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Objective: To identify prevalence of chloroquine resistance point mutation at (Pfcrt, K76T) and (Pfmdr1, N86Y) copy number variation. Methods: SYBR Green I based real time PCR was used. One hundred and thirty-three samples were analyzed for (Pfcrt, K76T) and (Pfmdr1, N86Y) copy number from dried blood spot. Parasite DNA was extracted using high pure DNA preparation kit. The amplification of DNA was done by using AccuPower 2× GreenStar™ qPCR Master mix. For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI. Absolute quantification method was used to determine the Pfmdr1-N86Y copy number variations. Standard curve was built from strain 3D7 gDNA since it has single copy of Pfmdr1 per haploid genome. The known positive controls with single and multi-copy number of Pfmdr1 gene were included in each experiment. The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation. Results: Out of 133 samples 73 (54.89%) were confirmed as mutant (Pfcrt, 76T) and the remaining 60 (45.11%) were genotyped as wild type (Pfcrt, K76). The (Pfmdr1, N86Y) copy number variation was determined for 133 clinical samples. Out of these samples 61 (45.86%) had single copy and the remaining 72 (54.14%) had multi-copy numbers higher than 1.5 copies per genome. Thirty-four (25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38 (28.57%) were more than 2.5 copies per genome. The minimum and maximum copies per genome were 0.474 and 4.741, respectively. Conclusions: The study showed high prevalence level and fixation of Pfcrt, 76T mutation after chloroquine withdrawal. The prevalence of Pfmdr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers. However, the prevalence level was not statistically significant.

Abstract Background Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches. Methods 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites. Results An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear. Conclusions The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.

Background: There is a dearth of data on eczema from the African continent despite the purported increasing burden., Objectives: To describe the prevalence of eczema at Kiambu Level 5 Hospital and patient skincare., Methods: A descriptive retrospective cross-sectional research design was performed describing the period between 2016 and 2020. Data analysis was done using STATA Version 13. Geospatial mapping of patient residence was also conducted., Results: Eczema was the most common skin condition diagnosed with a prevalence of 25.5%. Majority of the patients favored the use of non-conventional products such as bar soap and milking jelly as routine skincare products. Geospatial mapping demonstrated a higher prevalence of eczema in the urban areas and also revealed access to a specialist as a contributor to health-seeking behavior., Limitations: Subtypes of eczema were not described and this data only reflects 1 facility serving an entire county., Conclusion: The prevalence of eczema in Kiambu Level 5 Hospital mirrors the increasing global burden of eczema. Urban environments have a higher density of eczema in Kiambu. More research is needed to decipher the impact of the preferred non-conventional skin care products on eczema., Competing Interests: None disclosed., (© 2024 by the American Academy of Dermatology, Inc. Published by Elsevier Inc.)

Ethnopharmacological Relevance: Solanum incanum L. is commonly used in traditional herbal medicine (THM) in Kenya for treating various ailments. Recent developments in disease treatment have introduced the concept of host-directed therapy (HDT). This approach involves targeting factors within the host cell that can impede the growth or replication of a pathogen. One such host factor is delta aminolevulinate dehydratase (δ-ALAD), the second enzyme in the heme biosynthesis pathway utilized by Plasmodium for growth. Studies using mice models have shown an increase in δ-ALAD expression during Plasmodium berghei infection. Another plant in the Solanum genus, S. guaranticum, has been found to inhibit δ-ALAD in red blood cells in vitro and in the brain in vivo. Is it possible that the bioactive compounds in S. incanum extracts could also be effective in HDT for malaria treatment?, Aim of Study: To better assess the effectiveness of S. incanum leaf extracts as a curative and prophylaxis in malaria parasite infection, and to test the plant's ability to decrease δ-ALAD expression., Materials and Methods: The leaves of S. incanum were collected, dried, and pulverized before being subjected to a successive extraction protocol to obtain crude, hexane, ethyl acetate, and aqueous extract fractions. Phytochemical analysis was conducted on all extract fractions, followed by GC-MS analysis of the fraction with the most potent antimalarial activity. An acute toxicity study was also performed on the extracted fractions. The potency of the extract fractions as curative and prophylactic antimalarial was then evaluated in THM using Plasmodium berghei-infected mice at a dose of 100 mg/kg. The extract fraction with the highest activity was further evaluated at varying doses and its effect on δ-ALAD was measured using RT-qPCR. The percentage of parasitemia and chemosuppression, and mean survival time were used as indices of activity., Results: Phytochemical analysis revealed that the ethyl acetate and aqueous extract fractions contained high terpenoids, flavonoids, and phenols levels. However, alkaloids were only present in moderate quantities in the aqueous extract, and quinones were found in high levels only in the crude extract. Additionally, all extract fractions contained saponins in high levels but lacked tannins. While the plant extracts were found to be non-toxic, they did not exhibit curative antimalarial activity. However, all extract fractions showed prophylactic antimalarial activity, with the ethyl acetate extract having the highest percentage of chemosuppression even at doses of 250 and 1000 mg/kg. In the negative control, the expression of δ-ALAD was 5.4-fold, but this was significantly reduced to 2.3-fold when mice were treated with 250 mg/kg of the ethyl acetate fraction. GC-MS analysis of the ethyl acetate fraction revealed high percentages of 2-methyloctacosane, tetracosane, and decane., Conclusion: The fractions extracted from S. incanum leaves have been found to possess only antimalarial prophylactic properties, with the ethyl acetate extract fraction showing the most effective results. The activity of this fraction may be attributed to its ability to decrease the expression of δ-ALAD, as it contains an alkane compound implicated with enzyme-inhibitory activity., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest to any content of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lymphatic filariasis is a mosquito borne disease which leads to abnormal painful enlarged body parts, severe disability and social stigma. We screened Wuchereria bancrofti in Matayos constituency in Busia County. Blood samples were collected from 23 villages selected purposively based on clinical case reports. Finger prick and/or venous blood sampling and mosquito collections was carried out. Antigenaemia and filarial DNA prevalence were determined. Infection rates on mosquito pools were estimated and SPSS version 26 was used for descriptive statistics analysis. A total of 262 participants were recruited, 73.3% (n = 192) of the participants had no symptoms, 14.1% (n = 5.3) had swollen legs, 5.3% (n = 14) had painful legs and 3.8% (n = 10) with scrotal swellings. Average antigenemia prevalence was 35.9% (n = 94) and DNA prevalence was at 8.0% (n = 21). A total of 1305 mosquitoes were collected and pooled into 2-20 mosquitoes of the same species and from the same village. Two pools out of 78 were positive for filarial DNA with a minimum infection rate of 0.15%. From this study, antigenaemia and infected mosquitoes are an indication of active transmission. The clinical signs are evidence that filarial infections have been in circulation for over 10 years. The global climate change phenomenon currently happening has been shown to adversely affect the transmission of vector borne diseases and is likely to increase lymphatic filariasis transmission in the area. This study therefore recommends further screening before Mass Drug Administration, morbidity management and enhanced mosquito control Programmes are recommended in the study area., (© 2023. The Author(s).)

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA., Methods: We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages., Results: We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His, while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites., Conclusions: The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and their impact on drug responses require investigations using reverse genetics techniques in malaria parasites., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Chepngetich J et al.)

Background: Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya., Methods: Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections., Results: The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample., Conclusions: The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission., (© 2023. The Author(s).)

Introduction: malaria remains the leading cause of morbidity and mortality in developing tropical and subtropical nations. Due to the emergence and spread of drug resistance to currently available drugs, there is a need for the search of novel, safe, and reasonably affordable anti-malarial medications. The objective of this study was to assess the in vivoanti-malarial effectiveness of Avicennia marina stem bark extracts in a mice model., Methods: guidelines 425 of the Organization for Economic Cooperation and Development were used to determine the extracts' acute toxicity. Mice infected with chloroquine-sensitive Plasmodium berghei (ANKA strain) were tested for in vivoanti-plasmodial activity, and by giving oral doses of 100 mg/kg, 250 mg/kg, and 500 mg/kg body weight of extracts, the plant's suppressive, curative, and preventive effects were assessed., Results: mice treated with dosages of up to 5000 mg/kg showed no evidence of acute toxicity or mortality. Consequently, it was determined that the acute lethal dosage of Avicennia marina extracts in swiss albino mice was greater than 5000 mg/kg. All doses of the extracts exhibited significant (p<0.05) dose-dependent suppression of P. berghei in the suppressive tests compared to the control group. At the highest dose (500 mg/kg), Methanolic crude extracts exerted the highest (93%) parasitemia suppression during the 4-day suppressive test. The extracts also displayed significant (p<0.001) prophylactic and curative activities at all doses compared to the control., Conclusion: results from this study ascertained the safety and promising curative, prophylactic and suppressive anti-plasmodial capabilities of the stem bark extracts of Avicennia marina in mice model., Competing Interests: The authors declare no competing interests., (Copyright: Gitau Wilfred et al.)

Background: Prevention and treatment of malaria during pregnancy is crucial in dealing with maternal mortality and adverse fetal outcomes. The World Health Organization recommendation to treat all pregnant women with sulfadoxine-pyrimethamine (SP) through antenatal care structures was implemented in Kenya in the year 1998, but concerns about its effectiveness in preventing malaria in pregnancy has arisen due to the spread of SP resistant parasites. This study aimed to determine the prevalence of SP resistance markers in Plasmodium falciparum parasites isolated from pregnant women seeking antenatal care at Msambweni County Referral Hospital, located in coastal Kenya, between the year 2013 and 2015., Methods: This hospital-based study included 106 malaria positive whole blood samples for analysis of SP resistance markers within the Pfdhfr gene (codons 51, 59 and 108) and Pfdhps gene (codons 437 and 540). The venous blood collected from all pregnant women was tested for malaria via light microscopy, then the malaria positive samples were separated into plasma and red cells and stored in a - 86° freezer for further studies. Archived red blood cells were processed for molecular characterization of SP resistance markers within the Pfdhfr and Pfdhps genes using real time PCR platform and Sanger sequencing., Results: All samples had at least one mutation in the genes associated with drug resistance; polymorphism prevalence of Pfdhfr51I, 59R and 108N was at 88.7%, 78.3% and 93.4%, respectively, while Pfdhps polymorphism accounted for 94.3% and 91.5% at 437G and 540E, respectively. Quintuple mutations (at all the five codons) conferring total SP resistance had the highest prevalence of 85.8%. Quadruple mutations were observed at a frequency of 10.4%, and 24.5% had a mixed outcome of both wildtype and mutant genotypes in the genes of interest., Conclusion: The data suggest a high prevalence of P. falciparum genetic variations conferring resistance to SP among pregnant women, which may explain reduced efficacy of IPTp treatment in Kenya. There is need for extensive SP resistance profiling in Kenya to inform IPTp drug choices for successful malaria prevention during pregnancy.

It is unknown if surface bound toll-like-receptor (TLR) agonists activate cells via density or total molecular number. To answer this question, we developed a TLR agonist surface conjugated polystyrene microparticle (MP) system. Using a library of MPs with varying TLR agonist density and number, we simultaneously observed innate immune cell MP uptake and TNFα expression using ImageStream flow cytometry on a cell by cell basis. The data shows that total TLR number and not density drives cellular activation with a threshold of approximately 10 5 -10 6 TLR agonists. We believe that this information will be crucial for the design of particulate vaccine formulations., (Copyright © 2020 Deak, Kimani, Cassaidy and Esser-Kahn.)

The uptake of forensic DNA testing technologies in Africa has been slow despite the revolutionary technology being discovered and adopted 3 decades ago. African governments and partners have invested in construction and equipping of forensic laboratories in Africa but the benefits are yet to be realised as the laboratories are still faced with the challenge of shortage of adequately trained personnel. This paper describes an innovative multidisciplinary training approach that was developed and used to train officers from the Directorate of Criminal Investigations Kenya. We report on the structure, implementation and effectiveness of the training. It is expected that with the increased number of trained forensic DNA analysts, there will be an improvement in quality of forensic DNA evidence presented in courts and a reduction in backlog in the forensic biology laboratories in Kenya., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Background: Plasmodium parasite resistance to artemisinin-based combination therapies (ACTs) calls for development of new, affordable, safe, and effective antimalarial drugs. Studies conducted previously on soybean extracts have established that they possess antimicrobial, anti-inflammatory, anticancerous, and antioxidant properties. The activity of such extracts on Plasmodium parasite resistance to artemisinin-based combination therapies (ACTs) calls for development of new, affordable, safe, and effective antimalarial drugs. Studies conducted previously on soybean extracts have established that they possess antimicrobial, anti-inflammatory, anticancerous, and antioxidant properties. The activity of such extracts on., Objectives: The aim of this study was to determine the antiplasmodial activity of soybean extracts using Plasmodium falciparum cultures, followed by an in vivo evaluation of safety and antimalarial activity of the extracts in Plasmodium berghei ANKA strain-infected mice., Method: Aqueous, methanol, and peptide extracts of soybean seeds were prepared. An in vitro evaluation of the extracts for antiplasmodial activity was carried out using two P. falciparum strains: D6, a chloroquine-sensitive Sierra Leone 1 strain and W2, a chloroquine-resistant Indochina 1 strain. Following the in vitro evaluation of the extracts for antiplasmodial activity was carried out using two in vivo evaluation of safety and antimalarial activity of the extracts in P. berghei ANKA strain. The two extracts were tested for their therapeutic potential (curative test). The peptide extract was further assessed to determine whether it could prevent the establishment of a P. berghei ANKA strain. The two extracts were tested for their therapeutic potential (curative test). The peptide extract was further assessed to determine whether it could prevent the establishment of a P. berghei ANKA strain. The two extracts were tested for their therapeutic potential (curative test). The peptide extract was further assessed to determine whether it could prevent the establishment of a., Results: Peptide and methanol extracts showed good activity with IC 50 of 19.97 ± 2.57  μ g/ml and 10.14 ± 9.04  μ g/ml and 10.14 ± 9.04  μ g/ml and 10.14 ± 9.04  μ g/ml and 10.14 ± 9.04  P < 0.001) in suppression with lower doses., Conclusion: The results show the presence of antimalarial properties in soybean extracts with higher curative activity when compared to the prophylactic activity. However, more research needs to be conducted on this plant to possibly establish lead compounds., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Kevin Nyandwaro et al.)

Background: The efficacy and safety of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) against asexual parasites population has been documented. However, the effect of these anti-malarials on sexual parasites is still less clear. Gametocyte clearance following treatment is essential for malaria control and elimination efforts; therefore, the study sought to determine trends in gametocyte clearance after AL or DP treatment in children from a malaria-endemic site in Kenya., Methods: Children aged between 0.5 and 12 years from Busia, western Kenya with uncomplicated Plasmodium falciparum malaria were assigned randomly to AL or DP treatment. A total of 334 children were enrolled, and dried blood spot samples were collected for up to 6 weeks after treatment during the peak malaria transmission season in 2016 and preserved. Plasmodium falciparum gametocytes were detected by qRT-PCR and gametocyte prevalence, density and mean duration of gametocyte carriage were determined., Results: At baseline, all the 334 children had positive asexual parasites by microscopy, 12% (40/334) had detectable gametocyte by microscopy, and 83.7% (253/302) children had gametocytes by RT-qPCR. Gametocyte prevalence by RT-qPCR decreased from 85.1% (126/148) at day 0 to 7.04% (5/71) at day 42 in AL group and from 82.4% (127/154) at day 0 to 14.5% (11/74) at day 42 in DP group. The average duration of gametocyte carriage as estimated by qRT-PCR was slightly shorter in the AL group (4.5 days) than in the DP group (5.1 days) but not significantly different (p = 0.301)., Conclusion: The study identifies no significant difference between AL and DP in gametocyte clearance. Gametocytes persisted up to 42 days post treatment in minority of individuals in both treatment arms. A gametocytocidal drug, in combination with artemisinin-based combination therapy, will be useful in blocking malaria transmission more efficiently.

We report a study that seeks to find a correlation between the overall sensitization potential quantified by the expression of IL-8 by stimulated monocytes and the chemical structure of a model contact allergen, 2,4-dinitrochlorobenzene (DNCB). We show that structure and reactivity of the chemical compounds play an important role in activation of the monocytes and subsequent inflammation in tissue. However, we observed a non-linear correlation between the rate of reaction and biological activity indicating a required balance of stability and reactivity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Kenya has, in the last decade, made tremendous progress in the fight against malaria. Nevertheless, continued surveillance of the genetic diversity and population structure of Plasmodium falciparum is required to refine malaria control and to adapt and improve elimination strategies. Twelve neutral microsatellite loci were genotyped in 201 P. falciparum isolates obtained from the Kenyan-Ugandan border (Busia) and from two inland malaria-endemic sites situated in western (Nyando) and coastal (Msambweni) Kenya. Analyses were done to assess the genetic diversity (allelic richness and expected heterozygosity, [H e ]), multilocus linkage disequilibrium ( I S A ) and population structure. A similarly high degree of genetic diversity was observed among the three parasite populations surveyed (mean H e  = 0.76; P > 0.05). Except in Msambweni, random association of microsatellite loci was observed, indicating high parasite out-breeding. Low to moderate genetic structure (F ST   = 0.022-0.076; P < 0.0001) was observed with only 5% variance in allele frequencies observed among the populations. This study shows that the genetic diversity of P. falciparum populations at the Kenyan-Ugandan border is comparable to the parasite populations from inland Kenya. In addition, high genetic diversity, panmixia and weak population structure in this study highlight the fitness of Kenyan P. falciparum populations to successfully withstand malaria control interventions., (© 2019 John Wiley & Sons Ltd.)

Plasmodium falciparum histidine-rich proteins 2 (PfHRP2) based RDTs are advocated in falciparum malaria-endemic regions, particularly when quality microscopy is not available. However, diversity and any deletion in the pfhrp2 and pfhrp3 genes can affect the performance of PfHRP2-based RDTs. A total of 400 samples collected from uncomplicated malaria cases from Kenya were investigated for the amino acid repeat profiles in exon 2 of pfhrp2 and pfhrp3 genes. In addition, PfHRP2 levels were measured in 96 individuals with uncomplicated malaria. We observed a unique distribution pattern of amino acid repeats both in the PfHRP2 and PfHRP3. 228 PfHRP2 and 124 PfHRP3 different amino acid sequences were identified. Of this, 214 (94%) PfHRP2 and 81 (65%) PfHRP3 amino acid sequences occurred only once. Thirty-nine new PfHRP2 and 20 new PfHRP3 amino acid repeat types were identified. PfHRP2 levels were not correlated with parasitemia or the number of PfHRP2 repeat types. This study shows the variability of PfHRP2, PfHRP3 and PfHRP2 concentration among uncomplicated malaria cases. These findings will be useful to understand the performance of PfHRP2-based RDTs in Kenya.

Rapid diagnostic tests (RDT) are valuable tools that support prudent and timely use of antimalarial drugs, particularly if reliable microscopy is not available. However, the performance and reliability of these tests vary between and within geographical regions. The present study evaluated the performance of routine malaria RDT in Kenyan febrile patients in Busia County, Kenya. A cross sectional study design was employed to recruit febrile patients attending health facilities between August and November 2016. A total of 192 febrile patients who were slide positive and negative were evaluated for their infection status by nested PCR and RDTs (PfHRP2/pLDH). In addition, P. falciparum diversity of the histidine-rich proteins 2 and 3, that influences the RDT test results were determined. All individuals were P. falciparum positive. Among the investigated 192 febrile patients, 76 (40%) were positive by microscopy, 101 (53%) by RDTs and 80 (42%) were PCR positive. The performance of the CareStart™ HRP2/pLDH (pf) RDTs was better than microscopy (Sensitivity 94%; Specificity 75%) and Nucleic acid testing (sensitivity 95%, specificity 77%) with high negative predictive values, indicating the suitability of the RDT in routine practice. Specific pfhrp2/pfhrp3 deletions shown to associate with RDT false negativity was not observed. However, high genetic diversity among pfhrp2 gene was observed. Eleven new PfHRP2 and nine PfHRP3 repeats were observed. False positivity by microscopy and under reporting of infections may thus be a barrier in malaria control and elimination programs. The HRP2/pLDH(Pf) based RDT yet demonstrate to be an effective tool for malaria surveillance program., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4&#95;KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7&#95;KO and PM8&#95;KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs., Competing Interests: The authors have declared that no competing interests exist.

Background: The use of saliva in diagnosis of infectious diseases is an attractive alternative to procedures that involve blood drawing. It promises to reduce risks associated with accidental needle pricks and improve patient compliance particularly in malaria survey and drug efficacy studies. Quantification of parasitaemia is useful in establishing severity of disease and in assessing individual patient response to treatment. In current practice, microscopy is the recommended technique, despite its limitations. This study measured the levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and investigated the relationship with blood parasitaemia., Methods: Matched pre-treatment blood and saliva samples were collected from patients at Msambweni District Hospital, Kenya. Parasitaemia was determined and only those confirmed to be Plasmodium falciparum mono-infected were recruited. PfLDH was quantified in saliva using a commercial ELISA kit. A total of 175 samples were collected. Relationship between blood parasitaemia and concentration of PfLDH in saliva was determined using Pearson correlation statistics. F test was used to determine whether there is a significant difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia., Results: One-hundred and seventy-five patient samples were positive for malaria by microscopy. Of these, 62 (35%) tested positive for PfLDH in saliva, 113 (65%) were false negatives. For those that tested positive, (53) 85% were from patients with moderate to high parasitaemia while 9 (15%) were from patients with low parasitaemia. A correlation co-efficient of 0.18 indicated a weak positive relationship between the concentration of PfLDH in saliva and blood parasitaemia. There was a marginal difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia [F (1, 59) = 1.83, p = 0.1807]., Conclusion: The results indicate that there is a weak correlation between levels of PfLDH in saliva and blood parasitaemia. This is weak association could be as a result of low sensitivity of the assay used as well as presence of inhibitors and proteases in saliva. Further studies should be focused towards reducing the number of false negatives and developing a customised assay that is specific for detection of PfLDH in saliva.

Ethnopharmacological Relevance: Turraea robusta and Turraea nilotica are African medicinal plants used for the treatment of a wide variety of diseases, including malaria. The genus Turraea is rich in limonoids and other triterpenoids known to possess various biological activities., Materials and Methods: From the stem bark of T. robusta six compounds, and from various parts of T. nilotica eleven compounds were isolated by the use of a combination of chromatographic techniques. The structures of the isolated compounds were elucidated using NMR and MS, whilst the relative configuration of one of the isolated compounds, toonapubesin F, was established by X-ray crystallography. The antiplasmodial activities of the crude extracts and the isolated constituents against the D6 and W2 strains of Plasmodium falciparum were determined using the semiautomated micro dilution technique that measures the ability of the extracts to inhibit the incorporation of (G-(3)H, where G is guanine) hypoxanthine into the malaria parasite. The cytotoxicity of the crude extracts and their isolated constituents was evaluated against the mammalian cell lines African monkey kidney (vero), mouse breast cancer (4T1) and human larynx carcinoma (HEp2)., Results: The extracts showed good to moderate antiplasmodial activities, where the extract of the stem bark of T. robusta was also cytotoxic against the 4T1 and the HEp2 cells (IC50<10 μg/ml). The compounds isolated from these extracts were characterized as limonoids, protolimonoids and phytosterol glucosides. These compounds showed good to moderate activities with the most active one being azadironolide, IC50 2.4 ± 0.03 μM and 1.1 ± 0.01 μM against the D6 and W2 strains of Plasmodium falciparum, respectively; all other compounds possessed IC50 14.4-40.5 μM. None of the compounds showed significant cytotoxicity against vero cells, yet four of them were toxic against the 4T1 and HEp2 cancer cell lines with piscidinol A having IC50 8.0 ± 0.03 and 8.4 ± 0.01 μM against the 4T1 and HEp2 cells, respectively. Diacetylation of piscidinol A resulted in reduced cytotoxicity., Conclusion: From the medicinal plants T. robusta and T. nilotica, twelve compounds were isolated and characterized; two of the isolated compounds, namely 11-epi-toonacilin and azadironolide showed good antiplasmodial activity with the highest selectivity indices., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: Malaria is caused by five Plasmodium species and transmitted by anopheline mosquitoes. It occurs in single and mixed infections. Mixed infection easily leads to misdiagnosis. Accurate detection of malaria species is vital. Therefore, the study was conducted to determine the level of mixed infection and misdiagnosis of malaria species in the study area using SYBR Green I-based real time PCR., Methods: The study was conducted in seven health centres from North Gondar, north-west Ethiopia. The data of all febrile patients, who attended the outpatient department for malaria diagnosis, from October to December 2013, was recorded. Dried blood spots were prepared from 168 positive samples for molecular re-evaluation. Parasite DNA was extracted using a commercial kit and Plasmodium species were re-evaluated with SYBR Green I-based real time PCR to detect mixed infections and misdiagnosed mono-infections., Results: Among 7343 patients who were diagnosed for malaria in six study sites within the second quarter of the Ethiopian fiscal year (2013) 1802 (24.54%) were positive for malaria parasite. Out of this, 1,216 (67.48%) Plasmodium falciparum, 553 (30.68%) Plasmodium vivax and 33 (1.8%) mixed infections of both species were recorded. The result showed high prevalence of P. falciparum and P. vivax, but very low prevalence of mixed infections. Among 168 samples collected on dried blood spot 7 (4.17%) were P. vivax, 158 (94.05%) were P. falciparum and 3 (1.80%) were mixed infections of both species. After re-evaluation 10 (5.95%) P. vivax, 112 (66.67%) P. falciparum, 21 (12.50%) P. falciparum + P. vivax mixed infection, and 17 (10.12%) Plasmodium ovale positive rate was recorded. The re-evaluation showed high level of mixed infection, and misdiagnosis of P. ovale and P. vivax., Conclusions: The result shows that P. falciparum prevalence is higher than P. vivax in the study area. The results, obtained from SYBR Green I-based real time PCR, indicated that the diagnosis efficiency of microscopy is very low for species-specific and mixed infection detection. Therefore, real time PCR-based species diagnosis should be applied for clinical diagnosis and quality control purposes in order to prevent the advent of drug resistant strains due to misdiagnosis and mistreatment.

A new triterpenoid, 3-oxo-12β-hydroxy-oleanan-28,13β-olide (1), and six known triterpenoids 2-7 were isolated from the root bark of Ekebergia capensis, an African medicinal plant. A limonoid 8 and two glycoflavonoids 9-10 were found in its leaves. The metabolites were identified by NMR and MS analyses, and their cytotoxicity was evaluated against the mammalian African monkey kidney (vero), mouse breast cancer (4T1), human larynx carcinoma (HEp2) and human breast cancer (MDA-MB-231) cell lines. Out of the isolates, oleanonic acid (2) showed the highest cytotoxicity, i.e., IC50's of 1.4 and 13.3 µM against the HEp2 and 4T1 cells, respectively. Motivated by the higher cytotoxicity of the crude bark extract as compared to the isolates, the interactions of oleanonic acid (2) with five triterpenoids 3-7 were evaluated on vero cells. In an antiplasmodial assay, seven of the metabolites were observed to possess moderate activity against the D6 and W2 strains of P. falciparum (IC50 27.1-97.1 µM), however with a low selectivity index (IC50(vero)/IC50(P. falciparum-D6)<10). The observed moderate antiplasmodial activity may be due to general cytotoxicity of the isolated triterpenoids.

Objective: To assess the feasibility of utilizing a small-scale, low-cost, pilot evaluation in assessing the short-term impact of Kenya's emergency-hire nursing programme (EHP) on the delivery of health services (outpatient visits and maternal-child health indicators) in two underserved health districts with high HIV/AIDS prevalence., Methods: Six primary outcomes were assessed through the collection of data from facility-level health management forms-total general outpatient visits, vaginal deliveries, caesarean sections, antenatal care (ANC) attendance, ANC clients tested for HIV, and deliveries to HIV-positive women. Data on outcome measures were assessed both pre-and post-emergency-hire nurse placement. Informal discussions were also conducted to obtain supporting qualitative data., Findings: The majority of EHP nurses were placed in Suba (15.5%) and Siaya (13%) districts. At the time of the intervention, we describe an increase in total general outpatient visits, vaginal deliveries and caesarean sections within both districts. Similar significant increases were seen with ANC attendance and deliveries to HIV-positive women. Despite increases in the quantity of health services immediately following nurse placement, these levels were often not sustained. We identify several factors that challenge the long-term sustainability of these staffing enhancements., Conclusions: There are multiple factors beyond increasing the supply of nurses that affect the delivery of health services. We believe this pilot evaluation sets the foundation for future, larger and more comprehensive studies further elaborating on the interface between interventions to alleviate nursing shortages and promote enhanced health service delivery. We also stress the importance of strong national and local relationships in conducting future studies.

Introduction: Humoral immune responses play a pivotal role in naturally acquired immunity to malaria. Understanding which humoral responses are impaired among individuals at higher risk for malaria may improve our understanding of malaria immune control and contribute to vaccine development., Methods: We compared humoral responses with 483 Plasmodium falciparum antigens between adults in, Kisumu (high, year-long malaria transmission leading to partial immunity), and adults in Kisii (low, seasonal malaria transmission). Then within each site, we compared malaria-specific humoral responses between those at higher risk for malaria (CD4(+) ≤500) and those at lower risk for malaria (CD4(+) >500). A protein microarray chip containing 483 P. falciparum antigens and 71 HIV antigens was used. Benjamini-Hochberg adjustments were made to control for multiple comparisons., Results: Fifty-seven antigens including CSP, MSP1, LSA1 and AMA1 were identified as significantly more reactive in Kisumu than in Kisii. Ten of these antigens had been identified as protective in an earlier study. CD4(+) T-cell count did not significantly impact humoral responses., Conclusion: Protein microarrays are a useful method to screen multiple humoral responses simultaneously. This study provides useful clues for potential vaccine candidates. Modest decreases in CD4 counts may not significantly impact malaria-specific humoral immunity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches., Methods: 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites., Results: An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear., Conclusions: The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.