Malaria Transmission In Uganda Research Articles

Susceptibility of Anopheles gambiae to Natural Plasmodium falciparum Infection: A Comparison between the Well-Established Anopheles gambiae s.s Line and a Newly Established Ugandan Anopheles gambiae s.s. Line.

Much of our understanding of malaria transmission comes from mosquito feeding assays using Anopheles mosquitoes from colonies that are well adapted to membrane feeding. This raises the question whether results from colony mosquitoes lead to overestimates of outcomes in wild Anopheles mosquitoes. We successfully established an Anopheles colony using progeny of wild Anopheles gambiae s.s. mosquitoes (Busia mosquitoes) and directly compared their susceptibility to infection with Plasmodium falciparum with the widely used An. gambiae s.s. mosquitoes (Kisumu mosquitoes) using gametocyte-infected Ugandan donor blood. The proportion of infectious feeds did not differ between Busia (71.8%, 23/32) and Kisumu (68.8%, 22/32, P = 1.00) mosquitoes. When correcting for random effects of donor blood, we observed a 23% higher proportion of infected Busia mosquitoes than infected Kisumu mosquitoes (RR, 1.23; 95% CI, 1.10-1.38, P < 0.001). This study suggests that feeding assays with Kisumu mosquitoes do not overestimate outcomes in wild An. gambiae s.s. mosquitoes, the mosquito species most relevant to malaria transmission in Uganda.

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

Age-dependent carriage of alleles and haplotypes of Plasmodium falciparum sera5, eba-175, and csp in a region of intense malaria transmission in Uganda

BackgroundThe development of malaria vaccines is constrained by genetic polymorphisms exhibited by Plasmodium falciparum antigens. The project the age-dependent distribution of alleles or haplotypes of three P. falciparum malaria vaccine candidates, Circumsporozoite Protein (csp), Erythrocyte Binding Antigen 175 (eba-175) and Serine Repeat Antigen 5 (sera5) in a region of intense malaria transmission in Uganda.MethodsA cross-sectional study was carried out between August and November 2009 in which 250 study participants were selected from a population of 600. Finger prick blood samples were collected after informed consent from participants below 5 years, 5–10 years, and above 10 years of age. Blood was used for microscopy, RDT and dried blood spots. Plasmodium falciparum DNA was extracted by chelex method. Alleles of sera5 and eba-175 were determined by polymerase chain reaction (PCR) amplification followed by resolution of products by agarose gel electrophoresis. Allele calling was done using gel photographs from ethiduim bromide stained gels. Haplotypes of csp were identified by sequencing 63 PCR products using the P. falciparum 7G8 laboratory strain sequence as a reference. The data were analysed using SPSS 16, EQX for windows and Chi-square test was used to calculate associations (P-values), Excel was used to generate graphs. The BioEdit and NCBI blast software programs were used to analyse the sequences from which csp haplotypes map was constructed.ResultsEba-175 FCR3 (48/178) and CAMP (16/178) alleles were observed, the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older participants. Sera5 alleles ORI (6/204) and ORII (103/204) were observed in the population, ORII was more prevalent and was significantly associated with age (P values < 0.0001), parasite density (P-value < 0.0001) and clinical outcomes (P value = 0.018). There was marked csp diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar to a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. There was an age-dependent distribution of csp haplotypes with more haplotypes being harbored by children < 5-year of age, (10/16) compared to adults (2/16). Interestingly, the csp haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed.ConclusionThis data suggest that the eba-175 FCR3 allele, sera5 ORII allele, and csp haplotypes are targets of host immunity and under immune selection pressure in Apac District. These molecules could provide alternative malaria vaccine candidates as sub-unit vaccines.

Identification and characterization of immature Anopheles and culicines (Diptera: Culicidae) at three sites of varying malaria transmission intensities in Uganda

BackgroundOver the last two decades, there has been remarkable progress in malaria control in sub-Saharan Africa, due mainly to the massive deployment of long-lasting insecticidal nets and indoor residual spraying. Despite these gains, it is clear that in many situations, additional interventions are needed to further reduce malaria transmission. The World Health Organization (WHO) has promoted the Integrated Vector Management (IVM) approach through its Global Vector Control Response 2017–2030. However, prior roll-out of larval source management (LSM) as part of IVM, knowledge on ecology of larval aquatic habitats is required.MethodsAquatic habitats colonized by immature Anopheles and culicines vectors were characterized at three sites of low, medium and high malaria transmission in Uganda from October 2011 to June 2015. Larval surveys were conducted along transects in each site and aquatic habitats described according to type and size. Immature Anopheles, culicines and pupae from the described habitats were sampled using standard dipping methods to determine larval and pupae densities. Larvae were identified as anopheline or culicine, and counted. Pupae were not identified further. Binary logistic regression analysis was used to identify factors associated with the presence of immature Anopheles and culicines in each site.ResultsA total of 1205 larval aquatic habitats were surveyed and yielded a total of 17,028 anopheline larvae, 26,958 culicine larvae and 1189 pupae. Peaks in larval abundance occurred in all sites in March–May and August-October coinciding with the rainy seasons. Anopheles larvae were found in 52.4% (n = 251) of aquatic habitats in Tororo, a site of high transmission, 41.9% (n = 536) of habitats in Kanungu, a site with moderate malaria transmission, and 15.8% (n = 418) in Jinja, a site with low malaria transmission. The odds of finding larvae was highest in rice fields compared to pools in both Tororo (odds ratio, OR = 4.21, 95% CI 1.22–14.56, p = 0.02) and Kanungu (OR = 2.14, 95% CI 1.12–4.07, p = 0.02), while in Jinja the odd were highest in containers (OR = 4.55, 95% CI = 1.09–19.14, p = 0.03). In Kanungu, larvae were less likely to be found in containers compared to pools (OR = 0.26, 95% CI 0.09–0.66, p = 0.008) and river fringe (OR = 0.19, 95% CI 0.07–0.52, p = 0.001). Medium sized habitats were associated with high odds of finding larvae compared to small habitats (OR = 3.59, 95% CI 1.18–14.19, p = 0.039).ConclusionsThese findings show that immature Anopheles and culicines were common in areas of high and moderate transmission but were rare in areas of low transmission. Although immature Anopheles and culicines were found in all types of water bodies, they were most common in rice fields and less common in open drains and in river fringes. Methods are needed to reduce the aquatic stages of anopheline mosquitoes in human-made habitats, particularly rice fields.

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, PfRipr, as a highly conserved blood-stage malaria vaccine candidate

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine candidate for next-generation vaccines against P. falciparum.

Age-dependent carriage of alleles and haplotypes of Plasmodium falciparum sera5, eba-175, and csp in a region of intense malaria transmission in Uganda

Background: The development of malaria vaccines is constrained by genetic polymorphisms exhibited by Plasmodium falciparum antigens. We investigated the age-dependent distribution of alleles or haplotypes of three P. falciparum malaria vaccine candidates, circumsporozoite protein (csp), erythrocyte binding antigen 175 (eba-175) and serine repeat antigen 5 (sera5) in a region of intense malaria transmission in Uganda. Methods & Materials: A cross sectional study was carried out between August and November 2009. Blood samples were collected after informed consent from 250 individuals below 5 years, 5-10 years and above 10 years olds. P. falciparum DNA was extracted from all samples. Alleles of sera5 and eba-175 were determined by polymerase chain reaction (PCR) amplification followed by resolution of PCR products by agarose gel electrophoresis and allele calling using photographs of ethidium bromide-stained gels. Haplotypes of CSP were identified by sequencing 63 PCR products and using P.falciparum 7G8 strain sequence as a reference. Results: Both eba-175 FCR3 (48/178) and CAMP (16/178) alleles were observed with the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older individuals. Both sera5 alleles ORI (6/204) and ORII (103/204) were observed in the population but ORII was more prevalent. SERA5 ORII allele was significantly associated with age (P values < 0.0001), parasite density (P value < 0.0001) and clinical outcomes (P value = 0.018). There was marked CSP diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar with a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. Conclusion: There was an age-dependent distribution of CSP haplotypes with more haplotypes being harbored by < 5-year olds, (10/16) compared to adults (2/16). Interestingly, the CSP haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed. Our data suggest that eba-175 FCR3 allele, sera5 ORII allele, and CSP haplotypes are targets of host immunity and under immune selection pressure in Apac District.

The past, present and future use of epidemiological intelligence to plan malaria vector control and parasite prevention in Uganda.

BackgroundAn important prelude to developing strategies to control infectious diseases is a detailed epidemiological evidence platform to target cost-effective interventions and define resource needs.MethodsA review of published and un-published reports of malaria vector control and parasite prevention in Uganda was conducted for the period 1900–2013. The objective was to provide a perspective as to how epidemiological intelligence was used to design malaria control before and during the global malaria eradication programme (GMEP) and to contrast this with the evidence generated in support of the Roll Back Malaria (RBM) initiative from 1998 to date.ResultsDuring the GMEP era, comprehensive investigations were undertaken on the effectiveness of vector and parasite control such as indoor residual house-spraying (IRS) and mass drug administration (MDA) at different sites in Uganda. Nationwide malariometric surveys were undertaken between 1964 and 1967 to provide a profile of risk, epidemiology and seasonality leading to an evidence-based national cartography of risk to characterize the diversity of malaria transmission in Uganda. At the launch of the RBM initiative in the late 1990s, an equivalent level of evidence was lacking. There was no contemporary national evidence-base for the likely impact of insecticide-treated nets (ITN), no new malariometric data, no new national cartography of malaria risk or any evidence of tailored intervention delivery based on variations in the ecology of malaria risk in Uganda.DiscussionDespite millions of dollars of overseas development assistance over the last ten years in ITN, and more recently the resurrection of the use of IRS, the epidemiological impact of vector control remains uncertain due to an absence of nationwide basic parasite and vector-based field studies.ConclusionReadily available epidemiological data should become the future business model to maximize malaria funding from 2015. Over the next five to ten years, accountability, impact analysis, financial business cases supported by a culture of data use should become the new paradigm by which malaria programmes, governments and their development partners operate.

Effector Phenotype ofPlasmodium falciparum–Specific CD4+T Cells Is Influenced by Both Age and Transmission Intensity in Naturally Exposed Populations

Mechanisms mediating immunity to malaria remain unclear, but animal data and experimental human vaccination models suggest a critical role for CD4(+) T cells. Advances in multiparametric flow cytometry have revealed that the functional quality of pathogen-specific CD4(+) T cells determines immune protection in many infectious models. Little is known about the functional characteristics of Plasmodium-specific CD4(+) T-cell responses in immune and nonimmune individuals. We compared T-cell responses to Plasmodium falciparum among household-matched children and adults residing in settings of high or low malaria transmission in Uganda. Peripheral blood mononuclear cells were stimulated with P. falciparum antigen, and interferon γ (IFN-γ), interleukin 2, interleukin 10, and tumor necrosis factor α (TNF-α) production was analyzed via multiparametric flow cytometry. We found that the magnitude of the CD4(+) T-cell responses was greater in areas of high transmission but similar between children and adults in each setting type. In the high-transmission setting, most P. falciparum-specific CD4(+) T-cells in children produced interleukin 10, while responses in adults were dominated by IFN-γ and TNF-α. In contrast, in the low-transmission setting, responses in both children and adults were dominated by IFN-γ and TNF-α. These findings highlight major differences in the CD4(+) T-cell response of immune adults and nonimmune children that may be relevant for immune protection from malaria.

Mind the gap: house structure and the risk of malaria in Uganda.

BackgroundGood house construction may reduce the risk of malaria by limiting the entry of mosquito vectors. We assessed how house design may affect mosquito house entry and malaria risk in Uganda.Methods100 households were enrolled in each of three sub-counties: Walukuba, Jinja district; Kihihi, Kanungu district; and Nagongera, Tororo district. CDC light trap collections of mosquitoes were done monthly in all homes. All children aged six months to ten years (n = 878) were followed prospectively for a total of 24 months to measure parasite prevalence every three months and malaria incidence. Homes were classified as modern (cement, wood or metal walls; and tiled or metal roof; and closed eaves) or traditional (all other homes).ResultsA total of 113,618 female Anopheles were collected over 6,765 nights. 6,816 routine blood smears were taken of which 1,061 (15.6%) were malaria parasite positive. 2,582 episodes of uncomplicated malaria were diagnosed after 1,569 person years of follow-up, giving an overall incidence of 1.6 episodes per person year at risk. The human biting rate was lower in modern homes than in traditional homes (adjusted incidence rate ratio (IRR) 0.48, 95% confidence interval (CI) 0.37–0.64, p<0.001). The odds of malaria infection were lower in modern homes across all the sub-counties (adjusted odds ratio 0.44, 95%CI 0.30–0.65, p<0.001), while malaria incidence was lower in modern homes in Kihihi (adjusted IRR 0.61, 95%CI 0.40–0.91, p = 0.02) but not in Walukuba or Nagongera.ConclusionsHouse design is likely to explain some of the heterogeneity of malaria transmission in Uganda and represents a promising target for future interventions, even in highly endemic areas.

A lab-on-chip for malaria diagnosis and surveillance.

BackgroundAccess to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges.MethodsThe platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection.ResultsThis diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/μL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%.ConclusionsThese results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.

Impact of intermittent preventive treatment with dihydroartemisinin-piperaquine on malaria in Ugandan schoolchildren: a randomized, placebo-controlled trial.

Intermittent preventive treatment (IPT) in schoolchildren offers a promising option for malaria control. However, the optimal drug and dosing regimens for IPT remain to be determined. We conducted a randomized, double-blind, placebo-controlled trial in 740 schoolchildren aged 6-14 years living in a setting of high malaria transmission in Uganda. Enrolled children were randomized to dihydroartemisinin-piperaquine (DP) given once a month (IPTm), DP given once a school term (4 treatments over 12 months, IPTst), or placebo and followed for 12 months. The primary outcome was the incidence of malaria over 12 months. Secondary outcomes included parasite prevalence and anemia over 12 months. Analyses were conducted on an intention-to-treat basis. In the placebo arm, the incidence of malaria was 0.34 episodes per person-year and the prevalence of parasitemia and anemia was 38% and 20%, respectively. IPTm reduced the incidence of malaria by 96% (95% confidence interval [CI], 88%-99%, P < .0001), the prevalence of asymptomatic parasitemia by 94% (95% CI, 92%-96%, P < .0001), and the prevalence of anemia by 40% (95% CI, 19%-56%, P < .0001). IPTst had no significant effect on the incidence of symptomatic malaria or the prevalence of anemia, but reduced the prevalence of asymptomatic parasitemia by 54% (95% CI, 47%-60%, P < .0001). Monthly IPT with DP offered remarkable protection against clinical malaria, parasitemia, and anemia in schoolchildren living in a high-malaria-transmission setting. NCT01231880.

Malaria morbidity, mortality and pregnancy outcome in areas with different levels of malaria transmission in Uganda: a hospital record-based study

A retrospective hospital record-based study for Uganda was conducted in Hoima district (an area of stable malaria transmission) and in Kabale district (an area of unstable malaria transmission). In-patient and maternity ward registers for January 1997–December 1998 were reviewed and malaria admissions, mortality, abortions, stillbirths and birthweights were recorded. Data were analysed in relation to rainfall patterns. Admissions due to malaria were significantly higher at Hoima compared to Kabale hospital in 1997 ( P < 0·0001). However, the situation reversed in 1998 with significantly more malaria cases registered at Kabale compared to Hoima hospital ( P < 0·0001). The increase at Kabale hospital in 1998 was attributed to increased and prolonged rains during 1997 (El Niño). Pregnancy was a risk factor for admission with malaria at Kabale, but not at Hoima. Anaemia was significantly more common among patients admitted to hospital at Hoima compared to Kabale in 1997 and 1998 ( P < 0·0001 and P = 0·02, respectively). The rate of low birthweight (birthweight < 2·5 kg) was significantly higher among primigravidae and multigravidae at Hoima hospital compared to Kabale hospital ( P < 0·0001). There were significantly more stillbirths at Kabale compared to Hoima hospital ( P < 0·0001). Routine hospital data such as birthweight and number of malaria cases can provide information on the level of malaria transmission useful for the health services to target appropriate malaria interventions and allocate resources to control outbreaks of malaria epidemics.