Whole genome sequencing and genotyping Klebsiella pneumoniae multi-drug resistant hospital isolates from Western Kenya.

Objectives: Klebsiella pneumoniae are a frequent cause of nosocomial infections worldwide. Sequence type 147 (ST147) has been reported as a major circulating high-risk lineage in many countries, and appears to be a formidable platform for the dissemination of antimicrobial resistance (AMR) determinants. However, the distribution of this pathogen in Western African hospitals has been scarcely studied. The main objective of this work was to perform whole genome sequencing of K. pneumoniae isolates from a referral hospital in Kakamega (Kenya) for genotyping and identification of AMR and virulence determinants. Methods: In total, 15 K. pneumoniae isolates showing a broad spectrum antimicrobial resistance were selected for whole genome sequencing by Illumina HiSeq 2500 platform. Results: ST147 was the dominant lineage among the highly-resistant K. pneumoniae isolates that we sequenced. ST147 was associated with both community- and the hospital-acquired infections, and with different infection sites, whereas other STs were predominantly uropathogens. Multiple antibiotic resistance and virulence determinants were detected in the genomes including extended-spectrum ß-lactamases (ESBL) and carbapenemases. Many of these genes were plasmid-borne. Conclusions: Our data suggest that the evolutionary success of ST147 may be linked with the acquisition of broad host-range plasmids, and their propensity to accrue AMR and virulence determinants. Although ST147 is a dominant lineage in many countries worldwide, it has not been previously reported as prevalent in Africa. Our data suggest an influx of new nosocomial pathogens with new virulence genes into African hospitals from other continents.

Antimalarial activity assay of artesunate-3-chloro-4(4-chlorophenoxy) aniline in vitro and in mice models.

The global spread of multi-drug resistant P. falciparum, P. vivax, and P. malariae strains and absence of long-term effective vaccine makes chemotherapy the mainstay of malaria control strategies in endemic settings. The Mossman's assay and the Organization for Economic Co-operation and Development (OECD), 2001 guideline 423, were used to determine the cytotoxicity and acute oral toxicity of a novel hybrid drug, artesunate-3-Chloro-4(4-chlorophenoxy) aniline (ATSA), in vitro and in vivo, respectively. A modified Desjardins method was used to screen for antiplasmodial activity using P. falciparum (3D7 and W2) strains in vitro. The Peter's 4-day suppressive tests (4DTs) was used to evaluate the in vivo antimalaria activity using P. berghei ANKA strain, lumefantrine resistant (LuR), and piperaquine resistant (PQR) P. berghei lines. In silico prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles was assayed using PreADMET online prediction tool. The reference drug in all experiments was artesunate (ATS). Statistical significance between ATSA's activities in treated and control mice was evaluated by one-way analysis of variance (ANOVA). Results show that inhibitory concentrations-50 (IC50) of ATSA is 11.47 ± 1.3 (3D7) and 1.45 ± 0.26 (W2) against 4.66 ± 0.93 (3D7) and 0.60 ± 0.15 (W2) ng/ml of ATS with a selective index of 2180.91(3D7) and a therapeutic index (TI) of > 71). No mortalities were observed in acute oral toxicity assays and mean weight differences for test and controls were statistically insignificant (P > 0.05). The in vivo activity of ATSA was above 40% with effective dosage-50 (ED50) of 4.211, 2.601, and 3.875 mg/kg body weight against P. berghei ANKA, LuR, and PQR lines, respectively. The difference between treated and control mice was statistically significant (P < 0.05). ATSA has high intestinal absorption (HIA) > 95% and has medium human ether-a-go-go related gene (hERG) K+ channel inhibition risks. Preclinical and clinical studies on ATSA are recommended to evaluate its value in developing novel drugs for future management of multi-drug resistant malaria parasites.

Increased HIV Incidence in Wuchereria bancrofti Microfilaria Positive Individuals in Tanzania.

BACKGROUND: Infections with Wuchereria bancrofti are associated with reduced immunity against concomitant infections. Indeed, our previous study described a 2.3-fold increased HIV incidence among individuals with W. bancrofti infection, as measured by the circulating filarial antigen of the adult worm. This new study aimed to retrospectively determine microfilariae status of the participants to assess if the previously described increased HIV susceptibility was associated with the presence of MF in the same cohort. METHODS: CFA positive but HIV negative biobanked human blood samples (n = 350) were analyzed for W. bancrofti MF chitinase using real time PCR. RESULTS: The PCR provided a positive signal in 12/350 (3.4%) samples. During four years of follow-up (1109 person years (PY)), 22 study participants acquired an HIV infection. In 39 PY of W. bancrofti MF chitinase positive individuals, three new HIV infections occurred (7.8 cases per 100 PY), in contrast to 19 seroconversions in 1070 PY of W. bancrofti MF chitinase negative individuals (1.8 cases per 100 PY, p = 0.014). CONCLUSIONS: In the subgroup of MF-producing Wb-infected individuals, the HIV incidence exceeded the previously described moderate increased risk for HIV seen in all Wb-infected individuals (regardless of MF status) compared with uninfected persons from the same area.

Antimicrobial resistance patterns and characterisation of emerging beta-lactamase-producing Escherichia coli in camels sampled from Northern Kenya.

BACKGROUND: Animal husbandry practices in different livestock production systems and increased livestock-wildlife interactions are thought to be primary drivers of antimicrobial resistance (AMR) in Arid and Semi-Arid Lands (ASALs). Despite a tenfold increase in the camel population within the last decade, paired with widespread use of camel products, there is a lack of comprehensive information concerning beta-lactamase-producing Escherichia coli (E. coli) within these production systems. OBJECTIVES: Our study sought to establish an AMR profile and to identify and characterise emerging beta-lactamase-producing E. coli isolated from faecal samples obtained from camel herds in Northern Kenya. METHODS: The antimicrobial susceptibility profiles of E. coli isolates were established using the disk diffusion method, with beta-lactamase (bla) gene PCR product sequencing performed for phylogenetic grouping and genetic diversity assessments. RESULTS: Here we show, among the recovered E. coli isolates (n = 123), the highest level of resistance was observed for cefaclor at 28.5% of isolates, followed by cefotaxime at 16.3% and ampicillin at 9.7%. Moreover, extended-spectrum beta-lactamase (ESBL)-producing E. coli harbouring the blaCTX-M-15 or blaCTX-M-27 genes were detected in 3.3% of total samples, and are associated with phylogenetic groups B1, B2 and D. Multiple variants of non-ESBL blaTEM genes were detected, the majority of which were the blaTEM-1 and blaTEM-116 genes. CONCLUSIONS: Findings from this study shed light on the increased occurrence of ESBL- and non-ESBL-encoding gene variants in E. coli isolates with demonstrated multidrug resistant phenotypes. This study highlights the need for an expanded One Health approach to understanding AMR transmission dynamics, drivers of AMR development, and appropriate practices for antimicrobial stewardship in camel production systems within ASALs.

Antiplasmodial Activity Assay of 3-Chloro-4-(4-chlorophenoxy) Aniline Combinations with Artesunate or Chloroquine In Vitro and in a Mouse Model.

Malaria is the eighth highest contributor to global disease burden with 212 million cases and 429,000 deaths reported in 2015. There is an urgent need to develop multiple target drug to curb growing resistance by Plasmodia due to use of single target drugs and lack of vaccines. Based on a previous study, 3-chloro-4-(4-chlorophenoxy) aniline (ANI) inhibits Plasmodia enoyl acyl carrier protein reductase. This study aimed at evaluating the antiplasmodial activity of ANI combinations with artesunate (AS) or chloroquine (CQ) against P. falciparum in vitro based on the semiautomated microdilution assay and P. berghei in vivo based on Peters' 4-day test. Data were analysed by linear regression using version 5.5 of Statistica, 2000. From the results, on the one hand, a combination of 1.1 ng/ml AS and 3.3 µg/ml of ANI inhibited 50% growth of W2, while a combination of 0.8 ng/ml of AS and 2.6 µg/ml of ANI inhibited 50% growth of 3D7. On the other hand, a combination of 22 ng/ml CQ and 3.7 µg/ml of ANI inhibited 50% growth of W2, while a combination of 4.6 ng/ml CQ and 3.1 µg/ml of ANI inhibited 50% growth of 3D7. In in vivo assays, a combination of ED50 concentrations of AS and ANI cleared all parasites, while 1/2 and 1/4 ED50 combinations inhibited 67.0% and 35.4% parasite growth, respectively. ED50 combinations of CQ and ANI inhibited 81.0% growth of parasites, while 1/2 and 1/4 ED50 combinations inhibited 27.3% and 10.2% parasite growth. Assuming a linear relationship between percentage chemosuppression and combination ratios, only 0.88 mg/kg of AS combined with 1.68 mg/kg of ANI or 1.78 mg/kg of CQ with 3.15 mg/kg of ANI inhibited 50% parasite growth in vivo. ANI combinations with AS or CQ are thus potential antimalarial drug combinations if their clinical efficacy and safety are ascertained.

Mitochondrial DNA D-Loop Diversity of the Helmeted Guinea Fowls in Kenya and Its Implications on HSP70 Gene Functional Polymorphism.

We analyzed variations in 90 mitochondrial DNA (mtDNA) D-loop and heat shock protein 70 (HSP70) gene sequences from four populations of domesticated helmeted Guinea fowls (70 individuals) and 1 population of wild helmeted Guinea fowls (20 individuals) in Kenya in order to get information about their origin, genetic diversity, and traits associated with heat stress. 90 sequences were assigned to 25 distinct mtDNA and 4 HSP70 haplotypes. Most mtDNA haplotypes of the domesticated helmeted Guinea fowls were grouped into two main haplogroups, HgA and HgB. The wild population grouped into distinct mtDNA haplogroups. Two mtDNA haplotypes dominated across all populations of domesticated helmeted Guinea fowls: Hap2 and Hap4, while the dominant HSP70 haplotype found in all populations was CGC. Higher haplotype diversities were generally observed. The HSP70 haplotype diversities were low across all populations. The nucleotide diversity values for both mtDNA and HSP70 were generally low. Most mtDNA genetic variations occurred among populations for the three hierarchical categories considered while most variations in the HSP70 gene occurred among individuals within population. The lack of population structure among the domestic populations could suggest intensive genetic intermixing. The differentiation of the wild population may be due to a clearly distinct demographic history that shaped its genetic profile. Analysis of the Kenyan Guinea fowl population structure and history based on mtDNA D-loop variations and HSP70 gene functional polymorphisms complimented by archaeological and linguistic insight supports the hypothesis that most domesticated helmeted Guinea fowls in Kenya are related to the West African domesticated helmeted Guinea fowls. We recommend more molecular studies on this emerging poultry species with potential for poverty alleviation and food security against a backdrop of climate change in Africa.

In vitro selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemisinin.

BACKGROUND: Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance. METHODS: Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast. RESULTS: Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene. DISCUSSION: This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria. CONCLUSION: Artemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Safety and Analgesic Properties of Ethanolic Extracts of Toddalia Asiatica (L) Lam. (Rutaceae) Used for Central and Peripheral Pain Management Among the East African Ethnic Communities.

BACKGROUND: Although herbs are often perceived as "natural" and therefore safe, many different side effects have been reported. Additionally, there is limited scientific evidence to establish the safety and efficacy of most herbal products. The aim of this study was to evaluate the biochemical and haematological effects of Toddaliaasiatica (L) Lam. (Rutaceae) (T. asiatica (L.) in albino Wistar rats. MATERIALS AND METHODS: The phytochemicals present in the plant were determined. The analgesic activity was determined using the hot plate technique. The whole blood with anticoagulant was used for assay of the haematological parameters using the COULTERAc•T5diff AL Hematology Analyzer (Fullerton, CA, USA). The biochemical parameters determined with HumaLyzer 2000, a semi-automatic, microprocessor-controlled photometer fromchem-labs, Nairobi. RESULTS: The effect of extract on serum biochemical parameters after 14 days treatment with the crude ethanolic extract of T. asiatica (L.) revealed significant difference in the Cholesterol (P = 0.041), alanine transaminase (P = 0.007), gamma-glutamyl transferase (P = 0.045). There was no significance in the alkaline phosphatase (ALP), aspartate transaminase (AST) levels compared to the untreated controls. Peripheral blood films (PBFs) of the treated animals were performed and stained with leishman's stain. Major morphological changes were observed including anisocytosis, burr cells, anisochromia, hypochromia and reactive lymphocytes among others. CONCLUSION: The crude extract of T. asiatica (L.) showed better analgesic effect (28.2±13.16) than Acetylsalicylate used as control (4±0.31). The potential of T. asiatica (L.) asananalgesic was remarkable. However, the crude extract of T. asiatica (L.) induced nephrotoxicity and liver enzymes modulation and elevated total cholesterol in the test organisms compared to the untreated negative controls.

Molecular Detection and Characterization of Theileria Infecting Wildebeest (Connochaetes taurinus) in the Maasai Mara National Reserve, Kenya.

Theileria is a genus of tick-borne protozoan that is globally widespread and infects nearly all ungulates in which they cause either latent infection or lethal disease. Wild animals are considered reservoir hosts of many species of Theileria and their diversity in wildlife species is increasingly becoming of interest. The molecular characterization and identification of Theileria infecting wildlife has been studied in a few species including buffalo, which are considered reservoir host for Theileria parva infecting cattle. In this study, we sequenced Theileria species infecting wildebeest (Connochaetes taurinus) and used molecular-genetic and phylogenetic analysis of the 18 Small Subunit of the Ribosomal RNA (18S rRNA) to identify their relationships with known species of Theileria. Our results revealed three new Theileria haplotypes infecting wildebeest. Phylogenetic analysis revealed that haplotype 1 and 2 clustered in the same clade as Theileria separata and with Theileria sp. isolated from other small to medium sized antelopes. Haplotype 3 clustered close to the Theileria ovis clade. This is the first molecular description and characterization of Theileria species infecting blue wildebeest in East Africa. This study demonstrates the potential for Theileria transmission between wildebeest and small domestic ungulates, such as sheep and goats.

Efficacy of chloroquine, sulphadoxine-pyrimethamine and amodiaquine for treatment of uncomplicated Plasmodium falciparum malaria in Kajo Keji county, Sudan.

To provide advice on the rational use of antimalarial drugs, Médecins Sans Frontières conducted a randomized, an open label efficacy study in Kajo Keji, an area of high transmission of malaria in southern Sudan. The efficacy of chloroquine (CQ), sulphadoxine-pyrimethamine (SP) and amodiaquine (AQ) were measured in a 28-day in vivo study, with results corrected by PCR genotyping. Of 2010 children screened, 115 children aged 6-59 months with uncomplicated Plasmodium falciparum malaria were randomized into each group to receive a supervised course of treatment. Of these, 114, 103 and 111 were analysed in the CQ, SP and AQ groups, respectively. The overall parasitological failure rates at day 28 were 93.9% [95% confidence interval (CI) 87.3-97.3] for CQ, 69.9% (95% CI 60.0-78.3) for SP, and 25.2% (95% CI 17.7-34.5) for AQ. These results provide important missing data on antimalarial drug efficacy in southern Sudan. They indicate that none of the drugs could be used in monotherapy and suggest that even in combination with artemisinin, cure rates might not be efficacious enough. We recommend a combination of artemether and lumefantrine as first-line treatment for uncomplicated P. falciparum malaria cases in Kajo Keji county.