Infectious Diseases

Multiplex PCR Based Urinary Tract

Infection (UTI) Analysis Compared to
Traditional Urine Culture in Identifying
Significant Pathogens in Symptomatic
Patients
Kirk J. Wojno, David Baunoch, Natalie Luke, Michael Opel, Howard Korman, Colleen Kelly,
S. Mohammad A. Jafri, Patrick Keating, Dylan Hazelton, Stephany Hindu,
Bridget Makhloouf, David Wenzler, Mansour Sabry, Frank Burks, Miguel Penaranda,
David E. Smith, Andrew Korman, and Larry Sirls
OBJECTIVE To evaluate whether multiplex PCR-based molecular testing is noninferior to urine culture for
detection of bacterial infections in symptomatic patients.
METHODS Retrospective record review of 582 consecutive elderly patients presenting with symptoms of lower
urinary tract infection (UTI) was conducted. All patients had traditional urine cultures and PCR
molecular testing run in parallel.
RESULTS A total of 582 patients (mean age 77; range 60-95) with symptoms of lower UTI had both urine
cultures and diagnostic PCR between March and July 2018. PCR detected uropathogens in 326
patients (56%, 326/582), while urine culture detected pathogens in 217 patients (37%, 217/582).
PCR and culture agreed in 74% of cases (431/582): both were positive in 34% of cases (196/582)
and both were negative in 40% of cases (235/582). However, PCR and culture disagreed in 26%
of cases (151/582): PCR was positive while culture was negative in 22% of cases (130/582), and
culture was positive while PCR was negative in 4% of cases (21/582). Polymicrobial infections
were reported in 175 patients (30%, 175/582), with PCR reporting 166 and culture reporting 39.
Further, polymicrobial infections were identified in 67 patients (12%, 67/582) in which culture
results were negative. Agreement between PCR and urine culture for positive cultures was 90%,
exceeding the noninferiority threshold of 85% (95% conflict of interest 85.7%-93.6%).
CONCLUSION Multiplex PCR is noninferior to urine culture for detection and identification of bacteria. Further
investigation may show that the accuracy and speed of PCR to diagnose UTI can significantly
improve patient outcomes. UROLOGY 136: 119−126, 2020. © 2019 Elsevier Inc.

T
raditional urine culture is commonly regarded as fastidious, and therefore difficult to grow in culture. Further,
the gold standard for detection and identification PCR results can be obtained in a day or less, while culture
of pathogens. However, evidence has been accu- can require 2 or more days. Previous studies have reported
mulating to support use of molecular methods such as PCR. PCR to have both superior sensitivity and specificity, and
With antimicrobial resistance becoming both more com- have recommended PCR for rapid identification of patho-
mon and complex, effective treatment of (urinary tract gens in sepsis,1-3 and for diagnosis of genital infections and
infection) UTIs is even more dependent on the accurate sexually transmitted diseases,4-6 parasitic infections,7 tuber-
identification of pathogens. Some organisms can be culosis,8 and gastrointestinal infections.9
Few studies have compared multiplex PCR with urine
Financial Disclosures: Dr Larry Sirls received honoraria for presentations made at
culture for diagnosis of UTIs and acute cystitis. Although
AUA 2019 and SUFU 2019. several studies have compared performance of PCR with
From the Comprehensive Urology-A Division of Michigan Healthcare Professionals, urine culture for detection of a single pathogen, only 4
Royal Oak, MI; the Pathnostics, Irvine, CA; and the Kelly Statistical Consulting, Carls-
bad, CA
have tested multiplex PCR: one against 15 bacteria,10 a
Address correspondence to: Kirk Wojno, M.D., Comprehensive Urology - A Division second against 14 bacteria together with 6 fungi,11 a third
of Michigan Healthcare Professionals, 31157 Woodward Ave, Royal Oak, MI 48073. against 20,12 and the fourth against 9 bacteria.13 Polymi-
David Baunoch, Ph.D., Pathnostics, 17661 Cowan, Irvine, CA 92614. E-mails:
[email protected]; [email protected]
crobial infections may occur in as many of 39% of
Submitted: August 15, 2019, accepted (with revisions): October 10, 2019 UTIs14,15 and can display enhanced virulence and
© 2019 Pathnostics. Published by Elsevier Inc. https://doi.org/10.1016/j.urology.2019.10.018 119
This is an open access article under the CC BY-NC-ND license. 0090-4295
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
increased antibiotic resistance.16 Simultaneous detection fixed with methanol. The slides were covered in crystal violet
of a larger number of pathogens may confer benefits for solution for one minute, rinsed with water, covered with iodine
outcome of UTIs. The long-term objective is to determine for 1 minute, and then rinsed with water again. Staphylococcus
whether the speed and accuracy of multiplex PCR aureus 29213 was used as a positive control, and E. coli 35218
improves patient care and potentially reduces costs. This was used as a negative control.
Pathogen identification was conducted using the VITEK 2
study is the first step to confirm that PCR is noninferior to
Compact System (bioMerieux, Durham, NC) in accordance
traditional urine culture in detecting bacteria in symptom- with standard operating procedures. Briefly, a sterile swab was
atic patients. In this study, we compared a multiplex panel used to transfer morphologically similar colonies from positive
of 31 bacteria against urine culture for diagnosis of blood agar plates to prepared polystyrene test tubes containing
patients presenting with symptoms of UTI. Though this 3.0 mL of sterile saline. The sample was adjusted for density
PCR also reports resistance for each organism, these data (equivalent to McFarland No. 0.50 to 0.63). The sample tube
are not reported here. Although it may sound intuitive and an appropriate identification card were placed into the cas-
that the resistance of a single organism is different from sette and inserted into the VITEK 2 instrument. A GN card was
the susceptibility results of the polymicrobial “soup”, the used for Gram negative bacteria, and a GP card used for Gram
data are being analyzed to better understand the patterns positive bacteria. An YST card was used for yeast. Pathogen
of the results. identification was then read from the VITEK 2 instrument.

DNA Extraction and Analysis

DNA was extracted from urine samples using the KingFisher/
MATERIALS AND METHODS MagMAX Automated DNA Extraction instrument and the
Participants MagMAX DNA Multi-Sample Ultra Kit (ThermoFisher, Carls-
This was a single-site (Comprehensive Urology, Royal Oak, MI) bad, CA). Briefly, 400 mL of urine was transferred to wells in 96-
retrospective study. IRB approval was obtained prior to com- well deep well plates, sealed, and centrifuged to concentrate the
mencing the study (IRB protocol number: 20171870). All samples, after which supernatant was removed. Enzyme Lysis
patients meeting the inclusion criteria presenting to clinic Mix (220 mL/well) was added and incubated for 20 minutes at
between March and July 2018 (n = 582) were included in the 65C. Proteinase K Mix (PK Mix) was added (50 mL/well) and
study. Inclusion criteria: ≥60 years of age; symptoms of acute cys- incubated for 30 minutes at 65C. Lysis buffer (125 mL/well) and
titis or UTI; sufficient urine sample volume for urinalysis, tradi- DNA Binding Bead Mix (40 mL/well) were added and the sam-
tional culture, and PCR; all samples were shipped FedEx priority ples shaken for a minimum of 5 minutes. The 96-well plate was
overnight. All samples except 4 were received by the laboratory then loaded into the KingFisher/MagMAX Automated DNA
the day after collecting the samples. Four samples were received Extraction instrument, which was operated in accordance with
2 days after collection. standard operating procedures.
This study focused on patients ≥60 years of age. UTI is com- DNA samples were analyzed using the Pathnostics Guidance
mon in this age group and can be more difficult to diagnose. UTI Test. Samples were mixed with universal PCR master mix
Localized urogenital symptoms may not be present in this popu- and amplified using TaqMan technology on a Life Technologies
lation and differentiation between UTI and asymptomatic bacte- 12K Flex OpenArray System. DNA samples were spotted in
riuria can be difficult.17 Patients in this age group could benefit duplicate on 112-format OpenArray chips. Plasmids for each
significantly from better identification of UTI pathogens. organism being tested for were used as positive controls. Candida
tropicalis was used as an inhibition control. A data analysis tool
Urine Culture developed by Pathnostics was used to sort data, assess the quality
Study participants provided a urine sample obtained either by self- of data, summarize control sample data, identify positive assays,
administered clean catch or by catheterization. The urine was calculate concentrations, and generate draft reports. Probes and
mixed and a sterile plastic loop (1 mL) used to inoculate blood primers were used for the following pathogens:
agar plates. A sterile plastic loop (1 mL) was also used to inoculate Bacteria: Acinetobacter baumannii, Actinobaculum schaalii,
colistin and nalidixic acid agar/MacConkey agar (CNA/MAC) Aerococcus urinae, Alloscardovia omnicolens, Citrobacter freundii,
plates, one loop-full of urine on the CNA side of the plate and Citrobacter koseri, Corynebacterium riegelii, Enterobacter aerogenes,
another full loop-full on the MAC side of the plate. All plates Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Klebsiella
were incubated at 35C in 5% CO2 for ≥18 hours and then exam- pneumoniae, Morganella morganii, Mycobacterium tuberculosis,
ined for evidence of growth. Plates with <104 CFU/mL were Mycoplasma genitalium, Mycoplasma hominis, Pantoea agglomerans,
reported as normal urogenital flora. For plates with growth (≥104 Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Ser-
CFU/mL), the quantity and morphology of each organism was ratia marcescens, Staphylococcus aureus, Streptococcus agalactiae,
recorded. The maximum readable colony count using the 1 mL Ureaplasma urealyticum
loop is >105 CFU/mL. Colony counts were performed on the Bacterial Groups: Coagulase negative staphylococci (Staphy-
blood agar plates. Species identification and colony counts were lococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus
performed on CNA/MAC plates. For plates with ≤2 pathogens, lugdunesis, Staphylococcus saprophyticus); Viridans group strepto-
species identification and colony counts were reported for each cocci (Streptococcus anginosus, Streptococcus oralis, Streptococcus
pathogen with ≥104 CFU/mL. If ≥3 pathogens were present, and pasteuranus)
one or 2 were predominant, species identification, and colony
counts were reported. If ≥3 pathogens were present without pre-
dominant species, a mixed morphotype was reported. STATISTICAL ANALYSIS
Samples for Gram stain were prepared by applying a thin, Demographics and symptoms were compared for male and
even smear on microscope slides, allowed to air dry, and then female patients with 2-sample t tests or Fisher’s exact tests,

120 UROLOGY 136, 2020

as appropriate. Noninferiority of Guidance UTI to tradi- Sixty percent of patients (60%, 347/582) had positive results
tional culture in terms of detecting bacterial infections by PCR, urine culture, or both. PCR detected bacteria in 56% of
was assessed by comparing the lower 95% Wilson-score patients (326/582), while urine culture detected pathogens in
confidence interval (CI) for the positive percentage agree- 37% of patients (217/582) (Table 2). PCR and culture agreed in
ment to the noninferiority threshold of 85%. Incidences 74% of cases (431/582): both PCR and culture were positive in
34% of patients (196/582), and both were negative in 40%
of bacterial infections in males and females (according to
(235/582). There was disagreement between PCR and culture in
PCR and culture) were compared with Fisher’s exact tests. 26% of cases (151/582): PCR was positive while culture was neg-
The required sample size to yield 90% power to con- ative in 22% of patients (130/582), and PCR was negative while
clude noninferiority of Guidance UTI relative to tradi- culture was positive in 4% (21/582). The agreement between
tional urine C&S was calculated assuming a Guidance PCR and urine culture for positive cultures was 196/217 (90%),
UTI sensitivity (relative to culture) of 91% and assuming exceeding the noninferiority threshold of 85% (95% CI: 85.7%-
60% of patients would test positive by culture (based on 93.6%).
results in PGX-031 UTI TaqMan QuantStudio 12K Flex The multiplex panel used in this study tested for 31 bacteria,
Data Analysis). This sample size was calculated in the and PCR and urine culture together identified 29 different bacte-
Tests for One Proportion Procedure of NCSS Power rial pathogens. PCR detected 24 bacteria, while culture detected
Analysis Statistical Software, Version 14. A sample size of 21 different bacteria.
The most common organisms detected by PCR were E. coli
310 patients with positive culture tests yields 90% power
(29% of PCR positives, 93/326), Actinobaculum schaali (27% of
to conclude noninferiority. Assuming 60% of patients test PCR positives, 89/326) and Viridans group Streptococci (27% of
positive for culture, a total of 517 patients should be PCR positives, 89/326) (Fig. 1). Traditional culture failed to
enrolled in the study. detect Actinobaculum schaali (n = 0), and Viridans group Strepto-
cocci was isolated in culture only rarely (6% of culture positives,
14/217). E. coli was also the most common bacterium detected
RESULTS by culture (34% of culture positives, 74/217) and Enterococcus
A total of 582 patients, mean age 77 (range 60-95) with symp- faecalis was the second most common (21% of culture positives,
toms of lower UTI had both urine culture and diagnostic PCR 46/217).
between March and July 2018 (Table 1). Sixty percent (60%, There were 8 bacteria that were identified only by PCR and 5
347/582) were male and 40% (235/582) were female. Clinical bacteria that were only detected by culture (Table 3). These 5
symptoms included dysuria (38%, 221/582), incontinence bacteria identified only by culture were not included on the mul-
(33%, 192/582), urine that was cloudy or had an odor (23%, tiplex PCR panel. These 5 bacteria were only detected in 8
133/582), and pain or discomfort (7%, 40/582). patients by culture, accounting for 4% of the total positive

Table 1. Study participant demographics and symptoms

Parameter Total Male Female p Value*
Age (years)
n 582 347 235
Mean (SD) 77.2 (7.9) 76.9 (7.9) 77.7 (7.9) .01
Median 77 76 79
Min, Max 60, 95 60, 95 60, 95
Symptoms − n (%)
Dysuria 219 (37.6%) 110 (31.7%) 109 (46.4%) .0005
Urinary incontinence 196 (33.7%) 145 (41.8%) 51 (21.7%) <.0001
Cloudy or Strong-smelling urine 132 (22.7%) 75 (21.6%) 57 (24.3%) .48
Pain 18 (3.09%) 7 (2.02%) 11 (4.68%) .09
Abdominal 7 (1.20%) 3 (0.86%) 4 (1.70%) .45
Flank 5 (0.86%) 1 (0.29%) 4 (1.70%) .16
Lower back 4 (0.69%) 1 (0.29%) 3 (1.28%) .31
Penis/scrotum 2 (0.34%) 2 (0.58%) 0 (0.00%) 1.00
Pelvic discomfort 24 (4.12%) 6 (1.73%) 18 (7.66%) .001
Lower grade fever 1 (0.17%) 1 (0.29%) 0 (0.00%) 1.00
Agitation 2 (0.34%) 2 (0.58%) 0 (0.00%) .52
Frequency 10 (1.72%) 4 (1.15%) 6 (2.55%) .21
Nocturia 1 (0.17%) 0 (0.00%) 1 (0.43%) .40
Patient-reported hematuria 1 (0.17%) 0 (0.00%) 1 (0.43%) .40
Dipstick results − n (%)
Hematuria 246 (42.7%) 138 (40.4%) 108 (46.2%) .17
Atypical symptoms − n (%)
Increased falls/Tripping/Tired/ 7(1.2%) 6 (1.73%) 0 (0.00%) 1.00
Feeling Ill/Decline in ADLs
Antibiotic usage − n (%)
Antibiotic treatment in last 3 weeks 89 (15.3%) 30 (8.65%) 59 (25.1%) <.0001
* p values are from Fisher’s exact test or a t-test, as appropriate.

UROLOGY 136, 2020 121

 30/39 (76.9%)
94/178 (52.8%)
196/217 (90.3%)

The agreement between PCR and urine culture for positive cultures was 196/217 (90%), exceeding the noninferiority threshold. Polymicrobial infections are those having ≥2 organisms infections. PCR
culture samples (8/217): Enterobacter cloacae as monomicrobial
infection in 2 patients (1%, 2/217), Enterococcus faecium alone

Agreement
in 1 patient (0.5%, 1/217), Enterobacter cloacae and faecium in
1 patient (0.5%, 1/217), Enterobacter cloacae and E. coli in 1
patient (0.5%, 1/217), Proteus mirabilis and Streptococcus gallolyti-
cus in 1 patient (0.5%, 1/217), and Kocuria rosea and Kocuria
kristinae in 1 patient each (0.5%, 1/217), (Table 3). Three of
these 8 patients tested positive to other bacteria by PCR, 1 tested
178 (30.6%)
217 (37.3%)
235/365 (64.4%)
positive to Candida albicans, and 1 tested positive to JC virus by

(196+235)/582
39 (6.7%)

PCR. By contrast, there were 108 patients who tested positive

(74.1%)
for at least one of the 8 bacteria that were detected by PCR but
Total

not by culture. Only 45 of the 108 (42%) tested positive to other

bacteria by culture.
In 88 patients (15%, 88/582), both PCR and urine culture
identified the same pathogens and the same number of bacteria.
In 85/88 cases (97%) both culture and PCR reported a single
(196+235)/582

pathogen. The most common bacteria with total agreement

365 (62.7%)

were E. coli (35%, 30/85), Enterococcus faecalis (19%, 16/85),

6 (1.0%)
15 (2.6%)
21 (3.6%)

(74.1%)
Negative

and Klebsiella pneumoniae (14%, 12/85). Together, these 3 bacte-

582

ria accounted for 58 of 85 cases (68%) of single bacteria total

agreement, but only 29% (194/661) of the total bacteria
reported by PCR.
PCR

There were 175 cases of polymicrobial infection, defined as

≥2 bacteria. PCR detected 166 and culture detected 39
235/256 (91.8%)

(Table 2). There were 30 cases in which both PCR and culture
Total Positive

163 (28.0%)
196 (33.7%)
235 (40.4%)
256 (44.0%)
33 (5.7%)

reported polymicrobial infections. There were 3 cases in which

culture reported a polymicrobial infection while PCR reported a
monomicrobial infection, and an additional 6 cases in which
culture reported a polymicrobial infection and PCR was nega-
Table 2. Agreement of PCR and traditional urine culture in patients with clinical symptoms of UTI

tive. In 1 of these 6 cases, both bacteria detected by culture were

not included on the PCR panel; in another case, one of the bac-
teria detected by culture was not included on the PCR panel.
196/326 (60.1%)
Monomicrobial

Although it occurs rarely, molecular inhibition can cause PCR

94 (16.2%)
97 (16.7%)
130 (22.3%)
326 (56.0%)
3 (0.5%)
Positive

tests to be negative. This may have happened with the other 4

cases, which amount to less than 1% (4/582) of patients tested.
was significantly more sensitive than urine culture for detecting polymicrobial infections.

In 67 polymicrobial cases (12% of patients, 67/582; 38% of pol-

ymicrobial infections, 67/175) culture was negative. Of the
30 cases in which PCR and culture detected polymicrobial infec-
tions, 7 were labelled as “Mixed” by culture so there could be no
agreement on the organisms detected, 23 had at least one of the
69 (11.9%)
99 (17.0%)
63 (10.8%)
160 (27.5%)

94/160 (58.8%)

Monomicrobial percentage agreement (%, 95% CI) 52.8% (45.5, 60.0%).

30 (5.2%)

culture detected organisms detected by PCR and 17 had 2 of the

Polymicrobial

Polymicrobial percentage agreement (%, 95% CI) 76.9% (61.7, 87.4%).

culture detected organisms detected by PCR. There were 3 cases

Negative percentage agreement (%, 95% CI) 64.4% (59.3, 69.1%).

in which 2 pathogens were reported and there was total agreement

Overall percentage agreement (%, 95% CI) 61.7% (57.7, 65.5%).

between PCR and culture. In 2 cases, the pathogens were E. coli

and Enterococcus faecalis; in 1 case, the pathogens were Proteus
mirabilis and Enterococcus faecalis. Although there were 92 cases of
at least 3 pathogens, there were none in which PCR and culture
30/166 (18.1%)

agreed on the organisms detected, since culture did not identify

Monomicrobial

the organisms in most of these cases, but just labelled them as

Total Positive
Polymicrobial

166 (28.5%)

“Mixed”. Which bacteria cause infection and inflammation in

67 (11.5%)

polymicrobial situations is impossible to determine.

DISCUSSION
Agreement

This study showed that multiplex PCR is not inferior to

Negative
Positive

traditional urine culture, and in fact detected bacteria in

Total

36% of symptomatic patients who had a negative urine

culture. In addition, multiplex PCR detected more poly-
microbial infections than urine culture, 28% of patients,
compared to 7% of patients. In addition to higher detec-
Culture

tion rates, PCR can provide results in as little as 6 hours

compared to traditional culture which takes 48 or more

122 UROLOGY 136, 2020

Figure 1. Frequency of detection of bacteria by PCR and urine culture, ordered in decreasing frequency of detection by urine
culture.

hours. The rapid, accurate identification offered by PCR study may reflect that they were enrolled from a busy clini-
can facilitate more appropriate and efficacious treatment cal urology practice that may see more men patients in
and may improve clinical care and outcomes. general. Though women typically have more UTI’s than
UTI is a significant health concern in the United men, the sex differences in this study should not affect the
States, causing approximately 7 million visits to a doctor’s accuracy of PCR vs standard urine culture.
office, 1 million emergency department visits, and over Polymicrobial infections may be observed in about 39%
100,000 hospitalizations annually, with the cost exceeding of UTIs, a potential difficulty in determining appropriate
$2.6 billion each year.18,19 Although the patients in this treatment.14,15 Khasriya showed that urine sediment cul-
study were outpatients, all of them were at least 60 years tures in patients with LUTS were significantly different
old. About 40% of men and 28% of women 70-79 years of than voided urine cultures, and were more commonly pol-
age will have nonspecific lower urinary tract symptoms ymicrobial, arguing that intracellular bacterial communi-
(LUTS) that can be clinical difficult to differentiate from ties and bacteria adherent to the epithelial cells are
UTI.20,21 Thus, the diagnosis of UTI on the basis of clini- detected by PCR but not by culture.23 This may be clini-
cal criteria alone has been reported to have an error rate cally important, since each pathogen carries a unique pat-
of approximately 33%.22 The high numbers of men in this tern of antimicrobial resistance, and one bacterial species
can confer antibiotic resistance on other bacterial species
in a polymicrobial environment. This emphasizes an
Table 3. Bacteria detected only by PCR and only by culture important limitation of traditional urine culture: the iden-
Pathogens Detected Pathogens Detected tification of polymicrobial UTIs is poor, whereas the accu-
Only by PCR Only by Culture racy of PCR for polymicrobial infections was clearly
Actinobaculum schaalii Enterobacter cloacae better. PCR can detect the fastidious organisms that are
Alloscardovia omnicolens Enterococcus faecium part of the microbiome; some are clearly pathogens while
Corynebacterium riegelii Kocuria kristinae the role of others is under further investigation.
Mycobacterium tuberculosis Kocuria rosea While other studies have reported higher detection
Mycoplasma hominis Streptococcus gallolyticus
Pantoea agglomerans
rates using PCR compared to urine culture, most tested
Providencia stuartii against single pathogens, which is clearly not sufficiently
Ureaplasma urealyticum comprehensive for clinical use.24-26 A small number of
The bacteria detected only by culture did not have PCR probes in studies has examined the performance of multiplex PCR,
the multiplex PCR panel used for this study. testing for between 9 and 20 pathogens.10-13 In this study,

UROLOGY 136, 2020 123

our multiplex PCR tested for 31 bacteria and detected 24 Finally, the clinical impact of the rapid identification of
different bacteria; the number of bacteria detected the organism and the resistance are unknown and are the
exceeded the size of panels used in previous studies. subject an ongoing prospective trial.
The importance of multiplexing at this level is under- A potential weakness of this study is ascertaining the
scored by detection rates for polymicrobial infections, clinical significance of the pathogens identified. The uri-
defined as 2 or more pathogens. Multiplex PCR was better nary microbiome is still being identified and evaluated;
able to detect polymicrobial infections than culture and some organisms may be pathogens, some protective and
was also better able to identify the pathogens. Culture was some interdependent. Although many bacteria detected in
rarely able to detect cases of >3 pathogens and was unable this study are known pathogens for UTI (eg, E. coli, Klebsi-
to identify the bacteria in these cases. In this study culture ella pneumoniae, Enterococcus faecalis),27 their contribution
was unable to identify the pathogens in 85% of polymicro- to pathogenesis may be less definite in cases of polymicro-
bial infections. Such cases may prove to be clinically sig- bial infection. That is, UTI symptoms in polymicrobial
nificant, inasmuch as mutualism may increase resistance infections may be caused by a subset of the bacteria, or
and complicate treatment. some combination. Evidence suggests that polymicrobial
There were a small number of bacteria that were infections can form cooperative networks that enhance
detected by culture but not by PCR. Cases in which cul- antibiotic resistance, wherein one bacterial species can
ture results were positive and PCR was negative include confer antibiotic resistance on other bacterial species.28
on 3.6% (21/582) of patients involving 9 bacteria. In 8 of A strength of this study is the inclusion criteria for
these 21 patients, PCR was negative because the multiplex enrolling patients that experienced urologists felt were
panel did not include probes for the 5 bacteria infecting symptomatic of UTI and required urine culture with or
those patients. There are limits to multiplexing; it is not without empiric treatment. This should make the results
currently feasible to test for all known pathogens by PCR. of this study generalizable to the clinician who is tasked
However, judicious selection of PCR targets allows detec- with evaluating and managing the symptomatic patient
tion and identification of clinically significant bacteria. with clinical UTI symptoms. Another strength is the use
On the other hand, PCR detected 661 instances of 24 of multiplex PCR for detection and identification of poly-
different bacteria in 56% of patients with about 50% of microbial infection, allowing for the detection of a larger
those being polymicrobial infections. Another advantage number and broader range of different bacteria than tradi-
of PCR is that since no probe is used to detect lactobacil- tional culture. These data are being further evaluated to
lus, for example, this bacterium, commonly agreed to be a examine bacterial interdependence and patterns of bacte-
contaminant, is not detected and reported. rial combinations to identify new paradigms of pathogene-
E coli was the most common bacteria detected for both sis and potential treatment.
PCR and culture. However, the rest of the 5 most fre-
quently detected bacteria were very different between
PCR and culture. The rest of the top 5 for PCR were Acti-
CONCLUSIONS
nobaculum schaallii, Viridans group Streptococci, Aerrococcus This study shows that multiplex PCR is noninferior to tra-
urinae, and Coagulase negative Staphylococcus. Actinobacu- ditional urine culture for detection and identification of
lum schaalii was not detected at all by culture. The rest of bacteria in patients with clinical symptoms of UTI. PCR
the top 5 for culture were Enterococcus faecalis, Klebsiella exhibited greater accuracy than culture for pathogens
pneumoniae, Viridans group Streptococci, and Pseudomonas detected and identified bacteria in 36% of patients who
aeruginosa, all of which were detected by PCR at higher had a negative traditional urine culture. PCR was much
rates than in culture. Others have grown these pathogens more sensitive in detecting polymicrobial infections than
from the urine with enhanced urine culture techniques, urine culture. The accuracy and speed of PCR testing over
confirming their importance in the urinary microbiome. traditional urine culture, and the potential identification
Their failure to grow in traditional culture reflects their of polymicrobial infections with complicated resistance
fastidious nature and further demonstrates the limitations sharing mechanisms is exciting but requires further study
of the traditional culture. to determine the clinical importance.
The long-term objective is to determine whether the
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10.1128/JCM.01619-15. nostic methods for the detection of cytomegalovirus in recipients of
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gastrointestinal infections: challenges and opportunities. Expert Rev tional tests for the detection of Trichomonas vaginalis. Sex Transm
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1167599. 27. McLellan LK, Hunstad DA. Urinary tract infection: pathogenesis
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Real-time polymerase chain-reaction detection of pathogens is feasi- 10.1016/j.molmed.2016.09.003.
ble to supplement the diagnostic sequence for urinary tract infec- 28. de Vos MGJ, Zagorski M, McNally A, Bollenbach T. Interaction
tions. BJU Int. 2010;106:114–120. https://doi.org/10.1111/j.1464- networks, ecological stability, and collective antibiotic tolerance in
410X.2009.09017.x. polymicrobial infections. Proc Natl Acad Sci USA. 2017;114:10666–
11. Lehmann LE, Hauser S, Malinka T, et al. Rapid qualitative urinary 10671. https://doi.org/10.1073/pnas.1713372114.
tract infection pathogen identification by SeptiFastÒ Real-Time PCR.
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12. Cybulski Z, Schmidt K, Grabiec A, et al. Usability application
of multiplex polymerase chain reaction in the diagnosis of microor-
ganisms isolated from urine of patients treated in cancer hospital. EDITORIAL COMMENT
Radiol Oncol. 2013;47:296–303. https://doi.org/10.2478/raon-2013-
0044. Standard urine cultures typically result in 48 hours or longer, and
13. van der Zee A, Roorda L, Bosman G, Ossewaarde JM. Molecular in practice this often leads to empiric antibiotic treatment of pre-
diagnosis of urinary tract infections by semi-quantitative detection sumed infections, including the suboptimal use of antibiotics. In
of uropathogens in a routine clinical hospital setting. PLoS One.
one study, 23% of emergency room patients with positive urine
2016;11. https://doi.org/10.1371/journal.pone.0150755.
cultures received inappropriate empiric antibiotic treatment
14. Price TK, Dune T, Hilt EE, et al. The clinical urine culture:
enhanced techniques improve detection of clinically relevant micro-
requiring later intervention following discharge.1 In the current
organisms. J Clin Microbiol. 2016;54:1216–1222. https://doi.org/ era of rampant antibiotic resistance, rapid diagnosis of infections
10.1128/JCM.00044-16. and associated antibiotic resistance are desperately needed to
15. Hilt EE, McKinley K, Pearce MM, et al. Urine is not sterile: use of expedite targeted treatment, and minimize overuse and inappro-
enhanced urine culture techniques to detect resident bacterial flora priate use of antibiotics. Culture-independent assays, such as the
in the adult female bladder. J Clin Microbiol. 2014;52:871–876. one described in the accompanying paper, may result in as few as
https://doi.org/10.1128/JCM.02876-13. 6 hours. The current study is a timely investigation comparing
16. Croxall G, Weston V, Joseph S, Manning G, Cheetham P, McNally standard urinary cultures and a multiplex PCR-based assay in
A. Increased human pathogenic potential of Escherichia coli from poly- detecting bacteria in patients with UTI symptoms. The authors
microbial urinary tract infections in comparison to isolates from mono-
conclude that the multiplex PCR assay was noninferior to stan-
microbial culture samples. J Med Microbiol. 2011;60(Pt 1):102–109.
https://doi.org/10.1099/jmm.0.020602-0.
dard cultures in detection of bacteria in symptomatic patients.
17. Rowe TA, Juthani-Mehta M. Diagnosis and management of urinary This study is a step forward in the implementation of culture-
tract infection in older adults. Infect Dis Clin North Am. 2014;28: independent assays in the diagnosis and treatment of UTI.
75–89. https://doi.org/10.1016/j.idc.2013.10.004. The study raises a number of important points. First, in 22%
18. Suskind AM, Saigal CS, Hanley JM, Lai J, Setodji CM, Clemens JQ. of cases, multiplex-PCR identified organisms where standard cul-
Incidence and management of uncomplicated recurrent urinary tract ture was negative. Bacteria uniquely detected by PCR in this
infections in a national sample of women in the United States. Urol- study included a number of genera that were also detected in the
ogy. 2016;90:50–55. https://doi.org/10.1016/j.urology.2015.11.051. urinary tract of healthy patients without urinary tract infection
19. Simmering JE, Tang F, Cavanaugh JE, Polgreen LA, Polgreen PM. in another report.2 Such findings highlight the importance of
The increase in hospitalizations for urinary tract infections and the
further investigation into the urinary microbiome as well as the
associated costs in the United States, 1998-2011. Open Forum Infect
Dis. 2017;4:ofw281. https://doi.org/10.1093/ofid/ofw281.
uropathogenic capacity of noncultureable and other poorly-stud-
20. Coyne KS, Sexton CC, Thompson CL, et al. The prevalence of lower ied bacteria, and whether their presence in urine should guide
urinary tract symptoms (LUTS) in the USA, the UK and Sweden: therapy. Additionally, the enhanced capacity of PCR-based
results from the epidemiology of LUTS (EpiLUTS) study. BJU Int. assays to identify multiple bacterial species will play a particu-
2009;104:352–360. https://doi.org/10.1111/j.1464-410X.2009.08427.x. larly important role in the diagnosis and treatment of patients

UROLOGY 136, 2020 125

with complicated UTIs, including subgroups such as chronically The development of a culture-independent multiplex PCR
catheterized patients or those with prostatitis, in which multiple assay for the identification of uropathogens is an essential step in
species in the form of biofilms or intracellular bacterial commu- rapidly and accurately identifying uropathogens. However, the
nities may be present.3 Indeed, bacteria in such communities ability of multiplex PCR to identify organisms commonly missed
exhibit increased rates of resistance.4 by SUC raises the question of which of these organisms are path-
The authors make note of an arm of the study, as of yet ogenic and which is part of the urinary microbiome. Price et al
unpublished, designed to describe and analyze the resistance pro- (2018) cite several studies performed in their laboratory demon-
files of bacteria identified by multiplex PCR. These results will strating the “limitations of an Escherichia coli-centric view of
be critically important in comparing PCR to standard cultures, UTI” pointing out that the majority of patients in their UTI
as the accuracy of the assays in guiding appropriate antibiotic cohort had additional species present (35 out of 43 patients).1
therapy is paramount. Importantly, there is an ongoing prospec- In this paper, we present data showing associations between
tive, randomized clinical trial wherein both multiplex PCR and routinely missed suspect uropathogens and clinically significant
standard cultures will be performed on urine samples in patients urinary tract infections. Reviewers have commented that many
with UTI symptoms, but the treating physicians will have access of the organisms identified as potential uropathogens can be
to one, the other, or both sets of results (depending on the arm seen in asymptomatic patients. The converse is also true with
of the study).5 Outcome measures will include safety, recurrent many studies demonstrating the presence of common uropatho-
or persistent infections, and time to symptom resolution. These gens, such as E. coli and E. faecalis in patients with asymptomatic
eagerly awaited results will build on the present study in assessing bacteriuria.2 The significance of both commonly ascribed uro-
the clinical efficacy of multiplex PCR relative to traditional cul- pathogens such as E. coli and emerging uropathogens such a A.
tures in the diagnosis and treatment of UTI, and have the poten- baumani are best viewed as organisms which might be clinically
tial to shift the standard of care. significant in cases of dysbiosis. In these cases, the presence and
interaction of these organisms may play an important role in
Glenn T. Werneburg, Department of Urology, Glickman symptom development. This study also underscores high inci-
Urological and Kidney Institute, Cleveland Clinic dence of false-negative cases and inability to identify polymicro-
Foundation, Cleveland, OH bial infections. These failures can lead to treatment failure and
increase antibiotic resistance. Hence, the nature of complex
References UTI infections requires a renewed effort to develop a methodol-
1. Lingenfelter E, Drapkin Z, Fritz K, et al. ED pharmacist monitoring of ogy that identifies the multiple and culture elusive pathogenic
provider antibiotic selection aids appropriate treatment for outpatient organisms and then identify the appropriate antibiotic for all the
UTI. Am J Emerg Med. 2016;34:1600. pathogenic microorganims identified. This complexity has a
2. Bajic P, Van Kuiken ME, Burge BK, et al. Male bladder microbiome direct impact on the choice of therapy. In using PCR exclu-
relates to lower urinary tract symptoms. Eur Urol Focus. 2018. sively, there are difficulties in relying on the presence or absence
3. Delcaru C, Alexandru I, Podgoreanu P, et al. Microbial biofilms in of resistance genes in determining which therapy is best suited to
urinary tract infections and prostatitis: etiology, pathogenicity, and manage the infections. Since not all antibiotic resistance genes
combating strategies. Pathogens. 2016;5:65. have been identified, there are significant gaps with respect to
4. Trautner BW, Darouiche RO. Role of biofilm in catheter-associated
certain antibiotic modalities. Furthermore, the presence or
urinary tract infection. Am J Infect Control. 2004;32:177.
5. ClinicalTrials.gov. Bethesda (MD): National Library of Medicine
absence of antibiotic resistance genes could overestimate or
(US). 2019 April 30. Identifier NCT03931538, Rapid Molecular underestimate the susceptibility of the resistance pattern.
Organism Identification and Mixed Flora Antibiotic Resistance Pro- In that many of these complex infections are polymicrobial
filing (MixAR) Prospective Study [cited 2019 Sept 28]. Available in nature, the phenotypic antibiotic response is also impacted
from: https://clinicaltrials.gov/ct2/show/NCT03931538?term=Rapid by interactions occurring between different bacterial species.
+Molecular+Organism+Identification+and+Mixed+Flora+Antibi The ideal technology must have a quick turnaround time while
otic+Resistance+Profiling+%28MixAR%29+Prospective+Study also detecting slow-growing fastidious, Gram-positive organisms
+%28RapID+MixAR%29&rank=1., 2019. that may impact treatment decisions. This combination of uro-
pathogen identification, coupled with genotypic and pheno-
typic characterization would allow for a better understanding of
https://doi.org/10.1016/j.urology.2019.10.029
this complex process and potentially lead to better therapeutic
UROLOGY 136: 125−126, 2020. © 2019 Pathnostics.
Published by Elsevier Inc. outcomes.

David Baunoch, Pathnostics, Irvine, CA

References
1. Price TK, Dune T, Hilt EE, et al. The clinical urine culture:
AUTHOR REPLY enhanced techniques improve detection of clinically relevant micro-
organisms. J Clin Microbiol. 2016;54:1216–1222.
Accumulating evidence shows that Standard Urine Culture 2. Yu Y, Zielinski MD, Rolfe MA, et al. Similar neutrophil-driven
(SUC) misses a significant number of potential uropathogens. inflammatory and antibacterial responses in elderly patients with
SUC failure, in part, is due to the incorrect nutrient composition symptomatic and asymptomatic bacteriuria. Infect Immun. 2015;83:
4142–4153. https://doi.org/10.1128/IAI.00745-15.
of the growth media, pH, time allotted for growth to occur, and
the presence of competing organisms. Additionally, other SUC
https://doi.org/10.1016/j.urology.2019.10.030
inadequacies include long turnaround times, false negatives, and
UROLOGY 136: 126, 2020. © 2019 Pathnostics. Published
the inability to detect more than 2 organisms. The inadequacies by Elsevier Inc.
may result in treatment failure.

126 UROLOGY 136, 2020