Lower respiratory tract infections (LRTIs) remain a major cause of morbidity and mortality worldwide. Among the various pathogens implicated in community-acquired LRTIs, Mycoplasma pneumoniae is recognized as a significant atypical bacterial agent, particularly affecting older children and young adults. It accounts for 10%–40% of community-acquired pneumonia (CAP) cases globally and is capable of causing a wide spectrum of diseases, ranging from mild tracheobronchitis to severe, sometimes fulminant, pneumonia.1 In addition to respiratory involvement, M. pneumoniae infections can also lead to extrapulmonary manifestations including neurological, dermatological, and hematological complications.2 The clinical diagnosis of M. pneumoniae infection is challenging due to its non-specific symptoms and overlap with other respiratory pathogens. As a result, empirical treatment is often initiated without Microbiological confirmation. This has significant therapeutic implications, since M. pneumoniae is intrinsically resistant to β-lactam antibiotics and requires treatment with macrolides or other non-cell wall-targeting agents. However, the rising incidence of macrolide-resistant M. pneumoniae (MRMP), particularly due to mutations in the 23S rRNA gene, poses a growing threat to effective management and highlights the importance of early, accurate diagnosis.2 Laboratory confirmation of M. pneumoniae infection traditionally relies on serology or culture, but both methods have limitations, including delayed results, reduced sensitivity and lack of availability of selective culture media in resource limited laboratories. Molecular methods such as polymerase chain reaction (PCR) have emerged as valuable diagnostic tools owing to their high sensitivity, specificity, and rapid turnaround time, independent of organism viability.1 Despite the global significance of M. pneumoniae, there is limited data on its prevalence, and resistance patterns in Indian patients with community-acquired LRTIs, particularly in South Kerala which is critical for optimizing diagnostic strategies and therapeutic approaches. This study aims to determine the prevalence of M. pneumoniae in community-acquired LRTIs among children and adults in South Kerala using PCR. The findings are intended to contribute to regional epidemiological data and support evidence-based management of atypical pneumonia.

Detection of M. pneumoniae Using Real-Time PCR: A Real-Time PCR assay was employed for the detection of M.pneumoniae using an in-house primer and Type-it® HRM kit (Qiagen). All experimental procedures were conducted in accordance with standard biosafety protocols within a biosafety level 2 (BSL-2) cabinet. Genomic DNA was extracted from 387 respiratory clinical specimens received over a period of 6 months using the QIAamp® DNA Mini Kit. The extracted DNA was stored at -20 °C until further use in the PCR assay. Each 20 μl reaction mixture consisted of 10 μl of DNA template, 2 μl of primer mix, and 8 μl of Type-it® HRM buffer. PCR amplification was carried out using both the CFX96 Real-Time PCR System (Bio-Rad) and the Mic Real-Time PCR System IQ (Bio Molecular Systems®). The amplification protocol began with a reverse transcription step at 50°C for 15 minutes, followed by an initial denaturation and Taq polymerase activation at 95°C for 5 minutes. This was succeeded by 40 amplification cycles, each comprising denaturation at 95°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 15 seconds. After the amplification phase, High-Resolution Melt (HRM) analysis was conducted by gradually increasing the temperature from 70°C to 93°C in 0.1°C increments, with a 2-second hold at each step to accurately determine the melt profile. Each RT-PCR run included both positive and negative controls to ensure assay reliability. The amplification and melt curve data were analyzed using MIC PCR and Bio-Rad software, and M. pneumoniae was identified based on its specific melting profile (Figure 1). All results were appropriately documented.

Out of the 387 samples tested for M. pneumoniae from November 2024 - April 2025, by PCR, 142 (36.7%) were positive (Figure 2). Among the positive cases, 85 (59.8%) were from individuals in the 0–10 years age group, followed by 31 (21.8%) in the 11–20 years age group (Figure 3). M. pneumoniae was more frequently detected in females [75 (52.8%)] than in males (Figure 4). The majority of positive detections [98 (69%)] were from sputum samples, while the least number of positives (1 each) were from endotracheal secretions and nasopharyngeal swabs (Figure 5).

Our study findings demonstrate the continued relevance of M.pneumoniae as an important respiratory pathogen, especially in pediatric populations, and they align with global and regional patterns. In our study, M.pneumoniae was detected in 59.8% of cases using polymerase chain reaction (PCR), which is notably higher than the 7.3% positivity reported by Chacko et al., from Amala Institute, Thrissur, Kerala, who employed IgM ELISA for detection in children with lower respiratory tract infections. The discrepancy in detection rates can likely be attributed to the superior sensitivity and specificity of PCR, which allows for direct identification of M. pneumoniae DNA, even in the early phase of infection or before antibody production begins. In contrast, serological methods such as IgM ELISA may yield lower sensitivity due to delayed antibody responses, particularly in younger children.3 A study conducted in Delhi by Kumar et al., reported M. pneumoniae positivity in 26.1% of children under 5 years and 59.5% in those aged 5 years and above, using serological methods.2 These findings were concordant with our study. This age-dependent variability reflects the influence of immune maturity on serological test performance. Compared to these findings, the consistently high positivity rate observed in our study across age groups using PCR further emphasizes the diagnostic advantage of molecular techniques. These findings highlight the utility of PCR as a reliable tool for early and accurate detection of M. pneumoniae in children with community-acquired lower respiratory tract infections.4 Internationally, our findings align closely with those of the study from Belgium by Song et al., which underscored the significance of pathogen detection during epidemic seasons in hospitalized children with community-acquired pneumonia (CAP). The study also highlighted the age-specific disease burden, noting that school-aged children were disproportionately affected—a pattern similarly observed in our population analysis.5 The superior diagnostic performance of PCR observed in our study is supported by existing literature comparing various diagnostic modalities for Mycoplasma pneumoniae. According to a study published in the Brazilian Journal of Infectious Diseases (BJID, 2007), PCR offers a sensitivity ranging from 78–100% and specificity of 92–100%, with results available within hours. These parameters are significantly higher compared to serological methods such as ELISA (sensitivity 83–100%, specificity 79–100%) and complement fixation assays (sensitivity 71–90%, specificity 88–92%), which often require 1–2 weeks for results and are dependent on the host immune response. Culture, though highly specific (100%), has a sensitivity of only 61% and requires 2–6 weeks for results, making it impractical for acute clinical decision-making. The rapid turnaround time and high accuracy of PCR not only facilitates timely diagnosis and targeted treatment but also supports its integration as a frontline diagnostic tool in resource-equipped laboratories. These findings reaffirm the utility of PCR in enhancing the detection rate of M. pneumoniae, as demonstrated in our study.6 Our study adds to the growing body of evidence on the epidemiology of M. pneumoniae infections in the post-COVID era. Notably, a recent study from Wuhan was the first to characterize both the epidemic trends and genomic features of M. pneumoniae following the COVID-19 outbreak in 2020. Their findings underscore the importance of continued surveillance and genomic monitoring, especially as respiratory pathogens re-emerge or shift in prevalence patterns due to changes in public health dynamics post-pandemic. In this context, our study provides additional data on the detection rates and age distribution of M. pneumoniae infections, which may reflect evolving transmission dynamics and immunity landscapes. Together, these insights highlight the need for sustained vigilance and the integration of molecular diagnostics in routine clinical and epidemiological surveillance of atypical respiratory pathogens.1 From Kerala, a study by Cherian et al., reported M. pneumoniae in 22.4% of lower respiratory tract infection cases among children which was lower compared to our study. Their study also noted that there was clustering of positive cases during the pre-monsoon and monsoon seasons.7 A study by Sreenath et al., conducted in Dehi, stressed the emergence of macrolide resistance and advocated for molecular testing over empirical therapy. Although we did not conduct resistance profiling, this is a pertinent consideration for future work, especially in the context of rising antibiotic resistance globally.8 Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgement The authors would like to thank the staff of the Department of Molecular Biology, Microbiological Laboratory, Thiruvananthapuram and Coimbatore for their technical assistance and support during the course of the study.

The findings demonstrate a considerable burden of M. pneumoniae in South Kerala, underscoring the value of PCR in early diagnosis. Integration of molecular testing into routine diagnostics is recommended, along with continued surveillance and resistance profiling to inform antibiotic stewardship.

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