Patient-level performance evaluation of a smartphone-based malaria diagnostic application.

BACKGROUND: Microscopic examination is commonly used for malaria diagnosis in the field. However, the lack of well-trained microscopists in malaria-endemic areas impacted the most by the disease is a severe problem. Besides, the examination process is time-consuming and prone to human error. Automated diagnostic systems based on machine learning offer great potential to overcome these problems. This study aims to evaluate Malaria Screener, a smartphone-based application for malaria diagnosis. METHODS: A total of 190 patients were recruited at two sites in rural areas near Khartoum, Sudan. The Malaria Screener mobile application was deployed to screen Giemsa-stained blood smears. Both expert microscopy and nested PCR were performed to use as reference standards. First, Malaria Screener was evaluated using the two reference standards. Then, during post-study experiments, the evaluation was repeated for a newly developed algorithm, PlasmodiumVF-Net. RESULTS: Malaria Screener reached 74.1% (95% CI 63.5-83.0) accuracy in detecting Plasmodium falciparum malaria using expert microscopy as the reference after a threshold calibration. It reached 71.8% (95% CI 61.0-81.0) accuracy when compared with PCR. The achieved accuracies meet the WHO Level 3 requirement for parasite detection. The processing time for each smear varies from 5 to 15 min, depending on the concentration of white blood cells (WBCs). In the post-study experiment, Malaria Screener reached 91.8% (95% CI 83.8-96.6) accuracy when patient-level results were calculated with a different method. This accuracy meets the WHO Level 1 requirement for parasite detection. In addition, PlasmodiumVF-Net, a newly developed algorithm, reached 83.1% (95% CI 77.0-88.1) accuracy when compared with expert microscopy and 81.0% (95% CI 74.6-86.3) accuracy when compared with PCR, reaching the WHO Level 2 requirement for detecting both Plasmodium falciparum and Plasmodium vivax malaria, without using the testing sites data for training or calibration. Results reported for both Malaria Screener and PlasmodiumVF-Net used thick smears for diagnosis. In this paper, both systems were not assessed in species identification and parasite counting, which are still under development. CONCLUSION: Malaria Screener showed the potential to be deployed in resource-limited areas to facilitate routine malaria screening. It is the first smartphone-based system for malaria diagnosis evaluated on the patient-level in a natural field environment. Thus, the results in the field reported here can serve as a reference for future studies.

Correction: Antimalarial drug resistance molecular markers of Plasmodium falciparum isolates from Sudan during 2015-2017.

[This corrects the article DOI: 10.1371/journal.pone.0235401.].

Increased Serum Levels of Asymmetric Dimethylarginine and Symmetric Dimethylarginine and Decreased Levels of Arginine in Sudanese Patients with Essential Hypertension.

INTRODUCTION: Essential hypertension (EH) is a disease caused by various environmental and genetic factors. Nitric oxide (NO) is important for the functional integrity of the endothelium. It is produced in endothelial cells by endothelial NO synthase (eNOS) that mediates the conversion of the amino acid arginine into NO and citrulline. Asymmetric dimethylarginine (ADMA) acts as an inhibitor of eNOS. In contrast, symmetric dimethylarginine (SDMA) has no direct effect on eNOS but plays an important role competing with arginine for transport across the amino acid transporter. ADMA and SDMA have been found to play a central role in the development of cardiovascular diseases. Serum ADMA levels may serve as a future diagnostic marker and a target of therapy in hypertensive patients in the Sudanese population. This study aimed to investigate the relation between serum arginine, ADMA, and SDMA levels with EH in the Sudanese population. METHODS: Patients (n = 260) with established hypertension and controls (n = 144) with normal blood pressure were included in this case-control study. Serum blood samples were analyzed for arginine, ADMA, and SDMA, using high-performance liquid chromatography-tandem mass spectrometry. Other laboratory data were measured using routine methods. Mann-Whitney's U test and χ2 tests were used for continuous and categorical data, respectively. A multivariate logistic regression analysis was conducted to investigate the independent effect of multiple variables on the development of hypertension. RESULTS: Serum arginine levels were significantly lower in the patient group than in the control group (p < 0.001). ADMA and SDMA levels were significantly higher in the patient group than the control group (p < 0.001, p = 0.001, respectively). Multivariate logistic regression analysis showed that only older age, being a male, and arginine levels are independent factors controlling the development of hypertension (p < 0.001, p < 0.001, and p = 0.046, respectively). ADMA and SDMA levels were not independent factors for the development of hypertension. CONCLUSIONS: This study demonstrated increased serum levels of ADMA and SDMA and decreased arginine levels in Sudanese patients with EH. Lowering serum ADMA levels or increasing the arginine levels might be a novel therapeutic target in these individuals.

Antimalarial drug resistance molecular makers of Plasmodium falciparum isolates from Sudan during 2015-2017.

BACKGROUND: Current malaria control and elimination strategies rely mainly on efficacious antimalarial drugs. However, drug resistance is a major threat facing malaria control programs. Determination of drug resistance molecular markers is useful in the monitoring and surveillance of malaria drug efficacy. This study aimed to determine the mutations and haplotypes frequencies of different genes linked with antimalarial drug resistance in certain areas in Sudan. METHODS: A total of 226 dried blood spots (DBS) of microscopically diagnosed P. falciparum isolates were collected from Khartoum and three other areas in Sudan during 2015-2017. Plasmodium falciparum confirmation and multiplicity of infection was assessed using the Sanger's 101 SNPs-barcode and speciation was confirmed using regions of the parasite mitochondria. Molecular genotyping of drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, exonuclease, Pfk13, parasite genetic background (PGB) (Pfarps10, ferredoxin, Pfcrt, Pfmdr2)) was also performed. All genotypes were generated by selective regions amplicon sequencing of the parasite genome using the Illumina MiSeq platform at the Wellcome Sanger Institute, UK then genotypes were translated into drug resistance haplotypes and species determination. FINDINGS: In total 225 samples were confirmed to be P. falciparum. A higher proportion of multiplicity of infection was observed in Gezira (P<0.001) based on the Sanger 101 SNPs -barcode. The overall frequency of mutant haplotype Pfcrt 72-76 CVIET was 71.8%. For Pfmdr1, N86Y was detected in 53.6%, Y184F was observed in 88.1% and D1246Y was detected in 1.5% of the samples. The most frequently observed haplotype was YFD 47.4%. For Pfdhfr (codons 51, 59,108,164), the ICNI haplotype was the most frequent (80.7%) while for Pfdhps (codons 436, 437, 540, 581, 613) the (SGEAA) was most frequent haplotype (41%). The Quadruple mutation (dhfr N51I, S108N + dhps A437G, K540E) was the highest frequent combined mutation (33.9%). In Pfkelch13 gene, 18 non-synonymous mutations were detected, 7 of them were detected in other African countries. The most frequent Pfk13 mutation was E433D detected in four samples. All of the Pfk13 mutant alleles have not been reported to belong to mutations associated with delayed parasite clearance in Southeast Asia. PGB mutations were detected only in Pfcrt N326S\I (46.3%) and Pfcrt I356T (8.2%). The exonuclease mutation was not detected. There was no significant variation in mutant haplotypes between study areas. CONCLUSIONS: There was high frequency of mutations in Pfcrt, Pfdhfr and Pfdhps in this study. These mutations are associated with chloroquine and sulfadoxine-pyrimethamine (SP) resistance. Many SNPs in Pfk13 not linked with delayed parasite clearance were observed. The exonuclease E415G mutation which is linked with piperaquine resistance was not reported.

Assessment of Plasmodium falciparum drug resistance molecular markers from the Blue Nile State, Southeast Sudan.

BACKGROUND: Plasmodium falciparum malaria is a public health problem worldwide. Malaria treatment policy has faced periodic changes due to emergence of drug resistant parasites. In Sudan chloroquine has been replaced by artesunate and sulfadoxine/pyrimethamine (AS/SP) in 2005 and to artemether-lumefantrine (AL) in 2017, due to the development of drug resistance. Different molecular markers have been used to monitor the status of drug resistant P. falciparum. This study aimed to determine the frequency of malaria drug resistance molecular markers in Southeast Sudan. METHODS: The samples of this study were day zero dried blood spot samples collected from efficacy studies in the Blue Nile State from November 2015 to January 2016. A total of 130 samples were amplified and sequenced using illumina Miseq platform. The molecular markers included were Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13, exonuclease and artemisinin resistant (ART-R) genetic background (Pfmdr2, ferroredoxine, Pfcrt and Pfarps10). RESULTS: Resistance markers for chloroquine were detected in 25.8% of the samples as mutant haplotype Pfcrt 72-76 CVIET and 21.7% Pfmdr1 86Y. Pfdhfr mutations were detected in codons 51, 59 and 108. The ICNI double-mutant haplotype was the most prevalent (69%). Pfdhps mutations were detected in codons 436, 437, 540, 581 and 613. The SGEGA triple-mutant haplotype was the most prevalent (43%). In Pfdhfr/Pfdhps combined mutation, quintuple mutation ICNI/SGEGA is the most frequent one (29%). Six of the seven treatment failure samples had quintuple mutation and the seventh was quadruple. This was significantly higher from the adequately responsive group (P < 0.01). Pfk13 novel mutations were found in 7 (8.8%) samples, which were not linked to artemisinin resistance. Mutations in ART-R genetic background genes ranged from zero to 7%. Exonuclease mutation was not detected. CONCLUSION: In this study, moderate resistance to chloroquine and high resistance to SP was observed. Novel mutations of Pfk13 gene not linked to treatment failure were described. There was no resistance to piperaquine the partner drug of dihydroartemisinin/piperaquine (DHA-PPQ).

Absence of K13 gene mutations among artesunate/sulfadoxine-pyrimethamine treatment failures of Sudanese Plasmodium falciparum isolates from Damazin, southeast Sudan.

BACKGROUND: The emergence of resistant parasites to artemisinin poses a threat to malaria treatment. The study aimed to investigate K13 gene mutations in Plasmodium falciparum artesunate (AS)/sulfadoxine-pyrimethamine (SP) efficacy study in Sudan. METHODS: A total of 31 (14 failures and 17 adequate clinical and parasitological response [ACPR]) pretreatment dried blood samples from patients with uncomplicated P. falciparum malaria treated with AS/SP were examined. Nested polymerase chain reaction (PCR) and DNA sequencing of the K13 gene was performed. RESULTS: PCR products were obtained from 30 (96.8%) samples and sequencing was successful in 28 (90.3%). No mutation of the K13 gene was recorded in the treatment failure group. A single mutation (C>T; A621V) in one ACPR patient sample was detected. CONCLUSION: There is no evidence of K13 mutation among AS/SP treatment failure patients. A single mutation of the K13 gene not linked to treatment failure has been detected.

Association of NOS3 gene polymorphisms with essential hypertension in Sudanese patients: a case control study.

BACKGROUND: Essential hypertension (EH) is influenced by various environmental and genetic factors. Nitric oxide is important for the functional integrity of the vascular endothelium and is produced in endothelial cells by the enzyme endothelial nitric oxide synthase (eNOS). EH has a strong genetic component, and the NOS3 gene, which encodes eNOS, represents an interesting candidate for contribution to the phenotype. The most clinically relevant polymorphisms in the NOS3 gene are rs1799983 in exon 7 (encoding Glu298Asp), a variable number tandem repeat (VNTR) in intron 4, and rs2070744 (T-786C) in the promoter region. This study aims to investigate the association between these three polymorphisms in the NOS3 gene and EH in Sudanese patients. METHODS: Hypertensive patients (n = 157) > 18 years of age with established hypertension from various hospitals in Khartoum, and controls (n = 85) > 18 years of age and with blood pressure measurements <140/90, were included in this case control study. Genotypes at the NOS3 variants were determined using TaqMan and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses. Genotype and allele frequencies were compared between the two groups by χ2 analysis, and differences were expressed as odds ratios with 95% confidence intervals (CIs). P values <0.05 were considered statistically significant. RESULTS: The rs2070744 polymorphism in NOS3 was found to be associated with EH in the Sudanese population as the patients group had higher frequency of CC genotype compared with the controls (6.6% vs 6.1%, p = 0.02). Considering a dominant inheritance model, the frequency of TC + CC genotypes in patients was significantly higher than that in the control subjects (52.6% vs 34.1%, respectively; p < 0.01), with an odds ratio (95% CI) of 2.14 (1.23-3.74). In addition, the C allele was more frequent in the patients than the control group (29.6% vs 20%, p = 0.03, OR = 1.84 (1.15-2.93)). The c allele of intron 4 VNTR was reported in >1% of the Sudanese population under study. CONCLUSION: The results of this study indicated that the rs2070744 polymorphism in NOS3 may be a genetic susceptibility factor for EH in the Sudanese population. The c allele of intron 4 VNTR is not rare in the Sudanese population.

Association of lipocalin-2 level, glycemic status and obesity in type 2 diabetes mellitus.

BACKGROUND: Management of type 2 diabetes mellitus aims to maintain a normal glycemic status, which if not, it may lead to acute and/or chronic diabetic complications. Earlier studies found Lipocalin-2 elevated in complications associated with type 2 diabetes mellitus such as ischemic heart disease. These lipocalin-2 changes had been linked to obesity and uncontrolled diabetes. So, it could be useful to understand the effect of glycemic control and obesity on lipocalin-2. METHODS: This was a case control study. Fifty-seven patients with type 2 diabetes and 30 non-diabetic controls participated after getting a written consent. Weight (kg), height (m) and waist circumference (cm) were measured then the body mass index (kg/m2) was determined. Blood samples were collected after an overnight fasting. HbA1c, lipid profile and serum creatinine were measured using enzymatic methods. Lipocalin-2 was measured using sandwich ELISA. RESULTS: Lipocalin-2 was found significantly higher in patients with type 2 diabetes (P = 0.001). However, it had no significant correlation with any of the studied variables. Females had elevated BMI compared to males in the patients group (P < 0.001). HbA1c, serum creatinine, LDL and total cholesterol were elevated in patients with diabetes (P < 0.02). HDL was lower in the patients (P = 0.002). Significant elevation in HbA1c was found in male patients (P = 0.028) compared to female patients. Patients were further classified into controlled, uncontrolled diabetics, obese and non-obese. There was a significant elevation in waist circumference in uncontrolled diabetics compared to controlled ones. Lipocalin-2 had no significant changes between controlled and uncontrolled diabetics nor non-obese and obese patients. CONCLUSION: Patients with type 2 diabetes mellitus have elevated level of serum lipocalin-2. There was no significant association found between lipocalin-2 and glycemic control nor obesity.

Efficacies of DHA-PPQ and AS/SP in patients with uncomplicated Plasmodium falciparum malaria in an area of an unstable seasonal transmission in Sudan.

BACKGROUND: Artemisinin-based combination therapy (ACT), together with other control measures, have reduced the burden of falciparum malaria in sub-Saharan countries, including Sudan. Sudan adopted ACT in 2004 with a remarkable reduction in mortality due to falciparum malaria. However, emergence of resistance to the first-line treatment artesunate and sulfadoxine/pyrimethamine (AS/SP) has created new challenges to the control of malaria in Sudan. A search for an alternative drug of choice for treating uncomplicated malaria has become inevitable. The objective of this study was to evaluate the therapeutic efficacies of dihydroartemisinin/piperaquine (DHA-PPQ) and AS/SP in an area of unstable transmission in Blue Nile State, Sudan in 2015-16. METHODS: A total of 148 patients with uncomplicated malaria were recruited in the study from November 2015 to end of January 2016. Seventy-five patients received DHA-PPQ while 73 received AS/SP. Patients were monitored for clinical and parasitological outcomes following the standard WHO protocol for a period of 42 days for DHA-PPQ and 28 days for AS/SP; nested PCR (nPCR) was performed to confirm parasite re-appearance from day 7 onwards. RESULTS: Fifty-five patients completed the DHA-PPQ arm protocol with success cure rate of 98.2% (95% CI 90.3-100%) and one late clinical failure 1.8% (95% CI 0.0-9.7%). The AS/SP showed adequate clinical and parasitological response (ACPR) of 83.6% (95% CI 71.9-91.8%), early treatment failure was 1.6% (95% CI 0.0-8.8%) and late parasitological failure (LPF) was 14.8% (95% CI 7-26.2%). The respective PCR uncorrected LPF was 20%. CONCLUSION: DHA-PPQ is an efficacious ACT and candidate for replacement of first-line treatment in Sudan while AS/SP showed high treatment failure rate and must be replaced.

The 434(G>C) polymorphism within the coding sequence of Eosinophil Cationic Protein (ECP) correlates with the natural course of Schistosoma mansoni infection.

Schistosomiasis is a chronic parasitic infection with over 200 million people infected worldwide. In Schistosoma mansoni infections, parasite-derived eggs get trapped in the liver, causing the formation of granulomas, which may develop into periportal fibrosis and portal hypertension, and thus severe morbidity. Eosinophil cationic protein (ECP) is a secretory protein of eosinophil granulocytes that efficiently kills the larval stage of S. mansoni, but also affects fibroblast functions. We have investigated the prevalence of the ECP gene polymorphism 434(G>C) in two African populations, from an S. mansoni endemic area in Uganda (n=297) and from a non-endemic area in Sudan (n=78), and also compared these with a Swedish population (n=209). The genotype frequencies in the Ugandan population differed significantly from both the Sudanese and Swedish populations (P<0.001). In the Ugandan population there was a significant association between genotype and prevalence of infection (P=0.03), with lower prevalence in subjects with the GG genotype compared with GC (P=0.02) and CC (P=0.03). There was also a trend towards an association with periportal fibrosis (P=0.08) in the Ugandan population. This suggested association was confirmed when the predominant tribe (n=212) was analysed separately (P=0.004). Our results suggest that ECP may be an important protein, both in the immune response against S. mansoni and in the development of periportal fibrosis. The results also suggest genetic selection towards the ECP 434CC genotype in populations living in S. mansoni endemic areas.