A previously healthy 9-yr-old boy with a cough and sputum since 7 days and a fever since 3 days visited the outpatient department (OPD) of Wonju Medical Center. He took cough medication for 2 days before admission but did not take any antibiotics. He was an elementary school student, and there was no recent history of sick contacts, travel, or illness.

Auscultation of the lungs revealed coarse crackles with right side rhonchi. Laboratory results showed that complete blood cell count, white blood cell differential count, electrolyte level, liver function tests, and urinalysis were normal. C-reactive protein (CRP) was 12.0 mg/L (reference range: 0-5 mg/L). The chest radiograph showed pneumonic consolidation on the right upper lung field. Sputum culture showed growth of normal flora. Sputum smears were negative for acid-fast bacilli, and a tuberculin skin test was negative. Blood culture showed no growth after 7-day incubation. The serologic test for detecting anti-mycoplasma IgM antibody (SERODIA®-MYCO II, Fujirebio, Tokyo, Japan) was negative, but the patient's cold agglutinins titer was 1 :128. A PCR test of a nasopharyngeal swab specimen was positive for M. pneumoniae. Gene amplification of M. pneumoniae was performed by MyGenie 32 Thermal Block (Bioneer, Daejeon, Korea), and we used 20 µL of high fidelity PCR premix (AccuPower®Premix, Bioneer, Daejeon, Korea) as the reactive agent. PCR primers were designed for detecting known M. pneumoniae single point mutations (A2063G, A2064G, T2611C, and C2617A). In this case, the resistant strains displayed a point mutation in the 23S rRNA: A-to-G transition mutation at nucleotide 2063.

Initial treatment with flomoxef was given intravenously (70 mg/kg/day, divided into 3 doses). The result of PCR for M. pneumoniae was positive, and roxithromycin (7 mg/kg/day, divided into 2 doses) was added on the 2nd day of hospitalization. On the 5th day of hospitalization, neither respiratory symptoms nor follow-up chest radiography findings improved. Treatment was, therefore, switched from roxithromycin to azithromycin (10 mg/kg/day, once a day for 3 days). On the 9th day of hospitalization, clinical symptoms and chest radiography findings improved, so the patient was discharged. Four days later, he was symptom-free with no detectable complications on OPD follow-up. The M. pneumoniae isolated from this patient carried an A2063G mutation.

The second patient is the younger brother of the patient described in case 1. This patient was 5-yr-old boy who presented with cough and sputum for 10 days without fever. He visited a local pediatric clinic and also took roxithromycin for 7 days before the visit to our OPD. He had attended preschool and had been relatively healthy. On physical examination, his breath sounds were coarse with fine crackles, but chest radiography findings were normal. Roxithromycin (7 mg/kg/day, divided into 2 doses) was administered. However, 10 days later, the patient revisited the OPD with aggravated cough and newly developed fever. The chest radiography revealed pneumonic consolidation on the right upper lung field, similar to that seen on the chest radiographs of his elder brother. Although the respiratory symptoms of the siblings started at around the same time, fever with changes in chest radiography findings in case 2 (younger brother) developed 14 days later than those in case 1. The patient was admitted to the hospital, and azithromycin treatment was started immediately (10 mg/kg/day for 3 days). Laboratory results, including urinalysis, complete blood count, electrolyte level, and liver function tests, were all normal. CRP was 10 mg/L. The titer of anti-mycoplasma IgM antibody (SERODIA®-MYCO II) was 1:1,280 and the cold agglutinins titer was 1:256. Real-time PCR for M. pneumoniae was also positive and 23S rRNA sequencing showed A2063G mutation in the 23S rRNA gene, which was also present in M. pneumoniae isolated from this patient's brother. He underwent conservative management in hospital for 7 days, and then he was discharged.

M. pneumoniae DNA was extracted using an Exiprep Genomic DNA Kit (Bioneer, Daejeon, Korea). Extraction was performed according to the manufacturer's instructions.

AccuPower® MP Real-time PCR Kit (Bioneer) was used for detection of M. pneumoniae according to the manufacturer's instructions. Briefly, M. pneumoniae-specific DNA regions were detected with fluorescence of specific hydrolysis probes (5'-fluorescein carboxylic acid [FAM]; 3'-Black Hole Quencher-1 [BHQ1]) during thermal cycling (Exicycler™ 96 Real-Time Quantitative Thermal Block, Bioneer) [9]. PCR was carried out using a PCR premix kit for detection of M. pneumoniae (Bioneer). Five microliters of extracted DNA, positive control DNA, or distilled water (negative control) were added into individual premix tubes. The reaction mixture was then subjected to denaturation for 10 min at 95℃, then 40 PCR cycles, each of 20 sec at 95℃ and 30 sec at 55℃, followed by final cooling step of 1 min at 25℃.

A PCR assay followed by direct amplicon sequencing was developed to detect point mutations conferring resistance to macrolides in domain V of the M. pneumoniae 23S rRNA gene. After we obtained the DNA sequence of M. pneumoniae from Gen-Bank® (NIH, Bethesda, MD, USA), we designed primers specific for polymorphism. Forward primer (Target gene: 23s rRNA 1851-2675 bp): 5'-GAA GGT TAA AGA AGG AGG TTA GCG CAA-3'. Reverse primer: 5'-TCG GTC CTC TCG TAC TAG AAG CAA CA-3'. Amplification of the 23S rRNA gene of M. pneumoniae was performed using a MyGenie 32 Thermal Block (Bioneer) thermal cycler. PCR was carried out in 20 µL of high fidelity PCR premix (Bioneer) using the following profile: denaturation for 5 min at 94℃ and 35 cycles of 30 sec at 94℃, 30 sec at 55℃, and 1 min at 72℃. Final extension (10 min at 72℃) was followed by cooling at 4℃. The amplified products were separated by electrophoresis on a 1.5% agarose gel and visualized under ultraviolet light. The amplicons were purified using gel purification (Bioneer), and were directly sequenced on an ABI Prism 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA).