Jpn. J. Infect. Dis.

, 60, 370-373, 2007

Original Article
First Report on Clinical Features of Mycoplasma pneumoniae
Infections in Vietnamese Children
Phan L. T. Huong*, Ngo T. Thi1, Nguyen T. T. Nguyet1, Ta K. Van1, Dang T. Hang1,
Vu T. T. Huong, Dang D. Anh and Tsuguo Sasaki2
Department of Bacteriology, National Institute of Hygiene and Epidemiology; 1National Hospital of Pediatrics,
Hanoi, Vietnam; and 2National Institute of Infectious Diseases, Tokyo 208-0011, Japan
(Received April 27, 2007. Accepted August 31, 2007)

SUMMARY: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) in children,

but there has been no clinical report on M. pneumoniae infections in Vietnamese children. We investigated the
clinical features of M. pneumoniae infection when the pathogen was detected in the respiratory tract in hospitalized
children aged 1 - 15 years due to lower respiratory tract infections or CAP in Vietnamese children. Throat swabs
from 47 patients (18.6%) of 252 patients with a clinical diagnosis of CAP were PCR positive (male, 34; female,
13), and 21 throat swabs (8.3%) showed culture positive for M. pneumoniae. The M. pneumoniae pathogen
could be detected by PCR and/or culture in 52 patients (male, 36; female, 16). The major clinical signs in the 52
patients were fever (>38°C) in 100%, pharyngitis in 100%, tachypnea in 94%, dry cough in 86.5%, and rough
breathing in 83% of patients. The average term of illness prior to hospitalization was 7.5 ± 4.1 days, and the
average number of hospitalized days was 7.9 ± 3.5 days. β-lactam group antibiotics, which were ineffective
against M. pneumoniae infection, were used in 37 cases (71%).

a tendency to develop into severe pneumonia, which requires

INTRODUCTION
Mycoplasma penumoniae is a common pathogen causing
community-acquired respiratory tract infection such as upper
respiratory inflammation, bronchitis, and pneumonia, mainly
in children and young adults. Pneumonia develops in about
3 - 5% of cases of M. pneumoniae infection, which is gener-
ally not accompanied with purulent sputum often found in
other bacterial pneumonia, but is characterized by a persistent
nonproductive cough (1).
In Vietnam, community-based studies suggested that in
rural areas, every child aged less than 5 years suffered from
5 to 8 episodes of acute respiratory infection (ARI), while in
urban areas every child suffered from 3 to 5 episodes annually
(2). Among children under 1 year and 3 years of age, the
percentage of children with symptoms of ARI were 68.4%
(266/389) and 70.8% (923/1,304), respectively (3). Among
those patients, 20 - 30% develop pneumonia and 10 - 15%
develop severe pneumonia requiring hospitalization. In
Vietnam, laboratory diagnosis of M. pneumoniae infection
has not been common, which means that clinical doctors
have almost no microbiological information about ARIs,
especially lower respiratory tract infections (LRTIs) due to
M. pneumoniae. Therefore, empirical therapy is often applied
to patients with symptoms of ARI. In general, erythromycin
(ERY) and clarithromycin (CLR) among 14-membered
macrolides and the 15-membered macrolide azithromycin
(AZM) are used as the first-choice therapeutic agent against
M. pneumoniae infection in children, as well as in adults.
However, antibiotics of the β-lactam group are usually used
for the treatment of ARIs in Vietnam. An improper treatment
inevitably fails to cure ARI. Thereafter, the illness is showing
*Corresponding author: Mailing address: Department of Bacte-
riology, National Institute of Hygiene and Epidemiology, 1 Yersin
Street, Hanoi, Vietnam. Tel: +84-4-9714408, Fax: +84-4-8210853,
E-mail: thanhhuong@nihe.org.vn
hospitalization.
The present study was conducted to investigate the clinical
features of M. pneumoniae infection when the pathogen was
detected in the respiratory tract in hospitalized children aged
1 - 15 years due to LRTIs (or community-acquired pneumo-
nia [CAP]) in Vietnam.
MATERIALS AND METHODS
Patients and diagnosis: This study was carried out with
the approval of the National Institute of Hygiene and Epi-
demiology (NIHE) Research Ethics Committee. Patients
suspected as suffering from CAP were investigated for micro-
biological diagnosis based on respiratory samples (pharyngeal/
nasopharyngeal exudates) and serum. These clinical samples
were taken from 252 patients at the National Hospital of
Pediatrics from July 2004 through August 2006, in Hanoi,
Vietnam. At the time of admission, systematic recordings were
made of each patient’s medical history, including the period
of onset of the current illness, the underlying respiratory symp-
toms, and the presence of fever (temperature ≥38°C). After
a complete physical examination, chest X-rays were taken.
The laboratory samples taken at enrollment included blood
specimens for counting leukocyte and for the detection of
IgM antibody specific to M. pneumoniae, and throat swabs
for the isolation of M. pneumoniae and for the detection of
M. pneumoniae DNA.
Isolation of M. pneumoniae: Modified Hayflick’s media
were used (4). The broth medium was composed of 7.5 parts
of PPLO broth (Difco, Detroit, Mich., USA), 1.5 parts of
heat-inactivated horse serum, 1 part of aqueous extract (25%)
of bakers’ yeast, penicillin G (1,000 units/ml), thallium acetate
(0.025%), glucose (0.5%), and phenol red (0.002%). The
composition of agar medium was the same as that of the broth
medium, except that glucose and phenol red were omitted,
and 1.2% agar was added. A throat swab was immersed

370
several times in 1.0 ml of PPLO broth, and then 0.2 ml of patients (45/52) and, in one lung in 5.8% of patients, and
the suspension was transferred to the diphasic (agar/broth) bronchitis (both lungs) in 7.7% of patients. The average length
medium and 0.1 ml was transferred onto the agar medium. of illness prior to hospitalization was 7.5 ± 4.1 days (Table 2).
The agar medium was incubated under 5% CO2 in air with Table 3 shows the distribution of CAP cases on the basis
moisture, and the diphasic medium was incubated aerobically of PCR and/or culture positive for M. pneumoniae by age
at 37°C for 3 weeks. When a color change was observed in and sex group. The highest proportion of patients with M.
the diphasic medium, 0.1 ml of the broth was subcultured pneumoniae infections were 1 - 5 years old (52%; 27/52);
onto the agar medium. When typical colonies were observed 30.8% of patients (16/52) were 6 - 10 years old, 11.5% (6/52)
on the agar medium, a single colony was inoculated into the
broth medium. After cloning of colonies, M. pneumoniae was Table 1. Proportion of patients with M. pneumoniae acute respiratory in-
identified by PCR. fections (ARIs) on the basis of PCR, culture, and serological findings
Serological diagnosis: The sera samples were stored at Total no. of enrolled ARIs patients (%)
–20°C until testing. The particle agglutination (PA) antibody
titer for M. pneumoniae was assayed by using Serodia-MYCO PCR positive cases 47/252 (18.6)
II (Fuji Rebio, Ltd., Tokyo, Japan), which was manufac- Culture positive cases 21/252 (8.3)
tured by using artificial gelatin particles, sensitized with cell Total positive cases with PCR
52/252 (20.6)
and/or culture
membrane components of M. pneumoniae, according to the
Serological positive cases 64/252 (25.4)
manufacturer’s instructions.
PCR detection and typing of M. pneumoniae: Throat
swabs immersed in broth were kept at –70°C. At test time Table 2. Frequency of clinical findings in M. pneumoniae infections
they were thawed, and then DNA was extracted with a Reported
SepaGene Kit (Sanko Pharmaceutical Co., Tokyo, Japan) Finding Our findings (%)
findings
according to the manufacturer’s instructions. M. pneumoniae
Symptom/Sign
DNA was detected by the nested PCR method with primer
Fever (>38°C) 52/52 (100) 96 - 100% 1)
sets for amplification of the P1 gene as previously described
Pharyngitis 52/52 (100)
(5). The first primer set was ADH2F: 5´-GGC AGT GGC
Fatigued 52/52 (100) NR3)
AGT CAA CAA ACC ACG TAT-3´ and ADH2R: 5´-GAA
Dry cough 45/52 (86.5) 93 - 100% 1)
CTT AGC GCC AGC AAC TGC CAT-3´. The second primer
Tachypnea 49/52 (94.2) NR3)
set was ADH3F: 5´-GAA CCG AAG CGG CTT TGA CCG
Rough breathing (expiratory) 43/52 (82.7) 80 - 84% 1)
CAT-3´ and ADH3R: 5´-GTT GAC CAT GCC TGA GAA
Alveolar exudation rales 9/52 (17.3)
CAG TAA-3´. Typing of M. pneumoniae based on the P1
X-ray
gene was carried out by PCR with ADH/4F: 5´-GAC CGC
Bronchitis (both of lungs) 4/52 (7.7)
ATC AAC CAC CTT TGC GTT ACG-3´ and mixed reverse
Bronchopneumonia (both of lungs) 45/52 (86.5)
primers; N1: 5´-CCC GGT GGT GGA AGT ATT TT-3´ and
Bronchopneumonia (one lung) 3/52 (5.8)
2N2C: 5´-TGC CTT GGT CAC CGG AGT TG-3´ (6).
PMN (polymorphonuclear) cells
Detection of resistance point mutation in domain V of
7,475 - 18,160 PMN/μl (> 7,000) 8/52 (15.4)
23S rRNA: For M. pneumoniae isolates and PCR-positive
1,496 - 5,890/μl (< 7,000) 44/52 (84.6)
samples of M. pneumoniae DNA, identification of point
Duration (mean ± SD)
mutation in domain V of 23S rRNA for the ERY-resistant M.
Of illness 15.19 ± 8.862)
pneumoniae was performed according to the methods reported
Of illness (prior to hospitalization) 7.5 ± 4.1 days
by Matsuoka et al. (7). The detection of point mutation indi-
Of hospitalization 7.9 ± 3.5 5.77 ± 2.822)
cates the phenotype of resistance, because there is only a single
1)
rRNA operon in the genome (8). Neither plasmids of erm : See Reference 15.
2)
: See Reference 20.
genes to mediate ribosomal modification, nor any enzymes 3)
: Not reported.
that inactivate macrolides were found in M. pneumoniae.
Thus, the prevalence of macrolide-resistant M. pneumoniae
detected by the PCR methodology should reflect the true Table 3. Community-acquired pneumonia by PCR
and/or culture according age and sex
incidence of resistant strains.
No. of PCR- and culture-positive
Age (y)
Male Female Total (%)
RESULTS
<1 3 0 3 (5.8)
Clinical symptoms of M. pneumoniae patients: A total 1-5 19 8 27 (51.9)
of 252 pediatric patients hospitalized due to CAP were 6 - 10 11 5 16 (30.8)
enrolled on the basis of PCR, culture, and serological tests. 11 - 15 3 3 6 (11.5)
Forty-seven (18.6%) were PCR positive for M. pneumoniae
Total (%) 36 (69.2) 16 (30.8) 52 (100)
DNA, and 21 (8.3%) were culture positive (Table 1). The
total number of patients in which M. pneumoniae was
detected by PCR and/or culture methods was 52 (20.6%), Table 4 . Antibiotics used before hospitalization
and 64 (25.4%) were serologically positive for IgM specific
Antibiotic Patients’ population (%)
to M. pneumoniae (IgM titer ≥ 320).
Major clinical symptoms of the 52 PCR- and/or culture- β-lactam group antibiotics 71
positive cases were fever > 38°C (100%), dry cough (86.5%), Unknown antibiotics 25
and tachypnea (94%). Pharyngitis was found in all patients. Erythromycin 2
X-rays showed bronchopneumonia in both lungs in 86.5% of Amikacin 2

371
 Table 5. Relationship between PCR results and IgM antibody titer
PCR positive Cumulative PCR PCR negative Cumulative PCR
Antibody titer
47 cases positive (%) 205 cases negative (%)
<20 6 13 39 19
20 2 17 34 35.6
40 5 28 41 55.6
80 0 30 70.2
160 5 38 25 82.4
320 2 43 21 92.7
640 0 12 98.5
1,280 3 49 2 99.5
2,560 7 64 1 100
5,120 7 79
10,240 4 87
>10,240 6 100
Antibody titer < 320: 18/47 cases with PCR-positive (38%).
Antibody titer ≥ 320: 29/47 cases with PCR-positive (62%).

were 11 - 15 years old, and 5.8% (3/52) were less than 1 year
old. Males comprised 69.24% (36/52) of patients, and females
comprised 30.76% (16/52). In this study, the male/female ratio
of all 252 enrolled patients was 2.36 (177/75).
Antibiotics used before hospitalization: Table 4 shows
the proportion of patients receiving antibiotic treatment
before hospitalization. Of 52 CAP patients, 37 (71%) were
treated with ineffective β-lactam group antibiotics, 13 (25%)
were treated with unknown antibiotics, and only 1 (2%) was
treated with erythromycin. The average treatment period with
ineffective antibiotics was 7.5 days. Macrolides are gener-
ally considered the first-choice agents against M. pneumoniae
infection. However, in general, β-lactam group antibiotics are
used in the treatment of CAP in Vietnam.
Relationship between PCR results and IgM antibody
titer: A comparison of PCR results and PA titer is shown in
Table 5. Among 47 PCR-positive patients’ sera, PA titer
< 320 occurred in 18/47 cases (38.3%) and PA titer ≥ 320
occurred in 29/47 cases (61.7%). PA titers > 1:40 were seen
in 34 of 47 serum samples (72.34%). PA titers ≥ 5,120 were
found in 17 cases (36.17%).
Type of adhesion protein P1 and macrolide-resistant
genes of M. pneumoniae: The adhesin protein P1 is densely
localized at the surface of the attachment organelle and plays
a major role in binding to the receptor molecule of epithelial
cells (9-11). The P1 gene is present as a single copy in the M.
pneumoniae genome and can be classified in two groups
(group I and group II) according to nucleotide sequence poly-
morphism (12). Of 47 PCR-positive M. pneumoniae samples,
59.6% (28/47) were group I and 40.4% (19/47) were group II
(data not shown). None of these samples had macrolide-
resistant genes (data not shown).
DISCUSSION
In Vietnam, there is no national epidemiological surveil-
lance system for M. pneumoniae infections. Therefore, the
incidence of mycoplasmal pneumonia in Vietnamese children
is unclear, which may result in ineffective therapy to patients
with M. pneumoniae infections. For this reason, we investi-
gated the clinical features of M. pneumoniae infections in
Vietnamese children based on the epidemiological study
protocol used at the National Institute of Infectious Diseases
in Japan.
Isolation of M. pneumoniae from pharyngeal specimens was
conducted, but the isolation rate was low due to technicians
who were inexperienced in using the isolation technology
and contamination with bacteria and/or fungi. Contamina-
tion with bacteria and/or fungi was found in about 10% of
cultures. For isolation of M. pneumoniae from clinical speci-
mens, the essential points are to find good growth-promoting
horse serum and to prepare good-quality fresh yeast extract.
For laboratory diagnosis of M. pneumoniae infection, cultur-
ing is not practical because of the long period of incubation
needed. The most common laboratory diagnostic method is
serodiagnosis. A variety of antibody detection kits are avail-
able on the market and simple, rapid tests are applicable.
Negative results may often be obtained in primary infec-
tion of infants and school children within a week after falling
ill. Even if a high antibody titer is obtained with a single
serum sample, the possibility of past infection cannot be ruled
out. In our study, higher PA titers ≥ 5,120 were found in 17
cases (36.17%). In comparison with other reports (13,14),
our patients’ PA titers were relatively high. Obtaining this
kind of response might be the result of a lengthy onset (7.5 ±
4.1 days) due to the use of ineffective antibiotics before
hospitalization for some patients (Table 4). Yamazaki et al.
(14) reported that PA titer increases of >4-fold were seen in
30 of 36 paired serum samples among PCR-positive children,
but none of the paired serum samples showed increased PA
antibody titers of >4-fold among PCR-negative patients. This
indicates that PA using paired serum is clinically useful for
the diagnosis of M. pneumoniae infection and that PA anti-
body increases with the number of onset days. PCR for the
detection of M. pneumoniae DNA has been conducted as
a laboratory diagnosis. Although many PCR primers are
reported, our primer set is one of the most sensitive primers
for the detection of M. pneumoniae DNA. In our study, PCR
showed the most sensitive results (Table 1).
The clinical features of mycoplasmal pneumonia in our
cases were generally mild and showed good prognosis.
Major symptoms were mostly the same as those described in
other reports (Table 2). Fever (>38°C) and pharyngitis were
found in all patients, and dry cough, tachypnea, and rough
breathing were found in most patients. Clyde (15) showed
that positive correlations with M. pneumoniae infection
included having a large family and presence of ear infections,
headache, rash, and sore throat, but leukocytosis was signifi-
cantly more common in the group with pneumonia due to
causes other than M. pneumoniae. In general, the number of

372
polymorphonuclear (PMN) cells does not often increase in
cases of M. pneumoniae infection. In this study, among PCR-
and/or culture-positive cases, only 15.4% (8/52) of the pa-
tients had an abnormally high number of PMN cells, whereas
84.6% of positive cases had a normal number of PMN cells
(Table 2). Bronchopneumonia in both lungs (86.5%) or one
lung (5.8%) and bronchitis in both lungs (7.7%) were observed
by X-ray. In our results, 69.24% of the patients were male
and 30.76% were female. However, it is well known that there
is no difference in infection with M. pneumoniae between sexes.
Macrolides are generally considered as the first-choice
agents against M. pneumoniae infection. However, in gen-
eral, β-lactam group antibiotics are used in the treatment of
CAP in Vietnam. At the National Hospital of Pediatrics, the
patients were treated with Tazocin (composed piperacillin
and tazobactam) plus aminoglycoside group antibiotics such
as gentamicin or plus Zithromax (azithromycin dihydrate).
Patients with a history of failed treatment with β-lactam
antibiotics were mainly treated with Zithromax.
Since the study of M. pneumoniae infection has just
started in Vietnam, we do not have sufficient epidemiologi-
cal data such as seasonal incidence, yearly incidence, regional
incidence, shift of M. pneumoniae P1 type, appearance of
macrolide-resistance M. pneumoniae, and so on. In Japan, a
large epidemic occurred every 4 years until 1988, but such
large periodical epidemics have not recurred since 1992, per-
haps owing to early diagnosis of mycoplasmal pneumonia
and decreased familial infections and outbreaks at schools
(1). In Japan, no strain of M. pneumoniae resistant to
macrolides had been found before 1999, while about 15% of
M. pneumoniae isolated or detected by PCR were judged
as macrolide-resistant after 2000 (5,7). In Vietnam, no
macrolide-resistant M. pneumoniae was found in limited
numbers of PCR-positive specimens. When patients infected
with macrolide-resistant M. pneumoniae were treated with
macrolides, the total febrile period was 3 days longer than
that of patients with macrolide-sensitive M. pneumoniae (5).
Therefore, it is important to pay attention to the appearance
of macrolide-resistant M. pneumoniae in Vietnam. A molecu-
lar epidemiological study based on p1 gene polymorphism
showed that two groups of M. pneumoniae isolates shifted
about every 8 to 10 years (1,16). A similar tendency of epide-
miological patterns has also been reported from analyses of
clinical isolates in Germany, France, and Denmark (17-19).
To understand the features of M. pneumoniae infections in
Vietnam will require continuous nationwide epidemiological
studies.
ACKNOWLEDGMENTS
We would like to thank the Board of National Hospital of Pediatrics (NHP)
for their collaboration. Sincere thanks go to the doctors of the Department of
Respiratory Infections at NHP and the researchers of the Department of
Immunology and Molecular Biology of NIHE for their cooperations.
373
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