Molecular etiological profile of atypical bacterial pathogens, viruses and coinfections among infants and children with community acquired pneumonia admitted to a national hospital in Lima, Peru
Valle‑Mendoza et al. BMC Res Notes
Molecular etiological profile of atypical bacterial pathogens, viruses and coinfections among infants and children with community acquired pneumonia admitted to a national hospital in Lima, Peru
Juana del ValleM‑endoza 0 3
Wilmer Silva‑Caso 0 3
Angela CornejoT‑apia 0
Fiorella OrellanaP‑eralta 0
Eduardo Verne 2
Claudia Ugarte 2
Miguel Angel AguilarL‑uis 0 3
María del Carmen De Lama‑Odría 0
Ronald NazarioF‑uertes 3
Mónica EsquivelV‑izcarra 3
Verónica Casabona‑Ore 0
Pablo Weilg 0
Luis J. del Valle 1 4
0 School of Medicine, Research and Innovation Centre of the Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas , Av. San Marcos cdra. 2. Cedros de Villa, Chorrillos, Lima , Peru
1 Barcelona Research Center for Multiscale Science and Engineering , Departament d'Enginyeria Quıímica, EEBE , Universitat Politècnica de Catalunya (UPC), Barcelona Tech, C/Eduard Maristany , 10‐14, Ed. I2, 08019 Barcelona , Spain
2 Hospital Nacional Cayetano Heredia , Lima , Peru
3 Instituto de Investigación Nutricional , Av. La Molina 1885, Lima 12 , Peru
4 Barcelona Research Center for Multiscale Science and Engineering , Departament d'Enginyeria Quıímica, EEBE , Universitat Politècnica de Catalunya (UPC), Barcelona Tech, C/Eduard Maristany , 10‐14, Ed. I2, 08019 Barcelona , Spain
Objective: The main objective of this study was to detect the presence of 14 respiratory viruses and atypical bacteria (Mycoplasma pneumoniae, Chlamydia pneumoniae), via polymerase chain reaction in patients under 18 years old hospitalized due to community‑ acquired pneumonia (CAP) from Lima, Peru. Results: Atypical pathogens were detected in 40% (58/146); viral etiologies in 36% (52/146) and coinfections in 19% (27/146). The most common etiological agent was M. pneumoniae (n = 47), followed by C. pneumoniae (n = 11). The most frequent respiratory viruses detected were: respiratory syncytial virus A (n = 35), influenza virus C (n = 21) and parainfluenza virus (n = 10). Viral‑ bacterial and bacterium‑ bacterium coinfections were found in 27 cases. In our study population, atypical bacteria (40%) were detected as frequently as respiratory viruses (36%). The presence of M. pneumoniae and C. pneumoniae should not be underestimated as they can be commonly isolated in Peruvian children with CAP.
Respiratory viruses; Respiratory infection; Atypical pathogens; Community‑ acquired pneumonia; CAP
Community-acquired pneumonia (CAP) is defined as an
acute infection within the lungs diagnosed by clinical
features and lung imaging in a previously healthy person due
to an infection acquired outside of a healthcare setting
]. This illness is the leading cause of death worldwide
among children under 5 years old, representing 2 million
deaths per year [
]. According to the British Thoracic
Society, the clinical features associated with CAP within
this age group include fever, tachypnea, breathlessness,
cough, wheeze or chest pain .
In developing countries, the etiological data from
children with CAP were obtained from reports between
1980 and 1990 that mainly used serological methods [
and also some low-level evidence descriptive studies [
]. Most of the studies describing the causative agent of
CAP in children are limited by the low yield of cultures,
the difficulty of obtaining adequate sputum specimens
and the reluctance to perform lung aspirations and
bronchoalveolar lavages in this population .
The main objective of this study was to detect the
presence of 14 respiratory viruses and atypical bacteria
(Mycoplasma pneumoniae, Chlamydia pneumoniae) in
patients under 18 years old hospitalized due to CAP from
tube containing viral transport medium (minimal
essential medium with 2% fetal bovine serum, amphotericin
B 20 μg/ml, neomycin 40 μg/ml,). Two aliquots of each
fresh specimen were stored at – 20 °C to be later
analyzed for respiratory viruses and atypical bacteria.
Materials and methods
Patients and study design
A consecutive cross-sectional study was conducted in
patients under 18 years of age, admitted to Hospital
Cayetano Heredia in Lima-Peru with the diagnosis of
community acquired pneumonia (CAP). Patients who
fulfilled the selection criteria were studied from January
2009 to December 2010.
Inclusion criteria Patients who were hospitalized in the
pediatrics wards with the diagnosis of CAP during the
Exclusion criteria Patients who were diagnosed with
pneumonia 48–72 h after being admitted. Patients who
were admitted to the ICU service with the diagnosis of
pneumonia or severe pneumonia. Patients who were
transferred from other hospitals to the pediatrics wards
with the diagnosis of pneumonia.
For each patient, a questionnaire with clinical and
epidemiological features was completed by the physician
who admitted the patient. The questionnaire applied was
designed by the government for pneumonia surveillance
and includes the following information: age, gender and
relevant clinical information (onset, fever higher than
38 °C, cough, headache, ear pain, photophobia,
conjunctival congestion, rhinorrhea, wheezing, expectoration,
pharyngeal congestion, sore throat, malaise,
abdominal pain, nausea, vomiting, diarrhea, lymphadenopathy,
fatigue, arthralgias and myalgias).
This study has been approved by two independent
Ethics Committees from Hospital Cayetano Heredia and
Instituto de Investigación Nutricional. All samples were
analyzed after a written informed consent was signed by
parents or children’s caregivers.
Nasopharyngeal samples were obtained by inserting a
swab into both nostrils parallel to the palate (Mini-Tip
Culture Direct, Becton-Dickinson Microbiology System,
MD 21152, USA) and a second swab from the posterior
pharyngeal and tonsillar areas (Viral Culturette,
Becton-Dickinson Microbiology Systems, MD, USA). Both
nasal and pharyngeal swabs were placed into the same
Reverse transcription polymerase chain reaction (RT‑PCR) for the analysis of respiratory viruses
For the multiplex RT-PCR, viral genomic RNA and DNA
were extracted from a total volume of 200 µl of sample by
the guanidinium thiocyanate extraction method [
lysis buffer included 500 molecules of the cloned
amplified product used as internal control in each reaction
tube and then excluded false negative results due to
nonspecific inhibitors or extraction failure. Two independent
multiplex reverse transcription nested RT-PCR assays able
to detect from 1 to 10 copies of viral genomes were
]. One nested RT-PCR was performed using
specific primers for influenza viruses (Flu-A, Flu-B and
Flu-C), respiratory syncytial viruses (RSV-A and
RSVB) and adenovirus (ADV). Another, nested RT-PCR was
prepared with specific primers for detection of human
parainfluenza viruses (PIV-1, PIV-2, PIV-3 and PIV-4),
corona viruses (CoV-229E and CoV-OC43), human
rhinoviruses (HRV), and enteroviruses (HEV). For the PCR,
a single step combined RT-PCR amplification reaction,
henceforth called multiplex assay 2, was performed as
described previously [
] (Additional file 1).
Polymerase chain reaction (PCR) for the analysis
of Mycoplasma pneumoniae and Chlamydia pneumoniae
Polymerase chain reaction (PCR) was performed with
5 μl of template DNA, polymerase (GoTaq; Promega,
Madison, Wisconsin, USA). For M. pneumoniae, the
primers: Myco-f 5′-GAA GCT TAT GGT ACA GGT
TGG-3′ and Mico-r 5-ATT ACC ATC CTT GTT GTA
AGG-3′ were used; and for C. pneumoniae, we used
primers: Clam-1f-5′-TGC ATA ACC TAC GGT GTG
TT-3′ and Clam-1r 5′-TGC ATA ACC TAC GGT GTG
TT-3′, Clam-2f-5′-AGT TGA GCA TAT TCG TGA
TT-3′ and Clam-2r 5′-TTT ATT CCG TGT CGT CCA
G-3′. The PCR consisted of initial incubation at 95 °C for
2 min, followed by 40 cycles of 95 °C for 30 s; 58 °C for
30 s, and 72 °C for 30 s; with a final extension at 72 °C for
5 min. Amplicons were detected as 275 and 225 for M.
pneumoniae and C. pneumoniae respectively base pair
bands after gel electrophoresis and nucleic acid staining
In each PCR assay, negative (transport medium) and
positive control (cDNA) were prepared with the same
procedure. Amplified products were recovered from the
gel, purified (SpinPrep Gel DNA Kit; San Diego, CA) and
sent for commercial sequencing (Macrogen, Korea).
Qualitative variables were reported as frequencies and
A total of 146 patients under 18 years old hospitalized
with the diagnosis of CAP were studied. Most patients
were infants under 1-year-old (81.51%) followed by
children between 2 and 5 years old (11.64%). The most
frequent symptoms were cough (86.96%), fever (79.45%),
rhinorrhea (76.71%), and pharyngeal congestion (21.92%)
Atypical pathogens were detected in n = 58/146
(39.72%) cases, respiratory viruses in n = 52/146 (35.62%)
and coinfections in n = 27/146 (18.49%) samples; we
were unable to isolate pathogens in 36 (24.66%)
samples. M. pneumonia and RSV-A were the most common
etiologies detected in 32.19% and 23.97% respectively,
followed by C. pneumoniae (7.53%) (Table 2).
Coinfections were detected in 27 cases (18.49%), and
the most frequent association corresponded to M.
pneumoniae with VRS-A (9.59%). No viral-viral associations
were observed (Table 2).
A monthly distribution of the CAP cases was analyzed
according to their etiologies during the study period.
An even distribution of infections with C. pneumoniae
were observed across the year and a relative increase of
M. pneumoniae was observed from March to June. An
isolated peak of respiratory viruses was detected during
March being RSV-A the most common isolated virus
Establishing the etiology of CAP in children can be
challenging in developing countries due to many factors
including: the difficulty to obtain adequate samples, the
Frequency Prevalence (%)
(n = 146)
Table 2 Etiological diagnosis of CAP by PCR
Others (< 2% of cases: Ear pain, photophobia, conjunctival congestion,
abdominal pain, lymphadenopathy, fatigue, myalgia)
invasive characteristic of specific diagnostic tests and the
unavailability of reliable diagnostic methods in the primary
care setting. Without a sensitive and specific diagnostic
method, physicians have to rely on clinical criteria based
on signs and symptoms and epidemiological information
of CAP to determine the possible causative agent and
provide the patient with the proper treatment [
2, 4, 6, 10
Multiple studies have previously reported that
respiratory viruses are the leading cause of community acquired
pneumonia in children and can be detected in more than
50% of the cases [
]. However, this results may vary
between studies due to the differences in seasonal
patterns observed in distinct areas [
4, 6, 10, 12
]. In our study
population, atypical bacteria were slightly more frequently
detected (39.73%) than respiratory viruses (35.62%).
In the group of patients with pneumonia caused by
atypical pathogens, M. pneumoniae was the
predominant microorganism and was detected in 32.19% of the
samples. This finding correlates with some previous
studies that have detected M. pneumoniae in up to 36% of
children with community acquired pneumonia [
Moreover, we observed a similar M. pneumoniae
predominance in a previous study we conducted in children
with acute respiratory illness (ARI) around the same
study period. We found that in children with ARI, M.
pneumoniae was present in up to 25% (170/675) of
samples and C. pneumoniae in 10% (71/65) .
The most common pathogen isolated within the
group of patients with viral pneumonia was RSV type
A (23.97%), followed by Parainfluenza 2 (2.74%). Other
studies have reported a similar distribution of viral
etiologies in children with CAP [
4, 7, 11
]. However, seasonal
pattern variations and viral outbreaks can considerably
alter the prevalence of certain viruses between
surveillance studies, especially for RSV and influenza virus [
In our series, we observed Chlamydia pneumonia
infections evenly distributed through the year, whereas
a relative increase of M. pneumoniae was observed
from March to June. However, no clear seasonal
pattern can be concluded for both atypical bacteria or
respiratory viruses during our study period, probably due
to the limited number of cases. Nevertheless, our study
demonstrates the constant presence of atypical bacteria
throughout the year in patients with CAP.
In recent years, there has been an increasing interest
regarding the association between bacteria and viruses
in the pathogenesis of pneumonia. Studies have shown
patients that had a viral infection followed by a secondary
bacterial lower respiratory infection, had a higher
morbidity and mortality [
]. Coinfections between
bacterial and viral isolates have been detected in up to 45%
of pediatric patients with CAP; and the most common
association has been reported to be between
Streptococcus pneumoniae and respiratory viruses . However,
M. pneumoniae has also been described as a bacterium
commonly isolated in sputum samples from young
children with coinfections. Moreover, it has been proposed
that patients infected with M. pneumoniae may be more
susceptible to other infectious pathogens [
]. In our
study, coinfections between M. pneumoniae and other
microorganisms were observed in 15.73% of the samples,
and RSV was the most frequent co-infective agent
present in 9.59% of samples.
Several studies have demonstrated that the detection of
viruses in children with CAP has been underestimated,
primarily due to limited diagnostic methods and difficult
sample collection [
]. In this study, Nested RT-PCR
was used to simultaneously detect a wide variety of
viruses with a high sensitivity . Furthermore, a rapid
extraction method of genomic material was employed,
allowing a more efficient recognition of viral RNA and
even bacterial DNA.
In conclusion, our study revealed that both atypical
bacteria and respiratory viruses are among the most
frequent agents detected in children with CAP from Lima,
Peru. The incorporation of highly sensitive and
specific molecular techniques, such as RT-PCR [
be considered in order to achieve an accurate
etiological diagnosis and therapeutic management, avoiding
the empirical use of antibiotic therapy, particularly in
children with pneumonia of viral etiology. In addition,
an increase in macrolide resistance has been observed
worldwide among CAP patients infected with S.
pneumoniae and M. pneumoniae. This highlights the importance
of a precise etiological diagnosis during the management
of CAP in children [
A timely pathogen identification can prevent
nosocomial spread of the disease and provide epidemiological
information to healthcare networks [
], as well as
provide key data to reduce the inappropriate use of
]. Antibiotic choice for CAP can vary widely
across practices and an increasing use of broad-spectrum
antibiotics have been observed by clinicians at suburban
practices. In addition, factors not related to the
microbiologic etiology such as age, previous antibiotic receipt
or type of insurance are common arbitrary criteria used
for antibiotic choice increasing the risk for drug
]. Further investigations should be conducted in
Peru to have a better understanding of the role of atypical
agents in CAP and the risks for antibiotic resistance.
Our results have shown that RT-PCR is a more efficient
diagnostic technique since it can detect multiple viruses
that are not recognized by conventional methods.
Nevertheless, despite the improvement in the etiological
diagnosis of CAP in children, we could not identify an
etiology in a significant proportion of patients. In those
cases, S. pneumoniae could be the causative pathogen as
it is the main cause of pneumonia in most age groups.
However, we cannot rule out the presence of other
Additional file 1. Primers for Influenza Virus (Flu), respiratory syncytial
virus (RSV), Human Parainfluenza Viruses (Parainf.), Coronaviruses, Entero‑
viruses (Enterov.), and Rhinoviruses (Rhinov.) Used in the First Round
Multiplex RT‑PCR and in the Following Nested PCR.
JdVM, WSC, LJdV, EV and CU designed the study protocol ACT, FOP, MAL, RNF,
MEV and performed the PCR for virus and atypical germs. JdVM and LJdV were
responsible for obtaining funding and laboratory work supervision. WSC, RNF
and VCO was responsible for the clinical assessment, samples collection and
database completion. JdVM, WSC, MCdLO, PW and LJdV drafted the manu‑
script. All authors critically revised the manuscript for intellectual content. All
authors read and approved the final manuscript.
Pediatric health personnel from all Hospitals participating in the study.
On behalf of all authors, the corresponding author states that there are no
competing interest or funding related to this study.
Availability of data and materials
Abstraction format used in the study and dataset are available and accessible
from corresponding author upon request in the link: https://figshare.com/
Consent to publish
Ethics approval and consent to participate
This study has been approved by two independent Ethics Committees from
Hospital Cayetano Heredia and Instituto de Investigación Nutricional. All samples
were analyzed after a written informed consent was signed by parents or
This work was supported by Grants from Programa de Ciencia y Tecnología
(FINCyT‑ PIN‑071‑2008) from the Peru.
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
1. Daniel M , Musher MD , Anna R , Thorner MD . Community‑acquired pneumonia . N Engl J Med . 2014 ; 371 : 1619 - 28 .
2. Nair H , Simões EAF , Rudan I , Gessner BD , Azziz‑Baumgartner E , Zhang JS , et al. Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis . Lancet . 2013 ; 9875 : 1380 - 90 .
3. UNICEF/WHO. Pneumonia: the forgotten killer of children . Wkly Epidemiol Rec . 2008 ; 83 : 1 - 16 .
4. British Thoracic Society Standards of Care Committee. BTS guidelines for the management of community acquired pneumonia in childhood . Thorax . 2011 ; 66 ( Suppl 2 ): 1 - 23 .
5. Korppi M. Community‑acquired pneumonia in children: issues in optimizing antibacterial treatment . Paediatr Drugs . 2003 ; 5 : 821 - 32 .
6. Padilla J , Lindo F , Rojas R , Tantaleán J , Suárez V , Cabezas C , et al. Etiology of community acquired pneumonia in children 2-59 months old in two ecologically different communities from Peru . Arch Argent Pediatr . 2010 ; 108 ( 6 ): 516 - 23 .
7. Casas I , Powell L , Klapper PE , Cleator GM . New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assays . J Virol Methods . 1995 ; 53 ( 1 ): 25 - 36 .
8. Coiras MT , Pérez‑Breña P , García ML , Casas I. Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested‑PCR assay . J Med Virol . 2003 ; 69 ( 1 ): 132 - 44 .
9. Coiras MT , Aguilar JC , García ML , Casas I , Pérez‑Breña P. Simultane ‑ ous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested‑PCR assays . J Virol . 2004 ; 72 ( 3 ): 484 - 95 .
10. Sinaniotis CA. Viral pneumoniae in children: incidence and aetiology . Paediatr Respir Rev . 2004 ; 5 : S197 - 200 .
11. Stuckey‑Schrock K , Hayes BL , Georg C . Community‑acquired pneumonia in children . Am Fam Phys . 2012 ; 86 ( 7 ): 661 .
12. Xinfen Yu , Kou Yu , Daozong Xia, Jun Li , Xuhui Yang , Yinyan Zhou , et al. Human respiratory syncytial virus in children with lower respiratory tract infections or influenza‑like illness and its co ‑infection characteristics with viruses and atypical bacteria in Hangzhou, China . J Clin Virol . 2015 ; 2015 (05): 015 .
13. Somer A , Salman N , Yalcin I , Ağaçfidan A . Role of Mycoplasma pneumoniae and Chlamydia pneumoniae in children with community‑acquired pneumonia in Istanbul, Turkey . J Trop Pediatr . 2006 ; 52 : 173 - 8 .
14. Del Valle‑Mendoza J , Orellana‑Peralta F , Marcelo ‑Rodríguez A , Verne E , Esquivel‑ Vizcarra M , Silva‑ Caso W , et al. High prevalence of Mycoplasma pneumoniae and Chlamydia pneumoniae in children with acute respiratory infections from Lima, Peru . PLoS ONE . 2017 ; 12 ( 1 ): e0170787 .
15. Rhedin S , Lindstrand A , Rotzén‑ Östlund M , Ryd‑Rinder M , Öhrmalm L , Tolfvenstam T , et al. Respiratory viruses associated with community acquired pneumonia in children: matched case-control study . Thorax . 2015 ; 70 : 847 - 53 .
16. Ruuskanen O , Lahti E , Jennings LC , Murdoch DR . Viral pneumonia . Lancet . 2011 ; 377 : 1264 - 75 .
17. Chen K , Jia R , Li L , Yang C , Shi Y. The aetiology of community associated pneumonia in children in Nanjing, China and aetiological patterns associated with age and season . BMC Public Health . 2015 ; 15 : 113 .
18. Garau J , Nicolau D , Wullt B , Bassetti M. Antibiotic stewardship challenges in the management of community‑acquired infections for prevention of escalating antibiotic resistance . J Global Antimicrob Res . 2014 ; 2 ( 4 ): 245 - 53 .
19. Cooper NJ , Sutton AJ , Abrams KR , Wailoo A , Turner D , Nicholson KG . Effectiveness of neuraminidase inhibitors in treatment and prevention of influenza A and B: systematic review and meta‑analyses of randomised controlled trials . BMJ . 2003 ; 326 ( 7401 ): 1235 .
20. van den Broek d'Obrenan J , Verheij TJ , Numans ME , van der Velden AW. Antibiotic use in Dutch primary care: relation between diagnosis, consultation and treatment . J Antimicrob Chemother . 2014 ; 69 : 1701 - 7 .
21. Handy L , Bryan M , Gerber J , Zaoutis T , Feemster K. Variability in antibiotic prescribing for community‑acquired pneumonia . Pediatrics . 2017 ; 139 ( 4 ): e20162331 .