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Molecular diagnosis and epidemiology of Mycoplasma Pneumoniae

Dorigo-Zetsma, J.W.

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CHAPTERR 5

Molecularr detection of Mycoplasma pneumoniae among general practitionerr patients with acute respiratory infection and their

householdd contacts reveals children as human reservoir

J.Wendelienn Dorigo-Zetsma1,2, Berry Wilbrink2, Hans van der Nat2, Aadd I.M. Bartelds4, Marie-Louise A. Heijnen3, Jacob Dankert1

'Departmentt of Medical Microbiology, Academic Medical Center, Amsterdam, diagnostic Laboratoryy for Infectious Diseases and Perinatal Screening and department of Infectious Diseasess Epidemiology, National Institute of Public Health and the Environment (RIVM),

Bilthoven,, and Netherlands Institute of Primary Health Care (NIVEL), Utrecht, The Netherlands s

SubmittedSubmitted for publication

ChapterChapter 5

ABSTRACT T

Duringg a 30 months prospective study in the Netherlands, the distribution of Mycoplasma

pneumoniaepneumoniae and respiratory viruses among 1172 patients with acute respiratory infection

(ARI)) was studied in the outpatient general practitioner setting. Nose/throat samples from

householdd contacts of M. pneumoniae positive patients were collected to study the effect of

antibioticc treatment of index cases on M. pneumoniae transmission. Among the 1172 ARI

patientss M. pneumoniae, as detected by PCR, was present in 39 (3.3%) patients. The infection

ratee of M. pneumoniae was similar in all age groups. Of 79 household contacts, 12 (15%) had

M.M. pneumoniae. The frequency of M. pneumoniae among household contacts of index cases

treatedd with appropriate antibiotics and non-treated cases was similar. Nine of the 12 M.

pneumoniaepneumoniae positive contacts were younger than 16 years (P=0.Q2), and 4 (44%) of them did

nott develop ARI. Therefore, children are apparently a relevant reservoir for M. pneumoniae.

70 0

A// pneumoniae infection among outpatients

INTRODUCTION N

MycoplasmaMycoplasma pneumoniae, a common respiratory pathogen, causes usually mild upper

respiratoryy tract infections such as sore throat, pharyngitis, and tracheitis. In 5-10% of

patients,, M. pneumoniae infection results in tracheobronchitis or pneumonia. An estimated 2-

4%% of patients with M. pneumoniae infection requires hospitalisation (1). Data on the

incidencee and transmission of M. pneumoniae are derived mainly from targe scale population

studiess performed in the 1960s and 1970s, making use of classical methods like culture and

serologyy to diagnose M. pneumoniae (2,3,4,5). Modern techniques like PCR on easily

obtainablee specimens such as throat- and nasal swabs can be used to provide new insight into

thee epidemiology of M. pneumoniae infection in outpatient- and community settings (1,6,7,8).

Inn the Netherlands, respiratory infections, including those due to M. pneumoniae, arc being

studiedd in an outpatient general practitioner (GP) setting within the framework for monitoring

influenzaa virus outbreaks, as recommended by the World Health Organization (9). This

monitoringg is performed in a national surveillance network by GPs evenly distributed over the

country.. GPs identify patients with acute respiratory infection (ARI), and register those ARI

patientss who present with influenza-like illness (ILI) . GPs collect nose/throat swabs from

randomlyy selected ARI patients. These samples are analysed for the presence of respiratory

virusess and M. pneumoniae.

Wcc aimed to assess (i) the frequency of M. pneumoniae infection among ARI patients in

thee outpatient GP setting, (ii) the distribution of M. pneumoniae patients by patient age and

seasonn as compared to patients with ARI due to respiratory viruses, (lii ) the frequency of M

pneumoniaepneumoniae infection by age among household contacts of M. pneumoniae positive index

cases,, and (iv) the influence of antibiotic treatment of M. pneumoniae positive patients on

transmissionn of A/ pneumoniae in the household setting.

PATIENTS,, MATERIAL S AND METHODS

Designn of the surveillance

GPss from 45 practices, evenly distributed over the Netherlands, participate in a nation-

widee sentinel surveillance network co-ordinated by the Netherlands Institute of Primary

HealthHealth Care (NIVEL). The surveillance covers about I % of the population in the Netherlands

(15.77 million inhabitants). This sample of 150,000 people is representative for the national

populationn in terms of age, sex and degree of urbanisation (10). Since 1970, GPs of the

networkk register patients presenting with ILI to enable the calculation of the weekly incidence

off ILI . Since 1992 GPs collect nose/throat samples from patients presenting with ILI and

otherr ARI (hereafter referred to as ARI non-ILI) in order to assess the aetiology of such

71 1

ChapterChapter 5

infections.. ILI is defined as a respiratory infection with acute onset, fever (a rectal

temperaturee of at least 38 °C) and at least one of the following symptoms: coryza, sore throat,

cough,, frontal headache, retrosternal pain, myalgia. ARI-non ILI is defined as a respiratory

infectionn with acute onset and at least one of the above mentioned symptoms. Patients

presentingg with ARI-non ILI are not registered by the GPs participating in the surveillance.

M.M. pneumoniae infection among patients with ARI

Duringg a 30 months prospective study, from January 1, 1997 through June 30, 1999, GPs

inn each practice were requested to collect a nose/throat sample from randomly selected

patientss with ARI. Each week GPs sampled a maximum of two patients. One swab taken from

thee nose and one swab taken from the throat were placed together in 4 ml of Hanks' balanced

saltt solution containing gelatine, lactalbumin, yeast and antibiotics, and were sent to the

Virologyy Department of the Laboratory of Infectious Diseases and Perinatal Screening (LIS)

att the National Institute of Public Health and the Environment by routine mail. The sample,

referredd to as nose/throat sample, was processed at the day of arrival. For each patient, GPs

completedd a questionnaire including patient characteristics, time of onset of illness, clinical

signss and symptoms, and the presence of respiratory infections among direct contacts of the

patients. .

Detectionn of M. pneumoniae by PCR

Uponn receipt of the samples at the laboratory, the nose and throat swabs were twirled in the

transportt medium and removed. Four hundred p.] of this nose/throat sample was processed for

A// pneumoniae PCR, as described previously (11). In short, the suspension was centnfuged at

12,0000 x g for 30 min. DNA was isolated from the pellets using a proteinase K lysis protocol,

andd analysed for the presence of M. pneumoniae DNA using a nested PCR protocol with the

PII cytadhesin gene as the target. The samples were checked for the presence of PCR

inhibitors,, using an amplification control (11). In case of inhibition, the lysate was diluted

1:100 and retested in the PCR. Reaction products were analyzed by 2% agarose gel

electrophoresis. .

Detectionn of respiratory viruses

Specimenss were routinely processed for isolation and detection by PCR of various

respiratoryy viruses. Virus isolation was performed according to standard methods (12,13).

Tertiairyy cynomolgus monkey cells (tMK) and human diploid lung fibroblasts (local cell line

GaBi)) in tubes were each inoculated with about 500 u.1 of the nose/throat sample. Cell cultures

weree inoculated in roller drums at 33°C. Another aliquot of 500 u.1 of the sample was used to

inoculatee tMK cells in flat-bottom tubes, which were subsequently centnfuged at 5,000 x g at

RTT for 75 min, and incubated stationarily at 33°C. Influenza viruses were (sub)typed in

72 2

A// pneumoniae infection among outpatients

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hemagglutinationn inhibition assays (14). Other viruses were identified by standard methods

(12,13).. PCR was performed for RSV (15), rhinovirus and enterovirus (16) and coronaviruses

OC43and229E(17). .

Transmissionn of M. pneumoniae among household contacts of the patients

Householdd contacts of patients with a nose/throat sample positive for M. pneumoniae were

askedd to participate in the transmission study. From the household contacts a nose/throat

samplee was collected by the GP within 4 weeks after sampling of the index case. A

questionnairee with the characteristics of the sampled persons, the presence of signs and

symptomss of a respiratory infection and antibiotic usage was completed. Samples were

processedd for M. pneumoniae PCR.

Statisticall analysis

Thee x' lest was used to compare the frequency of M. pneumoniae infection in the different

agee categories and the frequency of M. pneumoniae among household contacts of index cases

treatedd with appropriate antibiotics and of non-treated cases. For comparison of the frequency

off M. pneumoniae in the winter period and the summer period the y} test with Yates

correctionn was used. The x~t c st vvith Mantcl-Haenszcl trend analysis was used to compare the

frequencyy of M. pneumoniae in the age categories of the household contacts.

RESULTS S

Distributio nn of M. pneumoniae and respiratory viruses among GP patients with ARI

Duringg the 30 months study period GPs in 32 (70%) of the 45 general practices participating

inn the surveillance collected nose/throat samples from 1172 ARI patients. The 1172 patients

weree a representative sample of the national population with respect to geographic and sex

distributionn (18). There was a slight ovcrrepresentation of the youngest age category and an

undcrrepresentationn of people over 60 years of age (Table 1).

Inn total, 631 respiratory pathogens were detected in nose/throat samples from 565 (48%)

patients.. Mixed infections occurred in 56 ARI patients, predominantly in the 0 to 4 years age

category. .

Off the 1172 ARI patients, 772 (65%) presented with ILI and 400 (35%) with ARI non-ILI.

M.M. pneumoniae DNA was present in samples from 39 (3.3%) of the 1172 patients, being 24

(3.1%)) ILI patients and 15 (3.7%) patients with ARI non-ILI. Of the 39 M. pneumoniae

positivee patients, 22 (56%) were males and 17 (44%) were females. The detection rate of M

pneumoniaepneumoniae was similar in the different age groups (T^O.05) (Table 1). Mixed infections due

74 4

M.M. pneumoniae infection among oulpalicnts

too M. pneumoniae and a viral agent were detected in 7 patients: in 4 with rhinovirus, in 2 with

enterovirus,, and in 1 with respiratory syncytial virus.

Thee vast majority of the samples (86%; 1012/1172) was obtained from patients presenting

withh ARI in the winter period, October through March (Fig 1). M pneumoniae infection

occurredd during the whole year. Although the frequency (5.6%; 9/160) was higher in the

summerr period than in the winter period (2.9%; 30/1012) (Fig 1), this difference was not

significant. .

Figuree 1 Totall number of samples and of M. pneumoniae positive samples from patients consulting their GP for ARI by monthh in the Netherlands, from January 1, 1997 until July 1, 1999. Frequency of M. pneumoniae infections duringg summer periods was not significantly different from that during winter periods (X test with Yates correction,, P>Q.Q5)

Frequencyy of M. pneumoniae among household contacts

Thee 39 M. pneumoniae positive index cases represented 39 households. In four households

samplingg of the household contacts was not possible within 4 weeks after samples from the

indexx had been collected, and three patients and two GPs refused to participate in this part of

thee study. In total, nose/throat samples from all the 79 household contacts of 30 index cases

weree obtained. Samples were collected at a mean of 22 days (range 14 to 30 days) after initial

samplingg of the index case. hi. pneumoniae was present in samples from 12 (15%) of the 79

householdd contacts (Fig. 2). Among these 12 M. pneumoniae positive contacts 9 were

childrenn (0-15 yrs) and 3 persons were older than 15 years (P=0.02). Of the 12 M.

pneumoniaepneumoniae positive contacts 8 had an ARI, of whom 3 had consulted their GP because of

75 5

ChapterChapter 5

ARI.. Four of the children contacts were asymptomatic 4 weeks before and at least 4 weeks

afterr sampling (Fig. 2).

M.M. pneumoniae infection among household contacts of antibioti c treated and non-treated

patients. .

Eightt of the 30 index cases had been treated with doxycycline or macrolide antibiotics,

whereass 3 had received B-lactam antibiotics and 19 had not used antibiotics. The frequency of

M.M. pneumoniae positive throat samples among the 23 household contacts of the 8 doxycycline

orr macrolide treated cases was not significantly different from that among the 9 household

contactss of the 3 B-lactam antibiotic treated cases and the 47 household contacts of the 19

casess who did not receive antibiotics (Z^O.05) (Table 2). The 3 M. pneumoniae positive

householdd contacts of the 8 index cases treated with doxycycline or macrolides had ARI,

whereass among the 9 M. pneumoniae positive household contacts of the B-lactam antibiotic

treatedd or non-treated cases 5 had ARI (Table 2).

Tabicc 2 Presence of Xf. pneumoniae and the occurrence of ARI among 79 household contacts of 11 antibiotic treatedd and 19 non-treated M. pneumoniae positive patients

Treatmentt of Xf.Xf. pneumoniae

positivee index cases

Doxycycline/macro o lides s G-lactamm antibiotics Noo antibiotics

No.. of index x cases s

8 8

3 3 19 9

No.. of household d contacts s

23 3

9 9 47 7

ARI I present t

3 3

0 0 5 5

Xf. Xf. pneumoniaepneumoniae posit

ARI I absent t

0 0

0 0 4 4

vee contacts

totall (% of all contacts)

33 (13%)*

00 * 9(19%)* *

*noo statistically significant differences (%2 test, P>0.05)

DISCUSSION N

Duringg a 30 months prospective study the distribution of M. pneumoniae and various

respiratoryy viruses among GP patients with ARI in the Netherlands was studied. M.

pneumoniaepneumoniae was sought for by PCR in nose/throat samples from 1172 such patients. Among

pneumoniaa patients the incidence of M. pneumoniae infection diagnosed by serology and

culturee has been estimated at 200 per 100,000 population per year in a 12 years population

studyy in the USA (2), The incidence of M, pneumoniae infection as diagnosed by PCR among

outpatientss with ARI and assessed in 5 successive winter periods in France ranged from 190

76 6

M.M. pneumoniae infection among outpatients

age e (years) )

75 5

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negativee household contacts s (n= = =67) )

A A A A

A A

'A' '

i i A A A A AA A A A A

M.M. pneumoniae positivee household contacts s (n=12) )

Figuree 2 Numberr and age distribution of M. pneumoniae positive index cases participating in the transmission study (•), theirr M. pneumoniae negative household contacts (o) and M. pneumoniae positive symptomatic (A) and asymptomaticc (A) household contacts. Age categories are indicated by the dotted lines '' More M. pneumoniae positive household contacts were detected in the youngest age categories (0-4 and 5-15 years)) than in the other age categories (16 years and older) (Mantei-Haenszel x2 trend analysis, P=0.02)

too 1,234 per 100,000 population (8). In our study we detected M. pneumoniae by PCR in

sampless from 39 (3.3%) of 1172 ARI patients, of whom 772 presented with ILI and 400 with

ARII non-ILI. The calculated incidence of M. pneumoniae among the ARI patients presenting

too the GP was 587 per 100,000 population per year in the Netherlands. The incidence of ILI

amongg ARI patients was 2,410 per 100,000 population per year (19). The frequency of M.

77 7

ChapterChapter 5

pneumoniaepneumoniae infection among these ILI patients was 3.1%. Therefore, the incidence of M.

pneumoniaepneumoniae infection in GP patients with ILI was calculated to be 75 per 100,000 population

perr year. Although patients presenting with ARI non-ILI were not registered during this

study,, various sources provide data on the frequency of ARI among GP patients in the

Netherlandss (20). From these data and the ILI incidence data, the calculated incidence of ARI

non-ILII was 13,840/100,000 population per year. Since we found M. pneumoniae present in

3.7%% of the samples from ARI non-ILI patients, the incidence of M. pneumoniae infection

amongg such patients was calculated to be 512/100,000 population per year, Thus, the

incidencee of M. pneumoniae among ARI patients presenting to the GP was 587 per 100,000

populationn per year. In the community, the incidence of M. pneumoniae infection is expected

too be much higher as follows from our finding that among M. pneumoniae positive household

contactss of hi. pneumoniae positive patients, only 25% consulted their GP. The incidence of

5877 M. pneumoniae infections in ARI GP patients per 100,000 population found in our study,

reflectss the incidence in a M pneumoniae endemic 30 months period in the Netherlands (21),

whereass the incidence of 1,234 per 100,000 population as reported in the French study was

estimatedd in a M. pneumoniae epidemic winter period (8).

ARII due to M. pneumoniae occurred during all seasons. The number of M. pneumoniae

positivee patients was highest during winter months. The proportion of M. pneumoniae

positivee patients among patients with ARI was highest during summer months because of the

loww frequency of respiratory virus infections during these months (not shown).

Inn the GP patient population, M. pneumoniae infection occurred at all ages and no

significantt difference between the different age groups was present, similarly to the results as

recentlyy reported by Layani et al. for a French outpatient population (8). Their and our

findingss are not in agreement with data from other studies (2,22,23,24) in which school-age

childrenn (5 to 15 yr) and adults (30 to 45 yr) were identified as preference age groups for M.

pneumoniaepneumoniae infection. A possible explanation could be that in these studies mainly

hospitalisedd patients with M. pneumoniae infection were surveyed, whilst our study was

performedd in a GP setting.

Amongg the 79 household contacts of 30 M. pneumoniae positive patients, significantly

moree Af. pneumoniae positive contacts were in the 0 to 4 and 5 tol5 years age groups than in

thee other age groups. The M. pneumoniae positive children had either no signs and symptoms

off ARI or ARI was so mild that these children did not visit their GP. This strongly suggests

thatt young children are a relevant reservoir for M. pneumoniae, and play an important role in

transmissionn as has been reported for respiratory viruses (25,26,27,28), The finding also

supportss the assumption that clinical signs and symptoms of a first infection due to M.

pneumoniaepneumoniae are absent or mild but that sensitisation occurs. A subsequent M. pneumoniae

infectionn at older age may then induce more severe clinical manifestations (29).

78 8

M.M. pneumoniae infection among outpatients

Thee frequency of M. pneumoniae positive contacts in the households of the 8 doxycycline

orr macrolide treated index cases, of the 3 6-lactam antibiotic treated index cases and of the 19

non-treatedd index cases was similar. As doxycycline and macrolides are antibiotics by which

M,M, pneumoniae infection is effectively treated (30), our finding suggests that M. pneumoniae

transmissionn from such effectively treated cases still occurs in the household setting. It may

bee that transmission can be prevented only by early treatment of the index case. Similarly,

spreadd of Bordeteila pertussis among household contacts was prevented only if effective

treatmentt of the index cases by erythromycin was initiated early in the disease (31,32).

Inn conclusion, our study shows a frequency of M. pneumoniae infection of 3.3% among

GPP patients presenting with ARI, and of 15% among their household contacts as detected by

FCR.. In the latter group, M. pneumoniae was mainly present in children aged 0 tol5 years in

whomm the infection was either mild or asymptomatic. The rather high infection rate among

thesee household contacts of M. pneumoniae positive patients justifies the conclusion that

childrenn may be an as yet unrecognised reservoir for M, pneumoniae.

Acknowledgements s

Wee thank the general practitioners from the NIVEL sentinel network for registering influenza-like illness and providingg us with nose/throat swabs; H. Boswijk, K. Bijlsma, and C. Verwey (RIVM) for laboratory analyses; wee thank J. Schellckens and S.A.J. Zaat for critically reading the manuscript.

79 9

ChapterChapter 5

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81 1