Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n = 300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospitalacquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.
The authors tested an alternative method for CD4 and CD8 T lymphocytes enumeration, the immunoalkaline phosphatase method (IA), in three African countries and in Denmark. The IA determinations from 136 HIV antibody positive and 105 HIV antibody negative individuals were compared to the corresponding results obtained by flow cytometry (FC) performed in the respective countries. The authors found good correspondence between the two methods for measurements of CD4 and CD8 T lymphocytes independent of serological status and geographical site. However, the CD4 and CD8 T lymphocyte values obtained by the two methods are not interchangeable as IA compared to FC consistently gives higher percentage of CD4 T lymphocytes, and lower percentages of CD8 T lymphocytes. Mean differences between the two methods did not differ between the three African countries indicating that the IA method provides systematic results. Replicate measurements suggested good correspondence between results obtained by IA. By using an IA level of < 300 CD4 T lymphocytes/ml, the sensitivity was 81% and specificity 96% for detecting an FC level of < 200 CD4 T lymphocytes/ml. Using an IA level of < 20% CD4 T lymphocytes, the sensitivity was 89% and specificity 95% for detecting an FC level of <14% CD4 T lymphocytes, The FC and IA methods had the same internal correspondence between low absolute CD4 T cell counts and low CD4 percentages; the sensitivity and specificity for detecting a low absolute CD4 T cell count with a low CD4 percentage was 92% and 68% for FC and 91% and 73% for IA, respectively. The IA method is 10-fold cheaper than FC, is independent of advanced laboratory facilities, and does not need immediate processing of samples as blood smears can be stored for long periods. The IA method is therefore suitable for use in areas with limited resources and laboratory facilities where there is a need for immunological surveillance in hospital or community studies.
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