Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study.

BACKGROUND: Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence. METHODS: We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models. RESULTS: Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), "K. quasivariicola" (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and "K. quasivariicola" (odds ratio = 0.17, p < 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53]). CONCLUSIONS: Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options.

Bicycle-vehicle interactions at mid-sections of mixed traffic streets: Examining passing distance and bicycle comfort perception.

This paper studies the relevant factors in mixed urban traffic that may impact the lateral spacing between bicycles and vehicles (passing distance, PD), and their resulting effect on a bicyclists' comfort based on a study of six sites in Ottawa, Canada. The observations are: [i] the average position of bicycles from the curb is 0.57m, and lesser (i.e. 0.35m) in the presence of parking; [ii] 90% of passes exceed 1.23m; [iii] PD is positively correlated with motor vehicle speed, lane width, and bicycle position from adjacent curb edge line, whiles inversely correlated to ambient traffic density and bicycle speed; [iv] motor vehicle speed has the highest prediction of PD variability; [v] PD and ambient traffic density (ATD) are found to be the most important factors to a bicyclists' comfort perception (BCP). Two linear regression models for PD and BCP were developed and significant variables are identified as: motor vehicle speed, bicycle speed, ATD, number of lanes, and lane width. The presence or absence of a grade slope is found to be significant to the PD model and not to BCP. The models both exhibit limited predictive ability, however residual plots and significance of included variables are indicative of correct assumptions for the models. It is recommended that speed calming, sharrows, road signs instructing road sharing, and educating road users against "dooring" crashes be considered in improving road sharing, especially for narrow lanes (i.e. less than 3.6m) and lanes wider than 4.5m. It is also prudent for designers to avoid installing parking zones on narrow shared roads.

Comparison of biomarker based Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and conventional methods in the identification of clinically relevant bacteria and yeast.

BACKGROUND: MALDI-TOF MS is an analytical method that has recently become integral in the identification of microorganisms in clinical laboratories. It relies on databases that majorly employ pattern recognition or fingerprinting. Biomarker based databases have also been developed and there is optimism that these may be superior to pattern recognition based databases. This study compared the performance of ribosomal biomarker based MALDI-TOF MS and conventional methods in the identification of selected bacteria and yeast. METHODS: The study was a cross sectional study identifying clinically relevant bacteria and yeast isolated from varied clinical specimens submitted to a clinical laboratory. The identification of bacteria using conventional Vitek 2™ automated system, serotyping and MALDI-TOF MS was performed as per standard operating procedures. Comparison of sensitivities were then carried out using Pearson Chi-Square test and p-value of <0.05 was considered statistically significant. Secondary outcomes analyzed included the major and minor error rates. RESULTS: Of the 383 isolates MALDI-TOF MS and conventional methods identified 97.6 and 95.7% (p = 0.231) to the genus level and 97.4 and 88.0% (p = 0.000) to the species level respectively. Biomarker based MALDI-TOF MS was significantly superior to Vitek 2™ in the identification of Gram negative bacteria and Gram positive bacteria to the species level. For the Gram positive bacteria, significant difference was observed in the identification of Coagulase negative Staphylococci (p = 0.000) and Enterococcus (p = 0.008). Significant difference was also observed between serotyping and MALDI-TOF MS (p = 0.005) and this was attributed to the lack of identification of Shigella species by MALDI-TOF MS. There was no significant difference observed in the identification of yeast however some species of Candida were unidentified by MALDI-TOF MS. CONCLUSION: Biomarker based MALDI-TOF MS had good performance in a clinical laboratory setting with high sensitivities in the identification of clinically relevant microorganisms.

Reference intervals for thyroid stimulating hormone and free thyroxine derived from neonates undergoing routine screening for congenital hypothyroidism at a university teaching hospital in Nairobi, Kenya: a cross sectional study.

BACKGROUND: In order to accurately interpret neonatal thyroid function tests (TFTs), it is necessary to have population specific reference intervals (RIs) as there is significant variation across different populations possibly due to genetic, environmental or analytical issues. Despite the importance of RIs, globally there are very few publications on RIs for neonatal TFTs primarily due to ethical and technical issues surrounding recruitment of neonates for a prospective study. To the best of our knowledge, this is the first report from Africa on neonatal RIs for TFTs. METHODS: We used hospital based data largely derived from neonates attending the wellness clinic at the Aga Khan University Hospital Nairobi (AKUHN) where screening for congenital hypothyroidism is routinely done. Specifically we derived age and gender stratified RIs for free thyroxine (fT4) and thyroid stimulating hormone (TSH) which had been analyzed on a Roche e601 analyzer from 2011 to 2013. Determination of reference intervals was done using a non-parametric method. RESULTS: A total of 1639 and 1329 non duplicate TSH and fT4 values respectively were used to derive RIs. There was a decline in TSH and fT4 levels with increase in age. Compared to the Roche RIs, the derived RIs for TSH in neonates aged 0-6 days and those aged 7-30 days had lower upper limits and narrower RIs. The fT4 lower limits for neonates less than 7 days and those aged 7-30 days were higher than those proposed by Roche. There was a significant difference in TSH RIs between male and female neonates aged less than 15 days. No gender differences were seen for all other age stratifications for both TSH and fT4. Appropriate age and gender specific RIs were subsequently determined. CONCLUSION: The AKUHN derived RIs for fT4 and TSH revealed similar age related trends to what has been published. However, the differences seen in upper and lower limits across different age stratifications when compared to the Roche RIs highlight the need for population specific RIs for TFTs especially when setting up a screening programme for congenital hypothyroidism. We subsequently recommend the adoption of the derived RIs by the AKUHN laboratory and hope that the RIs obtained can serve as a reference for the African population.

Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: a cross-sectional study.

BACKGROUND: The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines are the most popular breakpoint guidelines used in antimicrobial susceptibility testing worldwide. The EUCAST guidelines are freely available to users while CLSI is available for non-members as a package of three documents for US $500 annually. This is prohibitive for clinical microbiology laboratories in resource poor settings. We set out to compare antibiotic susceptibility determined by the two guidelines to determine whether adoption of EUCAST guidelines would significantly affect our susceptibility patterns. METHODS: We reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) isolates from an automated microbiology identification system (VITEK-2) at the Aga Khan University Hospital Nairobi's Pathology department. These MICs were then analyzed using both CLSI 2015 and EUCAST 2015 guidelines and classified as resistant, intermediate or susceptible. We compared the susceptibility and agreement between the CLSI and EUCAST categorizations. RESULTS: Susceptibility data from a total of 5165 E. coli, 1103 S. aureus and 532 P. aeruginosa isolates were included. The concordance rates of the two guidelines for E. coli, S. aureus and P. aeruginosa ranged from 78.2 to 100 %, 94.6 to 100 % and 89.1 to 95.5 % respectively. The kappa statistics for E. coli MICs revealed perfect agreement between CLSI and EUCAST for cefotaxime, ceftriaxone and trimethoprim-sulfamethoxazole, almost perfect agreement for ampicillin, ciprofloxacin, cefuroxime, gentamicin and ceftazidime, substantial agreement for meropenem, moderate agreement for cefepime and amoxicillin-clavulanate, fair agreement for nitrofurantoin and poor agreement for amikacin. For S. aureus the kappa statistics revealed perfect agreement for penicillin, trimethoprim-sulfamethoxazole, levofloxacin, oxacillin, linezolid and vancomycin, almost perfect agreement for clindamycin, erythromycin and tetracycline and moderate agreement for gentamicin. For P. aeruginosa the kappa analysis revealed moderate to almost perfect agreement for all the anti-pseudomonal antibiotics. CONCLUSION: The results show comparable antibiotic susceptibility patterns between CLSI and EUCAST breakpoints. Given that EUCAST guidelines are freely available, it makes it easier for laboratories in resource poor settings to have an updated and readily available reference for interpreting antibiotic susceptibilities.

Synthesis of potential pyrimidine derivatives via Suzuki cross-coupling reaction as HIV and kinesin Eg5 inhibitors.

A series of 4-amino-5-((4-chlorophenyl)diazenyl)-6-(alkylamino)-1-methylpyrimidin-2-one derivatives 7-16 were prepared by nucleophilic displacement of 6-chloro-pyrimidine 6 by various amines. 4-Amino-5-((aryl-4-yl)diazenyl)-6-aryl-1-methylpyrimidin-2-one analogs 19-27, as well as 4-amino-5-((aryl-[1,1'-biphenyl]-4-yl)diazenyl)-6-aryl-1-methylpyrimidin-2-one 29-31 and 4-amino-6-aryl-1-methylpyrimidin-2-one 34-34, were synthesized via Suzuki cross-coupling reaction, using Pd(PPh3)4 as a catalyst and arylboronic acids as reagents. All compounds were evaluated for their antiviral activity against the replication of HIV-1 and HIV-2 in MT-4. Compounds 6, 16, 27, and 29 showed a 50% effective concentration of >2.15, >3.03, >2.29, and >1.63 µM, respectively, but no selectivity was observed (selectivity index < 1). Two of the newly synthesized pyrimidines 12 and 29 exhibited moderate kinesin Eg5 inhibition.