1132 infection control & hospital epidemiology october 2015, vol. 36, no. 10


Acinetobacter spp. To minimize false positives, we defined a positive PCR reaction as 4 standard deviations above the cycle threshold of the negative controls. Lacking specific primers for Enterobacter spp. and Enterococcus spp., those organisms could only be detected through culture-based methods. Finally, as our laboratory does not have equipment needed to grow C. difficile, we could not confirmpositive C. difficile PCRresults by culturing. Investigators performing the culture and PCR procedures were blinded to each other’sresults.


300 µl of sterile water, and vortexed for 30 seconds. Afterward, 20 µl of the resulting supernatant was added to 40 µl of Lys and Go solution (Pierce Biotechnology, Rockford, IL), and 2 µl of this solution was ultimately used as template for RT-PCR amplification using the protocols of Clifford et al.16 This 16S rDNA PCR (16S PCR) assay, predicted to detect 94% of all bacterial species,16 can detect as few as 1×102 copies of purified genomicDNAin a reaction. Laboratory tests showed that the 16S PCR assay could detect bacteria on test surfaces treated with a solution containing as few as 3×103 organisms per milliliter. Additional species-specific PCR assays detected C. difficile, S. aureus, E.


coli,P.aeruginosa, K. pneumoniae,and


Genetic Relatedness of MDR-TOs and Correlation of Environmental Bio-Burden with Clinical Infections


All methicillin-resistant S. aureus (MRSA) and MDR K. pneumoniae, E. coli, P. aeruginosa, Enterobacter spp. and A. baumannii isolated from the environment or inpatient infections underwent pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS) was performed as previously described.17 Logistically, the FBCH microbiology department cannot store bacteria pathogens isolated from routine clinical infections longer than 7 days, so archiving was only feasible for MDR isolates. Therefore, only MDR isolates were available for PFGE and WGS comparison. Records for all TO-mediated infections occurring during


the 16-month observation period were extracted from central electronic medical records and laboratory information sys- tems. The number of TO-positive inpatient infections was compared with the incidence of environmental TO detection. Additional details of the methods and statistics sections,


which were performed using the R software package, are available in the supplemental section.18


results Detection of Species-Nonspecific (General) Biomaterial Surface, room, and swab totals. A total of 1,273 high-touch


surfaces were swabbed before and after terminal cleaning during 77 room visits to 49 unique rooms. Of these, 6 rooms had patients on isolation precautions. Altogether, 2,604 surface samples were obtained; only 2,546 paired swab tips were available for analysis, primarily due to damage during transportation. Of these, 3 swab pairs lacked associated DAZO results. Of the 2,546 paired swabs, 47% (1,197) and 42%


(1,069) had cultivable biomaterial and PCR-amplifiable 16S rDNA, respectively. The slightly lower frequency of positive swabs identified by 16S rDNA testing was due to the relative non-selectivity of BAP media. BAP medium accommodates the growth of molds and yeast commonly present in environmental samples; in contrast, the PCR assay is specific to bacteria. Patterns of contamination and removal. Cleaning was


thorough (ie, ≤10% DAZO gel remaining) for 42.6% of surfaces inspected. Cleaning was effective (ie, biomaterial undetected on post-cleaned surface) for 61.6% of surfaces for 16S rDNA, 61.2% for BAP, and 88.7% for MAC (Figure 1). These percentages apply to all room surveys (77 visits to 49 unique rooms). However, cleaning effectiveness varied by assay and surface type; the most and least contaminated surfaces (relative rank order) were very similar, regardless of efficacy measure used. Bathroom sinks were most likely to be contaminated by biomaterial, and side rails were among the least likely to be contaminated. Detection of biomaterial after terminal cleaning differed significantly among the assays used, and as anticipated, PCR assays were more sensitive (16S rDNA vs BAP: P<0.001; 16S rDNA vs MAC: P<0.001; and MAC vs BAP: P<0.001). Terminal cleaning removed cultivable growth on MAC from 71.3% of surfaces that harbored them prior to cleaning. In general, only a limited amount of non–species- specific bacterial 16S rDNA is removed (ie, only 47.2% of PCR-positive precleaned surfaces tested negative after cleaning) (Table 1).


Detection of Specific TO Species Types, ratios, and assay correlation of species-specific TOs.


More than 80 different species of cultivable aerobic bacteria were identified, with 10 constituting >55% of all organisms detected (Supplemental Table 1). Coagulase-negative Staphylococcus spp. (30.2%) and Acinetobacter spp. (12.6%) were the 2 most common genera present. MDR-TOs were rarely detected (ie, 3 of 2,546 swabs or 0.1%). The incidence of TO-positive swabs (≥1 TO detected) was 3.3% (84 of 2,546) using the culture method and 4.1% (104 of 2,546) using the PCR method (Table 2). On 1,273 surfaces swabbed, 85 distinct TOs were detected using the culture method and 106 were detected by PCR (Tables 3 and 4). There was no significant discord between TO detection by culture and PCR, suggesting that results from the 2 methods are comparable. On cleaned surfaces, detection of TOs by culturing correlated well with detection using species-specific PCR. The Pearson correlation coefficient for rates of detection by the 2 methods was 0.56 (P=.00947 for a 1-tailed test). The relative order of surfaces, from most to least contaminated, as determined by culturing and PCR was also similar, with a Spearman’s ρ of 0.58 (P=.00688 for a 1-tailed test). Prior to cleaning, however, TO detection by the 2 methods were not strongly correlated (r=0.39; P=.068). Overall location of TO contamination. TOs were found in 35 room surveys by culturing and 51 room surveys by PCR


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116  |  Page 117  |  Page 118  |  Page 119  |  Page 120  |  Page 121  |  Page 122  |  Page 123  |  Page 124  |  Page 125  |  Page 126  |  Page 127  |  Page 128  |  Page 129  |  Page 130  |  Page 131  |  Page 132  |  Page 133  |  Page 134  |  Page 135  |  Page 136  |  Page 137  |  Page 138  |  Page 139  |  Page 140