Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria

Abstract

Background
The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specifc PCR commercial kits, paired serology, and urinary antigen.

Results
A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS = 23.1 %, mPCR-IS = 57.1 %, Seeplex®-IS = 52.4 %, and Speed-oligo®-NPA/NPS = 11.1 %, and the specificities were mPCR-NPS = 97.1 %, mPCR-IS = 77.8 %, Seeplex®-IS = 92.6 %, and Speed-oligo®-NPA/NPS = 96.1 %. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs.

Conclusions
All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60 %; thus, better diagnostic techniques for these three bacteria are required.

Electronic supplementary material
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Full text

R E S E A R C H Open Access
Comparison of serological methods with
PCR-based methods for the diagnosis of
community-acquired pneumonia caused by
atypical bacteria
Mariana Herrera
1,2
, Yudy Alexandra Aguilar
1,2
, Zulma Vanessa Rueda
3
, Carlos Muskus
4
and Lázaro Agustín Vélez
1,5*
Abstract
Background: The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila,
Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which
have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these
techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP
patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs),
nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specifc PCR commercial kits,
paired serology, and urinary antigen.
Results: A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila
infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were
mPCR-NPS = 23.1 %, mPCR-IS = 57.1 %, Seeplex®-IS = 52.4 %, and Speed-oligo®-NPA/NPS = 11.1 %, and the
specificities were mPCR-NPS = 97.1 %, mPCR-IS = 77.8 %, Seeplex®-IS = 92.6 %, and Speed-oligo®-NPA/NPS = 96.1 %.
The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the
commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired
serology and in all PCRs.
Conclusions: All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR
and Seeplex® in IS was approximately 60 %; thus, better diagnostic techniques for these three bacteria are required.
Keywords: M. pneumoniae,L. pneumophila,C. pneumoniae, Multiplex PCR, Atypical pneumonia, Molecular diagnosis
Background
Infections by the atypical bacteria Mycoplasma pneumo-
niae, Chlamydophila pneumoniae, and Legionella pneu-
mophila are frequent causes of community-acquired
pneumonia (CAP) in both children and adults [1–3].
Latin America has reported CAP figures caused by these
bacteria ranging from 1.7 to 15.7 % for M. pneumoniae,
3.4 to 6.1 % for C. pneumoniae, and 1.1 to 4 % for L.
pneumophila [3, 4].
Diagnosis of these bacteria is traditionally based on
cultures and serology, which involve special technical re-
quirements that are costly and time-consuming, offer
delayed results, and in the case of serology, require a
second convalescent-phase sample, which limits the clin-
ical usefulness of these techniques [5–7]. This explains
why although the circulation of atypical bacteria in the
region is evident, these bacteria can only be diagnosed in
very specialized reference centers. Due to this aspect,
and because the clinical presentation does not differ
significantly from that caused by pyogenic bacteria or re-
spiratory viruses [8], the perception is that these agents
* Correspondence: velezlazaro@yahoo.com
1
Grupo Investigador de Problemas en Enfermedades Infecciosas (GRIPE),
Sede de Investigación Universitaria, Calle 62 # 52-59, Laboratorio 630,
Universidad de Antioquia, Medellín, Colombia
5
Infectious Disease Section, School of Medicine, Universidad de Antioquia
UdeA, Medellín, Colombia
Full list of author information is available at the end of the article
© 2016 Herrera et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3
DOI 10.1186/s12952-016-0047-y

are rare in these countries. The therapeutic consequence
of this omission is the prescription of insufficient treat-
ments in some cases or treatments that are excessive
and unnecessary in others.
Given these problems, nucleic acid amplification tech-
niques are often used, including conventional PCR, real-
time PCR (qPCR), and in-house or commercial mPCR
[9–11]. These are considered faster, more sensitive, and
more specific than cultures and serology [12]. However,
the possibility of contamination and the difficulties of
interpreting positive cases as disease or colonization are
the main limitations. Although several commercial kits
for the detection of M. pneumoniae, C. pneumoniae, and
L. pneumophila are now available [10, 13–15], limited
information is available in the literature regarding the
validation process of such tests. The existing studies
have limited information about the clinical condition of
the study population in which the tests were validated,
the samples used, and the molecular targets; some stud-
ies compared only the commercial kit with another in-
house or commercial molecular test, without using any
other accepted reference tests (culture or paired ser-
ology). Additional file 1 describes the heterogeneity of
the previously conducted studies.
To investigate a possible solution to these diagnostic
difficulties, our aim was to standardize and validate an
in-house mPCR for a quick and timely diagnosis of CAP
caused by these atypical bacteria in a single reaction. In
addition, we sought to evaluate the diagnostic perform-
ance of mPCR in different respiratory samples, namely,
nasopharyngeal aspirates (NPAs), nasopharyngeal swabs
(NPSs) and induced sputum (ISs), and to compare this
performance with that of existing PCR commercial kits,
paired serology, and urinary antigen.
Results
Standardization of multiplex PCR
The primers used allowed the amplification of the gene
fragments of interest: mip from L. pneumophila,pstI
from C. pneumoniae and p1 from M. pneumoniae, and
these primers showed no cross-reactions among the bac-
teria, either with related species or other microorgan-
isms, according to the specificity analysis of the reaction
obtained with the BLAST program. The conditions,
under which optimal mPCR amplification was achieved
in a final volume of 25 μL, were 0.05 U/μL Taq polymer-
ase (Fermentas St. Leon-Rot, Germany), 1X Taq buffer
with KCl, 2.0 mM MgCl
2
, 0.2 mM dNTPs, 0.3 μM con-
centrations of each primer, 0.1 mg/μL BSA, and 6 μLof
DNA (The median concentration of the extracted
DNA from each sample was 4.9 ng/μL, upper limit:
166.18 ng/μL, lower limit: 2.11 ng/μL). The cycling
conditions in the C1000 thermal cycler (BioRad, CA,
USA) were as follows: one cycle of DNA denaturation
at 95 °C for 5 min; 35 cycles of denaturation at 94 °C
for45s,primerannealingat58°Cfor60sandpri-
mer extension at 72 °C for 45 s; and a final extension
at 72 °C for 7 min.
Standardized PCR had a detection limit of 375 copies
for each gene, regardless of whether the PCR was set-up
to amplify a single gene or two or three genes simultan-
eously (Fig. 1); however, some amplification was observed
with 187 copies of DNA, especially when a DNA mixture
of two bacterial strains was run. No cross-amplification
with DNA from the 17 different pathogens and/or
colonizing microorganisms of the respiratory tract or with
human DNA (Fig. 2) was observed.
Standardized mPCR was reproducible using a concen-
tration of 750 copies of each gene when six PCR reactions
were run simultaneously (intra-assay reproducibility) and
on six different days (interassay reproducibility). Regard-
less of the test day, the intensity of the signal did not vary.
Clinical and epidemiological characteristics
A total of 205 individuals with CAP were analyzed in
three groups –68 adults in Group 1, 88 adults in Group
2 and 49 children in Group 3. Table 1 describes the
main characteristics of these three groups. The etiology
observed in Table 1 does not reflect the percentage dis-
tribution of the microorganisms found in the evaluated
cohorts but is due to the selection of patients required
to evaluate the techniques being studied.
Most of the 20 individuals in group 4 (control group)
were male (60 %) and had a median age of 50 years (Q1
to Q3 = 29 to 55). Eight of the 10 individuals who
suffered rheumatic diseases had been diagnosed with
rheumatoid arthritis; 1, with systemic lupus erythemato-
sus; and 1, with Sjögren’s syndrome. Three of them were
receiving tumor necrosis factor-alpha (anti-TNFα) antag-
onists at the time of inclusion in the study.
Test results
Among the 225 patients included in the 4 groups, 190
paired serologies were performed (46 in group 1, 77 in
group 2, 48 in group 3, and 19 in group 4). In addition,
88 mPCR were performed in NPAs, 137 mPCR in NPSs,
49 mPCR and 49 Seeplex® Pneumobacter in IS, and 161
Speed-Oligo® in NPAs or NPSs. The L. pneumophila
urinary antigen was positive in only one patient in group
2, who also exhibited a positive paired serology; because
of that, this urinary antigen was not considered as a gold
standard.
The results of the negative and positive controls of the
serology tests, the urinary antigen and the different
molecular tests were always negative and positive, re-
spectively. The inhibition control of the PCRs was posi-
tive in all samples tested, indicating the absence of PCR
inhibitors.
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 2 of 11

In samples obtained from hospitalized patients show-
ing CAP symptoms and distributed among groups 1, 2
and 3, mPCR was only positive for M. pneumoniae in
one sample in group 1 and in 25 samples of group 3 (7
samples of NSP and 18 IS samples). No amplification
was observed for C. pneumoniae and L. pneumophila in
any of the samples with mPCR. In contrast, with com-
mercial PCR (Speed-oligo or Seeplex), amplification was
achieved in a larger number of samples undergoing
mPCR. With commercial PCR, a total of 18 M. pneumo-
niae-positive samples were detected in the three groups
(4 in group 1, 1 in group 2, and 13 in the group 3). Only
1 sample in group 2 was positive for C. pneumoniae, and
2 samples in group 1 were positive for L. pneumophila
(Table 2).
When assessing the positivity of serology by quad-
rupling the antibody titers, the technique considered
the gold standard in this study, serology was observed
to detect a greater number of positive samples than
any of the 3 types of PCR used in this study. For M.
pneumonia, 30 samples were positive (5 in group 1, 4
in the group 2, and 21 in the group 3). For C. pneu-
moniae, 18 samples were positive (3 in group 1, 9 in
group2,and6ingroup3),whereas10sampleswere
positive for L. pneumophila,(3ingroup1,2ingroup
2,and5ingroup3).Ingroup3,twotypesof
samples (NPSs and IS) were evaluated by mPCR.
OnlythepresenceofDNAfromM. pneumonia was
detected in 25 samples. Of these, 18 samples were
positive for the IS, and 7, for the NPSs.
Interestingly, in samples from the control group and
without symptoms of CAP, 6 serologically positive sam-
ples were detected. Of these, 2 were positive for M.
pneumoniae, and 4, for C. pneumoniae. In addition, one
sample was positive for mPCR, and 4, for commercial
PCR for L. pneumophila.
Fig. 1 Analytical sensitivity of mPCR using 1,500; 750; 375; and 187 copies of L. pneumophila mip genes, p1ofM. pneumoniae, and PstlofC.
pneumoniae MW: 100 bp molecular weight marker; NC: negative control; Lines marked with arrows correspond to the amplicons from 375 copies
of each gene
Fig. 2 Analytical specificity of mPCR. 1. Molecular weight marker 100 bp; 2. Negative control; 3. Positive control (487 bp L. pneumophila,
360 bp M. pneumoniae, and 283 bp C. pneumoniae); Bacteria: 4.Streptococcus pneumoniae; 5.Haemophilus influenzae; 6.Klebsiella
pneumoniae; 7.Escherichia coli; 8.Pseudomonas aeruginosa; 9.Staphylococcus aureus; 10. Nocardia spp.; 11.Enterobacter cloacae; Fungi:
12.Histoplasma capsulatum;13.Aspergillus terreus;14.Cryptococcus neoformans;15.Candida tropicalis;16.Candida albicans;17.Candida
guilliermondii;18.Candida glabrata;19.Paracoccidioides brasiliensis;20.Mycobacterium tuberculosis (bacteria); 21. Human DNA
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 3 of 11

Finally, when analyzing the samples obtained from
individuals with CAP in a global and comprehensive
manner, that is, without division by groups, M. pneumo-
niae was the most detected bacteria by any of the three
methods. By serology, 30 samples were detected, and by
mPCR, 26 samples, whereas 18 samples were positive by
cPCR. For C. pneumoniae, only one sample was positive
by cPCR, and 18 were positive by serology, whereas for
L. pneumophila, 2 samples were positives by cPCR, and
10, by serology (Table 2).
Given that no positive cases of C. pneumoniae and L.
pneumophila were obtained by mPCR, and very few
cases, by commercial PCR, only the operating character-
istics of the PCRs to M. pneumoniae are presented
below.
Table 3 shows that the PCRs exhibit high specificity
with low sensitivity in the nasopharyngeal samples for
both the NPAs and NPSs. The sensitivity was higher in
the IS, but was only 57.1 % for mPCR and 52.4 % for
Seeplex® PneumoBacter. In turn, when the PCRs with
Table 1 Clinical and epidemiological characteristics of the population with CAP
Variables Group 1
(n= 68)
Group 2
(n= 88)
Group 3
(n= 49)
Age in years, Median (Q
1
-Q
3
) 65 (41–76) 63 (40–76) 3 (1–7)
Males, n (%) 28 (41.2) 49 (55.7) 29 (59.2)
Received antibiotics in last 3 months, n (%) 19 (27.9) 10 (11.4) 9 (18.4)
Symptom duration, in days, Median (Q
1
-Q
3
)7(4–15) 6 (3–10) 4 (2–8)
Presence of comorbidities, n (%) 37 (54.4) 50 (56.8) 2 (4.1)
Chronic obstructive pulmonary disease 24 (35.3) 37 (42) 0
History of convulsions in the last month 0 3 (3.4) 2 (4.1)
Severe pneumonia
a
, n (%) 15 (22.1) 26 (29.5) 1 (2.0)
Frequency of atypical bacteria, n (%)
M. pneumoniae 5 (10.9) 4 (5.2) 21 (43.8)
C. pneumoniae 3 (6.5) 9 (11.7) 6 (12.5)
L. pneumophila 3 (6.5) 2 (2.6) 5 (10.4)
Length of hospital stay, in days, Median (Q
1
-Q
3
)6(3–9) 7 (4–10) 4 (2–9)
In-hospital death, n (%) 0 8 (9.1) 0
a
In children (group 3), this corresponds to the WHO classification of very severe pneumonia
Table 2 Positive results of serology, in-house mPCR, and commercial PCR classified by atypical bacteria
Techniques Group 1
a
NPS
Group 2
b
NPA
Group 3
c
NPS and IS
Group 4
d
NPS
M. pneumoniae n (%) n (%) n (%) n (%)
Serology 5/46 (10.9) 4/77 (5.2) 21/48 (43.8) 2/19 (10.5)
mPCR 1/68 (1.5) 0/88 (0) NPS: 7 (14.3)IS: 18 (36.7) 0/20 (0)
Commercial PCR
[Trade mark]
4/68 (5.9)
[Speed-oligo]
1/88 (1.4)
[Speed-oligo]
13/49 (26.5)
[Seeplex]
0/20 (0)
[Speed-oligo]
C. pneumoniae
Serology 3/46 (6.5) 9/77 (11.7) 6/48 (12.5) 4/19 (21.1)
mPCR 0/68 (0) 0/88 (0) NPS: 0 (0)IS: 0 (0) 0/20 (0)
Commercial PCR 0/68 (0) 1/88 (1.4) 0/49 (0) 0/20 (0)
L. pneumophila
Serology 3/46 (6.5) 2/77 (2.6) 5/48 (10.4) 0/19 (0)
mPCR 0/68 (0) 0/88 (0) NPS: 0 (0)IS: 0 (0) 1/20 (5.0)
Commercial PCR 2/68 (2.9) 0/88 (0) 0/49 (0) 4/20 (21)
NPS Nasopharyngeal swab, NPA Nasopharyngeal aspirate, IS Induced sputum
a
Prospective adults with community-acquired pneumonia (CAP). Speed-Oligo® was run as commercial PCR on the NPSs
b
Retrospective adults with CAP. Speed-Oligo® was run as commercial PCR on the NPAs
c
Children with CAP. Seeplex® PneumoBacter was run as a commercial PCR on IS
d
Individuals without CAP. Speed-oligo® was run as a commercial PCR on NPSs
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 4 of 11

the highest sensitivity in IS were compared with each
other, PCR Seeplex® PneumoBacter exhibited higher spe-
cificity and positive predictive value than mPCR.
Under our PCR conditions, the concordance between
methods in a single sample and between samples with a
single method was very low (kappa coefficient <0.4).
Interestingly, in the IS, the concordance was better be-
tween mPCR and Seeplex (kappa = 0.67) (Fig. 3).
Discussion
The results of this study allow us to highlight three key
aspects: 1) the in-house PCR standardized in this study
and the commercial PCRs used had low sensitivity and
poor concordance compared with the paired serology; 2)
the IS sample had the best performance for the diagnosis
of M. pneumoniae by PCR compared with those ob-
tained by NPAs and NPSs; and 3) the quadrupling of
titers in the paired serology for C. pneumoniae and
M. pneumoniae could occur in individuals without
CAP. Among them, PCRs could also be positive for
L. pneumophila.
Regarding the first point, the literature has reported
good agreement between the in-house and commercial
PCRs performed on sputum, bronchoalveolar lavage
(BAL), and endotracheal aspirates with paired serology
results [16]; however, in our study, the concordance be-
tween the evaluated PCRs and serology was very low.
Templeton et al. reported similar findings in 2003; they
found that out of 106 samples tested, 12 were positive
by 3 methodologies other than PCR, but only 8 of these
were positive by paired serology [17]. The finding of
positive PCRs in respiratory secretions without quadru-
pling in antibody titers in patients with CAP may occur
because these patients are asymptomatic carriers of M.
pneumoniae or C. pneumoniae in the respiratory epithe-
lium or because of the persistence of these bacteria or
their nucleic acids in the respiratory tract following
previous infections [18–20]. Similarly, the false nega-
tive results of PCRs could be explained by a bacterial
load below the detection limit of the PCRs, previous
antibiotic treatment in patients, dilutions of samples
when added to the transport medium, degradation of
Table 3 Operational features of the PCRs used for M. pneumoniae
Test (N) Sensitivity (%) Specificity (%) PPV (%) NPV (%)
mPCR NPA (77) N/A N/A N/A N/A
mPCR NPS (94) 23.1 (4.9–41.2) 97.1 (92.3–100) 75 (38.7–100) 76.7 (67.2–86.2)
mPCR IS (48) 57.1 (33.6–80.7) 77.8 (60.2–95.3) 66.7 (42.1–91.2) 70 (51.9–88.1)
Seeplex® PneumoBacter IS (48) 52.4 (28.6–76.1) 92.6 (80.7–100) 84.6 (61.2–100) 71.4 (55.0–87.8)
Speed-oligo® NPA/NPS (111) 11.1 (0–37.2) 96.1 (91.8–100) 20 (0–65.1) 92.4 (86.9–97.9)
Values calculated with paired serologists as the gold standard. NPA Nasopharyngeal aspirate. NPS Nasopharyngeal swab. IS Induced sputum. PPV Positive
predictive value. NPV Negative predictive value. N/A Not applicable (these values cannot be calculated for this test due to the lack of positive results in serology
that coincide with the positive results in mPCR of NPAs)
Fig. 3 Concordance (kappa index) between in-house (mPCR) and commercial PCR for Mycoplasma pneumoniae. NPS: Nasopharyngeal swab; NPA:
Nasopharyngeal aspirate; IS: Induced sputum
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 5 of 11

significant amounts of DNA during the sample
storage process, or the presence of interfering DNA
coming from human cells or other colonizing micro-
organisms of the respiratory tract, which would affect
amplification. The positive and negative results in the
controls in all experiments (serology, urinary antigen,
and molecular techniques) ruled out the possibility of
experimental error, and the amplification of the inhibition
controls ruled out the presence of PCR inhibitors.
With regard to the sensitivity obtained for in-house
mPCR and commercial kits using the quadrupling of ti-
ters in paired serology as the gold standard, this was
lower than previous studies [15, 21], in which the sensi-
tivity ranged from 66.7 to 97.3 %. Even sensitivities and
specificities of up to 100 % have been described when
the gold standard used was a PCR monoplex assay and
the study population was comprised solely of positive
individuals confirmed by this technique [22] (53). This
feature highlights the importance of knowing the charac-
teristics of the study population, the type of respiratory
sample being used (sputum, NPS, NPA, or BAL) and the
inhibitors potentially present in each of them, the popu-
lation where the PCR (adults, children, or elderly) is be-
ing assessed, and the various molecular targets being
used.
NPSs and NPAs have been proposed as good choices
of sample type for the diagnosis of CAP when resorting
to non-invasive samples [23], but for the diagnosis of
atypical bacteria, sputum has a higher performance than
NPSs [24, 25], and in turn, these samples are superior to
NPAs [26]. The results of our study were consistent with
this claim; that is, we found that the PCR results varied
for the diagnosis of M. pneumoniae depending on the
type of respiratory specimen used, as the IS enabled the
identification of a greater number of cases. In this re-
gard, Collier and Clyde [27] and Kenny et al. [28] indi-
cated that sputum samples were superior for the
detection of M. pneumoniae because the number of bac-
teria are higher in the pulmonary alveolus than in the
epithelium of the upper respiratory tract of patients with
pneumonia. However, Reznikov et al. [26] reported that
the PCR for M. pneumoniae in NPAs and NPSs had
similar positivity percentages (45 and 50 %, respectively)
but a greater presence of inhibitors in NPAs (36 %) than
in NPSs (0 %).
The type of population also affects the operational fea-
tures of the PCRs in that the results of paired serology
vary according to patient age, prior exposure to these
bacteria, or the presence of comorbidities. Acute M.
pneumoniae infections in children are characterized by
significant increases in IgM antibodies but can only
increase titers of IgG or both immunoglobulins; also, the
IgM titers may remain high for several months or even
years [29], which constitutes the main limitation of this
test. However, adults can respond by increasing only
IgG, especially when a re-infection occurs by this germ,
or they may be unable to mount an appropriate sero-
logical response due to deficiencies in the immune
system, which are common in patients of certain ages
[30] or with underlying diseases. Examples include
immunocompromised individuals or those with rheu-
matologic diseases [31]. Therefore, the gold standard
against which these molecular diagnostic techniques
are being assessed is far from being the ideal test.
Furthermore, PCR detection of atypical bacteria also
has limitations; no consensus exists regarding which
molecular target should be amplified to achieve
higher sensitivity and specificity, nor does a clearly
defined standard protocol exist [21, 32–34]. Depend-
ing on the selected molecular target, in which one or
multiple copies could be in the investigated genome,
the amount of DNA of the microorganisms present in
the sample can vary significantly. Even when the pres-
ence of the same gene is studied using two different
molecular tests, such as Speed-oligo® and mPCR, the
results may show poor consistency. This may be due
to differences in the methodologies used (including
the type of PCR –monoplex versus multiplex –and
the revealing technique –oligochromatography versus
agarose gel electrophoresis) or because of the amplifi-
cation of the different regions of the same gene [16].
Although some authors reported similar results when
they used a single PCR or a duplex assay to detect
two of these pathogens [14], others argue that the
conventional format for some PCRs is more sensitive
than the multiplex [35], which may have contributed,
at least in part, to some false negatives obtained with
mPCR in our study.
Finally, the positive results obtained by serology and
PCR in individuals without CAP require a better defin-
ition of the role of the causative microorganisms in the re-
spiratory microbiome of these subjects and of the
usefulness of this serological test as the gold standard.
Both M. pneumoniae and C. pneumoniae are bacteria that
are known to colonize the respiratory tract [18–20]. Re-
cent studies show the presence of M. pneumoniae and C.
pneumoniae in asymptomatic individuals (by culture, ser-
ology, or detection of DNA). Therefore, detection of these
pathogens by PCR does not necessarily indicate disease,
and such studies make it clear that none of the methods
currently used for diagnosis make it possible to differenti-
ate the carrier state of symptomatic infection [18–20]. It is
possible that because many infections caused by these
pathogens are asymptomatic, some of the patients without
CAP who served as controls may have been recently in-
fected by the pathogens without developing the disease
[36], which potentially helps explain the serology conver-
sions observed in these individuals.
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 6 of 11

In addition, in 2010, Villegas et al. claimed that C.
pneumoniae serology can give false positives due to cross-
reactions in cases of acute infection because of the
presence of heterotypic antibodies [37]. A similar
phenomenon can be observed with M. pneumoniae,
whose acute infections are often characterized by the tran-
sient generation of autoantibodies, which are considered
responsible for many of their extrapulmonary manifesta-
tions, and, as shown by our results, some patients with
autoimmune diseases may yield false positive results.
In this study, Speed-oligo® for L. pneumophila was posi-
tive in four patients with rheumatic diseases, whereas
mPCR was positive in one of those cases. Although the
carrier state for this germ has not been described, several
possible explanations exist for this finding. Either people
were colonized or were at risk of becoming ill because of
the bacteria [38, 39], or these results were false positives
of the PCRs, results that cannot be attributed to cross-
contamination with other samples as the extraction con-
trols and amplification of PCRs were always negative.
One limitation of the study was the absence of cul-
tures as a gold standard for diagnosis, particularly be-
cause such cultures may help to clear up discordant
cases. Another possible limitation was that to complete
the sample size, we had to resort to various groups of
patients (adults and children admitted prospectively and
retrospectively). Although these groups were analyzed
separately and we were able to evaluate how the tests
behaved among themselves in different samples and dif-
ferent populations, the sample size per group was low.
Further studies that prospectively evaluate these aspects
are required.
Conclusions
This study demonstrates that the molecular tests (in-
house and commercial) and the reference tests evaluated
for the diagnosis of atypical bacteria in patients with
CAP have low sensitivity, and do not allow discrimin-
ation between those patients with acute or convalescent
infection and asymptomatic carriers. Thus, the develop-
ment of better techniques is needed for the diagnosis of
CAP caused by M. pneumoniae,C. pneumoniae, and L.
pneumophila. Such studies should include prospective
evaluations of different sample types and molecular tar-
gets, quantification of bacterial DNA, pediatric popula-
tions and healthy adults, individuals with suspected CAP
infection by these microorganisms, immunocompetent
and immunocompromised individuals, and different mo-
lecular techniques.
Methods
Standardization of mPCR
DNA from M. pneumoniae strain FH of Eaton Agent
(gene p1), C. pneumoniae strain CM-1 (gene PstI), and
L. pneumophila strain Philadelphia-1 (gene mip) from
the American Type Culture Collection (ATCC® Virginia,
USA) was used for the standardization of mPCR, accord-
ing to the protocol and primers described by McDonough
et al. [40] (Additional file 2). The specificity of the primers
was verified using the BLAST program, and the tendency
to form homo- and heterodimers, in addition to secondary
structures, was evaluated using the Oligo Analyzer pro-
gram (IDT Technologies, www.idtdna.com/calc/analyzer).
The optimal concentrations of the PCR reagents were
experimentally determined: primers (0.2–1.0 μM),
Taq poly m er as e (0.05–0.3U/μL), Magnesium chloride
(1.0–2.5 mM), and bovine serum albumin (BSA
(0.1–0.7 μg/μL) as adjuvant. The best annealing
temperature was selected by performing a temperature
gradient between 55 °C and 66 °C; in addition, primer an-
nealing and extension were evaluated between 30 and
60 s. The optimal conditions were selected according to
the points to achieve the sharpness of banding with the
lowest DNA concentration.
PCR reactions were revealed using 2 % agarose
gel electrophoresis (AMRESCO®, USA), stained with
EZ-VISION™(AMRESCO®, USA); the gel was run at
70 V for 50 min. Gel images were obtained using
the ChemiDoc XRS (BioRad) equipment and the
Quantity One® program.
Determination of analytical sensitivity and specificity
The determination of analytical sensitivity was per-
formed using serial dilutions of DNA from the strains
obtained from ATCC or with plasmids containing gene-
specific inserts. Each amplified fragment was ligated to
the pGEM®-Teasy plasmid (Promega®, Southampton,
USA) according to the manufacturer’s instructions.
Then, the recombinant plasmids were purified using the
Wizard® plus SV Miniprep DNA Purification System
(Promega®, Southampton, USA), linearized and quanti-
fied using NanoDrop®. The number of copies was calcu-
lated from the obtained nanograms [41, 42], and serial
dilutions were made. Analytical specificity was evaluated
with DNA from different sources at a concentration of
4 ng/μL. We evaluated human DNA from peripheral
blood cells and DNA from pathogenic and frequent
colonizers of the respiratory tract. These colonizers
included the bacteria Streptococcus pneumoniae, Staphylo-
coccus aureus, Haemophilus influenzae, Escherichia coli,
Enterobacter cloacae, Mycobacterium tuberculosis, Nocar-
dia spp., Klebsiella pneumoniae ATCC10031 and Pseudo-
monas aeruginosa ATCC PA01 and the fungi Candida
albicans, Candida tropicalis, Candida guilliermondii,
Candida glabrata, Histoplasma capsulatum, Cryptococcus
neoformans, Aspergillus terreus,andParacoccidioides bra-
siliensis. A dilution corresponding to 750 copies of each
gene was used to evaluate reproducibility. Furthermore,
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 7 of 11

mPCR was run six times in one day to determine intra-
assay reproducibility and on six different days to verify
inter-assay reproducibility.
Validation of multiplex PCR
To validate the mPCR technique, a sample size of 188
patients with CAP was calculated, taking into account
an expected sensitivity of 92 % for mPCR, a prevalence
of CAP in the city caused by these three atypical bacteria
of 24.4 %, and a confidence level of 92 %. All patients
had to be hospitalized.
Study population
The population consisted of four study groups; the first
three groups involved patients hospitalized with CAP
who were not severely immunosuppressed. Group 1
consisted of 68 patients who were prospectively enrolled
for this study, whereas the patients in group 2 (n= 88)
and 3 (n= 49) were taken from two previous studies
conducted by our group. Positive cases were selected by
quadrupling the titers for these atypical bacteria, and the
patients with CAP caused by other pathogens or of un-
known etiology were selected randomly until the esti-
mated sample size was attained. The fourth group
included individuals without pneumonia (controls) and
was divided into two subgroups of equal numbers of pa-
tients. One subgroup consisted of blood donors who
were completely healthy; the other included patients
with rheumatic diseases who were at a higher risk of
false positive reactions in paired serology (Table 4).
Ethics, consent and permissions
All individuals who met the inclusion criteria for the
four groups signed an informed consent form in which
they agreed to participate. For children, the consent
form was signed by the parents or caregivers. Addition-
ally, all children over six also signed the consent form.
This study was approved by the Ethics Committee of the
School of Medicine at the Universidad de Antioquia
(Approval bylaws of the ethics committee: 017 of No-
vember 2011, 040 of May 2003 and 005 of May 2011)
and by the Ethics Committee of participating institu-
tions: E.S.E. Metrosalud Unidad Hospitalaria San Javier,
Clínica Infantil Santa Ana, Clínica Sagrado Corazón,
Clínica León XIII, Hospital Universitario San Vicente
Fundación, Hospital General de Medellín, Hospital Pablo
Tobón Uribe, Clínica Las Américas, Hospital San Rafael
de Itagüí, Clínica CES, Hospital Marco Fidel Suárez,
Clínica SOMA and Hospital Manuel Uribe Ángel.
Clinical samples and data collection
Blood, urine, and respiratory secretion samples were
taken from all patients at the time of enrollment. Ac-
cording to the established protocol for each study group,
the NPSs of groups 1 and 4 were stored at −20 °C,
whereas the NPAs of Group 2 and the NPSs and IS of
group 3 were stored at −80 °C until processing. Blood
samples were taken again between four and eight weeks
after capture for convalescent-phase serologic testing.
Antibodies and antigen-based detection methods
All individuals included in this study underwent the fol-
lowing microbiological tests for the diagnosis of atypical
bacteria (following the manufacturer’s instructions):
Detection of antibodies in acute and convalescent
serum: total antibodies for L. pneumophila
(serogroups 1 to 6, IFI Kits FOCUS Diagnostics®
Cypress, CA, USA), IgM and IgG antibodies for M.
pneumoniae (EIA Pneumobact IgM and IgG
VIRCELL®, Granada, Spain), and IgG antibodies for
C. pneumoniae (Micro-IFI IgG FOCUS Diagnostics®,
Cypress, CA, USA).
Urinary antigen for L. Pneumophila, serogroup 1:
performed with concentrated urine (Binax NOW®,
Legionella Urinary Antigen Test, Scarborough, ME,
USA).
PCR-based molecular diagnosis
Each sample was evaluated using at least two different
molecular tests; one was standardized mPCR, which was
performed on all samples; the second test was performed
using at least one of the two commercial kits to employ
a similar, standardized, and validated technique to
allow comparison with mPCR. Speed-oligo® (VIRCELL,
Granada, Spain) was used in the NPAs or NPSs of groups
1, 2 and 4, and Seeplex® PneumoBacter ACE detection
(Seegene, Seoul, Korea) was used in the IS of Group 3
(Table 4).
For PCR testing, between 300 and 500 μl of the sam-
ples were used for DNA extraction. These respiratory
samples were thawed and homogenized by vortex for
5 min, centrifuged for 10 min at 10,000 rpm, and the
supernatant was discarded. DNA was extracted using
the DNeasy® Blood & Tissue Kit (QIAGEN®, Hilden,
Germany) and quantified using a NanoDrop® (Thermo
Scientific). The total DNA volume added to the reaction
was 6 μL (which we considered to be optimal after
evaluating different volumes between 3 and 8 μL). The
DNA concentration was not standardized. Additionally,
the presence of inhibitors was ruled out amplifying the
β-globin gen.
All samples were coded and processed blindly to avoid
selection and information bias.
Data analysis
For data analysis, a database was generated using Access®
and was subjected to quality control prior to analysis.
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 8 of 11

Table 4 Eligibility criteria of the study population
Group 1 (n= 68) Group 2 (n= 88) Group 3 (n= 49) Group 4 (n= 20)
Recruitment Prospective, recruited from
2010 to 2012
Retrospective, recruited from
2005 to 2006.
Prospective, recruited from
2011 to 2012
Prospective, recruited from
2011 to 2012
Inclusion Criteria 1. Adults ≥18 years
2. Hospitalized with CAP, with
radiologic evidence
3. Agreed to participate in the study
4. Available NPS, urine, and blood
samples
1. Patients ≥18 years
2. Hospitalized with CAP, with
radiologic evidence
3. Agreed to participate in the
study
1. Children between 1 month
and 17 years
2. Hospitalized with CAP, with
radiologic evidence
3. Agreed to participate in the
study
4. Under 15 days of symptoms
1. Adults ≥18 years
2. Agreed to participate in
the study
3. Completely healthy,
recruited from blood banks,
or individuals with rheumatic
conditions, without respiratory
infection, who could be receiving
tumor necrosis factor antagonists
(anti-TNF)
Exclusion Criteria 1. Hospitalization during the 2 weeks
prior to recruitment
2. Obstructive pneumonia due to
lung cancer
3. Had received antibiotics for over
72 continuous hours at the time of
admission
4. Severely immunocompromised by:
•Steroid treatment (prednisone
≥0.3 mg/kg/day for 3 weeks or more
or ≥1mg/kg/dayfor≥7 days; if using
other steroids, an equivalent dose was
considered)
•Treatment with cytostatics (except
low doses of methotrexate:
≤15 mg/week),5. AIDS diagnosis,
lymphocyte count CD4+ <200/mm
3
in patients over 5 years of age or less
than 15 % of patients under 5
granulocytopenia <500/mm
3
,or
hematologic neoplasia.
1. Hospitalization during the
2 weeks prior to recruitment
2. Obstructive pneumonia due
to lung cancer
3. Severely immunocompromised by:
•Steroid treatment (prednisone
≥0.3mg/kg/dayfor3weeksormore
or ≥1mg/kg/dayfor≥7 days; if using
other steroids, an equivalent dose was
considered)
•Treatment with cytostatics (except
low doses of methotrexate:
≤15 mg/week),
•AIDS diagnosis, lymphocyte count
CD4+ <200/mm
3
in patients over
5 years or less than 15 % patients
under 5
•granulocytopenia <500/mm
3
or
hematologic neoplasia.
1. Hospitalization during the
2 weeks prior to recruitment
2. Primary immunodeficiency or
severe acquired immunodeficiency
3. Cystic fibrosis4. Neurological
alterations (cerebral palsy or
neuromuscular disorders) or
psychiatric alterations that kept
the individual from signing the
consent form,5. Inborn errors of
metabolism,6. Bronchiolitis in
children under 27. Hematologic
neoplasia,8. Granulocytopenia
<500 cell/mm39. Non-infectious
chronic neumopathy10. Had AIDS
or lymphocyte count CD4 < 15 %
in children under 5 years11.
Individuals currently being treated
with:
•High-dose steroids (prednisone
≥0.3 mg/kg/day for 3 weeks or
more, or ≥1 mg/kg/day for
≥7 days; if using other steroids,
an equivalent dose was considered)
•Treatment with cytostatics12.
Had received antibiotics for over
72 continuous hours at time of
admission
1. Respiratory infections in the
last month
2. Hospitalization during the
2 weeks prior to recruitment
3. Healthcare workers
4. Heart disease or chronic lung
diseases
5. Cancer, granulocytopenia, or
infection by HIV/AIDS
6. Individuals currently being treated
with:
•High-dose steroids (prednisone
≥0.3 mg/kg/day for 3 weeks or more
or ≥1 mg/kg/day for ≥7 days; if using
other steroids, an equivalent dose was
considered)
•Treatment with cytostatics
•methotrexate with doses
>15 mg/week7. Had received
antibiotics for over 72 continuous
hours at time of admission
Sample Type NPS NPA stored at −80 °C NPS and IS NPS
Commercial PCR used Speed-Oligo® Speed-oligo® Seeplex® Pneumobacter Speed-Oligo®
Herrera et al. Journal of Negative Results in BioMedicine (2016) 15:3 Page 9 of 11

Statistical analyses were performed using SPSS, version
21.0. Frequency distributions were used to describe the
sociodemographic and clinical characteristics of the L.
pneumophila,M. pneumoniae,orC. pneumoniae cases
identified. Sensitivity, specificity, positive and negative pre-
dictive value of mPCR, Speed-oligo®, and Seeplex® Pneu-
moBacter were determined using the Epidat 3.1 program.
Quadruplicate antibody titers and/or urinary antigen were
used as a gold standard test. In addition, the concordance
among the molecular techniques (mPCR, Speed-oligo®
and Seeplex® PneumoBacter), between these techniques
and serology, and between the different samples was eval-
uated using the Cohen kappa test.
Additional files
Additional file 1: Nucleic acid amplification techniques and samples
used for atypical bacteria detection. (DOCX 39 kb)
Additional file 2: The mPCR primers used for the amplification of
M. pneumoniae, L. pneumophila and C. pneumoniae. (DOCX 13 kb)
Abbreviations
BAL: bronchoalveolar lavage; CAP: community-acquired pneumonia;
IS: induced sputum; mPCR: multiplex PCR; NPA: nasopharyngeal aspirate;
NPS: nasopharyngeal swab.
Competing interests
The authors declare that they have no conflicts of interest. No relation exists
with the commercial firm that produces or distributes the serology or PCR
tests for the detection of atypical bacteria, and no financial, academic or
personal interest exists, directly or indirectly, that may call into question the
validity of what is reported. This study was funded by Fundación Rodrigo
Arroyave, Universidad de Antioquia, and Fundación Investigando en Salud y
Enfermedades Infecciosas, and the Universidad de Antioquia, through the
Estrategia de Sostenibilidad CODI 2013–2014.
Authors’contributions
MH and YAA conducted the laboratory tests. MH drafted the manuscript. CM
participated in the design of the study and in the molecular interpretation
and development of multiplex PCR. YAA, ZVR, and LAV conceived the study;
participated in its design, coordination, and data analysis; and helped to
draft the manuscript. All authors read and approved the final manuscript.
Authors' information
MH: Microbiologist, Master’s student in Basic Sciences with an emphasis in
microbiology and parasitology. YAA: Bacteriologist, PhD student in Basic
Sciences with emphasis in microbiology and parasitology. ZVR: MD, PhD in
Epidemiology, Research Department, School of Medicine, Universidad
Pontificia Bolivariana. CM: Bacteriologist, MSc in Microbiology and
Parasitology, PhD in Basic Science, Coordinator Molecular Biology and
Computational Unit, Programa de Estudio y Control de Enfermedades
Tropicales (PECET), School of Medicine, Universidad de Antioquia. LAV: MD,
specialist in Internal medicine and Infectious Diseases, Department of
Internal Medicine, School of Medicine, Universidad de Antioquia.
Acknowledgments
Thanks to Fundación Rodrigo Arroyave, Universidad de Antioquia, and
Fundación Investigando en Salud y Enfermedades Infecciosas for funding
the study. Thanks to Clínica SOMA, IPS Universitaria sede Leon XIII, Hospital
Universitario San Vicente Fundación –institutions where the prospective
patients were recruited.
Thanks to the Corporación para Investigaciones Biológicas, the Clinical
Laboratory at Hospital Universitario San Vicente Fundación and the School of
Microbiology of the Universidad de Antioquia for providing clinical isolates
of bacteria and fungi to determine the analytical specificity of mPCR.
Author details
1
Grupo Investigador de Problemas en Enfermedades Infecciosas (GRIPE),
Sede de Investigación Universitaria, Calle 62 # 52-59, Laboratorio 630,
Universidad de Antioquia, Medellín, Colombia.
2
Corporación de Ciencias
Básicas Biomédicas, Universidad de Antioquia UdeA, Medellín, Colombia.
3
Universidad Pontificia Bolivariana, Medellín, Colombia.
4
Programa de Estudio
y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia
UdeA, Medellín, Colombia.
5
Infectious Disease Section, School of Medicine,
Universidad de Antioquia UdeA, Medellín, Colombia.
Received: 30 September 2015 Accepted: 5 February 2016
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