Mycoplasma pneumoniae (MP) is one of the most prevalent pathogens responsible for respiratory diseases. It is estimated to account for 10%–30% of cases of community-acquired pneumonia (CAP) (Atkinson et al., 2008). In addition to causing acute respiratory infections, MP can also induce chronic inflammatory responses in the respiratory tract, thereby contributing to the pathogenesis of chronic respiratory disorders such as asthma (Liu et al., 2021). Moreover, MP infections have been observed to result in damage to extrapulmonary organs, with encephalitis and myocarditis among such manifestations (D’Alonzo et al., 2018; Wandro et al., 2018; Poddighe, 2020; Aizawa et al., 2021; Katsumi et al., 2021; Wang et al., 2021). Although M. pneumoniae infection is typically self-limiting, severe M. pneumoniae pneumonia has been increasingly reported in recent years (Cillóniz et al., 2016; Waites et al., 2017; Ha et al., 2023; Lai et al., 2024). Despite extensive research, the precise pathogenic mechanism of MP remains unclear (Jiang et al., 2021). Due to the absence of a cell wall, MP is inherently resistant to β-lactam antibiotics (Waites et al., 2017; Lee et al., 2018). Furthermore, MP is responsible for regional outbreaks of epidemics with a periodicity of 2–6 years and presents with atypical signs and symptoms (Sauteur et al., 2024). Consequently, an early and accurate diagnosis is of great importance for the antibiotic treatment and prognosis of MP infection. In-vitro MP culture is a challenging and time-consuming process, serological methods require confirmation approximately one to two weeks after infection, and polymerase chain reaction (PCR) methods necessitate the utilization of expensive thermal cycling apparatus and well-established laboratories, which are challenging to implement in primary hospitals in regions with limited healthcare resources and infrastructure (Loens and Ieven, 2016; Waites et al., 2017). It is therefore imperative that a flexible and rapid method for field-deployable diagnostic tests for MP infections be developed.

The community-acquired respiratory distress syndrome toxin (CARDS TX), initially identified and named by Kannan et al, 2005, is a distinctive human surfactant protein A binding protein of MP. It plays a pivotal role in the pathogenesis of acute and chronic diseases associated with MP infection (Kannan et al., 2005; Maselli et al., 2018). In addition to promoting adhesion between MP and host target cells, CARDS TX also enters and diffuses into host cells by binding to cell surface receptors, thereby triggering the release of inflammatory factors that produce cytopathic effects. These effects ultimately result in a series of pathological changes (Su et al., 2021). Monitoring CARDS TX is of significant clinical importance for diagnosing MP infections and reflects the condition of MP pneumonia.

Loop-mediated isothermal amplification (LAMP) technology is a relatively new isothermal amplification technique for specific nucleic acid fragments initially described by Japanese researcher Notomi in 2000 (Mori and Notomi, 2020). The procedure is rapid and efficient, and the reaction can be accomplished in approximately 30–60 min. The complete amplification process necessitates only a thermostat and a timer. LAMP technology has gained considerable attention in recent years due to its advantageous characteristics, including rapidity, sensitivity, simplicity, good specificity, and low cost. The method has been successfully applied in the detection of pathogenic microorganisms, the identification of single-nucleotide polymorphisms, the surveillance of infectious disease, and other fields of scientific investigation (Cai et al., 2019). While there are existing methods for the detection of MP based on the LAMP method, there is currently no simple and rapid method targeting CARDS TX for clinical use. The development of a rapid test for the CARDS TX of MP based on the LAMP method has the potential to both clarify the diagnosis of MP infection and assist in the determination of the condition and prognosis.

This study aims to develop a rapid detection method for the CARDS TX based on the LAMP method, thus enabling more expedient diagnosis and assessment of MP infection.

This study describes the establishment of a LAMP method for the rapid, straightforward, sensitive, and specific detection of CARDS TX in MP. In comparison to the qPCR method, the detection performance of this method is not statistically significantly different, and the two methods demonstrated a high degree of concordance of 95%.

MP infection is a leading cause of a range of respiratory illnesses, including bronchitis, pneumonia, and asthma, as well as extrapulmonary conditions such as myocarditis and encephalitis (Fan et al., 2023; Zhu et al., 2024). It can cause regional epidemics or even pandemics, in which patients may exhibit no recognizable typical features or present with cases of severe disease (Sauteur et al., 2024; Cillóniz et al., 2016; Waites et al., 2017; Ha et al., 2023; Lai et al., 2024). Macrolides are a crucial class of antibiotics for the treatment of MP infections. However, in Asia, particularly in China and Japan, the macrolide resistance rate of MP exceeds 80% (Tanaka et al., 2017; Zhao et al., 2019; Wang et al., 2022a, Wang et al., 2022b). In these regions, fluoroquinolones and tetracycline antibiotics are more frequently recommended for the treatment of MP infections in adults. Furthermore, MP exhibits intrinsic resistance to β-lactams (Waites et al., 2017; Lee et al., 2018). The empirical use of β-lactams or macrolides in the absence of an identified etiology may result in ineffective treatment and a worsening of the patient’s condition. Timely diagnosis and precise treatment are therefore of paramount importance in the management of MP infection (Wang et al., 2023). Three principal methods are employed for the detection of MP: culture, serological, and PCR-based molecular biology. However, the culture and serological methods are time consuming, typically requiring at least 1 to 2 weeks, while the PCR method necessitates the availability of high-quality laboratory facilities (Loens and Ieven, 2016; Waites et al., 2017). Consequently, there is currently no straightforward and rapid method for bedside diagnosis of MP infection (Cai et al., 2019).

The LAMP technique has been employed for the detection of a range of pathogens due to its greater simplicity and lower cost in comparison to PCR. The technique is now recommended as the first-line diagnostic method for detecting acute MP infections in Japan (Saito et al., 2005; Kakuya et al., 2014). A LAMP assay targeting the P1 gene of MP has been established for the detection of MP in pharyngeal swabs (Wang et al., 2019), and there are also other clinical and rapid MP molecular assays from throat swabs-based targeting 16S rRNA or CARDS TX gene that are commercially available and FDA cleared (Waites et al., 2017). However, there are certain limitations to their use in a clinical setting. For instance, the performance of these methods has only been established in human pharyngeal swab specimens, or the conditions required for their use are more complex and challenging to implement in rapid, point-of-care testing. In some cases, it is challenging to determine whether a specimen from the upper respiratory tract is indicative of infection, colonization, or contamination (Peng et al., 2020; Gunasekaran et al., 2019). Bronchoalveolar lavage fluid (BALF) is a reliable specimen for the identification of pathogens associated with lower respiratory tract infections (Wang et al., 2020). In addition, it is noteworthy that Ieven et al. found that assays targeting the P1 gene were more sensitive than those targeting 16S rRNA (Ieven et al., 1996). Importantly, Peters et al. reported that the CARDS TX PCR was capable of detecting 10 times more patients with MP than a PCR utilizing the P1 protein gene as a target gene (Peters et al., 2011).

It has been demonstrated that CARDS TX plays a significant role in the pathogenesis of MP infection (Maselli et al., 2018; Li et al., 2019; Ramasamy et al., 2021). In the acute phase of MP infection, CARDS TX can aggravate the damage to the respiratory system by directly destroying respiratory epithelial cells or inducing an inflammatory response; in the chronic phase, the persistence of CARDS TX in the body can result in chronic airway inflammation, which may lead to the induction of asthma, acute exacerbations of chronic obstructive pulmonary disease (AECOPD), and other associated complications (Peters et al., 2011; Medina et al., 2012). Accordingly, the risk of developing asthma subsequent to MP infection can be evaluated by monitoring respiratory CARDS TX (Xu et al., 2024). In addition, there was a positive correlation between CARDS TX expression levels and clinical severity. A study demonstrated that the severity of MP infection was positively correlated with patients’ serum CARDS TX levels (Ma et al., 2018). Li et al. reported that refractory MP pneumonia had significantly higher levels of CARDS TX in alveolar lavage fluid than patients with nonrefractory MP (Li et al., 2019). It has also been found that CARDS TX persists in the body for more than 600 days and is associated with poor control of prolonged asthma (Peters et al., 2011). All these studies suggest that the detection of CARDS TX gene or protein is of considerable value in the diagnosis and evaluation of MP infection, especially in the early stage of infection, and not only this, but the concentration of CARDS TX may also indicate the clinical changes of MP pneumonia.

It is of significant clinical importance that the detection of CARDS TX in MP through the LAMP method serves not only for the diagnosis of MP infection but also for the estimation of the severity and prognosis of MP infection. The LAMP method utilized in this study to detect MP CARDS TX takes only one incubator, which is easy to operate and has the superiority of saving time (within 60 min). It is optimally suited for expeditious point-of-care testing and settings where medical resources are relatively scarce. Moreover, compared with the gold standard PCR method, there was no statistical difference in the detection efficiency. The LAMP reaction employed in this study utilized six oligonucleotide primers situated in disparate regions of the CARDS TX gene to amplify the target DNA. It is therefore highly specific. Furthermore, in-silico analysis of amino acid sequences indicated that CARDS TX shares high sequence similarity with the pertussis toxin S1 subunit from Bordetella pertussis. Compared to Xiao et al (Xiao et al., 2022), our method has the advantage of being tested for cross-reactivity against three additional toxins, including pertussis toxin, which validates the specificity of the method and makes the results more reliable. In a previous study, the PCR method targeting the MP CARDS TX gene demonstrated superior sensitivity compared to the PCR method targeting the P1 gene (Peters et al., 2011). This finding was further corroborated in the present study, in which the LAMP method was employed for the detection of CARDS TX, exhibiting a minimum detectable concentration of 0.4986 pg/μl. This value was markedly lower than that observed in the LAMP reaction targeting the P1 gene, as previously described by Wang et al (Wang et al., 2019). The aforementioned evidence substantiates the assertion that the methodology based on the LAMP reaction targeting MP CARDS TX, as delineated in this study, exhibits high sensitivity and specificity for the detection of MP infection. A further advantage of our work over that of Xiao et al (Xiao et al., 2022). is that the CARDS TX primers were designed for use with both LAMP and qPCR methods. The qPCR method targeting CARDS TX was used as the gold standard. This allows for a more direct comparison of the detection efficacy of the LAMP and PCR methods. The results of the two methods exhibited a high degree of consistency, with the qPCR method demonstrating a lower positive detection rate than the LAMP method. Furthermore, our established LAMP method has been successfully employed for the detection of BALF specimens, thereby further illustrating its broad clinical applicability. Therefore, this method is an effective detection method that has the potential to significantly impact clinical practice, particularly in settings with limited medical funding resources, enabling the monitoring of potential MP outbreaks and guiding diagnosis and treatment decisions.

This study is flawed in the following ways. First, the species of bacteria designed for specific detection was limited. Second, the number of clinical samples employed for comparison between the LAMP and qPCR methods was relatively small. Nevertheless, based on the available epidemiologic data, the majority of common bacteria and toxins susceptible to comorbid MP infections have been incorporated into the specific test, thereby rendering the use of this LAMP assay a feasible option for the diagnosis of respiratory tract infections in a broad range of clinical settings. Furthermore, the current method does not permit the quantification of CARDS TX in the specimens under examination. The monitoring of CARDS TX levels is a clinically significant objective, which can be achieved through the utilization of methodologies such as CRISPR/Cas12b-assisted LAMP and the quantitative LAMP colorimetric phenol red method. This is the direction we are going to delve into next.

Based on the LAMP technology and the CARDS TX gene, we have successfully established a method for rapid and accurate detection of MP. This method is characterized by its high specificity and sensitivity, as well as its simplicity and the ability to produce results that are discernible to the naked eye within less than an hour. The detection of CARDS TX in MP also facilitates the assessment of the severity and prognosis of MP infection. This assay enables early and precise diagnosis in various medical settings, especially for point-of-care testing and in resource-limited environments, which is crucial for proper antibiotic treatment and the prognosis of MP infection. The LAMP assay targeting MP CARDS TX is a promising tool for the early surveillance and diagnosis of MP infection.