1
Evaluation of Curetis UnyveroTM
, a multiplex PCR-based testing system, for 1
rapid detection of bacteria and antibiotic resistance and its impact on the 2
management of severe nosocomial pneumonia 3
4
Wafaa Jamal1,2#, Ebtehal Al Roomi2, Lubna R. AbdulAziz2, Vincent O. Rotimi1,2 5
6
1Department of Microbiology, Faculty of Medicine, Kuwait University and
2Microbiology Unit, 7
Mubarak Al Kabir Hospital, Jabriya, Kuwait 8
9
# Corresponding author: 10
Wafaa Jamal, MD, PhD 11
Department of Microbiology, Faculty of Medicine, 12
Kuwait University, P. O. Box 24923, Safat 13110 13
Kuwait 14
E-mail: [email protected] 15
Tel #: +965 2463 6781 16
Fax #: +965 2533 2719 17
Running title: PCR to detect bacteria and antibiotic resistance 18
19
JCM Accepts, published online ahead of print on 30 April 2014J. Clin. Microbiol. doi:10.1128/JCM.00325-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Summary 20
Healthcare-associated pneumonia due to multi-drug resistant organisms represents a major 21
therapeutic challenge. Unfortunately, therapy is dependent on empirical therapy which often 22
leads to improper and inadequate antimicrobial therapy. A rapid multiplex PCR-based Unyvero 23
Pneumonia Application (UPA) assay that could assist in timely decision making has recently 24
become available. In this study we evaluated the performance of UPA in detecting etiological 25
pathogens and resistance markers in patients with nosocomial pneumonia (NP). The impact of 26
this assay on the management of severe nosocomial pneumonia was also assessed. Appropriate 27
specimens were processed by UPA according to manufacturer’s protocol in parallel with 28
conventional culture method. Of the 56 patients recruited into the study, 49 (87.5%) were 29
evaluable. Of these, 27 (55.1%) and 4 (8.2%) harbored multiple bacteria by the PCR assay and 30
conventional culture, respectively. Single pathogen was detected in 8 (16.3%) and 4 (8.2%) 31
patients, respectively. Thirteen different genes were detected from 38 patients namely ermB gene 32
(40.8%), oxa51-like gene (28.6%), integrase gene, sul1, (28.6%), int1 (20.4%), mecA and ctx-M 33
(12.3% each). Sample-to-answer was 4 h versus 48-96 h by UPA and culture, respectively. 34
Initial empiric treatment was changed within 5-6 hours in 33 (67.3%) based on the availability of 35
UPA results. Thirty (62.2%) of the patients improved clinically. A total of 3 (6.1%) patients died 36
mainly from their comorbidities. This data demonstrates the potential of multiplex PCR-based 37
assay for accurate and timely detection of etiological agents of NP, MDR organisms and 38
resistance markers that should guide the clinicians for early antibiotic adjustment. 39
40
41
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Introduction 42
The respiratory tract is the most common source of infection in the acutely ill patients and is one 43
of the leading causes of death in these patients. Pneumonia, a lower respiratory tract infection, is 44
a life-threatening condition which carries high mortality. This infection can be acquired in the 45
community (community-acquired pneumonia; CAP) or hospital-acquired (HAP), also known as 46
nosocomial pneumonia (NP). NP refers to infection that developed while the patient was in an 47
inpatient setting (1). It is further differentiated into ventilator-associated pneumonia (VAP) 48
depending on whether or not the process arose after the patient had been receiving 24 h of 49
mechanical ventilation (2, 3). NP is a frequent and severe infection in the hospital setting, 50
particularly the intensive care unit (ICU), with important morbidity, mortality, and cost 51
implications (3-5). Treating a critically ill patient with severe pneumonia is challenging as it 52
involves taking important decisions based on incomplete clinical picture. Thus, selecting 53
appropriate antimicrobial therapy as quickly as possible is absolutely crucial for a successful 54
outcome as timely action seems to lead to decreased mortality. 55
With the current paradigm for treatment of pneumonia, results of conventional microbiology 56
culture of respiratory samples are not available for at least 48-72 hours. However, it is prudent 57
for the clinician to commence empiric treatment immediately without the benefit of knowing the 58
potential causative pathogen and antimicrobial susceptibility. Providing appropriate and adequate 59
antimicrobial therapy is a vital component of successful treatment for severe NP as many reports 60
have shown that inadequate antimicrobial therapy increases the mortality rate (6-8). The 61
therapeutic turnaround time (TTAT), that is, the time taken from sending the first specimen for 62
investigation and the results becoming available to initiate appropriate treatment, varies 63
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considerably as the clinical scenario for treating patients with pneumonia is complex. The shorter 64
the TTAT the better the outcome. 65
Conceivably, availability of robust rapid molecular diagnostic technology in the routine 66
laboratory that can provide accurate and reproducible pathogen detection in hours rather days 67
might prevent some inappropriate and inadequate therapies. The objectives of this study were to 68
evaluate the performance of a multiplex PCR-based UnyveroTM
Pneumonia Application (Curetis 69
AG, Holzgerlingen, Germany) assay for the detection of bacteria and resistance markers from 70
respiratory specimens and determine the impact of this assay on the management of severe 71
nosocomial pneumonia. 72
Materials and Methods 73
Patients and specimens 74
Immunocompetent and immunocompromized severely ill patients with clinically suspected 75
respiratory tract infections who had been admitted to the ICUs or medical wards of Mubarak Al 76
Kabeer Hospital, Kuwait for ≥48 h, during the months of January-April, 2013, were recruited 77
into the study. Non-repetitive respiratory samples, mainly sputum, bronchoalveolar lavage 78
(BAL) and endotracheal secretions (ET), were obtained from the patients who met the definition 79
of nosocomial pneumonia and sent directly to the hospital diagnostic Microbiology Laboratory. 80
Other specimens collected included blood for blood culture and blood gases analysis, 81
hematological and biochemical profiles. Our hospital is a 800-bed tertiary teaching hospital with 82
a 26-bed adult ICU and 9-bed pediatric ICU. The medical ethics committee of our Ministry of 83
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Health (No. WMTJ/2186/2013) approved the study and written informed consent was obtained 84
from the patients or relatives. 85
Definition of pneumonia 86
A patient was suspected of pneumonia if there was either (1) clinical criteria, i.e. new or 87
progressive radiological pulmonary infiltrate plus 2 or more of the following: temperature >38oC 88
or <35.5oC; leukocytosis (leukocyte count of ≥ 12,000 cells/mm
3); or purulent respiratory 89
secretions, as determined by Gram-stain (9, 10) or (2) a simplified Clinical Pulmonary 90
Infectious Score (SCPIS) >5 points (11); SCPIS was a measure of the following variables: 91
temperature in degree centigrade, blood leucocytes per mm3, tracheal secretion culture, 92
oxygenation PaO2/FIO2 in mmHg and chest radiograph. A diagnosis of VAP was made in 93
patients with previous invasive mechanical ventilation for ≥48 h. 94
95
Laboratory methods 96
PCR-based UnyveroTM
Pneumonia Assay 97
UnyveroTM
Pneumonia Application (UPA; Curretis AG) identifies 18 bacteria, based on 23S 98
rRNA sequences, and a fungus (Pneumocystis jirovecii) which represent over 90% of the 99
etiological agents of severe non-viral pneumonia and simultaneously detects 22 resistance 100
markers. For detection of multidrug-resistant (MDR) organisms, the multiplexed PCR targets 3 101
classical Ambler class A β-lactamases (tem, shv, ctx-M) and 2 families of plasmid encoded 102
ampC genes (Ambler class C). An integrase gene as surrogate marker for MDR is also included. 103
All specimens were processed with UPA assay according to manufacturer’s protocol 104
[www.curetis.com]. Specimens were processed immediately as they came, one or two at a 105
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time in sequence with no batching. Briefly, 180 µl of patient’s sample and master mix were 106
loaded into self-contained cartridge (Curetis AG) and then placed in the analyzer, where sample 107
preparation, DNA extraction and purification, amplification and specific detection took place, 108
generating complete diagnostic information within 4 h. To detect many analytes, 8 multiplexed 109
PCRs were run in parallel for detection with panel specific microarrays. 110
111
Conventional culture and susceptibility testing 112
100 µl aliquots of the same sets of samples were inoculated in parallel on a set of selective and 113
non-selective routine agar plates; MacConkey (Oxoid, Basingstoke, UK), Blood agar (Oxoid), 114
Chocolate agar and Sabouraud agar (Oxoid), and incubated in appropriate atmospheric 115
conditions for 24 h or re-incubated for 48 h as necessary. Relevant clinically significant bacterial 116
isolates were identified by the VITEX 2 ID system (bioMerieux, Marcy, I’Etoile, France) and 117
VITEK MS (bioMerieux) (when necessary). Antimicrobial susceptibility testing (AST) was 118
performed using VITEK 2 AST cards and E test (bioMerieux) as required. Penicillin and 119
carbapenem susceptibility of Streptococcus pneumoniae and vancomycin susceptibility of 120
methicillin-resistant Staphylococcus aureus (MRSA) were determined using the E test. 121
Phenotypic detection of resistance mechanisms was carried out using GeneXpert (Cepheid AB, 122
Röntgenvägen, Solna, Sweden) for detection of mecA gene in MRSA. Detections of extended-123
spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) were carried out with the 124
cefotaxime/cefotaxime-clavulanic acid (CT/CTL) and ceftazidime/ceftazidime-clavulanic acid 125
(TZ/TZL) E test (bioMerieux) and imipenem-EDTA E test methods, respectively. The following 126
control strains were included in each run as appropriate: MRSA ATCC 43300 (mecA+ve
), MRSA 127
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ATCC 25923 (mecA-ve
), Escherichia coli ATCC 25922 (ESBL+ve
), MBL-producing Klebsiella 128
pneumoniae ATCC BAA-1705 (MBL+ve
) and K. pneumoniae ATC-1706 (MBL-ve
). 129
130
Impact on patient management 131
The patients were classified into 3 groups (12) as follows: (1) mild-to-moderate NP, i.e. no usual 132
risk factors, onset any time, or early-onset, and severe NP; (2) mild-to-moderate NP with risk 133
factors and onset any time; and (3) late-onset, severe NP or early-onset NP with risk factors. 134
Those whose empiric antimicrobial therapy remained the same after result of etiological agents 135
became known, and those in whom antibiotic therapy needed adjustment were analyzed to 136
determine the direct impact of the test system on the management of patients. 137
138
Statistical analysis 139
The EpiCalc 2000, version 1.02 (Brixton Health, Llanibloes, Powys, Wales, UK) was used to 140
compare the counts and sample size. 141
142
Results 143
Of the 56 patients recruited into this study, 49 (87.5%) were evaluable and 7 (12.5%) who did 144
not meet the definition of NP were excluded. The bio-data of the patients are shown in Table 145
1. The patients were aged 3-92 years (mean=55.6 year). Of these, 27 were in the adult ICUs, 2 in 146
Pediatric ICU (PICU) and 20 on the medical wards. There were 34 males to 15 females. 147
Endotrachial (ET) specimens were obtained from 30 patients, sputum 12 and BAL 7. They all 148
had shift in peripheral WBC count to the left, elevated median CRP and increased oxygen 149
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requirement. Sample-to-result time was c.4.3 h for UPA assay versus 48-96 h for conventional 150
cultural methods. Results obtained by the UPA were immediately conveyed in person by 151
either WJ or EAR to the ward/ICU and the interpretation and significance of each test 152
result discussed with the treating clinician. 153
Bacterial etiology and resistance genes detected by UPA assay versus culture 154
Summary of performance of the test assay and culture shows that clinically significant multiple 155
bacteria were detected in 27 (55.1%) cases by UPA and 4 (8.2%) by conventional 156
bacteriological culture. Eight (16.3%) and 4 (8.2%) cases yielded single pathogens by UPA and 157
culture, respectively, a statistically significant finding with P value <0.0001 [CI 29.00, 64.88]. 158
In 11 (22.5%) and 37 (75.5%) cases there were no significant pathogens detected by both UPA 159
and culture, respectively. The difference in lack of detection power by UPA and culture 160
reached a statistically significant level (P <0.0001 [CI 34.24, 71.88]; 3 of the 11 negative 161
specimens by UPA were acid-fast bacilli (AFB)-positive by Zeihl Neelson stain and grew 162
Mycobacterium tuberculosis by culture. Three (6.1%) specimens yielded “error/not valid run” by 163
UPA, meaning a number of criteria not being fulfilled such as cartridge not properly 164
processed, control within the system failing or there are holes/cracks in the array 165
membrane, dust on the array membrane and difficulties during array detection by the 166
optic module. 167
The microbial etiology according to UPA assay and culture is shown in Table 2. Overall, 168
statistically significant pathogens detected by UPA versus culture were: Acinetobacter 169
baumannii 13 and 3, respectively (P <0.007; CI 4.30, 36.52), Klebsiella pneumoniae 10 and 0 170
(P <0.0013; CI 7.08, 33.73), Pseudomonas aeruginosa 10 and 2 (P <0.015; CI 1.71, 30.94), 171
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Stenotrophomonas maltophilia 11 and 1 (P <0.0027; CI 6.03, 34.78) and Streptococcus 172
pneumoniae 10 and 2 (P <0.015; CI 1.71, 30.94). The etiological agents are further analyzed 173
according to the severity of the pneumonia into group 1, 2 and 3. In group 1 (8 patients) the 174
causative agents detected by UPA versus culture were S. pneumoniae in 6 (75%) cases versus 2 175
(25%), S. maltophilia 2 (25%) versus 0, A. baumannii 1 (12.5%) versus 0 and P. aeruginosa 1 176
(12.5%) versus 0, respectively. In group 2 NP (26 patients), detection by UPA versus culture 177
were: A. baumannii 4 (15.4%) and 2 (7.7%), K. pneumoniae 4 (15.4%) and 1 (3.9%), P. 178
aeruginosa 4 (15.4%) and 1 (3.9%), and S. maltophilia 3 (11.5%) and 0, respectively. Analysis 179
of microbial agents from group 3 NP (15 patients) indicated that the majority of the etiological 180
agents were detected by UPA assay. A. baumannii, K. pneumoniae, S. maltophilia and P. 181
aeruginosa were detected by UPA assay versus culture in 8 (53.3) and 1 (6.7%), 6 (40%) and 0, 182
6 (40%) and 1 (6.7%), and 5 (33.3%) and 1 (6.7%) patients, respectively. 183
A total of 13 different genes were detected by the UPA assay from 38 infected patients. Analysis 184
of the frequency of genes detected showed that ermB gene (40.8%) was the commonest gene 185
detected, followed by oxa51-like gene (28.6%), integrase gene, sul1, (28.6%), int1 (20.4%), 186
mecA and ctx-M (12.3% each). Other genes, such as tem, shv and ermC were each detected in 187
8.2% cases. 188
The degree of agreement between UPA and culture 189
There was no agreement in 18 (36.7%) cases but there were the same growth in 3 (6.1%). At 190
least one organism was common in 14 (28.6%) and both yielded no growth in 9 (18.4%). 191
Treatment changed based on UPA assay 192
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As shown in Table 3, initial empiric treatment was changed within 5-6 h after specimen 193
collection in 33 (67.3%) patients based on the results of UPA assay becoming available soon 194
after 4 h. Thirty (62.2%) of the patients improved clinically and microbiologically compared 195
with 8 (16.3%) in whom there was no improvement. A total of 3 (6.1%) patients died mainly 196
from their comorbidities (1 chronic myeloid leukemia, 1 severe myocardial infarction and 1 197
wild spread tuberculosis). 198
Impact on outcome of severe NP by Unyvero 199
Fifteen (30.6%) patients with severe pneumonia (group 3), from whom multiple bacteria, 200
including multidrug-resistant (MDR) strains were detected, were evaluated specifically for the 201
impact of Unyvero on timely intervention and outcome of therapy. All had leukocytosis, elevated 202
C-reactive protein (CRP) and increased oxygen requirement. Detection of resistance genes 203
influenced modification of therapy in all 15 cases with multiple MDR bacteria. In 3 of these 204
patients, Unyvero and culture identified MDR pathogens with good correlation between 205
phenotype and genotype for third-generation cephalosporins, carbapenems, macrolide and gyrase 206
inhibitor resistance leading to modification in antibiotic regimes to appropriate therapy. Thirteen 207
(86.7%) of these 15 patients improved clinically and bacteriologically and 2 (13.3%) died, 208
mainly from their co-morbidities. Ten (66.7%) would have been inappropriately treated if based 209
on results of conventional testing or empiric therapy only. 210
Discussion 211
Pneumonia is a medical challenge. NP, that is hospital-acquired pneumonia and ventilator-212
associated pneumonia (VAP), represents one of the most important causes of morbidity 213
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and mortality with high attributable mortality rates of between 25-50%. The results of large 214
multicenter studies demonstrate a clearer picture of the severity of the problem on worldwide 215
scale (8, 9). A confounding issue in the successful management of pneumonia is the long delay 216
in determining the identity of the causative agents and their antimicrobial susceptibility. Current 217
guidelines for the treatment of pneumonia are based on the standard of care, which delays the 218
identification of the etiological agent by 48-72 h due to the time it takes to culture the pathogens. 219
One way to improve the TTAT is to implement new diagnostic technologies in the microbiology 220
laboratories that would speed up pathogen and resistance identification. These concerns were 221
found to be addressed by the UPA assay. The assay allowed detection and correct identification 222
of clinically relevant etiological agents of NP and important resistance profiles in patients on the 223
ward and ICU settings within 4 h of specimens reaching the laboratory. All specimens were 224
processed immediately upon receipt in the laboratory thereby eliminating the need for 225
batching which could have impacted the TTAT in contrast to cultural method where 226
specimens were batched. Thus, the limitations in the area of rapid testing for multiple 227
pathogens and ability to incorporate molecular testing into clinical microbiology laboratories 228
were removed. In addition, not only could the assay be performed in a single day, it provided 229
valuable relevant results in just a fraction of the time it took by the conventional culture 230
technique. Furthermore, the results of UPA were conveyed directly in person to the 231
wards/ICU by members of our team who also discussed the interpretation and significance 232
of the results in relation to the clinical condition of the patient. This careful interpretation 233
of the results was undertaken to prevent overtreatment of possible replacement colonizers. 234
This UPA assay system is akin to a “Lab-on-a-Chip” or “micro total analysis system (uTAS)” 235
previously described by Yager el al (13) and Whitesides (14) as it utilizes highly sensitive and 236
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specific technique of nested multiplex PCR in an enclosed disposable cartridge equipped with 237
integrated reagent containers, a DNA purification column, eight PCR chambers and an according 238
number of detection arrays. As a result, it permits enormous benefits of this form of PCR to be 239
feasible in settings where even moderate contamination risk of pathogen or of amplicon is 240
unacceptable. Ultimately, this type of system could allow complex molecular methods to be 241
adopted in point-of-care settings where even community-acquired pneumonia patients present 242
initially to the doctor. Another advantage of the system is that it is automated which reduces 243
operator work-load and error; the process is rapid and reactions from one step to the other 244
measured in seconds. Pathogen identification and antibiotic susceptibility were simultaneously 245
measured at the same time and was able to provide real-time, relevant information about the 246
presence or absence of pathogen and their antibiotic resistance genes. 247
The UPA assay system is also an efficient solution to the “sample-to-assay” problem as it uses 248
small volumes of material without losing its sensitivity. Typically for few abundance of 249
pathogens sensitivity is correlated with testing large sample volume. UPA assay system uses all 250
the nucleic acid recovered from the small input material in the first step multiplex PCR and 251
second stage amplification then allows specific detection of the analytes in very small volume 252
PCRs without losing sensitivity common in small volume PCRs (15). 253
The testing of UPA assay with clinical samples demonstrates a successful real-world application 254
of this technology. In comparison to the cultural technique the system showed high percent 255
cumulative agreement (c.70%). The most common discordance was the detection of pathogens 256
by the UPA assay system in culture-negative samples apparently due to superior sensitivity of 257
the PCR assay. Furthermore, as shown in this study, the UPA assay system has ability to test for 258
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a large panel of pathogens and consequently leading to decrease in the number of negative 259
samples when compared with the conventional culture method and increase in instances when 260
multiple pathogens were detected in the same sample in this study as a result of limited number 261
and types of selective and non-selective culture media used in routine clinical microbiology 262
laboratory for culturing respiratory specimens or previous antibiotic use. 263
UPA assay system, in this study, demonstrated the importance of providing appropriate and 264
adequate therapy as a vital component of successful treatment of severe nosocomial pneumonia 265
as many published reports have shown that inadequate antibiotic therapy increase the mortality 266
rate as well as the mean duration of hospital stay (8, 16-18). It is an accurate and rapid test 267
system which enabled the treating doctor adequate decision making process to select the 268
appropriate antimicrobial therapy and thus improve medical outcome. This PCR-based rapid 269
assay detected many pathogens that were not detectable by conventional culture and provided 270
resistance markers in a timely manner. 271
It is worthy of note that while UPA detected S. pneumoniae, a known respiratory pathogen, 272
in specimens of 12 patients only 2 of these yielded the same organism in conventional 273
culture method. Failure to isolate S. pneumoniae on culture has been observed previously 274
by other workers and this may be attributable to prior antibiotic therapy that may impair 275
diagnostic validity of respiratory culture in addition to delay of sample processing that 276
could reduce the isolation rates and increase indigenous flora (19). Several studies have 277
reported that sputum and other respiratory cultures became rapidly negative for S. 278
pneumoniae during antibiotic treatment unlike PCR which remained positive in spite of 279
ongoing therapy (20, 21). Perhaps, in general, another reason for PCR positivity in culture-280
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negative samples could be its higher sensitivity compared to culture (22). The inclusion of 281
Pneumocystis jirovecii and L. pneumophila in the UPA panel offers valuable information. 282
UPA was positive for L. pneumophila in 2 patients with moderate to severe NP with 283
supportive clinical and laboratory findings but negative by culture. The patients improved 284
dramatically on appropriate antibiotic therapy. 285
In this study, UPA assay was used to assess the presence of multidrug-resistance (MDR) in 76 286
isolates defined as resistance to 3 classes of antibiotics. The majority of the patients in the group 287
2 and group 3 category harbored MDR Gram-negative bacterial pathogens, mainly A. baumannii, 288
K. pneumoniae, P. aeruginosa and S. maltophilia. Detection of MDR was achieved in this assay 289
by 3 classical Ambler class A beta-lactamases (tem, shv, ctx-M), 2 Ambler class C (plasmid-290
mediated ampC genes) and integrase gene (int1) and was indicated in c.55% of pathogens. Based 291
on these results, treatment regimens were changed in 33 (67%) of patients within 6 h of 292
specimen collection and laboratory processing (sample-to-answer timeframe) without waiting for 293
results of conventional culture which became available approximately 48-96 h later. Sixty-two 294
percent improved clinically and microbiologically; 3 patients died mainly because of their 295
comorbidities. 296
Direct impact of UPA assay compared with conventional culture was further analyzed in details 297
in the 15 patients in group 3. There was no doubt that the rapid molecular diagnostic platform 298
(UPA assay) was instrumental to the accurate pathogen identification and selection of 299
appropriate targeted antibiotic therapy with good benefits for the patients. In addition, by 300
improving the turnaround time to hours, instead of days, it provided clinicians opportunity to 301
change empiric therapy to definitive therapy at the shortest possible timeframe, thus impacting 302
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positively patient management and outcome. In our hospital treatment guideline for empirical 303
treatment of NP entails administering ceftazidime plus ciprofloxacin or meropenem. 304
Treatment was changed from the broad-spectrum regimen to targeted antibiotic therapy 305
involving the use of intravenous colistin and/or tigecycline or vancomycin when MRSA was 306
involved, in our patients with moderate to severe NP, with satisfactory outcome. This 307
decision was influenced by the results of UPA showing pathogens with multiple resistance 308
to the empirical broad-spectrum antibiotics. The patient that died of widespread 309
tuberculosis (TB) had pulmonary TB that was missed by UPA which also did not detect 310
any other microorganism in the specimen of this patient; routine bacteriological culture did 311
not yield any growth as well. Based on demonstration of AFB by ZN stain and radiological 312
findings, he was treated with standard quadruple anti-TB drugs but succumbed to his 313
infection. 314
An important limitation of this study is the relative small number of patients and the time 315
constraints for carrying out the study. A larger number of patients is required to demonstrate 316
more conclusively the impact of UPA assay on patient management. This will also permit 317
consistent performance at high level of technical proficiency, determination of high positive and 318
negative predictive values that can discriminate between a true infection and mere colonization, 319
and importantly help to determine its proper place in an algorithm of clinical laboratory 320
diagnosis of respiratory pathogens. It must be said though that because UPA assay consists of 321
finite panel, as it is with all multiplexed PCR, it may miss some organisms which might 322
grow on culture. Correct interpretation of these UPA results must be carefully made and 323
weighed against the clinical condition of the patient in order to avoid over-treating mere 324
colonizers. M. tuberculosis is not included in the UPA panel hence it failed to detect the 3 325
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cases of tuberculosis which were positive on ZN stain and later grew on culture. The 326
explanation for this omission might be based on the assumption that M. tuberculosis is an 327
unusual etiological agent of NP. Another important cautionary note when making 328
comparison between UPA and culture is that PCR-based identification will also detect dead 329
or treated organisms that may not grow on culture particularly if the patient has been on 330
medication with previous antibiotics. 331
Conclusion 332
The UPA assay was able to detect etiological agents of mild through moderate to severe 333
nosocomial pneumonia and their resistance markers in respiratory samples consistent with 334
standard clinical microbiology within a time period of approximately 4.3 hours versus 48-96 h 335
by conventional culture. This assay holds promise for the future where rapid detection of 336
pathogens and resistance mechanisms are determined in standardized assays that will allow 337
clinicians to diagnose pneumonia in real time and initiate appropriate antimicrobial therapy as 338
early as possible. The study also showed that antibiotic resistance with a complex genetic 339
background can be successfully predicted by the carefully selected markers on the assay panel. 340
However, as promising as the assay looks, care must be taken in the interpretation of test 341
results which should tally with the clinical presentation to avoid treating colonizers instead 342
of the patients. 343
Conflict of interest 344
WJ and VOR are members of the Regional Advisory Board of Astellas. VOR has received Pfizer 345
travel grants in the past and he is a member of Pfizer Middle East Advisory Board. 346
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419
420
421
422
423
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Table 1: Basic characteristics of the patients admitted into the study. 425
Characteristics No. (%)
Demographic data:
Age range (years)
Mean age
Male
Female
Location of care:
Adult intensive care unit (ICU)
Pediatric ICU
Medical ward
Comorbidities:
Diabetes mellitus
Chronic respiratory disease
Chronic cardiovascular disease
Chronic renal disease
Chronic hepatic disease
Solid cancer
Valid sample type:
Endotracheal secretion
Sputum
Bronchoalveolar lavage
Clinical laboratory findings:
Median CRP level (mg/dl)
Median WBC count (x109/l)
Median platelet count (x109/l)
Bacteremia
Mechanical ventilation
Pleural effusion
3-92
55.6±21.927*
34 (69.4)
15 (30.6
27 (55.1)
2 (4.1)
20 (40.8)
12 (24.5)
17 (34.7)
8 (16.3)
4 (8.2)
7 (14.3)
10 (20.4)
30 (61.2)
12 (24.5)
7 (14.3)
20.8
13.4
127.0
8 (16.3)
18 (36.7)
11 (22.5)
426
*±=standard deviation; CRP=C-reactive protein; WBC=white blood cells 427
428
429
430
431
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Table 2: Distribution of microorganisms according to severity of pneumonia 432
Microorganism (n)
No. of patient infected
P value
Group 1
(n=8)
Group 2
(n=26)
Group 3
(n=15)
PCR Culture PCR Culture PCR Culture
Acinetobacter baumannii (13)
Haemophilus influenzae (2)
Klebsiella pneumoniae (10)
Klebsiella oxytoca (2)
Legionella pneumophila (2)
Moraxella catarrhalis (1)
Proteus sp. (1)
Pseudomonas aeruginosa (12)
Serratia marcescens (3)
Staphylococcus aureus (2)
MRSA (3)
Stenotrophomonas maltophilia (12)
Streptococcus pneumoniae (12)
1 0
0 0
0 0
0 0
0 0
0 0
0 0
1 0
0 0
0 0
0 0
2 0
6 2
4 2
1 0
4 1
2 0
2 0
0 0
0 0
4 1
1 0
1 0
1 0
3 0
2 0
8 1
1 0
6 0
0 0
0 0
1 0
1 0
5 1
2 0
1 0
2 0
6 1
2 0
0.007
0.24
0.0013
0.24
0.24
0.5
0.5
0.015
0.12
0.24
0.12
0.0027
0.015
MRSA=methicillin-resistant Staphylococcus aureus; PCR=polymerase chain reaction. Group 1= 433
mild-to-moderate nosocomial pneumonia (NP), no usual risk factors, onset any time, or early-434
onset, and severe NP; Group 2= mild-to-moderate NP with risk factors and onset any time; 435
Group 3= late-onset, severe NP or early-onset NP with risk factors. P value of <0.05 is 436
statistically significant. 437
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Table 3: Treatment changed based on Unyvero results 438
Cases
No Percentage Comments
Treatment changed
Missing
Total
Improved:
Yes
No
Died
33
16
49
30
8
3
67.3
32.7
100.0
62.2
16.3
6.1
Within 6 hours of specimen collection
No pathogen detected plus invalid readings (errors)
Clinical parameters and CXR
CXR and clinical parameters unchanged
Died of comorbidities: 1 CML; 1 MTb; 1 severe MI
439
CXR= chest X ray; CML= chronic myeloid leukemia; MTb= wild spread tuberculosis; MI= 440
myocardial infarction 441
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