six clinical isolates in the CMB area. Furthermore, we estimated the nucleotide diversity, Watterson’s estimator and Tajima’s D value for the whole genome mutation rate in slide window.This study reported P. falciparum clustered generally according to their geographic origin. A total of 91 genes were identified as positive selection with Ka/Ks ratio significantly higher than 1, and most of them were multigene families encoding variant surface antigens (VSAs) such as var, rif and stevor. The enrichment of the positive selection on VSA genes implied that the environment complexity subjected CMB’s P. falciparum to more pressure for survival.Our data were aligned onto 96.05–98.61% of the reference 3D7 genome in high fold coverages. Principal component analysis result showed that P. falciparum to fit the host immune system well and aggravate the difficulty of treatment. Meanwhile, results obtained from this study will provide the fundamental basis for P. falciparum population genomic research in CMB area.Our research suggests that greater genetic diversity in CMB area and the positive selection signals in VSA genes, which allow The online version of this article (10.1186/s40249-018-0493-5) contains supplementary material, which is available to authorized users. Please see Additional file 2 and has a population of about 278 million. Nearly 70% of the local population were at risk of malaria infection [Plasmodium falciparum malaria also fuel concerns of re-introduction, and this had been shown to be partly attributed to increases in the intrinsic potential for malaria transmission [Malaria was one of the most prevalent parasitic diseases in the Greater Mekong sub-region (GMS) historically. The GMS countries are Cambodia, China, Laos, Myanmar, Thailand and Vietnam, bounded together by the Mekong River. The area considered covers 2.4 million kmnfection . Howevernfection , while tnfection . It shownfection . In addiP. falciparum isolates whole genome had been sequenced in the past, most of them were meant to identify the artemisinin resistance loci [P. falciparum isolates from CMB to provide information and biological insights that would accelerate the pursuit of effective malaria control in CMB area.Whole genome studies provide us with the opportunity to develop new control methods, including new drugs and vaccines, improved diagnostics and effective vector control techniques . Severalnce loci , 11. TheP. falciparum in the CMB area. Genomic DNA was extracted from the whole blood of malaria cases that were microscopically positive for P. falciparum and also confirmed by PCR for single infection. Samples with high parasite density (40 000–260 000 parasites/μl) were selected to ensure the integrity of sequencing. Among hundreds of samples that were collected from CMB area since 2011, only 6 sequenced results were good enough for this study and also provided enough coverage. Genomic DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit , and sheared into 350 bp fragments using Covaris instrument. The fragmented DNA molecules were used to construct the sequencing libraries with Illumina TruSeq DNA LT Sample Prep Kit , and the direct sequencing approach was used in our previous study [http://plasmodb.org) was downloaded [High importation risk from Myanmar and wide distribution of malaria vectors in the CMB region sustain risk for secondary infections among local populations. Tengchong County experienced a decreasing malaria prevalence period, and less than 100 confirmed malaria cases were recorded per year from 2011 . Case maus study . We filtus study . In addiwnloaded .P. falciparum 3D7 genome using Burrows-Wheeler Aligner and Sequence Alignment/Map (SAMtools-1.3) [P. falciparum isolates collected worldwide [ω) for the whole genome mutation rate in 4 kb sliding window and 2 kb step across each chromosome were estimated in ARLEQUIN-Ver3.5 [Sequencing reads were mapped from all the six samples of ols-1.3) , which iols-1.3) . We perforldwide , 19. TheN-Ver3.5 . Also, tN-Ver3.5 , 22 in KN-Ver3.5 , which hP. falciparum clustered generally according to their geographic origin and the Asian samples exhibited greater genetic diversity than African. The second and third principal components (F2 and F3) defined a distinct South-American cluster and distinguished the African samples better  and generated 78 to 248 M paired-end reads of 150 bp. Illumina sequencing reads have been submitted to the NCBI Short Read Archive (Bio-Project no. PRJNA393218). A variable proportion of reads (4.08–27.08%) from all the isolates were mapped to the reference, and aligned onto 96% of the reference genome in high fold coverage (20.57–121.78×). A total of 369 700 SNPs were captured and only 18 953 common loci were available for analysis after quality filtering Table . The majter Fig. . It is iω to be 0.0287, and genetic diversity was lower in exonic regions but higher in intronic and intergenic regions. As expected, the Watterson’s estimator (ω) is higher than global samples  [On genome scale, we estimated Oceania) . Mean paentclass1pt{minimavar genes, the repetitive interspersed family (rif) and the subtelomeric variant open reading frame (stevor) was found among these 91 positive selection genes. The variant antigens P. falciparum erythrocyte membrane protein 1 (PfEMP1) and repetitive interspersed families of polypeptides (RIFINs) are adhesins implicated in severe P. falciparum malaria, and STEVORs are erythrocyte binding protein that mediates merozoite invasion and resetting [Of the 5566 genes analyzed, 3485 have valid Ka/Ks value, while only 91 genes were identified as positive selection with Ka/Ks ratio significantly higher than 1 Fig. . Howeveresetting –28.P. falciparum isolates is higher than the global samples. The genetic structure of CMB parasites was similar with other Asian countries despite the vector species abundance generally enhance the environmental compatibility of parasites [P. falciparum in Africa [P. falciparum in CMB area and play an important role in the process of diversification.Recent studies in Myanmar revealed that malaria incidence and mortalities showed significant decreasing trend even in artemisinin-resistant areas . The effarasites –35. Meann Africa . The gren Africa  and alsoP. falciparum malaria pathogenesis and in immune evasion by the malaria parasite. Studies in Africa have shown that severe malaria is associated with the ability of erythrocytes infected with the parasite to bind uninfected erythrocytes and form rosettes [P. falciparum to fit the host immune system better. For example, STEVOR plays a role in creating antigenic diversity of schizont stage parasites, thereby adding additional complexity to the immunogenic properties of the infected red blood cell [rif genes family which contribute to the rosetting of P. falciparum mediated by blood antigen and help to express clonally variant antigens at the surface of the infected erythrocyte. Among all the 158 rif genes downloaded from plasmoDB, 66 genes have got valid value higher than 1, with 12 of them under significant positive selection. The signatures of positive selection suggested that the local environment complexity subjects CMB’s P. falciparum to more pressure for survival [On the other, it is advantageous to apply positive selection test where environment factors apply consistent pressure over generations in favor of specific beneficial trait. Early analyses identified loci showing evidence of recent positive directional selection and balancing selection confirmed that antimalarial drugs and host immunity have been major selective agents . In our rosettes . It is wood cell . In our survival , 27.P. falciparum in the CMB area. We found greater genetic diversity in CMB area and the positive selection signals in variant surface antigens genes. As gene flow is relatively unrestricted in East-south Asia, highly recombining populations of the P. falciparum are closely related, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Findings from this study provide more insight on the current epidemiological scenario of malaria in China. Lastly, our results will help deepen our understanding of P. falciparum evolution, and also provide the fundamental basis for further studies.Our study assessed the genome sequences of six clinical isolates of Additional file 1:Multilingual abstracts in the five official working languages of the United Nations. (PDF 899 kb)


Plasmodium parasites that are responsible for this disease is required to guide the most effective distribution of control measures. Serological surveillance will be particularly important in areas of low or periodic transmission because patient antibody responses can provide a measure of historical exposure. While methods for detecting host antibody responses to Plasmodium falciparum and Plasmodium vivax are well established, development of serological assays for Plasmodium knowlesi, Plasmodium ovale and Plasmodium malariae have been inhibited by a lack of immunodiagnostic candidates due to the limited availability of genomic information.Malaria remains a global health problem and accurate surveillance of P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.Using the recently completed genome sequences from Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.Using sera from European travellers with diagnosed Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.Using the recent availability of genome sequences to all human-infective  Plasmodium and several species are known to regularly infect humans. The vast majority of deaths occur in sub-Saharan Africa and are caused by Plasmodium falciparum, whereas outside of Africa, Plasmodium vivax is responsible for over half of all malaria infections leading to significant morbidity and mortality [Plasmodium species, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi both in terms of their global distribution and clinical impact. Plasmodium knowlesi, a parasite typically found in macaques, is a significant cause of human malaria in Southeast Asia, causing a spectrum of disease ranging from mild to fatal infections [P. knowlesi malaria with over 6700 cases reported in the last 2 years compared to only 85 cases of indigenous human malaria .Malaria is an infectious disease that remains a global health problem causing an estimated 219 million clinical cases resulting in 435,000 deaths in 2017 [Plasmodium infections and epidemiological surveillance is important for guiding the distribution of resources into intervention measures and establishing their clinical impact over time [Plasmodium infections include microscopy, rapid diagnostic tests (RDTs) and PCR-based approaches, each differing in their sensitivity, infrastructure requirements, and ability to diagnose the different species. Serological assays can provide a historical record of infection and because of the specificity of antibody-antigen binding, could also potentially discriminate between different Plasmodium spp. infections. Host antibodies appear rapidly after initial infection and can persist for months and even years after the parasites have been cleared [Diagnosis of ver time . Methods cleared , 6. Sero cleared –10, and  cleared , 12. It  cleared .Plasmodium proteins in a soluble recombinant form has proved challenging, perhaps because of the high A:T content of the genome and lack of recognisable protein domains in many Plasmodium proteins [Plasmodium proteins was developed that retained many of their biochemical functions [P. falciparum [P. vivax [Plasmodium. Here, the recent availability of high-quality genome sequences from the three other human-infective Plasmodium parasites: P. knowlesi, P. ovale and P. malariae were used to extend the available panel of proteins and were tested for reactivity to sera from individuals infected with different Plasmodium parasites.Many antibodies recognise epitopes that are only formed in the context of an antigen in its native conformation . To deteproteins . This prunctions . Centralunctions . Previoulciparum , 17 and P. vivax . Using sP. vivax , 12. ExpP. knowlesi [n = 81). Of these, 53 were from travellers of European origin and 28 from travellers born in malaria-endemic regions as follows: Angola , Burundi (1), Cameroon (1), Democratic Republic of the Congo (1), Eritrea (4), Ethiopia (1), The Gambia (1), India (3), Ivory Coast (2), Kenya (5), Pakistan (1), Thailand (1), and Uganda (4). Plasma from an endemic region with diagnosed infections were from a longitudinally followed population from Nyamisati, in the Rufiji region, Tanzania (n = 21) in 1994 when transmission was high [n = 28). Ethical approval was granted by the Ethical Review Board of the National Institute for Medical Research in Tanzania, and the Regional Ethical Review Board in Stockholm, Sweden . Serum samples from adult malaria patients with PCR-confirmed P. knowlesi mono-infection were collected at Kapit Hospital in Malaysian Borneo (n = 50) and from uninfected adult controls from Kapit Division (n = 66) after informed consent had been obtained. Approval to conduct this study was obtained from the Medical Research and Ethics Committee of the Ministry of Health, Malaysia and the Medical Ethics Committee, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak.Collection of sera from Malawian adults that were previously used to determine its effectiveness as an adjunct therapy to treat cerebral malaria was approved by the National Health Sciences Research Committee of Malawi . Plasma knowlesi  after reania n =  in 1994 P. knowlesi, P. malariae and P. ovale were identified from their respective genome sequences [P. ovale, proteins from Plasmodium ovale curtisi were selected since the manual gene annotation of this genome resulted in complete open reading frames compared to the draft genome available for Plasmodium ovale wallikeri [P. malariae P38, this analysis helped improve automated gene prediction by identifying missing signal peptides. Based on these predictions, the ectodomain regions were determined by removing signal sequences and transmembrane domains. All potential N-linked glycosylation sites were systematically mutated by substituting the serine/threonine in the context of an N-linked glycosylation sequon for alanine to prevent inappropriate glycosylation when expressed in mammalian cells as described previously [5 cells mL−1 (HEK293E) or 1.0 × 106 cells mL−1 (HEK293-6E). Cells are routinely cultured in volumes of 50 mL in Freestyle293 media following the manufacturer’s recommendations; for HEK293E cells, the media is supplemented with 1% FCS. To ensure efficient biotinylation, the cell culture media used to produce bait proteins with D-biotin to a final concentration of 100 μM. The following day, cells were transfected as described [Escherichia coli BirA enzyme (Addgene plasmid number 64395), at a 10:1 ratio, essentially as described [g for 20 min followed by filtration of the supernatant through a 0.22 μM filter. His-tagged proteins were purified from spent tissue culture supernatant with either a 1 mL HisTrap HP column  using an ÄKTAxpress or ÄKTApure instrument  or a bespoke purification instrument for parallel protein purification [The orthologues of immunoreactive blood-stage antigens from equences –24. For allikeri , 26. In eviously . All seqeviously  and a raeviously . Proteineviously  and HEK2eviously , essentieviously . In brieescribed  using eiescribed . Culturefication  in a HisTo determine protein integrity, 10 µL of transfection supernatant was resolved by SDS-PAGE using Novex NuPAGE 4–12% Bis Tris precast gels (Life Technologies) under reducing conditions, transferred to nitrocellulose membrane (Invitrogen), blocked with 2% BSA in phosphate-buffered saline (PBS)/0.1% Tween-20 (PBST), and probed with 0.02 µg/mL of streptavidin-HRP (Jackson Immunoresearch) diluted in PBS-2% BSA. After washing, biotinylated proteins were detected by addition of SuperSignal West Pico Chemiluminescent substrate (PIERCE) and developed on photographic film .−1 and used 1:1000. Plates were washed 3× in PBS-T before incubating with 1:10,000 dilution of peroxidase-conjugated AffiniPure goat anti-human IgA + IgG + IgM (H + L) (Jackson ImmunoResearch) in PBS-2%BSA for 1 h. Plates were washed in PBS-T and the HRP substrate ABTS (KPL) was added and absorption at 405 nm determined using an automated plate reader .ELISAs were performed by capturing biotinylated bait proteins into individual wells of streptavidin-coated 384-well plates (Greiner Bio-one). Plates were washed for 30 min with 50 μL PBS-T (0.2% Tween) and blocked with PBS-2% BSA for a minimum of 3 h. 20 μL of a bait protein diluted in PBS-2% BSA at a concentration previously determined as the amount required to saturate the biotin binding capacity of the well were added in triplicate and incubated for at least 16 h at 4 °C. Antisera were centrifuged at 13,000 rpm for a minimum of 1 h at 4 °C, diluted in PBS-2% BSA and incubated with rotation for at least 16 h at 4 °C before adding to the antigen-coated plates for 1 h. Serum dilutions used were: native Tanzanians 1:5000, native Malaysians 1:1000, imported malaria 1:500–1:1000, European travellers 1:100–1:500, and Malawian pooled sera resuspended to 20 mg mLPfHPzz, PvHPzz, PmP41, PmMSP5, PvMSP5). Logistic regression models to predict exposure were computed using the generalized linear models “glm” function in R. One model was fitted per species, making a total of five classifiers. For every model, each patient was designated as either: diagnosed for that particular species (either by qPCR or microscopy); a negative control from a non-endemic region with no infection history; or, as ambiguous, if the patient lived in an endemic region, but was not diagnosed for that particular species. Ambiguous cases were omitted in model training, since they could not be classified reliably. Once labelled, the data sets were randomly divided approximately 50:50 into a testing set and training set. Logistic regression models were fit to the training set, then evaluated on the testing sets. Receiver operating characteristic (ROC) curves were calculated using the model predictions on the testing set and their known diagnosed labels. Curves were coloured by an arbitrary cost function, summing the number of false positives and false negatives at each threshold to highlight the relative optima [Data are available in Additional file e optima , 33. Thee optima . To detePlasmodium parasites that infect humans, 12 proteins from P. falciparum and 10 proteins from P. vivax which were previously shown to be highly immunoreactive to sera from patients living in endemic regions were selected [P. knowlesi [, P. ovale and P. malariae [P. knowlesi and 2 paralogues from both the MSP3 and MSP7 multigene family from P. ovale and P. malariae were also selected since orthologues of these proteins are highly immunoreactive in P. falciparum , P. vivax (n = 17), but access to serum samples of travellers’ malaria with diagnosed infections for the rarer parasites was limited: P. ovale (n = 7), P. malariae (n = 3). For P. knowlesi, responses in the 10 Malaysian patients with PCR-confirmed single P. knowlesi infections were used. These patient sera were screened against the panel of all 22 antigens from five Plasmodium species. Two patients exhibited a broad reactivity to antigens from two species: a Swedish traveller diagnosed with P. vivax but whose serum exhibited broad reactivity across antigens from three Plasmodium species  and one of the Malaysian patients diagnosed with P. knowlesi but whose serum reacted strongly with four out of six P. vivax antigens; this patient was subsequently identified as a migrant worker from Indonesia where P. vivax is endemic. Because it is likely that these patients had prior exposure to another Plasmodium species other than their diagnosed infection, these patients were removed from this analysis. The pairwise correlation of the responses to each of the antigens in the panel was examined in the remaining patients , and yet patient responses showed little evidence of cross-reaction , and endemic malaria from a cohort of Tanzanian adults (n = 21) were tested. For P. knowlesi, sera from the 10 patients diagnosed with P. knowlesi infections from Kapit Hospital in Malaysian Borneo were used. The antibody responses between the different sources were investigated by segregating the samples into three different categories: travellers of European origin (n = 53), travellers with an origin in a malaria-endemic area (n = 28), and individuals residing in an endemic area (n = 31). The responses to each antigen corresponding to the species diagnosis was plotted within each category, and while differences between the different patient groupings were observed on a per antigen basis . The relatively small number of available diagnosed samples for the rarer parasites P. ovale (n = 18) and P. malariae (n = 17), limited the performance of their classifiers, as well as in the case of P. malariae, the reliance on just a single antigen (PmMSP10) after removing those that showed evidence of cross-reactivity.The performance of each diagnostic model was evaluated, and the random sampling procedure into training and testing sets was iteratively repeated. To quantify the models’ performance, the averaged receiver operating characteristic (ROC) curves for predicting exposure to each species Fig. a was firPlasmodium species , P. knowlesi infections could be identified with high confidence (AUC > 91%)  Fig. a. If a t 3% Fig. b.Fig. 4APlasmodium parasites although few have attempted to distinguish infections by the different species [P. ovale and P. malariae, making the cloning and expression of many candidate proteins from these species difficult. Recent sequencing of P. ovale and P. malariae [Plasmodium parasites that infect humans, together with the approach of expressing extracellular parasite proteins in a functionally active form using a mammalian expression system. After removing those proteins which showed evidence of cross-reactive responses, it was found that immunoreactivity to the antigens from P. ovale, , P. malariae (MSP10) and P. knowlesi , together with logistic regression modelling had moderate power to predict prior exposure to these species, and performed especially well for P. knowlesi.Malaria continues to be a significant global health problem although cases have dropped in recent years due to the deployment of effective intervention measures . Spurred species . This hamalariae , 24 has Plasmodium proteins is known to be technically challenging, possibly due to the unusual codon bias or highly repetitive amino acid sequences often found in Plasmodium proteins [Escherichia coli) or cell-free systems, such as wheat germ extracts [Plasmodium proteins, it was previously shown that these recombinant proteins can retain conformational epitopes, and shown that for the majority of antigens, a large fraction of the immunoreactivity to sera from Plasmodium-exposed patients is heat labile, demonstrating the benefit of using proteins that retain native folding for serological assays [Plasmodium blood-stage proteins expressed using this approach have been useful in sero-epidemiological studies for P. falciparum [P. vivax [P. falciparum and P. vivax orthologues of the HPzz protein between patients diagnosed with these parasites, despite there being only ~ 40% amino acid shared sequence identity. By contrast, very little cross-reactivity was observed in patient serological responses between the P. vivax and P. knowlesi P12 orthologues which are relatively well conserved, sharing > 70% amino acid sequence identity. One likely possibility is that the informative epitopes for these proteins are mainly composed of the precise arrangement of solvent-exposed amino acids, making overall linear sequence identity less important, whereas repeats of just a few amino acids, for example the repetitive “NANP” motifs in the circumsporozoite protein can be highly immunogenic [Expressing proteins , 38. Mosextracts –41. Whilextracts , 42. Whiextracts . When usl assays . Plasmodlciparum , 44 and unogenic .PkMSP10, PkP12, PkP38), was found to accurately detect exposure to P. knowlesi using a serological assay. This compares well with a recent study where the authors also selected candidates that were orthologous to known immunoreactive proteins in P. falciparum, and selected four antigens that were associated with P. knowlesi exposure [P. falciparum and P. vivax as the major cause of malaria [A panel of three recombinant antigens  Immunoreactivity to antigens for patients diagnosed with an infection from the corresponding species, grouped by patient background. (b) Average immunoreactivity to antigens for each species in patients diagnosed for that species. (c) Background immunoreactivity to antigens for patients not diagnosed with the corresponding Plasmodium species.


Malaria and HIV/AIDS infections are among the major public health concerns in sub-Saharan Africa, where they are associated with high morbidity and mortality. Recent findings indicate that individual people living with HIV/AIDS (PLWHA) with lower levels of CD4 T-cell count below 200/mm The study was conducted to assess the pattern of malaria parasite density at different levels of CD4 T-cells among people living with HIV/AIDS in Western part of Kenya. A randomized antimalarial treatment study among 126 people living with HIV/AIDS was conducted at Chulaimbo Sub-County Hospital, Western Kenya. All the participants enrolled into the study had their blood samples assessed for malaria parasite densities before commencement of antimalarial therapy and the results correlated with their CD4 T-cells levels obtained from their respective files.μL of blood, median was 17,720, and mode was 4,000. Male participants had a higher geometrical mean parasite density  compared to females'  (p = 0.03). Low CD4 counts were associated with high density malaria parasitaemia and consequently, very high CD4 counts seemed to exhibit low malaria parasite density among PLWHA. An insignificant negative correlation, however, between CD4 T-cells count and malaria parasite densities was noted (p = 0.169). Mean malaria parasite density on pretreatment samples was 43,168 parasites /μL than their counterparts with >200 cells/μL of CD4 T-cell levels in PLWHA resident in Western Kenya. Secondly, males significantly had a higher geometrical mean parasite density than females regardless of their CD4 status. It is anticipated that the results from this study could be used/applied in developing interventional measures to address malaria/HIV-AIDS coinfections aimed at saving life, particularly in the sub-Saharan African region where the two infections are rampant. The study was able to establish higher parasite density among individuals with ≤200 cells/ In sub-Saharan African countries, HIV/AIDS and malaria coinfection are among the most important public health burden/concern evidently contributing to high morbidity and mortality in the region. Over the years, the prevalence, morbidity, and mortality associated with the two coinfections have been changing globally and the trend has been varying from one region to another. Globally, it was estimated that 212 million cases of malaria occurred in 2015, representing a 22% decline from the figures reported in the year 2000 and a further decline of 14% since 2010. The African and Asian regions/continents where 90% of world malaria cases were reported during the same period had experienced minimal disease decline by between 2 adn 7% [In sub-Saharan Africa, malaria cases decreased from 146 million in 2005 to 114 million in 2015. However, high infection rates in children aged 2–10 have been widely reported, though mortality rates declined to 29% [The pandemic of HIV/AIDS infection has persistently remained a major health concern since its discovery in the 1980s. Globally, people living with HIV/AIDS as of December 2015 were estimated at 36.7 million  with new infection  of 2.1 million. This represents an increase of 6.4% from 33 million in 2007 estimates [Malaria and HIV/AIDS overlapping geographical distribution has raised a lot of concerns and challenges in public health, with HIV infection increasing malaria susceptibility and reducing the efficacy of antimalarial drugs, while malaria infection has been found to increase the risk of HIV disease progression and mother-to-child transmission of HIV .In endemic malaria areas of sub-Saharan Africa, HIV has been found to increase the risk of malaria infection especially in subjects with increased immunosuppression, while in unstable malaria areas, HIV-infected ones are at high risk of developing complicated and severe malaria that may result in death .μL) in HIV positive subjects were associated with high parasite density levels [Malaria and HIV/AIDS coinfections are increasingly reported worldwide  and the y levels . SeveralThe study was conducted at Chulaimbo Health Centre, located some 24.1km north of Kisumu City in Western part of Kenya . The stu et al. (2011) [A prospective study was conducted at a health facility between November 2014 and October 2015 among people living with HIV/AIDS (PLWHA) in a malaria endemic zone within 10 km radius. Western Kenya, particularly the former Nyanza Province, is considered both malaria and HIV/AIDS endemic area [. (2011) , residen P. Falciparum; asexual parasitaemia of ≥1000 and ≤200,000 parasites/μL; febrile state with an auxiliary temperature of ≥37.5°C; haemoglobin (Hb) of ≥5 g/dL of blood and willing to give consent. Ineligibility to the study was based on multiple infections with Plasmodium species; aged below 2 years; severe anaemia (Hb ≤5.0 g/dL); females with a positive pregnancy test; history of taking antimalarial drugs within 48 hours prior to visiting the health facility; severe opportunistic infections (OI) and recipient of second-line regimen of ARV treatment .A total of 13,057 suspected malaria patients were screened at the facility and 5,867 had malaria. Of the positive malaria cases, 126 individuals with HIV/AIDS coinfection, aged between 2 and 72 years, met the inclusion criteria to participate in the study. They comprised both male and female subjects drawn from among those attending HIV management care within the health facility. Eligibility to the study was based on malaria positive slide with monoinfection of et al. (1990) formula. The minimum sample size (n) was thus calculated asThe sample size required for this study was calculated based on 95% and 90% confidence levels and margin of error of 5% and 10%, respectively, for the two drugs in accordance with Lemeshow1−∝/2 is the standard error of first drug (Quinine) = 1.96(95%), Zβ1- is standard error of second drug (Coartem®) = 1.282 (90%), δ is standard deviation of Quinine (=12), [1 is mean parasite clearance of Quinine (41 hr), M2 is mean parasite clearance of Coartem® (36 hr), and n = 2(1.96 +1.282)2 × 122/(41-36)2 = 121 patients.where n is the minimum sample size, Ze (=12), , M1 is mBlood smears (both thick and thin) were collected from each participant on day zero (D0) prior to commencement of treatment. Thin blood smears were fixed using absolute methanol prestaining and both slides were stained with 10% buffered Giemsa stain (pH 7.2) for 10 minutes. Thin blood smears were examined for speciation while thick ones were quantitatively examined for parasitaemia from which parasite density was calculated. Additional blood sample (0.02 ml) was taken from the same prick for the estimation of haemoglobin (Hb) using HemoCue AB, Angelhom, Sweden equipment.μL) of blood using McKenzie et al. (2005) [μL blood (range 4000–11000 WBC/μL blood) was used as the standard for the study population.Malaria parasites were counted in all positive thick blood smears and parasitaemia quantified per 200 white blood cells (WBC) counted in all fields. Ultimate parasitaemia value was calculated and given per microlitre (. (2005)  mathematRoutine CD4 cells count was conducted among all the PLWHA attending the facility as part of management care and their data were available in individual files kept at the clinic.Raw data was stored in Microsoft Excel Spreadsheet program and back-ups in flash discs and hand written copy in analysis notebook. After validation, data analysis was done using computer software SPSS  version 12 to generate means, standard deviation, median, and frequency distributions. To test for significant difference between groups, t-test was used for continuous variables such as age and parasitaemia that were normally distributed.This study was granted approval by Institutional Research & Ethical Committee (IREC) of Moi Teaching & Referral Hospital /Moi University, School of Medicine, Eldoret–No: FAN: IREC 000421 and the Scientific Steering Committee (SSC)/National Ethical Research Committee (NERC), KEMRI, Nairobi–No: SSC No 1495. All confidentiality regarding the study was professionally observed throughout the period of data collection. Access to participants' data at the study site was prohibited to persons other than the Principal Investigator. All participants to the study were assigned a study number at the point of recruitment to conceal their identity.Both good laboratory and clinical practices were observed at study site by professionally qualified personnel through continuous monitoring by the Safety Monitor, Principal Investigator (PI), and the clinical consultant. Giemsa stain and chemicals such as methanol and buffer salts were sourced from Sigma-Aldrich . Training and quality assurance in microscopy for study site technologists was done continuously by the PI. All subjects' slides were taken to Malaria Unit, Centre for Biotechnological Research and Development, KEMRI, Nairobi, for reexamination and validation. Random microscopy checks of malaria slides were regularly done at the site by the PI for quality control.A total of 126 participants were recruited into the study, 83.3% (105/126) of which were adults while children participants were 16.7% (21/126). Mean age of the participants was 31.3 and median was 31, the most frequent age was 30 years, and standard deviation was 14.7 in a range of between 2 and 72 years .μL of blood while the highest recorded density was 199,000 parasites /μL of blood. The mean parasite density pretreatment was 43,168 parasites /μL of blood and the median was 17,720 while the mode was 4,000. The mean temperature on recruitment, prior to commencement of antimalarial therapy, was 37.6°C (slightly febrile), and median temperature was 37.4°C , while the mode was 37.0°C. Temperature range was 34.8°C-40.9°C. The mean blood haemoglobin of participants was 11.7g/dl while median and mode were 11.9g/dl and 12.5g/dl, respectively. Mean CD4 cells count was 519.3 with a median of 478 and the mode was 449 with a range of between 12 and 1567.The lowest recorded parasite density pretreatment among the participants was 1,000 parasites/The mean age of the participants was 32.1 and 30.2 for females and males, respectively (p > 0.524) . Data reThe ages below 5 years of age and those between 10 and 19 years had higher mean parasite density compared to the other age groups .3 had a higher SD and Std error than their counterparts whose CD4 counts were above 200/mm3. When Hb versus parasitaemia data were analyzed and correlated, the correlation, however, was not statistically significant, p=0.543.Participating individuals' CD4 values were analyzed against parasitaemia prior to commencement of antimalarial therapy on day 0  and resuIt is apparent that association between the two infections  at the biological, immunological, and drug levels does exist. A significant variation in parasite density has further been observed among malaria/HIV coinfected groups . Other sμL regardless of their CD4 T-cells status. The current results seem to resonate well with data obtained from General Hospital; Makarfi Kaduna, Nigeria, where children aged 5–15 years had a statistically significant higher parasitaemia density compared to other age groups [μL  than the values below was observed [μL (p=0.03) [The current study findings showed that individuals living with HIV within age brackets below 5 years and those between 10 and 19 years had the highest malaria density of about 95,000/ e groups . The hige groups , which cobserved . In areaobserved . And amo(p=0.03) . There iThe added immune deficiency syndrome due to HIV depletion of CD4 T-cells may explain the high parasite density among people living with HIV/AIDS as indicated in the current study. The infection of HIV/AIDS has been known not only to induce depletion of CD4 T-cells but also to reduce CD8 T-cells causing downmodulation, reduction in T-cell subpopulation, and defective cell mediated immunity against any microbial infection , 24.Further, this study also documented a general higher geometrical mean malaria parasite density in males than in females with a significant difference within gender (p = 0.03), which was comparable with data obtained from Kaduna State, Nigeria .μL than their counterparts with cells above 200/μL) levels. Further, males significantly had a higher geometrical mean parasite density than females regardless of their CD4 status. It is anticipated that the results from this study could be used/applied in developing interventional measures to address malaria/HIV-AIDS coinfections aimed at saving life, particularly in the sub-Saharan African region where the two infections are rampant.This study established a higher parasite density among individual PLWHA with CD4 T-cells below 200/


Plasmodium falciparum populations and multiplicity of infection (MOI) on Bioko Island 7 years after BIMCP.Malaria is still a serious public health problem on Bioko Island , although the number of annual cases has been greatly reduced since 2004 through the Bioko Island Malaria Control Project (BIMCP). A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate the genetic diversity of P. falciparum malaria diagnosed with microscopy were collected from Bioko Island from January 2011 to December 2014. Parasite DNA was extracted using chelex-100 and species were identified using a real-time PCR followed by high-resolution melting. Plasmodium falciparum msp1 and msp2 allelic families were determined using nested PCR.A total of 181 patients with uncomplicated msp1 alleles  and two msp2 alleles (FC27 and 3D7) were analysed in all samples. In msp1, the MAD20 allelic family was predominant with 96.69% (175/178) followed respectively by the K1 allelic family with 96.07% (171/178) and R033 allelic family with 70.78% (126/178). In msp2, the FC27 allelic family was the most frequently detected with 97.69% (169/173) compared to 3D7 with 72.25% (125/173). Twenty-six different alleles were observed in msp1 with 9 alleles for K1, 9 alleles for MAD20 and 8 alleles for R033. In msp2, 25 individual alleles were detected with 5 alleles for FC27 and 20 alleles for 3D7. The overall MOI was 5.51 with respectively 3.5 and 2.01 for msp1 and msp2. A significant increase in overall MOI was correlated with the age group of the patients (P = 0.026) or parasite densities (P = 0.04).Three P. falciparum population in the study, reflecting both the high endemic level and malaria transmission on Bioko Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.The present data showed high genetic diversity and MOI values among the  Despite substantial efforts to eliminate or control malaria, this disease remains the leading cause of morbidity and mortality worldwide . Bioko Imsp1 gene located on chromosome 9 and contains 17 blocks of sequences flanked by conserved regions [msp2 gene located on chromosome 2 and is composed of five blocks, of which the central block 3 is the most polymorphic. The alleles of msp2 are commonly divided into FC27 and 3D7 [Genotyping of malaria parasite populations remains an important tool to determine the types and number of parasite clones in an infection. In molecular epidemiological studies of malaria, this approach is used to investigate the genetic diversity of infections with consideration of various factors, including transmission intensity and host immunity . The mos regions , 6. This regions , 9. Bloc regions . MSP2 is and 3D7 .P. falciparum populations in the region as well as worldwide and link parasite genotypes to the disease phenotypes are crucially important [msp1 and msp2 genetic diversity on Bioko Island, Equatorial Guinea. This study aimed to characterize the genetic diversity of P. falciparum populations and MOI in malaria parasites isolated from symptomatic patients on Bioko Island 7 years after BIMCP.Several studies have linked high multiplicity of infection (MOI) to the severity of malaria, especially in areas with high transmission rates . In thismportant , 13, 14.P. falciparum is still the major public health problem on the island [The study was carried out in the clinic of the Chinese medical aid team to the Republic of Equatorial Guinea, Malabo Regional Hospital in Malabo, located on the north coast of Bioko Island, Equatorial Guinea. This study was approved by the ethics committees of Malabo Regional Hospital. Bioko Island, the largest island of Equatorial Guinea, is located in the Gulf of Guinea, approximately 100 km off the coast of southern Nigeria and 160 km northwest of continental Equatorial Guinea .A total of 181 blood spot samples were collected from the patients with uncomplicated malaria enrolled during therapeutic efficacy monitoring of artesunate + amodiaquine  between January 2011 and December 2014. Included patients were aged between 4 months and 80 years, and were residents on Bioko Island. Malaria patients were classified into uncomplicated malaria states according to the WHO criteria , which w® filter paper  for future use. Microscopy thick and thin blood film slides were prepared using 10% Giemsa solution for 30 min. The stained slides were examined under a light microscope using 100 × oil immersions by an experienced laboratory technician. Parasite density was calculated per 200 white blood cells (WBC) assuming 8000 WBC/μl of blood stage. Based on the microscopy results, the study subjects were classified into three groups: group with low parasite density , group with moderate parasite density  and group with high parasite density . For quality control, archived malaria-positive microslides were re-examined, and parasite density was recorded; Plasmodium species were identified by a real-time PCR followed by high-resolution melting (HRM) [Plasmodium species including P. falciparum, Plasmodium ovale, Plasmodium malariae and Plasmodium vivax, which were kindly provided by Dr. Cao J  [Consent was obtained from all participating subjects or their parents. Dried blood spots were collected on day zero of enrollment through finger prick bleeding spotted onto Whatman 903ng (HRM) . The pGE, China) , were us® 100  method as previously described [Genomic DNA was extracted from the dried blood spots using 0.5% saponin  to free parasites from red blood cells followed by the Chelexescribed  and stormsp1 (block 2) and msp2 (block 3) were previously described [msp1  and msp2 (FC27 and 3D7) genes were amplified with a nested PCR amplification . For the first reaction, 1.0 μl of DNA was amplified with 12.5 μl 2× Taq PCR MasterMix , 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), and sterile ultrapure water to a final volume of 25 μl. For the second round of PCR, 1.0 μl of first-round PCR products was amplified with a 50 μl reaction system, which included 25 μl 2× Taq PCR MasterMix , 2.0 μl forward primer (10 μM), 2.0 μl reverse primer (10 μM), and water (up to 50 μl). The primary PCR amplification of the msp1 and msp2 genes comprised an initial step of 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension of 72 °C for 5 min. The nested PCR cycling parameters of the second round were the same as those of the primary reaction. Allelic specific positive control and DNA negative control were included in each set of reactions.Primers specific for the polymorphic regions of escribed , 17. The® Green I nucleic acid gel stain  and resolved by gel electrophoresis in 2.5% agarose gel. DNA sizes were determined using a low molecular weight ladder marker  and photographed using a Tanon 2500/2500R Gel Imaging System . Alleles of msp1 and msp2 were categorized according to their molecular weights and regarded as different when the molecular weight difference was equal to or greater than 20 bp.The resultant PCR products were stained with SYBRMultiplicity of infection (MOI) was determined by calculating the number of different alleles at each locus; single infections were those with only one allele per locus at all of the genotyped loci. Multiclonal infections were defined as those having more than one allele in at least one locus out of the loci genotyped , 19.msp1 and msp2 allelic frequency was calculated. The mean MOI was calculated for msp1, msp2 and overall MOI. The overall MOI was determined by dividing the total number of alleles detected in both msp1 and msp2 by the total number of samples. The proportions of allele comparisons were assessed by Chi square tests, and normally distributed continuous data were assessed by analysis of variance (ANOVA). The Spearman’s rank correlation coefficient was calculated to evaluate relationships between MOI, parasite densities or age groups in the patients. Statistical significance was defined as P < 0.05.All statistical analyses were performed using the software Statistical Package for Social Sciences version 17.0 . The P. falciparum monoinfection by PCR-HRM [Overall, 181 patients with uncomplicated malaria diagnosed with microscopy were enrolled in the study. They were confirmed with  PCR-HRM . Among tP. falciparum isolates, 178 (98.34%) samples showed successful amplification of msp1, and 173 (95.58%) showed amplification of msp2. Detailed information on msp1 and msp2 is shown in Table msp1, the MAD20 allelic family was predominant at 98.31% (175/178), followed by the K1 allelic family at 96.07% (171/178) and the R033 allelic family at 70.78% (126/178). Almost all samples positive for msp1  were classified as polyclonal infections with K1/MAD20, K1/R033, MAD20/R033 and K1/MAD20/R033, which represented 27.6, 1.7, 2.8 and 65.2%, respectively. MAD20 was the only allelic type with a monoclonal infection frequency of 1.10% (2/181). For msp2, the FC27 allelic family was the most frequently detected, with 97.68% (169/173), compared to 3D7 with 72.25% (125/173).Among these 181 msp1 including 9 K1 type alleles with a size ranging from 100 to 320 bp, 9 MAD20 type alleles (100–280 bp) and 8 R033 type alleles (140–520 bp). For the 140–160 bp fragment range, the frequencies of RO33 were significantly higher than those of K1 (P < 0.001) and MAD20 (P < 0.001). The different fragment sizes of each family of msp1 were shown in Fig. msp2 gene analysis, a total of 25 individual alleles including 20 3D7 type alleles with a size ranging from 200 to 860 bp and 5 FC27 type alleles (320–420 bp) were identified . When the msp1 and msp2 genes were considered separately, the MOI values were 3.5 and 2.01, respectively. There was a significant correlation between MOI for msp1 and age groups of patients . However, no significant correlation between MOI for msp2 and age groups  was found. As shown in Table msp1 and msp2 allelic families and their different combinations showed a positive impact on the parasite density. The results show that the overall MOI were significantly different among groups with different parasite densities . The overall MOI in the group with low parasite density (< 1000 parasites/μl) was significantly lower than that of the groups with moderate (P = 0.008) or high parasite density (P = 0.001).Almost all malaria patients in all age groups were found to have multiclonal infections. The MOI values for P. falciparum parasites impacts malaria transmission and malaria control strategies. Therefore, it is important to resolve the genetic population structure of P. falciparum parasites in epidemic areas. However, very few studies have investigated the genetic diversity of msp1 and msp2 in malaria parasites circulating in many endemic countries, including Equatorial Guinea. This study aimed to evaluate the genetic diversity and allelic frequency of msp1 and msp2 in malaria parasites isolated from the patients with uncomplicated malaria on Bioko Island.The genetic diversity of msp1 and msp2 genes, the high diversity is compatible with the high level of malaria transmission on the island. A total of 26 and 25 different alleles for msp1 and msp2, respectively, were obtained from the parasite isolates. This genetic diversity was higher than that in the neighbouring countries of Central Africa, such as the south-western region of Nigeria  [msp1: 15; msp2: 20) [msp1: 17; msp2: 25) [msp1: 39; msp2: 27) [P. falciparum msp1 and msp2 since 13 years ago. In their report, only 16 and 8 alleles, respectively, were identified [P. falciparum may be due to high transmission intensity and the possible impact of different control measures through BIMCP, mainly IRS, introduction of LLIN and changes in anti-malarial drugs [For the sp2: 15) , Brazzavsp2: 20)  and Bangsp2: 25) , but lowsp2: 27) . In ordesp2: 27)   was observed in the investigation, with an overall MOI of 5.46. This value is higher than that reported in many African countries, including southern Ghana (1.17–1.48) , the RepP < 0.05)  after the deployment of many malaria control measures through the BIMCP since 2004. Malaria caused by P. falciparum on the island was primarily multiple infections with generally high parasite variation, together with a high predominance of the K1 and MAD20 allelic families of msp1 and 3D7 and FC27 families of msp2. These findings also indicate that a methodical exploration of malaria prevalence, with full-scale drug resistance surveillance, is essential for effective malaria prevention and eradication countermeasures.This study provides foundational information on the genetic diversity of