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.

Genomic evaluation of novel Kenyan virulent phage isolates infecting carbapenemase-producing Klebsiella pneumoniae and safety determination of their lysates in Balb/c mice.

This study aimed to evaluate the genomic features of novel Kenyan virulent phage isolates infecting carbapenemase-producing Klebsiella pneumoniae and to determine the safety of their lysates using mice model in a preclinical study. The genomics showed that the Klebsiella phages vB_KpM_CPRSA and vB_KpM_CPRSB belonged to the genus Slopekvirus with a similarity index of less than 92% compared to the most closest relative species. Their genomes did not contain antimicrobial resistance and toxin genes. Then endotoxin levels in the Klebsiella phage lysates were statistically significant (p value ˃ 0.05). The serum activities of aspartate aminotransferase, alanine aminotransferase and urea in the group of balb/c mice injected with bacteriophage lysates through the intravenous route were higher compared to that of the intranasal route. Unexpectedly, there was mild congestion of the central veins of kidneys and liver without damage to renal tubules and hepatocytes and a lack of physical discomfort and pain in the mice. Our study isolated and characterised Klebsiella phages against carbapenem-resistant K. pneumoniae, which are promising therapeutic agents for the treatment of respiratory tract infections using the topical mode of administration as the preferred route of bacteriophage delivery.

Glucose-6-phosphate dehydrogenase deficiency allelic variants and their prevalence in malaria patients in Eritrea.

INTRODUCTION: Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy with a relatively high frequency in malaria-endemic regions. In Eritrea, there is scanty knowledge of G6PD deficiency. The aim of the study was to characterize and determine the prevalence of four common G6PD allelic variants. METHODS: Three hundred and fourteen dried blood spot samples from unrelated microscopically diagnosed malaria patient Eritrean ethnic groups living in five zobas (regions) of Eritrea were analysed by PCR-RFLP method to identify the G6PD B, G6PD A (A376G), G6PD A-(G202A), and G6PD Mediterranean (C563T) variants. To confirm the RFLP results, samples positive for A376G but negative for G202A variants were subjected to Sanger sequencing and a subset of PCR products (exon 5) directly sequenced to identify A376G and other mutations. RESULTS: For G6PD genotyping, G6PD B was detected in 87.5% and A376G detected in 12.5% of malaria patients, whereas G202A and C563T were absent. Bivariate Statistical analysis showed a statistically significant association between G6PD genotypes and zoba (P < 0.004 < 0.05). Sequencing revealed the expected A376G variant. In exon 5, four common (A376G) mutations, three uncommon mutations rs782669677 (535G→A) and one potentially new mutation (451G→C), relative to the reference, mRNA NM_001042351 were detected. Bioinformatic analysis of these mutations' potential functional impact suggests minimal effect on protein function. CONCLUSION: This is the first report indicating that G6PD B and G6PD A genotypes are prevalent in Eritrea. Similar findings were reported in neighboring countries. Further studies including phenotype analysis are needed to corroborate the observed results.

Effectiveness of sequential v. standard triple therapy for treatment of Helicobacter pylori infection in children in Nairobi, Kenya.

BACKGROUND: Once the diagnosis of Helicobacter pylori is confirmed, treatment requires at least two antibiotics and an acid inhibitor for a minimum of seven days. Unfortunately, treatment failures are being frequently reported. Treatment regimens that include sequential administration of antibiotics with acid inhibitors have been developed to try and increase the rate of eradication. OBJECTIVE: To determine the effectiveness of a novel 10-day sequential therapy compared with the standard 10-day triple therapy for treatment of H. pylori infection in children. METHODS: A double-blinded, randomised, controlled trial was conducted. Children under the age of 16 years with recurrent abdominal pain associated with dyspepsia and diagnosed with H. pylori by histology were randomly allocated either to a 10-day sequential treatment regimen or to a 10-day conventional triple therapy. Analysis of the outcome of this study was based on clinical improvement and confirmed H. pylori eradication based on stool H. pylori antigen detection and/or repeat endoscopy. RESULTS: Of the 71 patients included in the analysis, 45 (63.4%) were given the 10-day conventional treatment while 26 (36.6%) received the 10-day sequential treatment. There was no difference in clinical improvement after treatment in the two therapies. However, there was a significant difference in the eradication of H. pylori between the conventional v. sequential regimens (48.8% v. 84.6%, respectively; p=0.02, odds ratio 0.19). CONCLUSION: The sequential treatment had a significantly higher H. pylori eradication rate than the conventional treatment.

Prevalence of Helicobacter Pylori Caga and Vaca Allelic Variants and Associated Disease Outcomes in Kenyan Patients with Dyspepsia

Although Helicobacter pylori has been linked to various gastric disorders in Western countries and Asia; its aetiopathological role in African populations is controversial. The aim of this study was to investigate the role of H. pylori and its virulence genotypes in gastrointestinal diseases in Kenyan patients with dyspepsia. Gastric biopsy specimens were obtained for DNA isolation and histopathological analysis. Amplification was performed using specific oligonucleotide primers. H. pylori positivity was determined by H. pylori stool antigen test; rapid urease test; and histology and molecular diagnostic tools. H. pylori was detected with high frequency in patients with gastritis; peptic ulcer disease (PUD) and gastro-oesophageal reflux disease. This implies a significant risk of the development of these pathologies (p-value = 0.0000 in all cases). H. pylori strains with cagA occurred more frequently in PUD (65.2). vacA s1a genotype appeared to play a more significant pathological role (82.6 PUD) than the other variants (p-value = 0.0142). The prevalence of vacA m1 was significantly higher in gastritis cases (p-value = 0.0253). vacA m2 was found to be significantly associated with gastritis (p-value = 0.0253). This finding may point to the fact that H. pylori vacA m1 and vacA m2 are independently associated with an increased risk for gastritis. Indications are that H. pylori prevalence in Kenya may be declining. The independently occurring H. pylori genotypes; as opposed to simultaneous carriage; could be the reason for the low distribution of H. pylori pathologies

IL-1B-511 Allele T and IL-1RN-L/L Play a Pathological Role in Helicobacter Pylori (H. Pylori) Disease Outcome in the African Population.

BACKGROUND: Many of the pathogenic effects of Helicobacter pylori infection are related to chronic active inflammation, which is controlled and maintained by the complex interplay of pro-inflammatory and anti-inflammatory mediators. Pro-inflammatory genetic polymorphisms tend to increase the risk of development of gastric cancer. In Africa, the data are scarce regarding the effects of these polymorphisms on gastric pathology. The objective of this study is therefore to investigate the pro-inflammatory genetic polymorphisms and their role in H. pylori-related gastric disorders in a select African population. METHODS: This cross-sectional prospective study recruited six hundred and ninety six adult subjects with a history of uninvestigated dyspepsia. The H. pylori status was determined by tissue Giemsa staining, Rapid Urease Test (RUT), H. pylori stool antigen test (HpSAT), and PCR using the 16s-rRNA gene. The polymorphisms in IL-1B (511 C/T), TNF-A (_308 G/A) and IL-1RN were assessed by the PCR-restricted fragment length polymorphism (RFLP). RESULTS: H. pylori was significantly associated with gastric pathologies investigated (P = 0.0000). Heterozygous allele TC of IL-1 ß -511 was significantly associated with H. pylori infection (p = 0.003815). Similarly, allele IL-1 RN*2/2 and allele IL-1 RN-L/L were associated with H. pylori infection (p = 0.0025 and p = 0.0203). Allele T of IL-1 ß -511 and IL-1 RN-L/L are more frequent in H. pylori associated gastric pathologies in this series. CONCLUSION: Allele T of IL-1 ß -511 and long allele IL-1 RN-L/L play a role in H. pylori disease in this population.

A situational analysis of antimicrobial drug resistance in Africa: are we losing the battle?

BACKGROUND: The first arrival of a sizable shipment of penicillin at the North African Theatre of Operations for USA military use in 1943 was a landmark that turned a new chapter of antibiotic use in Africa. Over the past decade the expansion of resources and the technological advances have meant that much larger quantities of drugs are available in developing countries than ever before. As a result, many more individuals are receiving necessary treatment or therapy than just ten years ago. This very welcome event is accompanied by the terrible irony that increases in drug availability and use can promote drug resistance and render the same life-saving drugs ineffective. METHODS: The study focused on bacterial pathogens. One hundred and three relevant literatures were identified from the PubMed online database. The coverage included research articles concerning antimicrobial resistance involving subjects of an African country. RESULTS: Resistant bacteria are on a war path and evidently have acquired an edge over us. Our actions are evidently fuelling the resistance. The indiscriminate use of antibiotics in humans and livestock, wrong and substandard prescriptions by unqualified 'medical personnel' together with poor diagnosis or lack of it are all adding fuel to the already fired train of resistant microbes. CONCLUSION: To win the war and turn tables as we did with the discovery of penicillin and other antimicrobials in the 1940s, then we must all act now. Antimicrobial stewardship programs-Education, training of laboratory personnel and investment in laboratory infrastructure development are desirable in these situations.

Helicobacter pylori: prevalence and antibiotic susceptibility among Kenyans.

BACKGROUND: Helicobacter pylori infection in Kenya is staggeringly high. Evidence links infection of the gastric mucosa by H. pylori with subsequent development of gastric pathologies. AIM: We investigated the prevalence of H. pylori in dyspeptic patients, its relationship with gastric pathologies, and associated antibiotic susceptibility profiles, and compared two media to find the appropriate medium that enhances growth and expedites culture and isolation. METHODS: Rapid urease and histological tests were used to screen for H. pylori. Culture was performed to test sensitivity and evaluate media. Selective and nutritional supplements were added to culture media (Colombia blood agar and brain-heart infusion agar) for growth enhancement. E-test strips for metronidazole, amoxicillin and clarithromycin were used for susceptibility testing. RESULTS: The prevalence of H. pylori infection in children was 73.3%, and 54.8% in adults. All the H. pylori investigated in this study were largely sensitive to clarithromycin (100%, minimum inhibiting concentration (MIC) <2 microg/ml), amoxicillin (100%, MIC <2 microg/ml) and metronidazole (95.4%, MIC <8 microg/ml). There was, however, occasional resistance to metronidazole (4.6%, MIC >8 microg/ml). Both Colombia blood and brain-heart infusion agar, with the supplements, effectively supported H. pylori growth. Growth was achieved in an average of 36 hours for primary isolations and 24 hours for subcultures. CONCLUSION: The media described here reduce the time required to culture and isolate bacteria and perform susceptibility testing. Despite the high prevalence of H. pylori infection, the associated pathology is low and does not parallel H. pylori prevalence in the population.