Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

Colonization with extended spectrum beta-lactamase and carbapenemases producing Enterobacteriaceae among hospitalized patients at the global level: A systematic review and meta-analysis.

BACKGROUND: Gut commensal bacteria can mediate resistance against pathogenic bacteria. However, exposure to antibiotics and hospitalization may facilitate the emergence of multidrug resistant bacteria. We aimed to conduct a systematic review and meta-analysis to provide comprehensive evidence about colonization rate of extended spectrum beta-lactamase and carbapenemases producing Enterobacteriaceae. METHOD: We used PubMed, Google Scholar and Web of Science data bases to search studies from January 1, 2016 to August10, 2022 about colonization rate of extended spectrum beta-lactamase and carbapenemase producing Enterobacteriaceae. Data were extracted from eligible studies and analyzed using Stata version 16 software. The quality of included studies was assessed using the Joanna Briggs Institute Critical Appraisal tools, and publication bias was assessed using funnel plot and eggers test. RESULTS: We identified 342 studies from the comprehensive data search and data were extracted from 20 studies. The pooled estimate of extended spectrum beta-lactamase and carbapenemase producing Enterobacteriaceae were 45.6%(95%CI: 34.11-57-10) and 16.19% (95% CI: 5.46-26.91) respectively. The predominant extended spectrum beta-lactamase producers were E. coli,32.99% (95% CI: 23.28-42.69) and K. pneumoniae, 11.43% (95% CI:7.98-14.89). Prolonged hospitalization was linked to carbapenemase producing Enterobacteriaceae colonization with the odds of 14.77 (95% CI: -1.35-30.90) at admission and 45.63 (95% CI: 0.86-92.12) after ≥7 days of admission. CONCLUSION: The pooled estimate of extended spectrum beta-lactamase and carbapenemase producing Enterobacteriaceae were high. This indicates the need for strong mitigation strategies to minimize the spread of multidrug-resistant bacteria at the healthcare facilities.

Novel Escherichia coli-Infecting Bacteriophages Isolated from Uganda That Target Human Clinical Isolates.

Background: The antimicrobial resistance catastrophe is a growing global health threat and predicted to be worse in developing countries. Phages for Global Health (PGH) is training scientists in these regions to isolate relevant therapeutic phages for pathogenic bacteria within their locality, and thus contributing to making phage technology universally available. Materials and Methods: During the inaugural PGH workshop in East Africa, samples from Ugandan municipal sewage facilities were collected and two novel Escherichia coli lytic phages were isolated and characterized. Results: The phages, UP19 (capsid diameter ∼100 nm, contractile tail ∼120/20 nm) and UP30 (capsid diameter ∼70 nm, noncontractile tail of ∼170/20 nm), lysed ∼82% and ∼36% of the 11 clinical isolates examined, respectively. The genomes of UP19 (171.402 kb, 282 CDS) and UP30 (49.834 kb, 75 CDS) closely match the genera Dhakavirus and Tunavirus, respectively. Conclusion: The phages isolated have therapeutic potential for further development against E. coli infections.

Nasopharyngeal Staphylococcus aureus colonization among HIV-infected children in Addis Ababa, Ethiopia: antimicrobial susceptibility pattern and association with Streptococcus pneumoniae colonization.

Background: Staphylococcus aureus and Streptococcus pneumoniae are common inhabitants of the nasopharynx of children. HIV-infected children have higher risk of invasive diseases caused by these pathogens. With widespread use of pneumococcal conjugate vaccines and the emergence of methicillin-resistant S. aureus , the interaction between S. aureus and S. pneumoniae is of a particular significance. We sought to determine the magnitude of colonization by methicillin-sensitive and -resistant S. aureus and colonization by S. pneumoniae ; associated risk factors and antimicrobial susceptibility pattern among HIV-infected children in Addis Ababa, Ethiopia. Method: A prospective observational study was conducted among 183 HIV-infected children at ALERT hospital Addis Ababa, Ethiopia from September 2016 to August 2018. S. aureus and S. pneumoniae were identified using standard bacteriological techniques, antimicrobial susceptibility testing was performed on S. aureus and screening for methicillin resistance was carried out by amplifying the mecA gene. Risk factors were analysed by using binary logistic regression. Results: The prevalence of nasopharyngeal S. aureus , MRSA and S. pneumoniae colonization were 27.3, 2.7 and 43.2 %, respectively. Multivariable analysis indicated an inverse association between S. aureus and S. pneumoniae nasopharyngeal colonization [aOR, 0.49; CI, (0.24, 0.99); P=0.046]. The highest level of resistance in both methicillin-sensitive S. aureus (MSSA) and MRSA was observed against tetracycline. Conclusions: . We found an inverse association between S. aureus and S. pneumoniae colonization among HIV-infected children. Continued assessment of the impact of pneumococcal conjugate vaccines and antiretroviral therapy on nasopharyngeal bacterial ecology is warranted.

Mortality among non-severely under nourished children with pneumonia globally: protocol for a systematic review and meta-analysis.

Background: Pneumonia remains the commonest cause of ill health and mortality among children worldwide. Severe undernutrition increases the mortality risk among children with pneumonia. While children with pneumonia are at increased risk of developing malnutrition, the impact of pneumonia on mortality and nutritional status of non-severely undernourished children is not well described. The impact of nutritional supplementation on mortality and nutritional status in this population is not well understood. This review will collate available evidence on the all-cause mortality and anthropometric indices outcomes following pneumonia, as well as the impact of nutritional supplementation on mortality and anthropometry among non-severely malnourished children with pneumonia. Methods: The review will be done using a priori criteria developed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. Data will be obtained from data bases, grey literature, and bibliographies. An experienced librarian will conduct article search in PUBMED, MEDLINE, EMBASE, Web of Science, Google scholar, and Scopus. Retrieved articles will be entered in Endnote ver 9.0, duplicates removed, and transferred to Epi-reviewer for screening and data abstraction. Risk of bias in the included articles will be assessed using standard criteria. Heterogeneity will be assessed using I 2-statistic and sub-group analysis will be done. Data will be analysed using both narrative and quantitative synthesis. Quantitative synthesis will be done using DeSimonian and Laird Random-effects model in STATA ver 15.0. Conclusions: The results will provide baseline information about the mortality and anthropometric outcomes of pneumonia among non-severely malnourished children as well as the potential effect of nutritional supplementation on these outcomes. This will provide a basis to explore the potential for nutritional supplementation improving clinical outcomes like mortality and occurrence of severe acute malnutrition among children with severe pneumonia worldwide. Registration: The review has been registered in PROSPERO (CRD42021257272; 15 July 2021).

Whole-Genome Sequences of SARS-CoV-2 Isolates from Ethiopian Patients.

Three complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Ethiopian patients were compared with deposited global genomes. Two genomes belonged to genetic group 20A/B.1/GH, and the other belonged to genetic group 20A/B.1.480/GH. Enhancing genomic capacity is important to investigate the transmission and to monitor the evolution and mutational patterns of SARS-CoV-2 in this country.

Phenotypic and Molecular Characterization of Penicillin and Macrolide-Resistant Streptococcus pneumoniae Serotypes Among Pediatric Patients in Addis Ababa, Ethiopia.

BACKGROUND: In several countries, introduction of the pneumococcal conjugate vaccine (PCV) has led to a decline in antimicrobial-resistant pneumococcal disease but has also resulted in a concomitant increase in antimicrobial-resistant, non-vaccine serotypes of Streptococcus pneumoniae. We sought to determine the magnitude of penicillin and macrolide resistance among pneumococcal serotypes and the mechanisms of macrolide resistance in Ethiopia, 5 years after the introduction of PCV10 in the country. METHODS: Susceptibility to penicillin and erythromycin of 119 pneumococcal isolates collected from pediatric patients aged 0-15 years in Addis Ababa, Ethiopia, was tested using disc diffusion, and minimum inhibitory concentration (MIC) was also determined by Etest. Pneumococcal serotypes were determined by sequencing the cpsB gene and using Quellung reaction. Polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analysis were used to detect and differentiate the macrolide resistance genes erm(B), mef(A), and mef(B). RESULTS: Among the 119 isolates, 2.5% (3/119) were resistant to penicillin, while 58% (69/119) were intermediate. Resistance to erythromycin was observed in 33.6% (40/119) of the isolates with the highest level of resistance among isolates from middle ear discharge, i.e., 53.3% (8/15). Half (19/40) of the erythromycin resistant isolates were serotype 19A and among serotype 19A isolates, the majority i.e., 54.3% (19/35) were resistant to erythromycin. The most common macrolide resistance determinant was mef(E) with a prevalence of 50% (20/40). CONCLUSION: Five years after introduction of PCV10 in Ethiopia, we observed that the prevalence of penicillin-resistant S. pneumoniae was low. However, there was a high level of macrolide resistance which was mostly in serotype 19A, and the resistance was mainly mediated by efflux pumps. Introduction of PCV13 (which covers serotype 19A) would significantly improve coverage of the macrolide-resistant serotypes. Continued surveillance of pneumococcal serotype distribution and their antibiotic resistance pattern in Ethiopia is warranted.

Etiology, antibiotic susceptibility and prognostic factors of pediatric community-acquired sepsis in Addis Ababa, Ethiopia.

INTRODUCTION: There is a scarcity of data on pediatric community-acquired sepsis (CAS) in Ethiopia. We sought to determine the etiology, role of Streptococcus pneumoniae, antibiotic susceptibility pattern, and prognostic factors in children with CAS in Addis Ababa, Ethiopia. METHODOLOGY: A prospective cross-sectional study of 101 children aged 0-15 years with suspected CAS was performed at two major hospitals in Addis Ababa, Ethiopia. Blood culture, antibiotic susceptibility testing, amplification of the autolysin (lytA) gene and typing S. pneumoniae by sequencing and Quellung reaction were performed. Data were analyzed using descriptive statistics and logistic regression. RESULTS: The prevalence of culture-positive CAS was 18.81% (19/101). S. pneumoniae (21.1%) (Serotypes 19A (n = 2), 33C and 12F) and Klebsiella pneumoniae (21.1%) were the most common causes of CAS. Half of K. pneumoniae isolates were resistant to gentamicin and ceftriaxone. The most common antibiotics used for treatment were a combination of ampicillin with gentamicin (47.5%). The presence of lower respiratory tract infections (LRTIs) in the preceding 3 months was an independent predictor associated with culture-proven sepsis (adjusted odds ratio (AOR), 7.02; 95% confidence interval (CI), 1.42 - 34.64; P = 0.02). The case-fatality rate was 11.9% (12/101). Presence of underlying comorbidity (AOR, 6.8; 95% CI, 1.59-28.7; P = 0.009) was an independent predictor of mortality. CONCLUSIONS: S. pneumoniae and K. pneumoniae were the major causes of CAS and there was a substantial level of antibiotic resistance. Presence of LRTIs in the preceding 3 months was a predictor of culture-proven CAS whereas underlying comorbidity was a predictor of mortality.

Pneumococcal serotype 19A is the major cause of pediatric acute otitis media with ruptured tympanic membrane in Addis Ababa, Ethiopia, 5 years after the introduction of the ten-valent pneumococcal conjugate vaccine.

INTRODUCTION: In Ethiopia, there is a lack of data on pneumococcal serotypes causing acute otitis media (AOM) in children. We aimed to study the etiology, pneumococcal serotypes and antimicrobial resistance patterns of isolates from children with AOM with spontaneous perforation of the tympanic membrane (SPTM). METHODS: We carried out a prospective observational study in children with AOM with SPTM, aged 0-15 years in Addis Ababa, Ethiopia. Middle ear fluid was collected using sterile swabs, cultured and antibiotic susceptibility testing was performed. Serotypes of Streptococcus pneumoniae were determined by sequencing the cpsB gene and by the Quellung reaction. RESULTS: A total of 55 children were enrolled. Out of 55 samples that were cultured, 52 (94.5%) were culture positive for a total of 66 bacterial species, and 56.4% (31/55) samples were positive for 41 (62.1%) known pathogenic bacterial species. The most common pathogenic bacterial isolates were S. pneumoniae (36.6%), Staphylococcus aureus (19.5%), Streptococcus pyogenes (14.6%) and Haemophilus influenzae (12.2%). Serotype 19A (73.3%) was the predominant pneumococcal serotype. There was a high rate of non-susceptibility to penicillin (86.6%) and trimethoprim/sulfamethoxazole (80%) among pneumococcal isolates. Out of 21 different isolates tested for amoxicillin susceptibility, 15 (71.4%) were resistant. CONCLUSIONS: Pneumococcal serotype 19A was the predominant cause of AOM with SPTM in children in Addis Ababa, Ethiopia, 5 years after introduction of PCV10. There was a high rate of resistance to commonly prescribed antibiotics. The study highlights the need for wide scale surveillance of the etiology and antimicrobial susceptibility of AOM in Ethiopian children.

Pneumococcal Carriage, Serotype Distribution, and Risk Factors in Children With Community-Acquired Pneumonia, 5 Years After Introduction of the 10-Valent Pneumococcal Conjugate Vaccine in Ethiopia.

BACKGROUND: There is a scarcity of data on pneumococcal serotypes carried by children in Ethiopia. We studied pneumococcal nasopharyngeal carriage rate, serotypes, and risk factors among children with community acquired pneumonia (CAP). METHODS: A prospective observational cohort study was performed in children with CAP, aged 0-15 years, in 2 pediatric emergency departments in Addis Ababa, Ethiopia. Nasopharyngeal swabs were cultured, and serotypes of Streptococcus pneumoniae were determined by sequencing the cpsB gene and by the Quellung reaction. Risk factors were analyzed by using binary logistic regression. RESULTS: Nasopharyngeal swabs were collected from 362 children with CAP. Pneumococcal carriage rate was 21.5% (78 of 362). The most common serotypes were 19A (27%), 16F (8.5%), and 6A (4.9%). In addition, 8.5% of the pneumococcal isolates were nontypeable. In bivariate analysis, children with a parent that smokes were more likely to carry pneumococci (crude odds ratio, 3.9; 95% confidence interval [CI], 1.2-12.3; P = .023) than those with parents that do not smoke. In multivariable analysis, living in a house with ≥2 rooms (adjusted odds ratio [AOR], 0.48; 95% CI, 0.28-0.82; P = .007) and vaccination with ≥2 doses of 10-valent pneumococcal conjugate vaccine (PCV10) (AOR, 0.37; 95% CI, 0.15-0.92; P = .033) were protective of pneumococcal carriage. CONCLUSIONS: Five years after introduction of PCV10 in Ethiopia, the vaccine-related serotype 19A was predominant in the nasopharynx of children with CAP. Continued evaluation of the direct and indirect impact of PCV10 on pneumococcal serotype distribution in Ethiopia is warranted.

Bacteremic Community-Acquired Pneumonia in Ethiopian Children: Etiology, Antibiotic Resistance, Risk Factors, and Clinical Outcome.

BACKGROUND: Community-acquired pneumonia (CAP) remains a leading cause of morbidity and mortality. We sought to determine the magnitude, etiology, and risk factors of CAP in children 5 years after introduction of pneumococcal conjugate vaccine (PCV) 10 in Ethiopia. METHODS: We conducted a prospective observational study on the bacterial etiology and risk factors of CAP among children aged 0-15 years in 2 pediatric emergency departments in Addis Ababa, Ethiopia. Blood culture, antibiotic susceptibility testing, and amplification of pneumococcal lytA and cpsB genes were performed. Serotypes of Streptococcus pneumoniae were determined by Quellung reaction and sequencing the cpsB gene. RESULTS: Out of 643 eligible children, 549 were enrolled. The prevalence of bacteremic pneumonia was 5.6%. Staphylococcus aureus (26.5%) was the predominant pathogenic species, followed by Enterococcus faecium (11.8%), Escherichia coli (11.8%), and Klebsiella pneumoniae (11.8%). In univariate analysis, parental smoking and nonvaccination with PCV10 were associated with bacteremic CAP. In multivariable analysis, female sex (adjusted odds ratio [aOR], 2.3; 95% confidence interval [CI], 1.1-4.9), weight-for-age z-score (WAZ) <-2 SDs (aOR, 2.2; 95% CI, 1.1-4.8), and lower chest indrawing (aOR, 0.44; 95% CI, 0.2-0.95) were independently associated with bacteremic CAP. The overall in-hospital case fatality rate was 2.37% (13/549), and WAZ <-3 SDs (OR, 13.5; 95% CI, 3.95-46.12) was associated with mortality. CONCLUSIONS: Five years after the introduction of PCV10 in Ethiopia, S. aureus was the main cause of bacteremic CAP in children, the contribution of S. pneumoniae was low, and there was a high level of antibiotic resistance among isolates.