J Antimicrob Chemother 2020; 75: 2752–2759 doi:10.1093/jac/dkaa256 Advance Access publication 12 July 2020 Macrolide resistance and molecular typing of Mycoplasma pneumoniae infections during a 4 year period in Spain 1 Belén Rivaya †, Elena Jordana-Lluch1†‡, Gema Fernández-Rivas1, Sònia Molinos1, Roi Campos2§, Marı́a Méndez-Hernández2 and Lurdes Matas1,3* 1 Microbiology Department, Laboratori Clinic Metropolitana Nord, Hospital Universitari Germans Trias i Pujol, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Barcelona, Spain; 2Paediatric Department, Hospital Universitari Germans Trias i Pujol, Badalona, Spain; 3CIBER in Epidemiology and Public Health (CIBERESP), Madrid, Spain *Corresponding author. E-mail: lmatas.germanstrias@gencat.cat †Belén Rivaya and Elena Jordana-Lluch contributed equally to this work. ‡Present address: Antibiotic Resistance and Pathogenicity of Bacterial Infections Group, Balearic Islands Health Research Institute (IdISBa), Palma de Mallorca, Spain. §Present address: Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK. Received 20 December 2019; returned 17 March 2020; revised 21 April 2020; accepted 12 May 2020 Background: Mycoplasma pneumoniae (MP) causes community-acquired pneumonia affecting mainly children, and tends to produce cyclic outbreaks. The widespread use of macrolides is increasing resistance rates to these antibiotics. Molecular tools can help in diagnosis, typing and resistance detection, leading to better patient management. Objectives: To assess the MP genotypes and resistance pattern circulating in our area while comparing serological and molecular diagnosis of MP. Methods: Molecular and serological diagnosis of MP was performed in 821 samples collected in Badalona (Barcelona, Spain) from 2013 to 2017. Multiple locus variable number tandem repeat analysis (MLVA) and macrolide resistance detection by pyrosequencing were performed in those cases positive by PCR. Presence of respiratory viruses and relevant clinical data were also recorded. Results: MP was detected in 16.8% of cases by PCR, with an overall agreement with serology of 76%. Eleven different MLVA types were identified, with 4-5-7-2 (50.1%) and 3-5-6-2 (29.2%) being the most abundant, with the latter showing a seasonal increase during the study. A total of 8% of the strains harboured a point substitu- tion associated with macrolide resistance, corresponding mainly to an A2063G 23S rRNA mutation and directly related to previous macrolide therapy. Analysis of respiratory viruses showed viral coinfections in most cases. Conclusions: Serological and molecular tools combined could improve MP diagnosis and the analysis of its infection patterns. Macrolide resistance is associated with previous therapy. Given that MP pneumonia usually resolves spontaneously, it should be reconsidered whether antibiotic treatment is suitable for all cases. Introduction (RFLP)2 has been substituted by methods with higher discrimin- Mycoplasma pneumoniae (MP) is a prevalent respiratory pathogen atory power, such as multiple locus variable number tandem that causes about 20%–40% of cases of community-acquired repeat analysis (MLVA),3 allowing more accurate outbreak analysis pneumonia (CAP) in children and young adults, with epidemic and epidemic strain monitoring worldwide. peaks at 3–7 year intervals worldwide.1 Serology is the gold stand- Macrolides (MLs) are the first-line treatment, due to their low ard, but molecular techniques offer a rapid and reliable alternative side effects and convenient administration. MLs inhibit protein syn- for MP diagnosis and epidemiological surveillance. Molecular typ- thesis by binding to domain V of 23S rRNA.4 Mutations in domain II ing by P1 restriction fragment length polymorphism analysis or V of 23S rRNA or in L4 and L22 ribosomal proteins have been C The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. V This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecom- mons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 2752 Macrolide-resistant M. pneumoniae in Spain JAC described to be involved in ML resistance (MLr) in other species.5,6 was detected were further investigated for MLVA typing and MLr by pyrose- In MP, the MLr in vivo is mainly driven by specific mutations in the V quencing. Respiratory viruses were also tested for in all MP-positive sam- R domain of 23S rRNA gene, leading either to a high level resistance ples, using three different methods: BinaxNOWV Influenza A&B and R profile (mutations at positions 2063 or 2064) or to a low level MLr BinaxNOWV Respiratory Syncytial virus immunochromatographic assay (Abbott-Alere Healthcare, USA), direct immunofluorescence D3 Ultra8 DFA (transition at position 2067 or a transversion at position 2617).4,7–11 Respiratory virus Screening & Identification Kit (Quidel Corporation, USA), However, although no mutations in domain II could be linked to used until April 2014, and Anyplex II RV-16 Detection (Seegene Inc., Korea), MLr, mutations in ribosomal proteins L4 and L22 have been found used from May 2014, when Anyplex was integrated into our diagnosis rou- both in vitro4 and in vivo,9 although their real impact in MLr is still tine for adults and also for children with negative immunochromatographic unclear, so further research should be performed. results. As MP culture is mainly performed in reference centres, MLr de- termination by broth dilution tests11 is not accessible to most clin- MLVA typing ical microbiology laboratories. Molecular techniques targeting the DNA from PCR-positive samples was typed using the culture-independent single-copy 23S rRNA gene (such as PCR followed by high reso- MLVA approach described by Dumke and Jacobs,27 performing a nested lution melting curve analysis,12–14 Sanger sequencing,15 RFLP8 or PCR in order to increase the sensitivity. Products from the secondary PCR pyrosequencing analysis16–18) have been described. As a conse- were analysed by high-resolution capillary electrophoresis (QIAxcelV R quence of widespread ML use, resistance rates have risen from Advanced System, Qiagen, Germany) and subsequently purified (QIAquick 40% in 2008 to 90%–100% in China recently.19,20 Concurrently, in PCR Purification Kit, Qiagen). Purified PCR products were then Sanger Europe, rates vary from 1% in Slovenia to up to 26% in Italy.21–26 sequenced and the number of repeats was found by uploading the FASTA Due to the increase in the prevalence of MLr observed recently and file to the web resource Tandem Repeat Finder.28 Only four out of the five the lack of data in Spain, it is imperative to improve technical tools loci (Mp13, Mp14, Mp15 and Mp16) initially described by Dégrange et al.3 for detecting resistant strains without delay and to adjust the were analysed due to the high variability found in locus Mp1, as described patients’ therapy as necessary. elsewhere.29 The MLVA type is depicted as the number of repeats within the Mp13, Mp14, Mp15 and Mp16 loci, respectively, separated by a As there are no published data about the MP MLVA types circu- hyphen. Optimization of the protocol was performed using the DNA from lating in our area, this study aimed to provide insights about the M. pneumoniae M129, acquired from DSMZ (German Collection of genotypes and yearly distribution found in Catalonia (Spain). Microorganisms and Cell Cultures, Germany). Furthermore, the evaluation of the presence of single-base muta- tions in domain V of the 23S rRNA gene that provide MLr in our set- ting is described, in comparison with the results described in other Resistance detection by pyrosequencing countries. Additionally, the clinical performance of the molecular The pyrosequencing protocol was modified from Spuesens et al.16,17 Two technique used for MP pneumonia diagnosis was compared with different assays were performed in order to cover all known mutations routine serological testing. (550 bp apart) conferring ML-resistant genotypes found in vivo: the first assay was able to detect single base mutations at positions 2063, 2064 and 2067, while the second detected the mutation at position 2617. The proto- Materials and methods col described17 was modified by increasing the number of cycles to 45 for both primary and secondary PCR and by adding 2 lL of template for the Ethics, patients included, samples and collection secondary PCR. The presence of the targeted DNA fragment was detected R Written informed consent was obtained from patients’ guardians. This by high-resolution capillary electrophoresis (QIAxcelV Advanced System, study was approved by the Clinical Research Ethics Committee at Germans Qiagen), obtaining an approximately 165 bp fragment. Immobilization of Trias i Pujol University Hospital (‘Comité Ético de Investigación Clı́nica’, the biotinylated product and the pyrosequencing reaction were also per- CEIC). Samples collected included nasopharyngeal aspirates and sera. A formed following the protocol of Spuesens et al.16 with the following modi- total of 821 nasopharyngeal samples from 774 individuals were obtained fications: a total of 4 lL of streptavidin beads and 26 lL of H2O per sample between April 2013 and January 2017 in the Pediatric Emergency room at were used, and the plates were mixed for 10 min instead of 15 min. the University Hospital Germans Trias i Pujol (Badalona, Spain). Samples Pyrosequencing reactions and sequence analysis were also performed were divided into different groups: children (<18 years old) clinically diag- using the PyromarkTMQ96MD sequencer (Biotage, Sweden) and associated nosed with CAP, household or school contacts from a diagnosed pneumo- software. nia case and healthy volunteer subjects. Clinical data were registered by the Pediatric Department, and microbiological results were also recorded at Statistical analysis the Microbiology Department. Qualitative and categorical data were summarized with number and per- centage, and were compared using a v2 test. Statistical analyses were per- M. pneumoniae serological and molecular diagnosis formed with the statistical free software PSPP (http://www.gnu.org/ Detection of IgG or IgM antibodies from sera was performed by a particle- software/pspp/, last accessed April 2020). Statistical significance was R agglutination assay (SerodiaV-MYCO II, Fujirebio Europe) either singly or in accepted at P < 0.05. two samples separated by at least 21 days in order to demonstrate a sero- conversion. A positive result with clinical significance was defined as anti- body levels 1/160 for single samples. For the molecular diagnosis, DNA Results R was extracted from nasopharyngeal aspirates using the nucliSENSV Molecular detection of M. pneumoniae R easyMagV platform (bioMérieux, France). MP molecular diagnosis was per- formed using two different assays: during the first stage of the project, Real A total of 821 specimens (described in Table 1) were collected Time PCR Realcycler M. pneumoniae/Chlamydophila pneumoniae (Progenie from 774 individuals, corresponding to children in 769 cases (766 R molecular, Spain) was employed, whereas AnyplexV 2 RB5 Detection patients with suspicion of pneumonia or relatives with pneumonia, (Seegene Inc., Korea) was used later on. Those samples in which MP DNA and 3 healthy children) and adults in 52 cases (29 patients with a 2753 Rivaya et al. Table 1. Classification of the patients and specimens included in this study Number of Positive MP Complementary Samples tested for MP Age group Group of individuals samples tested detection (number) data 821 specimens Children Children with suspicion of 766a 17.5% (134)a 131 patients (774 individuals) (<18 years old) pneumonia or relatives with 2 household pneumonia contacts 1 classmate Healthy children 3 0.0% (0) Adults Adults with diagnosis of 29 13.8% (4) 2 patients pneumonia or close relatives 2 relatives with pneumonia Healthy adults (volunteer 23 0.0% (0) healthcare professionals) a For one specimen where MP was detected by PCR, it was not possible to perform the MLVA typing due to insufficient DNA. Thus, the total number of specimens from children typed was 133. pneumonia diagnosis or their household contacts, and 23 volun- 29.2%), 3-6-6-2 (11/137, 8%), 4-5-6-2 (4/137, 2.9%), 3-5-7–2 teer health professionals). A positive detection of MP was obtained (3/137, 2.2%), 4-5-5-2 (2/137, 1.5%), 4-5-7-3 (2/137, 1.5%), 4-6- in 138 (16.8%) specimens, corresponding to 127 individuals. One 7-2 (2/137, 1.5%), 3-3-6-2 (1/137, 0.7%), 3-4-6-2 (1/137, 0.7%) positive specimen was excluded from the study due to insufficient and 3-4-7-2 (1/137, 0.7%). The proportions of the types varied DNA; thus, the final count of positive specimens was 137 (16.7%, widely between the years of the study (Figure 1b). Comparing the 3 by Progenie, 134 by Anyplex). Positive detections were mostly 2 years with higher numbers of positive cases (2014 and 2015, from children (133, 97.1%), with only 4 (2.9%) from adults (close Figure 1a), the proportion of type 4-5-7-2 during those years was relatives). The median age of children was 6.6 years and the high- similar, while an increase of type 3-5-6-2 and a decrease of type est proportion of positive samples was detected in children aged 3-6-6-2 was observed from 2014 to 2015. Interestingly, the distri- >5 years (53.3%, 73/137), followed by children aged between 1 bution of the types also varied depending on the season and 5 years (36.4%, 50/137) and 1 year old (7.3%, 10/137) (Figure 1c), with genotype 4-5-7-2 mostly detected during sum- (Table 2). The majority of specimens were collected during the mer (both 2014 and 2015) in contrast to type 3-5-6-2, which was middle years of the project (2014 and 2015), and the positivity more frequent from winter 2014 to spring 2015. Genotype 3-6-6-2 rates per year were as follows: 26% in 2013 (19/73), 19% in 2014 also showed a peak during spring 2014, whereas the other minor- (49/258), 13.2% in 2015 (56/423) and 22.4% in 2016 (15/67), as ity genotypes were detected in similar numbers among all seasons depicted in Figure 1(a). from spring 2013 to spring 2016. The four positive specimens detected from adults had the Performance of molecular detection in comparison with same MLVA profile as the children, suggesting transmission of MP serological diagnosis within the same household. In parallel to PCR, serological diagnosis was also performed in 376 Twelve out of the 137 cases were reinfections after 1 or cases, including 105 patients with a positive MP detection by PCR 2 months from the primary specimen. In 9 specimens, the MLVA and 271 with a negative MP result. Among the 105 MP PCR-positive type detected was the same as the initial sample; however, for subjects, serology results in the acute phase (first serum sample) 2 patients, the MLVA changed. In one case, the initial MLVA was were also positive (antibody levels 1/160) in 75 (71.4%) cases, 3-6-6-2 (March 2015) but the MLVA from the specimens obtained whereas in the remaining 30 cases with negative serology (anti- in April and May 2015 was 3-5-6-2. For the other case, the initial body levels <1/160), seroconversion was observed in 13 (43.3%) type was 3-5-6-2 (March 2016) whereas the specimen obtained patients in convalescence phase (second serum sample), leading on April 2016 was 3-5-7-2. Given that both isolates had only a to 88 out of 105 patients (83.8%) with both positive serology and single repeat change from the initial profile, the second detected PCR, and an overall agreement between techniques of 76%. specimen could be a variant of the first isolate. Among the 271 MP PCR-negative subjects, serology was also nega- tive in 199 cases (73.4%). The remaining 72 cases showed a posi- tive serology result in a single sample. Thus, sensitivity and Clinical findings specificity of the PCR compared with serology was 55% and 92%, Several parameters such as chest radiography, axillary temp- respectively (Table 3). erature, respiratory rate, presence of adenopathy, arthralgia and pleural effusion were recorded, and a statistical analysis was M. pneumoniae MLVA typing performed to determine if there was a relationship to the MLVA Eleven different MLVA types were identified; the most abundant type. Results are shown in Table 2; however, none of the clinical was 4-5-7-2 (70/137, 50.1%), followed by 3-5-6-2 (40/137, symptoms evaluated was related to a specific MLVA type. 2754 Macrolide-resistant M. pneumoniae in Spain JAC Table 2. Characteristics and clinical symptoms distributed by MLVA type identified MLVA type % (n) from total of Characteristic or 4-7-5-2 3-5-6-2 3-6-6-2 Other positive specimens clinical symptoms (N = 70) (N = 40) (N = 11) (N = 16) (N = 137) P value Sex male (n) 58.6% (41) 52.5% (21) 45.5% (5) 56.3% (9) 55.5% (76) 0.834 female (n) 41.4% (29) 47.5% (19) 54.5% (6) 43.7% (7) 44.53% (61) Age group 1 year (n) 5.7% (4) 5.0% (2) 0.0% (0) 25.0% (4) 7.3% (10) 0.252 1–5 years (n) 38.6% (27) 40.0% (16) 36.4% (4) 18.6% (3) 36.4% (50) >5 years (n) 51.4% (36) 52.5% (21) 63.6% (7) 56.2% (9) 53.3% (73) adult (n) 2.2% (3) 0.73% (1) 0.0% (0) 0.0% (0) 2.9% (4) Body temperature afebrile (n) 54.3% (38) 57.5% (23) 81.8% (9) 50.0% (8) 57.0% (78) 0.348 fever/slight fever (n) 45.7% (32) 42.5% (17) 18.2% (2) 50.0% (8) 43.0% (59) Respiratory rate eupnoea (n) 70% (49) 72.5% (29) 81.82% (9) 68.75% (11) 71.5% (98) 0.857 tachypnoea (n) 30% (21) 27.5% (11) 18.18% (2) 31.25% (5) 28.5% (39) Lung auscultation normal (n) 45.7% (32) 47.5% (19) 72.73% (8) 37.5% (6) 47.5% (65) 0.316 altered (n) 54.29% (38) 52.5% (21) 27.27% (3) 62.5% (10) 52.5% (72) Chest X-ray normal (n) 2.9% (2) 10.0% (4) 0.0% (0) 6.2% (1) 5.1% (7) 0.655 pathological (n) 80.0% (56) 72.5% (29) 90.91% (10) 81.3% (13) 78.8% (108) not performed (n) 17.1% (12) 17.5% (7) 9.1% (1) 12.5% (2) 16.1% (22) Adenopathies absent (n) 97.1% (68) 97.5% (39) 100.0% (11) 100.0% (16) 97.8% (134) 0.857 present (n) 2.9% (2) 2.5% (1) 0.0% (0) 0.0% (0) 2.2% (3) Arthralgia absent (n) 97.1% (68) 97.5% (39) 100.0% (11) 100.0% (16) 97.8% (134) 0.857 present (n) 2.9% (2) 2.5% (1) 0.0% (0) 0.0% (0) 2.2% (3) Stevens–Johnson syndrome absent (n) 98.57% (69) 97.5% (39) 100.0% (11) 100.0% (16) 98.5% (135) 0.873 present (n) 1.43% (1) 2.5% (1) 0.0% (0) 0.0% (0) 1.5% (2) Viral coinfection absent (n) 58.6% (41) 42.5% (17) 72.7% (8) 56.25% (9) 54.7% (75) 0.232 present (n) 41.4% (29) 57.5% (23) 27.3% (3) 43.75% (7) 45.3% (62) Viral infections were also evaluated in MP-positive three of them in region 1 and one in region 2: A2063G (N = 7), patients. Respiratory viruses were found in 45.3% of patients A2063T (N = 1), A2064G (N = 2) (region 1) and C2617A (N = 1) (N = 62), with two or more viruses detected in 12.4% of the (region 2). cases. A total of 79 virus-positive samples were detected, Among the resistant strains, the most frequent MLVA type was listed in Table 4. The most frequently detected was Rhinovirus 4-5-7-2 (N = 8), followed by 3-5-6-2 (N = 3), and all of them were (37.9%, 30) followed by Parainfluenza virus (15.2%, 12) and detected between 2014 and 2016. All the resistant strains Respiratory Syncytial Virus (13.9%, 11). The genotypes 4-5-7-2 detected were linked to a previous treatment with MLs. In six and 3-5-6-2 were the most frequently co-infected with virus cases, patients developed the resistance during the treatment (P = 0.033). of the studied episode, with a specimen before treatment being susceptible to MLs and having the same MLVA profile as the after-treatment and resistant specimen. In four cases, re- MLr detection sistant strains were collected after ML treatment during the The MP PCR-positive samples were further screened for point ongoing or a previous CAP episode. The last case was a patient mutations known to confer MLr, and they were detected in 11 sam- whose brother previously had CAP caused by MP and was sub- ples (8%, 10 patients), showing four different types of mutation, sequently treated with MLs. 2755 Rivaya et al. (a) 500 (b) 60 N = 56 423 N = 49 4-5-7-2 400 Specimens tested 3-5-6-2 Detection of 40 N of cases 3-6-6-2 N of cases 300 M. pneumoniae 258 Other 200 20 N = 17 N = 15 100 73 56 67 49 19 15 0 0 13 14 15 16 13 14 15 16 20 20 20 20 20 20 20 20 Proportion of M. pneumoniae cases detected distributed MLVA types identified distributed by year by year during the timeframe of the study during the timeframe of the study (c) 30 4-5-7-2 3-5-6-2 N = 21 3-6-6-2 20 N = 18 Other N of cases N = 17 N = 14 N = 13 N = 10 N = 10 10 N=8 N=8 N=3 N=4 N=4 N=3 N=1 N=1 N=1 0 3) 3) 4) 5) 3) 4) 4) 4) 5) 5) 6) 5) 6) 6) 6) 7) 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 (2 (2 (2 (2 -2 (2 (2 (2 -2 (2 (2 (2 -2 (2 (2 -2 13 14 15 16 g er g g n er n er g n er n rin rin rin rin m m m m m m m m 20 20 20 20 tu tu tu tu Sp Sp Sp Sp m m m m r( r( r( r( Au Au Au Au Su Su Su Su te te te te in in in in W W W W MLVA types identified distributed by seasons during the timeframe of the study Figure 1. Molecular detection and typing of M. pneumoniae. (a) Detected cases of M. pneumoniae distributed by year. (b) MLVA types identified dis- tributed by year. (c) MLVA types identified distributed by season during the time frame of the study. Table 3. Performance of molecular diagnosis compared with serology for the detection of Mycoplasma pneumoniae in our region has not been widely studied yet, and, to the best of our knowledge, this is the first study to document the MLVA types Serology circulating in our area as well as the MLr prevalence. During the study period, a total of 137 (16.7%) specimens were positive Positive Negative Total and could be further analysed for MLVA typing and MLr detection. PCR Positive 75/88 a 30/17 a 105 The proportion of positive samples detected is similar to that in Negative 72 199 271 other European countries.21,30 During our study, a higher number of positive cases was Total 147/160a 229/216a 376 detected during 2014 and 2015 (Figure 1a), probably related to a Sensitivity 51%/55% a higher number of specimens processed. In accordance with similar Specificity 87%/92%a studies found in the literature,21 the highest proportion of positive Overall agreement 73%/76%a cases was in children older than 5 years (N = 73). Culturing MP is usually performed in reference centres. Given a Patients that showed seroconversion (second serology with a positive the prompt presence of IgM antibodies, especially in children, sero- result). PCR performed from nasopharyngeal aspirates. logical tests are the most often applied methods for MP diagnosis. Different techniques have been used, such as complement fixation tests, enzyme immunoassays or passive agglutination with latex or gelatin particles, which have been extensively reviewed.1 Discussion Usually, two samples (from initial and convalescent phases) are M. pneumoniae is known to cause between 20% and 40%1 of required to demonstrate the seroconversion and to increase the CAP in children and young adults, with increasing resistance to diagnostic sensitivity, although some authors have found that MLs,13–20 which are the first-line treatment. Prevalence of MP CAP using only an acute phase sample could lead to a diagnosis 2756 Macrolide-resistant M. pneumoniae in Spain JAC Table 4. Virus detecteda distributed by MLVA type identified MLVA type Virus 4-7-5-2 (N = 70) 3-5-6-2 (N = 40) 3-6-6-2 (N = 11) other (N = 16) Total Influenza virus 28.6% (2) 28.6% (2) 0.0% (0) 42.8% (3) 8.9% (7) Metapneumovirus 0.0% (0) 0.0% (0) 0.0% (0) 100% (1) 1.3% (1) Parainfluenza virus 66.7% (8) 25.0% (3) 0.0% (0) 8.3% (1) 15.2% (12) Respiratory Syncytial Virus 27.3% (3) 36.3% (4) 18.2% (2) 18.2% (2) 13.9% (11) Rhinovirus 53.3% (16) 36.7% (11) 3.3% (1) 6.7% (2) 37.9% (30) Adenovirus 50.0% (5) 50.0% (5) 0.0% (0) 0.0% (0) 12.7% (10) Bocavirus 0.0% (0) 50.0% (1) 50.0% (1) 0.0% (0) 2.5% (2) Coronavirus 0.0% (0) 100% (2) 0.0% (0) 0.0% (0) 2.5% (2) Enterovirus 50.0% (2) 0.0% (0) 25.0% (1) 25.0% (1) 5.1% (4) Total 45.6% (36) 35.4% (28) 6.3% (5) 12.7% (10) 100% (79) a Results are shown as the percentage with virus detected (n). in up to 50% of cases.31,32 In our comparison of serology and during winter and spring. Minority genotypes were evenly distrib- molecular methods, we found an overall agreement of 73% uted within seasons and the years of the study. between both techniques during the acute phase (first serum Clinical aspects in MP pneumonia can be subtle and shared with sample positive, antibody titre 1/160). After the seroconver- other viral or bacterial entities. However, some clinical findings or sion of 13 patients, the overall agreement increased to 76%. an altered X-ray test, showing only bilateral pneumonia (20% of Discrepancies between these methods can be explained due cases), could help in its diagnosis.1,41 Thus, the most reliable diag- the nature of the techniques.33–35 Immunosuppressed patients nostic is direct or indirect pathogen detection. Respiratory viruses or collecting the serology specimen too early during the acute were also tested for in patients with a confirmed MP pneumonia. phase could lead to false-negative results.36 On the other In most cases we detected multiple viral coinfections, which com- hand, in the event of a previous MP infection, IgM can be plicated distinguishing whether the observed clinical symptoms detected for months or even years.34,35 Kim et al.36 suggested were due to the virus or MP. This has been observed before, as to set the positivity cut-off to a titre 1/640 for the diagnosis of some studies have shown that different viruses, such as acute-phase CAP. In our study, in 61 out of the 72 serology- Rhinovirus, could be found in children without any symptoms;42 positive–PCR-negative cases, their antibody titres were really thus their detection should be taken into consideration with some close to the cut-off value (22 with a titre of 1/160 with no in- caution. In our study, this virus was the most frequently detected. However, it must be kept in mind that infections with other viruses crease during the convalescent phase and 46 cases with a titre such as Parainfluenza or Respiratory Syncytial Virus could be of 1/320), which could indicate previous contact with MP and related to the clinical symptoms shared with MP. thus be false positives for the current acute infection. On the In our study, no correlation was found between the MLVA type other hand, 17 patients had negative serology but positive mo- and the different symptomatology, in contrast to what was lecular detection of MP. It has been reported that MP can colon- described by Yan et al.,43 where the MLVA was linked to the clinical ize the nasopharynx for a long time without any clinical outcome, although this group also found that some MLVA types, ir- manifestation. Meyer Sauteur et al.37 detected MP DNA in 48% respective of their susceptibility profile, showed a higher risk of pro- of healthy children tested as controls during their study, and gression to severe MP pneumonia. Nilsson et al.33 were able to detect MP DNA for 7 weeks after the MLs are the first-line treatment for children with MP pneumonia clinical symptoms, even though the patients were correctly in our country.44 Fluoroquinolones and tetracyclines are generally treated with MLs. Further research would be necessary to clarify avoided in this population due to their side effects,45 although the impact of carriers in the community on infection transmis- countries with high rates of ML-resistant strains (such as Japan) sion. Molecular techniques can be valuable tools for MP diagno- have approved tosufloxacin or minocycline as second-line drugs sis, and further allow the study of epidemic clusters by for children with MP pneumonia who respond poorly to MLs.46 performing molecular typing or the detection of resistance pat- Several studies reporting MLr have been published. The first resist- terns. Thus, combining serological methods with molecular ant strains were detected in Asia and were linked to broad ML tools should improve MP diagnosis and avoid misinterpretation. use,47 with resistance rates that vary between 13% and 100% in MLVA is a powerful genotyping tool that can be used to better China19,20,48–50 or Japan.8,51–53 Similarly, antibiotic pressure seems distinguish among strains.38 The genotypes 4-5-7-2 and 3-5-6-2 to be the cause of the high resistance rate detected in Italy were the most abundant, as has been described in other (25.6%).26 Lower resistance rates have been described in other geographic areas such as Europe,39 the USA40 and China.10 European countries, such as 9.8% in France,13 9.3% in England,23 Furthermore, those genotypes were seasonal, with 4-5-7-2 mainly 1.2% in Germany24 and 1% in Slovenia.21 Interestingly, no resist- detected during summer whereas 3-5-6-2 was mainly circulating ant strains were detected in northern countries, such as Sweden.25 2757 Rivaya et al. Eight percent of the strains tested had a point substitution in domain V of the 23S rRNA gene that conferred resistance to MLs. References Interestingly, in 6 out of 11 patients, a shift to a resistant pattern 1 Atkinson TP, Balish MF, Waites KB. Epidemiology, clinical manifestations, was detected from the initial to the second sample collected for pathogenesis and laboratory detection of Mycoplasma pneumoniae infec- the clinical follow-up after ML treatment. It is important to high- tions. FEMS Microbiol Rev 2008; 32: 956–73. light that one of the resistant cases was resistant only after two 2 Cousin-Allery A, Charron A, de Barbeyrac B et al. Molecular typing of doses of MLs, as the treatment had to be interrupted due to an al- Mycoplasma pneumoniae strains by PCR-based methods and pulsed-field gel lergic event. MLVA typing confirmed that it was probably the same electrophoresis. Application to French and Danish isolates. Epidemiol Infect strain and not a reinfection with an already resistant strain. Given 2000; 124: 103–11. that the MLr found in vivo is mainly linked to point mutations in do- 3 Dégrange S, Cazanave C, Charron A et al. Development of multiple-locus main V of the 23S rRNA,45 only that region was analysed. However, variable-number tandem-repeat analysis for molecular typing of the real impact on the MLr phenotype of mutation on L4 and L22 Mycoplasma pneumoniae. J Clin Microbiol 2009; 47: 914–23. ribosomal proteins remains unclear,4,9 and it should be addressed 4 Pereyre S, Guyot C, Renaudin H et al. In vitro selection and characterization with further studies. of resistance to macrolides and related antibiotics in Mycoplasma pneumo- The rising incidence of ML-resistant strains favoured by a previ- niae. Antimicrob Agents Chemother 2004; 48: 460–5. ous ML treatment is a significant finding. Interestingly, symptom 5 Davies TA, Dewasse BE, Jacobs MR et al. 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Mutations in 23S rRNA account for intrinsic resistance to macrolides in Mycoplasma hominis and Mycoplasma accurate comparison between the two techniques. Secondly, even fermentans and for acquired resistance to macrolides in M. hominis. though the number of specimens investigated was high (N = 821), Antimicrob Agents Chemother 2002; 46: 3142–50. the final number of specimens positive for MP was relatively small 8 Matsuoka M, Narita M, Okazaki N et al. Characterization and molecular for statistical purposes (N = 137). Thirdly, this study only covers a analysis of macrolide-resistant Mycoplasma pneumoniae clinical isolates small area of Spain. It would be interesting to perform a multi- obtained in Japan. Antimicrob Agents Chemother 2004; 48: 4624–30. centre study to include several regions of our country in order to 9 Liu X, Jiang Y, Chen X et al. Drug resistance mechanisms of Mycoplasma corroborate our findings, preferably during an extended period pneumoniae to macrolide antibiotics. 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