Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda.

BACKGROUND: Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete. METHODS: Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2. RESULTS: Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. CONCLUSION: False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

Glucose-6-phosphate dehydrogenase status and risk of hemolysis in Plasmodium falciparum-infected African children receiving single-dose primaquine.

Glucose-6-phosphate dehydrogenase (G6PD) enzyme function and genotype were determined in Ugandan children with uncomplicated falciparum malaria enrolled in a primaquine trial after exclusion of severe G6PD deficiency by fluorescent spot test. G6PD A- heterozygotes and hemizygotes/homozygotes experienced dose-dependent lower hemoglobin concentrations after treatment. No severe anemia was observed.

The impact of stopping and starting indoor residual spraying on malaria burden in Uganda.

The scale-up of malaria control efforts has led to marked reductions in malaria burden over the past twenty years, but progress has slowed. Implementation of indoor residual spraying (IRS) of insecticide, a proven vector control intervention, has been limited and difficult to sustain partly because questions remain on its added impact over widely accepted interventions such as bed nets. Using data from 14 enhanced surveillance health facilities in Uganda, a country with high bed net coverage yet high malaria burden, we estimate the impact of starting and stopping IRS on changes in malaria incidence. We show that stopping IRS was associated with a 5-fold increase in malaria incidence within 10 months, but reinstating IRS was associated with an over 5-fold decrease within 8 months. In areas where IRS was initiated and sustained, malaria incidence dropped by 85% after year 4. IRS could play a critical role in achieving global malaria targets, particularly in areas where progress has stalled.

In vitro sensitivities of Plasmodium falciparum to different antimalarial drugs in Uganda.

The control of malaria is challenged by resistance of Plasmodium falciparum to multiple drugs. New combination regimens are now advocated for the treatment of uncomplicated falciparum malaria, but the extent of resistance to newer agents is incompletely understood. We measured the in vitro sensitivity of P. falciparum parasites cultured from children enrolled in a drug efficacy trial in Kampala, Uganda, from 2006 to 2008. Sensitivities were measured by comparing levels of histidine-rich protein-2 in parasites incubated with different concentrations of drugs with those in untreated controls. The cultured parasites exhibited a wide range of sensitivities to chloroquine (CQ); monodesethylamodiaquine (MDAQ), the major active metabolite of amodiaquine; and quinine (QN). Mean 50% inhibitory concentration (IC(50)) results were above standard cutoffs for resistance for CQ and MDAQ. Parasites were generally sensitive to dihydroartemisinin (DHA), lumefantrine (LM), and piperaquine (PQ). For CQ, MDAQ, and QN but not the other drugs, activities against individual strains were highly correlated. We also assessed known resistance-mediating polymorphisms in two putative transporters, pfcrt and pfmdr1. When parasites that were least and most sensitive to each drug were compared, the pfmdr1 86Y mutation was significantly more common in parasites that were most resistant to CQ and MDAQ, and the pfmdr1 D1246Y mutation was significantly more common in parasites that were most resistant to MDAQ and QN. In summary, we demonstrated in parasites from Kampala a range of sensitivities to older drugs; correlation of sensitivities to CQ, MDAQ, and QN; and good activity against nearly all strains for DHA, LM, and PQ.

Selection of parasites with diminished drug susceptibility by amodiaquine-containing antimalarial regimens in Uganda.

BACKGROUND: Amodiaquine (AQ) is paired with artesunate (AS) or sulfadoxine-pyrimethamine (SP) in recommended antimalarial regimens. It is unclear how readily AQ resistance will be selected with combination chemotherapy. METHODS: We collected 61 Plasmodium falciparum samples from a cohort of Ugandan children randomized for treatment with AQ-SP, AS-AQ, or artemether-lumefantrine (AL) for uncomplicated malaria. In vitro susceptibility to monodesethylamodiaquine (MDAQ) was measured with a histidine-rich protein 2-based enzyme-linked immunosorbent assay, and potential resistance-mediating polymorphisms in pfmdr1 were evaluated. RESULTS: Parasites collected from patients treated with AQ-SP or AS-AQ within the prior 12 weeks were less susceptible to MDAQ (n = 18; mean of the median inhibitory concentration [IC(50)], 62.9 nmol/L; range, 12.7-158.3 nmol/L) than were parasites from those not treated within 12 weeks (n = 43; mean IC(50), 37.5 nmol/L; range, 6.3-184.7 nmol/L; P=.009) or only from those patients in the treatment arm that did not receive AQ (n = 12; mean IC(50), 28.8 nmol/L; range, 6.3-121.8 nmol/L; P = .004). The proportion of strains with polymorphisms expected to mediate diminished response to AQ (pfmdr1 86Y and 1246Y) increased after AQ therapy, although differences were not statistically significant. CONCLUSIONS: Prior therapy selected for diminished response to MDAQ, which suggests that AQ-containing regimens may rapidly lose efficacy in Africa. The mechanism of diminished MDAQ response is not fully explained by known mutations in pfmdr1.

Marked variation in prevalence of malaria-protective human genetic polymorphisms across Uganda.

A number of human genetic polymorphisms are prevalent in tropical populations and appear to offer protection against symptomatic and/or severe malaria. We compared the prevalence of four polymorphisms, the sickle hemoglobin mutation (ß globin E6V), the α-thalassemia 3.7kb deletion, glucose-6-phosphate dehydrogenase deficiency caused by the common African variant (G6PD A-), and the CD36 T188G mutation in 1344 individuals residing in districts in eastern (Tororo), south-central (Jinja), and southwestern (Kanungu) Uganda. Genes of interest were amplified, amplicons subjected to mutation-specific restriction endonuclease digestion (for sickle hemoglobin, G6PD A-, and CD36 T188G), reaction products resolved by electrophoresis, and genotypes determined based on the sizes of reaction products. Mutant genotypes were common, with many more heterozygous than homozygous alleles identified. The prevalences (heterozygotes plus homozygotes) of sickle hemoglobin (28% Tororo, 25% Jinja, 7% Kanungu), α-thalassemia (53% Tororo, 45% Jinja, 18% Kanungu) and G6PD A- (29% Tororo, 18% Jinja, 8% Kanungu) were significantly greater in Tororo and Jinja compared to Kanungu (p<0.0001 for all three alleles); prevalences were also significantly greater in Tororo compared to Jinja for α-thalassemia (p=0.03) and G6PD A- (p<0.0001). For the CD36 T188G mutation, the prevalence was significantly greater in Tororo compared to Jinja or Kanungu (27% Tororo, 17% Jinja, 18% Kanungu; p=0.0004 and 0.0017, respectively). Considering ethnicity of study subjects, based on primary language spoken, the prevalence of mutant genotypes was lower in Bantu compared to non-Bantu language speakers, but in the Jinja cohort, the only study population with a marked diversity of language groups, prevalence did not differ between Bantu and non-Bantu speakers. These results indicate marked differences in human genetic features between populations in different regions of Uganda. These differences might be explained by both ethnic variation and by varied malaria risk in different regions of Uganda.

Distinguishing recrudescences from new infections in antimalarial clinical trials: major impact of interpretation of genotyping results on estimates of drug efficacy.

The use of molecular genotyping to distinguish recrudescence from new infections has become common in antimalarial clinical trials. However, methods used to interpret genotyping results have not been standardized. We analyzed data from 3,000 patients enrolled in clinical trials at seven sites in Uganda. Late treatment failure requiring genotyping occurred in 51% of the patients. Among samples successfully genotyped, 21% were definitive new infections (no recrudescent strains present on day of failure), 35% were definitive recrudescences (only recrudescent strains present), and 44% were mixed (new and recrudescent strains present). The probability of having a mixed genotyping result increased as transmission intensity increased. At the highest transmission site, the estimated risk of treatment failure increased from 34% to 84% for chloroquine plus sulfadoxine-pyrimethamine, from 18% to 45% for amodiaquine plus sulfadoxine-pyrimethamine, and from 12% to 57% for amodiaquine plus artesunate, depending on whether mixed genotyping results were classified as new infections or recrudescences, respectively. The method used to classify treatment outcomes can have a major impact on estimates of drug efficacy, especially in areas of high transmission intensity.

Principal role of dihydropteroate synthase mutations in mediating resistance to sulfadoxine-pyrimethamine in single-drug and combination therapy of uncomplicated malaria in Uganda.

Antimalarial resistance to sulfadoxine-pyrimethamine (SP) is mediated by mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. However, the relative importance of different mutations is incompletely understood and has not been studied with combination therapy. Samples from 812 patients treated for uncomplicated malaria in Kampala, Uganda were tested for the presence of mutations commonly found in Africa. The dhps Glu-540 mutation was the strongest independent predictor of treatment failure. The dhfr Arg-59 mutation was only predictive of treatment failure in the presence of the dhps Glu-540 mutation. Comparing combination regimens with SP monotherapy, the addition of chloroquine to SP did not improve efficacy, the addition of artesunate lowered the risk of treatment failure only for infections with both the dhfr Arg-59 and dhps Glu-540 mutations, and the addition of amodiaquine lowered this risk for all dhfr/dhps mutation patterns. The dhps Glu-540 mutation played a principal role and the dhfr Arg-59 mutation a secondary role in mediating resistance to SP alone and in combination.

Evaluation of a comprehensive refresher training program in malaria microscopy covering four districts of Uganda.

Microscopy remains the gold standard for malaria diagnosis. However, quality microscopy services are severely lacking in most African countries. To improve capacity for malaria microscopy in Uganda, a 3-day refresher training program was conducted in four districts. Training impact was measured through a written examination and evaluation of the quality of blood-slide preparation and accuracy of field microscopy. A total of 184 of 192 (96%) identified laboratory personnel participated in the training. Average test scores improved from 41% to 75% (P < 0.001). A total of 1,079 and 1,190 routinely made thick blood smears were collected before and after the training, respectively. Sensitivity improved from 84% to 95% (P < 0.001), and specificity improved from 87% to 97% (P < 0.001). The proportion of well-prepared blood smears improved from 6% to 75% (P < 0.001). Supplemental training can have a significant impact on the knowledge of staff, accuracy of microscopy, and quality of blood-slide preparation.

Complexity of Plasmodium falciparum clinical samples from Uganda during short-term culture.

We cultured Plasmodium falciparum parasites from 98 Ugandan children with malaria and determined the complexity of infection (COI) on the basis of msp-2 polymorphisms daily for 9 days. The mean COI decreased during culture from 1.73 to 1.56. New strains appeared after day 0 in 20 cultures. Strains disappeared after day 0 in 56% of 45 cultures that were initially mixed; persisting strains more commonly had wild-type dhfr (C59) and dhps (K540) sequences and mutant pfmdr1 (86Y) sequences. Thus, initial genotypes offer an imperfect representation of clinical COI. Loss of strains in culture may be due to diminished fitness of some drug-resistant strains.

Multiplicity of Plasmodium falciparum infection predicts antimalarial treatment outcome in Ugandan children.

BACKGROUND: In areas with intense malaria transmission, individuals are often simultaneously infected with multiple parasite strains. This study assessed the effect of multiple infections on treatment response in Ugandan children with uncomplicated malaria. METHODS: Four hundred and seventy six blood specimens were analysed for parasite genetic diversity. The P.falciparum merozoite surface protein-2 (msp-2) was analysed to establish multiplicity of infection for pre and post treatment specimens. RESULTS: There were 32 different msp-2 alleles, 15 corresponding to the IC/3D7 and 17 to the FC27 allelic family. The majority of the isolates (343, 72 %) were multiple infections resulting into an overall mean multiplicity of infection of 2.15 (SD+/-1.02). Children infected with multiple strains had nearly a 3-fold increase in treatment failure (Hazard Ratio = 2.8, 95 % CI: 1.5-5.3) compared to their age mates infected with a single strain. CONCLUSION: Multiple-strain infection reduced response to antimalarial therapy. Strategies that reduce multiple-strain infections (intermitted presumptive treatment, indoor residual spraying, insecticide treated nets and efficacious drugs) are likely to improve antimalarial drug efficacy and reduce rate of spread of drug resistance.

Improved malaria case management after integrated team-based training of health care workers in Uganda.

Malaria case management in Africa is characterized by presumptive treatment and substantial overtreatment. We evaluated an integrated team-based training program on malaria case management. Surveillance data 120 days before and after training were compared at eight health facilities in Uganda. After training, the proportion of patients with suspected malaria referred for blood smears increased from 38.3% to 54.6% (P=0.04) in persons<5 years of age years and from 34.1% to 53.4% (P=0.02) in those>or=5 years of age. The proportion of patients with negative blood smears prescribed antimalarial drugs decreased from 47.9% to 19.6% (P<0.001) in persons<5 years of age and from 38.8% to 15.6% (P<0.001) in those>or=5 years of age. Training did not improve the proportion of patients with positive blood smears prescribed antimalarial drugs, the proportion of patients prescribed appropriate antimalarial drugs, or the diagnostic accuracy of microscopy. Integrated team-based training may improve malaria case management and reduce the number of unnecessary antimalarial treatments.

HIV-1 infection in patients referred for malaria blood smears at government health clinics in Uganda.

BACKGROUND: HIV is associated with an increased incidence of malaria in adult African populations. In children, the relationship between HIV and malaria is less clear. We investigated the relationship between malaria and HIV-1 infection among adults and children referred for malaria blood smears at government health clinics in Uganda. METHODS: This was a cross-sectional study in which 1000 consecutive patients referred for malaria blood smears over the course of 1 to 2 months at each of 7 government clinics (N = 7000) were tested for HIV-1 from dried blood spots using enzyme-linked immunosorbent assay (ELISA) screening and nucleic acid-based confirmatory testing. Risk factors for HIV-1 infection were identified using multivariate logistic regression. RESULTS: Among 4467 children aged 16 years or younger, 77 (1.7%) were HIV-1 infected. Of 2533 adults, 270 (10.7%) were HIV-1 infected. In children, having a negative malaria blood smear was associated with higher odds of HIV-1 infection (odds ratio [OR] = 1.90, 95% confidence interval [CI]: 1.18 to 3.06) after controlling for age and gender. In adults, having a positive malaria blood smear was moderately associated with higher odds of HIV-1 infection (OR = 1.41, 95% CI: 1.01 to 1.97) after controlling for age and gender. CONCLUSIONS: In Ugandans evaluated for suspected malaria, associations between malaria smear results and HIV infection differed between children and adults. Although further operations research is needed, our results suggest that counseling and testing for HIV may be of particular importance in children suspected of malaria but with negative malaria smears and in adults with positive malaria smears.

False-positive results of enzyme immunoassays for human immunodeficiency virus in patients with uncomplicated malaria.

Malaria may impact upon human immunodeficiency virus (HIV) test results. We evaluated two HIV enzyme immunoassays (EIAs) by testing 1,965 Ugandans with malaria. We found poor positive predictive values (53% and 76%), particularly with younger age. Combining EIAs eliminated false positives but missed 21% of true positives. Performance of HIV EIAs in malaria may be unsatisfactory.