Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

Mycoplasma pneumonia in a patient with X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA), also referred to as Bruton's tyrosine kinase deficiency, is a rare genetic disorder that affects the immune system. We conducted genetic analysis on patients suffering from immunodeficiency by utilizing Next-Generation Sequencing techniques, as well as their closest relatives, to facilitate accurate diagnosis, offer genetic counseling services, and enhance our comprehension of XLA.

Epidemiological characteristics of mycoplasma pneumoniae in hospitalized children before, during, and after COVID-19 pandemic restrictions in Chongqing, China.

Background: Mycoplasma pneumoniae (MP) is a significant cause of community-acquired pneumonia with high macrolide resistance rates. Various COVID-19 pandemic restrictions have impacted the prevalence of MP. Objective: To assess the changes in the pattern of MP infections among children before, during, and after the COVID-19 pandemic. Methods: A total of 36685 enrolled patients, aged 0-18 years, diagnosed with pneumonia and admitted to Children's Hospital of Chongqing Medical University from January 2019 to December 2023, were retrospectively reviewed in this study. The epidemiological characteristics of pediatric MP infection were analyzed. Results: Among 36685 patients, 7610 (20.74%) tested positive for MP. The highest positive rate was observed among children aged over 6 years (55.06%). There was no gender disparity in MP infection across the three phases of the COVID-19 pandemic. Hospital stays were longest for children during the COVID-19 pandemic (P <0.001). MP infection was most prevalent in the summer (29.64%). The lowest positive rate was observed during the pandemic, with the highest rate found after easing the measures across all age groups (P <0.001). There was a surge in the positive rate of MP in the third year after the COVID-19 pandemic. Regression analyses demonstrated a shift in the age range susceptible to MP infection, with children aged 3.8 to 13.5 years post-pandemic compared to the pre-pandemic range of 5.3 to 15.5 years old. Additionally, the average macrolide resistance rate was 79.84%. We observed a higher resistance rate during the pandemic than in the pre- and post-pandemic phases (P <0.001). Conclusion: The restrictive measures implemented during the COVID-19 pandemic have influenced the spread of MP to some extent and altered demographic and clinical characteristics, such as age, age group, season, length of stay, and macrolide resistance. We recommend continuous surveillance of the evolving epidemiological characteristics of MP infection in the post-pandemic period when restrictions are no longer necessary.

Routine blood parameters as auxiliary diagnostic tools for Mycoplasma pneumoniae infection in children.

Introduction. Recently, the incidence of Mycoplasma pneumoniae (M. pneumoniae) infection in children has been increasing annually. Early differential diagnosis of M. pneumoniae infection can not only avoid the abuse of antibiotics, but also is essential for early treatment and reduction of transmission.Gap statement. The change of routine blood parameters may have important clinical significance for the diagnosis of M. pneumoniae infection, but it has not been reported so far.Aim. This study aims to establish a predictive model for M. pneumoniae infection and explore the changes and clinical value of routine blood parameters in children with M. pneumoniae infection, serving as auxiliary indicators for the diagnosis and differentiation of clinical M. pneumoniae infection.Methodology. A total of 770 paediatric patients with respiratory tract infections were enrolled in this study, including 360 in the M. pneumoniae group, 40 in the SARS-CoV-2 group, 200 in the influenza A virus group, and 170 in the control group. The differences of routine blood parameters among all groups were compared, and risk factors were analysed using multivariate logistics analysis, and the diagnostic efficacy of differential indicators using ROC curves.Results. This study revealed that Mono% (OR: 3.411; 95% CI: 1.638-7.102; P=0.001) was independent risk factor associated with M. pneumoniae infection, and Mono% (AUC=0.786, the optimal cutoff at 7.8%) had a good discriminative ability between patients with M. pneumoniae infection and healthy individuals. Additionally, Mono% (OR: 0.424; 95% CI: 0.231-0.781; P=0.006) and Lymp% (OR: 0.430; 95% CI: 0.246-0.753; P=0.003) were independent risk factors for distinguishing M. pneumoniae infection from influenza A virus infection, and the Lymp% (AUC=0.786, the optimal cutoff at 22.1%) and Net% (AUC=0.761, the optimal cutoff at 65.2%) had good discriminative abilities between M. pneumoniae infection and influenza A infection. Furthermore, platelet distribution width (OR: 0.680; 95% CI: 0.538-0.858; P=0.001) was independent risk factor for distinguishing M. pneumoniae infection from SARS-CoV-2 infection. Meanwhile, the ROC curve demonstrated that PDW (AUC=0.786, the optimal cutoff at 15%) has a good ability to differentiate between M. pneumoniae infection and SARS-CoV-2 infection.Conclusion. This study demonstrates that routine blood parameters can be used as auxiliary diagnostic indicators for M. pneumoniae infection and provide reference for the diagnosis and differentiation of clinical M. pneumoniae infection.

The correlation between the level of cellular classification in bronchoalveolar lavage fluid of children with severe Mycoplasma pneumoniae and clinical characteristics.

Mycoplasma pneumoniae pneumonia (MPP) is a common respiratory tract infection disease in children. To date, there have been few studies on the relationship between cytological changes in bronchoalveolar lavage fluid (BALF) and clinical features. The objective of this study is to investigate the correlation between changes in the proportion of cell classifications in BALF and the clinical features in children with severe MPP (SMPP). In total, the study included 64 children with SMPP requiring bronchoalveolar lavage who were admitted to our hospital between March and September 2022 (study group) and 11 children with bronchial foreign bodies without co-infection (control group), who were admitted during the same period. The proportion of cell classifications in BALF was determined by microscopic examination after performing Wright-Giemsa staining. Patients were grouped according to different clinical characteristics, and between-group comparisons were made regarding the variations in the proportion of cell classifications in BALF. The levels of blood routine neutrophil percentage (GRA%), C-reactive protein, D-dimer and lactate dehydrogenase in the study group were higher than those in the control group (P < 0.05). There were differences in the GRA% and macrophage percentage in the BALF between the two groups (P < 0.05). The GRA% and blood lymphocyte percentage were associated with pleural effusion. Multiple indicators correlated with extrapulmonary manifestations (P < 0.05). Moreover, the percentage of lymphocytes in the BALF correlated with pleural effusion, extrapulmonary manifestations and refractory MPP (RMPP) (P < 0.05). Logistic regression showed that BALF lymphocytes were protective factors for RMPP, while serum amyloid A and extrapulmonary manifestations were risk factors (P < 0.05). CONCLUSION: The BALF of children with SMPP is predominantly neutrophilic. A lower percentage of lymphocytes is related to a higher incidence of pleural effusion, extrapulmonary manifestations and progression to RMPP, as well as a longer length of hospitalisation. WHAT IS KNOWN: • Mycoplasma pneumonia in children is relatively common in clinical practice. Bronchoalveolar lavage (BAL) is a routine clinical procedure. WHAT IS NEW: However, there are relatively few studies focusing on the cytomorphological analysis of cells in BAL fluid.

Mycoplasma pneumoniae infection outbreak in Guangzhou, China after COVID-19 pandemic.

BACKGROUNDS: Mycoplasma pneumoniae (M. pneumoniae) is a common pathogen causing respiratory diseases in children. This study aimed to characterize epidemiological and disease severity shifts of M. pneumoniae: infections in Guangzhou, China during and after the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Throat swab samples were obtained from 5405 hospitalized patients with symptoms of acute respiratory infections to detect M. pneumoniae. Differences in epidemiological and clinical characteristics of M. pneumoniae: infections were investigated during 2020-2022 and after COVID-19 pandemic (2023). RESULTS: M. pneumoniae were detected in 849 (15.6%, 849/5405) patients. The highest annual positive rate was 29.4% (754/2570) in 2023, followed by 5.3% (72/1367) in 2022, 1.2% (12/1015) in 2021, and 2.0% (11/553) in 2020, with significantly increasing annual prevalence from 2020 to 2023. M. pneumoniae incidence peaked between July and December post-COVID-19 pandemic in 2023, with the highest monthly positive rate (56.4%, 165/293). Clinical characteristics and outcomes of patients with M. pneumoniae did not vary between periods during and after COVID-19 pandemic except that patients with M. pneumoniae post-COVID-19 pandemic were more likely to develop fever. Patients with severe M. pneumoniae pneumonia (SMPP) were more likely to develop respiratory complications, myocardial damage, and gastrointestinal dysfunction than those with non-SMPP. Patients with SMPP had lower lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and higher IL-4, IL-6, IL-10 levels than those with non-SMPP. Bronchoalveolar lavage fluid specimens from infected patients were obtained to identify macrolide resistance mutations. Macrolide-resistant M. pneumoniae (MRMP) proportion in 2023 was 91.1% (215/236). CONCLUSION: Outbreaks of M. pneumoniae: occurred in Guangzhou, China in 2023 upon Non-pharmaceutical interventions easing. Despite the increasing incidence of M. pneumoniae, the disease severity remained similar during and after the COVID-19 pandemic.

An accurate and convenient method for Mycoplasma pneumoniae via one-step LAMP-CRISPR/Cas12b detection platform.

Introduction: Mycoplasma pneumoniae (MP) is the major cause of respiratory infections that threaten the health of children and adolescents worldwide. Therefore, an early, simple, and accurate detection approach for MP is critical to prevent outbreaks of MP-induced community-acquired pneumonia. Methods: Here, we explored a simple and accurate method for MP identification that combines loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b assay in a one-pot reaction. Results: In the current study, the whole reaction was completed within 1 h at a constant temperature of 57°C. The limit of detection of this assay was 33.7 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100%, without any cross-reactivity with other pathogens. Overall, 272 clinical samples were used to evaluate the clinical performance of LAMP-CRISPR/Cas12b. Compared with the gold standard results from real-time PCR, the present method provided a sensitivity of 88.11% (126/143), specificity of 100% (129/129), and consistency of 93.75% (255/272). Discussion: Taken together, our preliminary results illustrate that the LAMP-CRISPR/Cas12b method is a simple and reliable tool for MP diagnosis that can be performed in resource-limited regions.

Real-time fluorescent multiple cross displacement amplification for rapid and sensitive Mycoplasma pneumoniae detection.

Mycoplasma pneumoniae is a significant pathogen responsible for community-acquired pneumonia, predominantly affecting children and adolescents. Here, we devised a rapid method for M. pneumoniae that combined multiple cross displacement amplification (MCDA) with real-time fluorescence technology. A set of ten primers, which were specifically designed for M. pneumoniae detection, were employed in a real-time fluorescence MCDA reaction. Of these, one primer incorporated a restriction endonuclease recognition sequence, a fluorophore, and a quencher, facilitating real-time fluorescence detection. The real-time (RT)-MCDA reactions were monitored in a simple real-time fluorescence instrument and conducted under optimised conditions (64°C for 40 min). The detection limit of the M. pneumoniae RT-MCDA assay for genomic DNA extracted from M. pneumoniae culture was down to 43 fg/µl. This assay accurately identified M. pneumoniae strains without cross-reacting with other bacteria. To validate its practical application, we tested the M. pneumoniae RT-MCDA assay using genomic DNA extracted from clinical samples. The assay's detection capability proved comparable with real-time PCR, MCDA-based biosensor detection, and visual inspection under blue light. The entire process, including rapid DNA extraction and real-time MCDA detection, was completed within 1 h. Overall, the M. pneumoniae RT-MCDA assay reported here is a simple and effective diagnostic tool for rapid M. pneumoniae detection, which holds significant potential for point-of-care testing and in resource-limited regions.

Persistent elevation in incidence of pneumonia in children in England, 2023/24.

Since November 2023, the absolute number of attendances at emergency departments for pneumonia among children aged 5-14 years in England have been above expected levels for the time of year. This increased signal peaked during March 2024 but then persisted into early summer 2024 despite decreases in prevalence of seasonal respiratory pathogens. Record linkage between emergency department and laboratory databases points to this unusual activity being driven largely by Mycoplasma pneumoniae.

The Epidemiology of Pathogens in Community-Acquired Pneumonia Among Children in Southwest China Before, During and After COVID-19 Non-pharmaceutical Interventions: A Cross-Sectional Study.

OBJECTIVE: This study aimed to investigate the pathogen epidemiology of community-acquired pneumonia (CAP) among children in Southwest China before, during and after the COVID-19 non-pharmaceutical interventions (NPIs). METHODS: Pathogen data of hospitalised children with CAP, including multiple direct immunofluorescence test for seven viruses, bacterial culture and polymerase chain reaction (PCR) for Mycoplasma pneumoniae, were analysed across three phases: Phase I (pre-NPIs: 1 January 2019 to 31 December 2019), Phase II (NPI period: 1 January 2020 to 31 December 2020) and Phase III (post-NPIs: 1 January 2023 to 31 December 2023). RESULTS: A total of 7533 cases were enrolled, including 2444, 1642 and 3447 individuals in Phases I, II and III, respectively. M. pneumoniae predominated in Phases I and III (23.4% and 35.5%, respectively). In Phase II, respiratory syncytial virus (RSV) emerged as the primary pathogen (20.3%), whereas detection rates of influenza A virus (Flu A) and M. pneumoniae were at a low level (1.8% and 9.6%, respectively). In Phase III, both Flu A (15.8%) and M. pneumoniae epidemic rebounded, whereas RSV detection rate returned to Phase I level, and detection rates of Streptococcus pneumoniae and Haemophilus influenzae decreased significantly compared to those in Phase I. Detection rates of adenovirus and parainfluenza virus type 3 decreased phase by phase. Age-stratified analysis and monthly variations supported the above findings. Seasonal patterns of multiple pathogens were disrupted during Phases II and III. CONCLUSIONS: COVID-19 NPIs exhibited a distinct impact on CAP pathogen epidemic among children, with post-NPIs increases observed in M. pneumoniae and Flu A prevalence. Continuous pathogen monitoring is crucial for effective prevention and control of paediatric CAP.

Predictive signature of murine and human host response to typical and atypical pneumonia.

BACKGROUND: Pneumonia due to typical bacterial, atypical bacterial and viral pathogens can be difficult to clinically differentiate. Host response-based diagnostics are emerging as a complementary diagnostic strategy to pathogen detection. METHODS: We used murine models of typical bacterial, atypical bacterial and viral pneumonia to develop diagnostic signatures and understand the host's response to these types of infections. Mice were intranasally inoculated with Streptococcus pneumoniae, Mycoplasma pneumoniae, influenza or saline as a control. Peripheral blood gene expression analysis was performed at multiple time points. Differentially expressed genes were used to perform gene set enrichment analysis and generate diagnostic signatures. These murine-derived signatures were externally validated in silico using human gene expression data. The response to S. pneumoniae was the most rapid and robust. RESULTS: Mice infected with M. pneumoniae had a delayed response more similar to influenza-infected animals. Diagnostic signatures for the three types of infection had 0.94-1.00 area under the receiver operator curve (auROC). Validation in five human gene expression datasets revealed auROC of 0.82-0.96. DISCUSSION: This study identified discrete host responses to typical bacterial, atypical bacterial and viral aetiologies of pneumonia in mice. These signatures validated well in humans, highlighting the conserved nature of the host response to these pathogen classes.

Laboratory diagnosis and treatment of Mycoplasma pneumoniae infection in children: a review.

Mycoplasma pneumoniae (MP) is the cause of Mycoplasma pneumoniae pneumonia (MPP) in children and adolescents, with the clinical manifestations highlighted by intermittent irritating cough, accompanied by headache, fever and muscle pain. This paper aimed to study the research status and focal points in MP infection, especially the common laboratory diagnostic methods and clinical treatment of Mycoplasma pneumoniae. Laboratory diagnostic methods include molecular assay, serological antibody detection, rapid antigen detection and isolation and culture. Polymerase chain reaction (PCR) is the gold standard with high sensitivity and specificity. The serological antibody can detect various immune antibodies qualitatively or quantitatively in serum. Rapid antigen can be detected faster, with no equipment environment requirements, which can be used for the early diagnosis of MP infection. While the culture growth cycle is long and insensitive, not recommended for routine diagnosis. Macrolides were the preferred drug for children with MPP, while the drug resistance rate was rising in China. Tetracycline can be substituted but was not recommended for children under 8 years of age, quinolone drugs are not necessary, severe MPP can be combined with glucocorticoids, involving the nervous or immune system can choose gamma globulin. Other treatments for MPP including symptomatic treatment which can alleviate symptoms, improve lung function and improve prognosis. A safe and effective vaccine needed to be developed which can provide protective immunity to children and will reduce the incidence of MPP.

[Analysis of epidemiological characteristics of respiratory pathogens in children with influenza-like illnesses in a children's hospital in Beijing from 2022 to 2023].

To investigate the status and epidemiological characteristics of respiratory pathogens infections in children with influenza-like illnesses (ILI) in Beijing Children's Hospital from 2022 to 2023. A dual amplification technique was used to detect nucleic acids of seven common respiratory pathogens, including influenza A virus (Flu A), influenza B virus (Flu B), mycoplasma pneumoniae (MP), respiratory syncytial virus (RSV), parainfluenza virus (PIV), adenovirus (ADV), and Chlamydia pneumoniae (CP), in outpatient and inpatient children (aged 0-18 years) with influenza-like symptoms who sought medical care at Beijing Children's Hospital, from January 2022 to March 2023. A total of 43 663 children were included in the study, of which 27 903 tested positive for respiratory pathogens with a total detection rate of 63.91%. Flu A had the highest detection rate of 69.93% (27 332/39 084), followed by MP about 13.22% (380/2 875). The total detection rate of RSV, PIV and ADV was 7.69% (131/1 704). Flu B had a detection rate of 0.16% (64/39 084). No CP was detected in this study. A total of 7 cases of dual infections were detected, with a detection rate of 0.41% (7/1 704). The Chi-square test was used to analyze the differences in detection rates of pathogens among different genders, age groups, and different seasons. Among the seven pathogens, only Flu A had statistically significant differences in gender (χ2=16.712, P<0.001). The detection rates of Flu A and MP showed an increasing trend with age (both P trend<0.001), while the detection rates of RSV and PIV showed a decreasing trend with age (both P trend<0.001). Flu A had its epidemic peak in winter and spring, with detection rates of 61.30% (3 907/6 374) and 77.47% (23 207/29 958) respectively; MP and PIV had higher detection rates in autumn (25.14% and 7.64% respectively); RSV showed a relatively higher detection rate in winter (8.69%); Flu B and ADV had lower detection rates throughout the study period (0.16% and 1.17% respectively). In conclusion, children with ILI in 2022-2023 were mainly infected with a single respiratory pathogen, and occasionally dual pathogen infections were observed. Among them, the detection rate of Flu A was the highest, and only Flu A showed a gender difference in detection rate. As the age of the children patients increased, the detection rate of Flu A and MP showed an increasing trend, while RSV and PIV showed a decreasing trend. The prevalence of Flu A, Flu B, MP, PIV, and RSV were seasonal.

Application of peripheral blood routine parameters in the diagnosis of influenza and Mycoplasma pneumoniae.

OBJECTIVES: Influenza and Mycoplasma pneumoniae infections often present concurrent and overlapping symptoms in clinical manifestations, making it crucial to accurately differentiate between the two in clinical practice. Therefore, this study aims to explore the potential of using peripheral blood routine parameters to effectively distinguish between influenza and Mycoplasma pneumoniae infections. METHODS: This study selected 209 influenza patients (IV group) and 214 Mycoplasma pneumoniae patients (MP group) from September 2023 to January 2024 at Nansha Division, the First Affiliated Hospital of Sun Yat-sen University. We conducted a routine blood-related index test on all research subjects to develop a diagnostic model. For normally distributed parameters, we used the T-test, and for non-normally distributed parameters, we used the Wilcoxon test. RESULTS: Based on an area under the curve (AUC) threshold of ≥ 0.7, we selected indices such as Lym# (lymphocyte count), Eos# (eosinophil percentage), Mon% (monocyte percentage), PLT (platelet count), HFC# (high fluorescent cell count), and PLR (platelet to lymphocyte ratio) to construct the model. Based on these indicators, we constructed a diagnostic algorithm named IV@MP using the random forest method. CONCLUSIONS: The diagnostic algorithm demonstrated excellent diagnostic performance and was validated in a new population, with an AUC of 0.845. In addition, we developed a web tool to facilitate the diagnosis of influenza and Mycoplasma pneumoniae infections. The results of this study provide an effective tool for clinical practice, enabling physicians to accurately diagnose and differentiate between influenza and Mycoplasma pneumoniae infection, thereby offering patients more precise treatment plans.

Current Mycoplasma pneumoniae outbreak: Don't forget its role in Bickerstaff encephalitis.

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in children and young adults. It is responsible of a broad array of extrapulmonary manifestations, the most severe affecting the central nervous system. We report a challenging diagnosis of macrolide-resistant M. pneumoniae-induced Bickerstaff encephalitis in a 16-year-old man.

The bacterial and fungal profiles of patients hospitalized with non-COVID-19 lower respiratory tract infections in Wuhan, China, 2019-2021.

AIMS: A severe lockdown occurred in Wuhan during the COVID-19 pandemic, followed by a remission phase in the pandemic's aftermath. This study analyzed the bacterial and fungal profiles of respiratory pathogens in patients hospitalized with non-COVID-19 lower respiratory tract infections (LRTIs) during this period to determine the pathogen profile distributions in different age groups and hospital departments in Wuhan. METHODS AND RESULTS: We collected reports of pathogen testing in the medical records of patients hospitalized with non-COVID-19 LRTI between 2019 and 2021. These cases were tested for bacterial and fungal pathogens using 16S and internal transcribed spacer sequencing methods on bronchoalveolar lavage fluid samples. The study included 1368 cases. The bacteria most commonly identified were Streptococcus pneumoniae (12.50%) and Mycoplasma pneumoniae (8.33%). The most commonly identified fungi were Aspergillus fumigatus (2.49%) and Pneumocystis jirovecii (1.75%). Compared to 2019, the S. pneumoniae detection rates increased significantly in 2021, and those of M. pneumoniae decreased. Streptococcus pneumoniae was detected mainly in children. The detection rates of almost all fungi were greater in the respiratory Intensive Care Unit compared to respiratory medicine. Streptococcus pneumoniae and M. pneumoniae were detected more frequently in the pediatric department. CONCLUSIONS: Before and after the COVID-19 outbreak, a change in the common pathogen spectrum was detected in patients with non-COVID-19 in Wuhan, with the greatest change occurring among children. The major pathogens varied by the patient's age and the hospital department.

A preliminary prediction model of pediatric Mycoplasma pneumoniae pneumonia based on routine blood parameters by using machine learning method.

BACKGROUND: The prevalence and severity of pediatric Mycoplasma pneumoniae pneumonia (MPP) poses a significant threat to the health and lives of children. In this study, we aim to systematically evaluate the value of routine blood parameters in predicting MPP and develop a robust and generalizable ensemble artificial intelligence (AI) model to assist in identifying patients with MPP. METHODS: We collected 27 features, including routine blood parameters and hs-CRP levels, from patients admitted to The Affiliated Dazu's Hospital of Chongqing Medical University with or without MPP between January, 2023 and January, 2024. A classification model was built using seven machine learning (ML) algorithms to develop an integrated prediction tool for diagnosing MPP. It was evaluated on both an internal validation set (982 individuals) and an external validation set (195 individuals). The primary outcome measured the accuracy of the model in predicting MPP. RESULTS: The GBDT is state-of-the-art based on 27 features. Following inter-laboratory cohort testing, the GBDT demonstrated an AUC, accuracy, specificity, sensitivity, PPV, NPV, and F1-score of 0.980 (0.938-0.995), 0.928 (0.796-0.970), 0.929 (0.717-1.000), 0.926 (0.889-0.956), 0.922 (0.727-1.000), 0.937 (0.884-0.963), and 0.923 (0.800-0.966) in stratified 10-fold cross-validation. A GBDT-based AI Lab was developed to facilitate the healthcare providers in remote and impoverished areas. CONCLUSIONS: The GBDT-based AI Lab tool, with high sensitivity and specificity, could help discriminate between pediatric MPP infection and non-MPP infection based on routine blood parameters. Moreover, a user-friendly webpage tool for AI Lab could facilitate healthcare providers in remote and impoverished areas where advanced technologies are not accessible.

Position statement of the Spanish Society of Paediatric Infectious diseases on the diagnosis and treatment of Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae (M. pneumoniae) is a bacterium with particular characteristics that give rise to a broad clinical spectrum, being respiratory infection the most frequent presentation. Infection by M. pneumoniae occurs in cyclical epidemics, and paediatricians in Spain have noticed an increase in cases since January 2024, establishing hospital registers to collect surveillance data (as it is not a notifiable disease in Spain). The diagnosis of infection by M. pneumoniae is made through serological testing and/or the detection of genetic material by means of polymerase chain reaction (PCR). Neither methods can differentiate between colonization and active infection, so a precise diagnosis is not possible and testing should only be requested in the case of high clinical suspicion. The role of antibiotherapy in infection by M. pneumoniae in its different clinical variants is not well defined. Most infections are self-limiting and mild, and there is insufficient evidence to support the use of antibiotherapy in these cases. Antibiotic treatment is justified in patients with risk factors for the development of severe disease (Down syndrome, anatomical or functional asplenia, immunosuppression), in hospitalized patients with respiratory infection and in patients with moderate or severe extrapulmonary forms. Taking into account aspects concerning the rational use of antimicrobials, the treatment of choice would be clarithromycin, with azithromycin as an alternative, reserving the use of doxycycline and levofloxacin for cases of antimicrobial resistance and/or infections of the central nervous system.

Graphene oxide/Cu-MOF-based electrochemical immunosensor for the simultaneous detection of Mycoplasma pneumoniae and Legionella pneumophila antigens in water.

The combination of copper-metal organic framework (Cu-MOF) with graphene oxide (GO) has received growing interest in electrocatalysis, energy storage and sensing applications. However, its potential as an electrochemical biosensing platform remains largely unexplored. In this study, we introduce the synthesis of GO/Cu-MOF nanocomposite and its application in the simultaneous detection of two biomarkers associated with lower respiratory infections, marking the first instance of its use in this capacity. The physicochemical properties and structural elucidation of this composite were studied with the support of XRD, FTIR, SEM and electrochemical techniques. The immunosensor was fabricated by drop casting the nanocomposite on dual screen-printed electrodes followed by functionalization with pyrene linker. The covalent immobilization of the monoclonal antibodies of the bacterial antigens of Mycoplasma pneumoniae (M. pneumoniae; M. p.) and Legionella pneumophila (L. pneumophila; L. p.) was achieved using EDC-NHS chemistry. The differential pulse voltammetry (DPV) signals of the developed immunosensor platform demonstrated a robust correlation across a broad concentration range from 1 pg/mL to 100 ng/mL. The immunosensor platform has shown high degree of selectivity against antigens for various respiratory pathogens. Moreover, the dual immunosensor was successfully applied for the detection of M. pneumoniae and L. pneumophila antigens in spiked water samples showing excellent recovery percentages. We attribute the high sensitivity of the immunosensor to the enhanced electrocatalytic characteristics, stability and conductivity of the GO-MOF composite as well as the synergistic interactions between the GO and MOF. This immunosensor offers a swift analytical response, simplicity in fabrication and instrumentation, rendering it an appealing platform for the on-field monitoring of pathogens in environmental samples.