Study Isolates.

<p>Study isolates (plus WC-31, which was isolated from a patient on Hospital Day 1 after transfer from an outside hospital). <b>Legend WC</b> = Weill Cornell; <b>PT</b> = Patient age and gender; <b>Date:</b> All dates are from 2008; <b>Floor:</b> Each letter represents a different hospital floor; Antimicrobial susceptibility to the various agents was determined following CLSI guidelines, or the Mean Inhibitory Concentration is reported directly for polymyxin B and tigecycline (µg/mL): A/S: ampicillin-sulbactam, Carb: imipenem or meropenem; Cef: cefepime; Gent; gentamicin; Amik: amikacin; S/T: sulfamethoxazole-trimethoprim; Levo: levofloxacin; PM: polymyxin B; Tig: tigecycline; Source: Resp = respiratory. Susceptibility interpretation based on 2008 CLSI cutoff points.</p

Integron Content in Select Study Isolates.

<p>Integron PCR gene products of WC 13–24 are shown. The block arrow marks the 550 base pair PCR product of the prevalent strain <i>A. baumannii</i>. The water control is labeled “neg.” The cartoon shows the layout of a typical Class I integron. The PCR primers are presented by CS-F and CS-R.</p

Rep-PCR Analysis of Study Isolates.

<p>Isolates with more than 90% similarity were considered related. Study isolates (WC 1–30) are shown, plus one isolate (WC-31) isolated on hospital day 1 after transfer from an outside hospital. Results show the majority of the study isolates are closely related.</p

Details of viral cultures and nucleic acids used in the study: RNA viruses.

<p>* The exact sequence of the stock used is not known. The GenBank sequence of the parent strain is indicated.</p><p>Details of viral cultures and nucleic acids used in the study: RNA viruses.</p

The limit of detection of the PCR/LDR/Universal Array assay using <i>in vitro</i> transcribed RNA or whole virus.

<p>ND = Not determined</p><p>Pfu/ml = plaque forming units/ml</p><p>ffu/ml = focus forming units/ml.</p><p>^a PCR/LDR was performed on cloned RNA fragments for all viruses except EBOV and DENV while</p><p>^bdilutions of culture supernatants were used for the latter two viruses. <i>Zaire ebolavirus</i>’95 was used for determination of LOD.</p><p>The limit of detection of the PCR/LDR/Universal Array assay using <i>in vitro</i> transcribed RNA or whole virus.</p

Comparison of universal array profile of viral RNA/DNA tested for the corresponding zip-codes.

<p>Normalized average signal intensity for the zip-codes assigned to each virus are presented. The color bars are the signals obtained with the indicated virus (positives). The black bars are the signals produced by the other ten viruses. A signal was considered positive if the intensity of the zip-code spot was at least 10-fold higher than the uniform background level of fluorescence of the array slide. Although a few other viruses produced low-level positive signals for zip18, this did not result in any false positive results since positive signals from at least two addresses was required for a positive identification. In the future, this issue would be rectified by switching to a different zip-code. The average signal intensity for the positives ranged from 31.2 to 123.4, depending on the virus. The average signal intensity for the negatives ranged from 0.3 to 6.2, thus they were not considered positive signals.</p

Schematic of the PCR/LDR assay for detection of VHF viruses.

<p>For each virus (ebolavirus is shown as a representative virus), 1–2 different regions are amplified by RT-PCR using forward and reverse primers, each with minimal degeneracy and all containing universal tails to prevent the formation of primer dimers. Cy-3 labeled downstream LDR primers and single base-discriminating upstream primers with unique zip-code complements (20-30-mers) are targeted to specific sequences/SNPs within the PCR amplicons. Ligation of two adjacent oligonucleotides annealed to a complementary DNA target occurs in the presence of thermostable ligase only if the nucleotides are perfectly matched at the junction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref055" target="_blank">55</a>]. The zip-code complements on the 5’ end of fluorescently labeled LDR products anneal to specific complementary zip-code addresses on a universal array [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref056" target="_blank">56</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref057" target="_blank">57</a>]. A positive signal on the universal array is detected as a fluorescent spot. Primers for the ligation reaction were designed targeting 2 or 3 areas within each PCR amplicon. Each virus could produce a maximum of six ligation products, except for VAR and VACC, for which there were a maximum of 5 each. The detection of 2 or more ligation products was required for the detection and identification of a virus. Representative arrays that detect and identify <i>Ebola Zaire</i>, Lassa and Yellow fever viruses are shown.</p