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Sample type is crucial to the diagnosis of Mycoplasma pneumoniae pneumonia by PCR

Laboratory of Respiratory Viruses and Mycoplasmas, Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland

Correspondence Riitta Räty riitta.raty{at}ktl.fi

Received September 10, 2004
Accepted November 30, 2004

Sensitive and specific methods for rapid laboratory diagnosis of Mycoplasma pneumoniae were not available until nucleic acid amplification methods were developed. The choice of sample type and method of sampling are crucial to optimal diagnostic efficacy. Three types of respiratory samples from 32 young military conscripts with pneumonia were collected during an outbreak of M. pneumoniae infection. Sputum, nasopharyngeal aspirate and throat swab specimens were tested by 16S rRNA gene-based PCR with liquid-phase probe hybridization, and the results were compared with serology. The PCR result was positive for 22 (69 %) of the sputa, 16 (50 %) of the aspirates and 12 (37.5 %) of the swabs. Serology with increasing or high titres supported the positive findings in all instances. Sputum, when available, is clearly the best sample type for young adults with pneumonia.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Mycoplasma pneumoniae is an important human respiratory tract pathogen and, during epidemic activity, is second only to Streptococcus pneumoniae as the most common aetiologic agent of community-acquired pneumonia (Heiskanen- Kosma et al., 1998; Jokinen et al., 2001). In addition to lower respiratory tract infections, M. pneumoniae also causes milder symptoms such as sore throat, pharyngitis or tracheobronchitis (Clyde, 1993) and the symptomless carrier status is not uncommon (Foy, 1993). Children and young adults are easily affected by M. pneumoniae, but no age group is protected and reinfections occur frequently (Foy et al., 1977). Localized outbreaks are common in closed communities such as army garrisons (Feikin et al., 1999).

An early laboratory diagnosis of M. pneumoniae infection would help the clinician to decide upon the choice and initiation of an appropriate antimicrobial treatment as beta-lactam antibiotics are not effective against this micro-organism. In children and in a fraction of adult cases, specific IgM antibodies in acute-phase sera may help establish a diagnosis early during the illness. Due to previous exposure to the organism, however, many adult patients do not produce IgM antibodies upon reinfection with M. pneumoniae (Petitjean et al., 2002). Molecular techniques applied directly to respiratory tract specimens are nowadays widely used for the rapid diagnosis of respiratory tract infections; therefore, obtaining a representative specimen from the patient from the actual site of infection is most important. Throat swabs and nasopharyngeal aspirates (NPAs) are the specimen types most often used for M. pneumoniae PCR (Nadal et al., 2001; Reznikov et al., 1995). In pneumonia, sputum when available, offers an option to test material straight from the lungs. Evaluations of the suitability of sputum for M. pneumoniae PCR and comparisons with other respiratory tract specimens from the same individuals have not previously been reported to our knowledge.

During an outbreak of M. pneumoniae infection in the Finnish army, we carried out a study comparing three types of respiratory tract samples from a group of patients with radiologically confirmed pneumonia. From each patient we received throat swabs, NPAs and sputum samples for analysis of M. pneumoniae by PCR. In addition, paired blood samples were collected from most of the patients for the measurement of antibody levels in the acute- and convalescent-phase sera.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Patients and specimens.

Between October 2001 and January 2002, during a wide-spread outbreak of M. pneumoniae infections in army garrisons, a comparative sampling study was conducted in the Central Military Hospital in Helsinki. All patients with acute X-ray-verified pneumonia and without prior antimicrobial treatment were enrolled in the study but only those with a complete set of respiratory specimens were eligible for the final analysis. These patients formed a group of 32 conscripts with an age range of 22–29 years. The NPAs routinely used for diagnosis of respiratory viruses were collected from all patients and, in addition, a throat swab and a sputum sample were collected, after informed consent in each case, during the acute phase of the illness. The patients were first seen 4.5 days (median; range 1–35 days) after the onset of respiratory symptoms. The specimens were transported to the Laboratory of Respiratory Viruses and Mycoplasma on the same day and were stored at 4 °C until DNA isolation and PCR were performed, at most within 4 days. Paired sera with an interval of 2 weeks on average (range 7–22 days) were obtained from 28 of the patients.

Sample treatment for PCR.

In the laboratory, prior to DNA extraction, the NPAs and sputum specimens were homogenized by adding an equal volume of a mucolytic agent (dithiotreitol, final concentration 4–6 mM). On arrival at the laboratory the throat swabs were immersed in 1 ml PBS. After agitating the swabs for 3 min and squeezing them against the walls of the tubes, the swabs were discarded and the suspensions were stored until further processing.

An aliquot of 200 µl of each of the specimens was taken and subjected to DNA extraction utilizing a commercial kit (QiaAmp DNA Blood Mini Kit, Qiagen) using the ‘blood and body fluid spin protocol (01/99)'.

DNA amplification.

Amplifications were performed using as primers two oligonucleotides with sequences from the variable regions of the 16S rRNA gene (Tjhie et al., 1994). The sequences specific for M. pneumoniae were 5'-AAG GAC CTG CAA GGG TTC GT-3' and 5'-CTC TAG CCA TTA CCT GCT AA-3'. With these primers the length of the PCR product was 277 bp. The second primer was biotinylated at its 5' end.

PCR was carried out in 96-well plates (Advanced Biotechnologies, Thermo-Fast 96) in a total volume of 50 µl, and the volume of the DNA sample was 5 µl. The PCR mixture contained 50 mM Tris/HCl (pH 8.8), 15 mM (NH₄)₂SO₄, 1.5 mM MgCl₂, 0.01 % gelatin, 0.1 % Triton X-100, 200 µM of each deoxynucleoside triphosphate, 0.5 µM of each primer and 2.5 U Taq polymerase (MBI Fermentas). The plates were placed in a thermocycler (Mastercycler, Eppendorf) and heated at 95 °C for 2 min. After the predenaturation, 35 cycles of amplification were performed. Each cycle consisted of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and elongation at 72 °C for 2 min. After the last cycle, an additional incubation of 72 °C for 7 min was performed for completion of the polymerization reactions.

Detection of the PCR products.

The assay utilized a biotinylated primer, which was incorporated during the PCR into the amplification product and enabled its capture and subsequent denaturation in the streptavidin-coated microtitre-plate well. A specific digoxigenin-labelled detection probe was then hybridized to the target sequence. The hybridization assay was performed following the instructions of the manufacturer of the detection kit (PCR-ELISA DIG Detection, Roche Diagnostics).

The probe (GPO-1; Tjhie et al., 1994) had the following sequence: 5'-ACT CCT ACG GGA GGC AGC AGT A-3'. Digoxigenin was detected with a peroxidase-conjugated anti-digoxigenin antibody and the colorimetric substrate ABTS. Optical densities from colour-forming reactions were measured at 405 nm (Multiskan MS 3.0, Labsystems). The negative controls had a mean optical density of 0.137 and a SD of 0.099. The cut-off value for positivity was defined as the mean for the negative controls plus five times the SD of the mean, resulting in a cut-off value of 0.632.

Controls and sensitivity determination.

Purified chromosomal DNA from M. pneumoniae was diluted in 10-fold steps. As a positive control we used the highest dilution of DNA that after amplification still showed a clear band on the ethidium bromide-stained gel. Distilled water served as a negative control, and five negative controls were always included per plate. All clinical samples were tested for inhibitory factors in a second PCR in which a standardized amount of a plasmid containing an adenovirus hexon gene construct was added (Räty et al., 1999).

The analytical sensitivity of the PCR method was determined by a series of 10-fold dilutions. The highest template dilution resulting in a significant colour-forming reaction after the hybridization, contained 13 fg of the mycoplasmal DNA, corresponding to 15 genome copies. (Himmelreich et al., 1996).

Serology.

Paired serum samples were tested for antibodies against M. pneumoniae using a standard complement-fixation test (Casey, 1965). A fourfold or greater increase in antibody titres between the paired sera was considered as definitely diagnostic, but constant high titres (128) were also scored as positive.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The present study was conducted to examine which type of respiratory tract specimen, sputum, NPA or throat swab, is most suitable for PCR diagnosis of M. pneumoniae pneumonia. All three specimen types were obtained from 32 young adults with pneumonia. Sputum samples proved clearly to be the best respiratory specimens for this purpose, yielding a positive PCR result in 69 % (22) of cases, while the positivity rate was 50 % (16) and 37.5 % (12) for NPAs and throat swabs, respectively (Table 1, Fig. 1). It is not known in which order the specimens were collected, and it is therefore possible that positivity in upper respiratory tract samples may be due to contamination after producing a sputum sample.

View larger version (22K): [in this window] [in a new window]   Fig. 1. The overlapping circles represent the three types of respiratory tract specimens collected from 32 pneumonia patients for M. pneumoniae diagnostics. A throat swab, an NPA sample and a sputum sample from each patient was tested by PCR. In 10 cases, all three sample types were positive, in six NPA and sputum were positive, in two sputum and throat swab were positive and in four cases sputum was the only positive sample type. In 10 cases all the respiratory tract samples tested negative.

Inhibition of the PCR may occur in respiratory specimens as reported by Reznikov et al. (1995) who found NPA samples were inhibitory more often than throat swabs. For DNA extraction we used a commercial kit reported to reduce the effect of inhibitory substances (Fahle & Fischer, 2000), and total inhibition was not seen in any of the specimens. Weak inhibition of the amplification of the internal control was noted in some of the sputum samples, a few of the NPAs and two of the throat swabs. However, this phenomenon did not prevent the M. pneumoniae-specific PCR from giving strong positive results, presumably due to a high target load in sputum and also in NPA specimens. Theoretically, some of the sputum and NPA specimens might have contained such a low target load that even weak inhibition could have prevented a positive PCR result. Nevertheless, the positivity rate in these two specimen types was clearly higher than in the throat swabs.

Our results suggest that sputum is the best specimen type. Sixty-nine percent of the sputum specimens were PCR-positive while only 50 % of the NPAs and 38 % of the throat swabs were positive. Since it is apparent that the specimens from the same patients are not independent of each other, a method for paired binomial samples (Zhou & Qin, 2003) was applied for calculation of differences in the proportions of positive specimens with the associated 95 % confidence intervals. According to these differences, it was obvious that the proportion of positive specimens was significantly higher for the sputum PCR, compared with NPA PCR or the throat swab PCR.

The superiority of sputum as a specimen type in the present study is in agreement with our earlier findings when M. pneumoniae was detected by direct nucleic acid detection from sputum and throat swabs (Kleemola et al., 1990) and by antigen detection from sputum and NPAs (Kleemola et al., 1993). In these two studies, specific antibody responses between paired sera supported the detection of the M. pneumoniae infection. The draw-back with sputum is that it can seldom be obtained from young children or old and frail patients and other sampling methods must be applied for such patients.

The outstanding diagnostic sensitivity of sputum PCR can be simply explained by the higher numbers of M. pneumoniae organisms in the pulmonary alveoli than on the epithelium of the upper respiratory tract of pneumonia patients. This has been demonstrated in experimentally infected hamsters by Brunner (1991) who quantified M. pneumoniae organisms in different parts of the respiratory tract by culture. In this animal model of human M. pneumoniae pneumonia, the number of c.f.u. from lung culture was 100–1000 times higher than from throat culture. Collier and Clyde (1974) quantified M. pneumoniae from human sputum specimens. A range of 10²–10⁷ c.f.u. was recovered per ml of sputum while the number of c.f.u. from the throat was estimated by Kenny et al. (1990) as 60–2000 per ml.

Serological results of paired sera were available from 28 patients. Fifteen of them showed a significant titre rise and an additional 10 patients showed persistent high titres (Table 1). Of the 15 cases with significant titre rises, the throat swab was positive in eight, the NPA in 11 and the sputum in 15 cases (53 %, 73 % and 100 %, respectively). When the constant high titres were regarded as positive results too, the respective percentages were 44 %, 56 % and 80 %. Five patients had constant high antibody levels but PCR-negative respiratory tract specimens (Table 1).

In the present study, the results by the two approaches – PCR on samples from the respiratory tract and serology on paired sera – were surprisingly concordant. The reason might be that the patients had a distinct lower respiratory tract infection, pneumonia, and health care systems in the Finnish army warranted that the patients were admitted to hospital shortly after the onset of the symptoms. Consequently, all specimens could be collected in the proper time related to the beginning of the illness. Paired sera were available in 20 out of the 22 sputum PCR-positive cases, and in all of them either a significant titre rise or a constant high titre was detected (Table 1). The other way round, all 15 cases with a diagnostic antibody increase were also identified as M. pneumoniae infections by a positive sputum PCR result. The five cases with constant high antibody levels but negative PCR results could represent M. pneumoniae infections in the past; it is well known that high complement-fixation titres may persist for several months. In none of the cases was PCR positive alone without an accompanying serological response, a situation which might have indicated carrier status.

Tests based on nucleic acid amplification methods are becoming more rapid to perform and available to an increasing number of microbiological laboratories. Thus the impact of molecular diagnostics on the management of patients should be increasing. However, the choice of the best sample type and thorough sampling are essential prerequisites for optimal performance of these tests, too. The present study proved that as far as respiratory tract specimens from young and basically healthy pneumonia patients are concerned, sputum is clearly superior to both NPAs and throat swabs for reliable detection of M. pneumoniae by PCR.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We are grateful to Dr Jouko Karjalainen, who was in charge of the patients participating in this study, and the personnel of the Central Military Hospital for collecting the samples. We thank Annamari Harberg and Anja Waroma from our laboratory for their skilful technical work. We are indebted to statistician Jukka Jokinen for expert help and to Dr Thedi Ziegler for critical reading of the manuscript.

This work was presented in part at the Fifth Nordic–Baltic Congress of Infectious Diseases, May 22–25 2002, St Petersburg, Russia.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES