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.

New-generation tetracyclines for severe macrolide-resistant Mycoplasma pneumoniae pneumonia in children: a retrospective analysis.

BACKGROUND: Macrolide-resistant Mycoplasma pneumoniae (MRMP) strains are increasingly prevalent, leading to a rise in severe Mycoplasma pneumoniae pneumonia incidence annually, which poses a significant threat to children's health. This study aimed to compare the effectiveness and safety of oral minocycline and doxycycline for the treatment of severe MRMP pneumonia in children. METHODS: This retrospective analysis included children treated for severe MRMP pneumonia at the Pediatric Department of Tongji Hospital, Shanghai, China, between September 2023 and January 2024 using minocycline and doxycycline. The patients were divided into four groups according to treatment: oral doxycycline alone (DOX group), oral minocycline alone (MIN group), oral doxycycline with intravenous glucocorticoids (DOXG group), and oral minocycline with intravenous glucocorticoids (MING group). Student's t-test, Mann-Whitney U test, and χ2 or Fisher's exact tests were used for group comparisons. RESULTS: A total of 165 patients were included in this study: 84 received minocycline, and 81 received doxycycline. The DOX group had higher fever resolution rates within 24, 48, and 72 h compared to the MIN group (63.2% vs. 31.8%, 79.0% vs. 63.6%, and 100% vs. 90.9%, respectively; all p < 0.05). The DOXG group showed higher fever resolution rates within 24 and 48 h than the MING group (92.3% vs. 83.4%, 100% vs. 92.7%, all p > 0.05). There were no statistically significant differences in time to imaging improvement, cough improvement, and disappearance of wet rales between groups, regardless of glucocorticoid combination. The longer the duration of fever prior to tetracycline therapy, the greater the likelihood of hypoxemia (p = 0.039) and a greater than two-fold elevation in the D-dimer level (p = 0.004).Univariate binary logistic regression model analysis revealed that CRP and erythrocyte sedimentation rate at disease onset were associated with defervescence within 24 h after treatment with tetracyclines alone (p = 0.020, p = 0.027), with erythrocyte sedimentation rate also influencing defervescence within 48 h (p = 0.022). CONCLUSION: Doxycycline treatment resulted in a higher rate of defervescence than minocycline. Prompt treatment reduced the probability of pleural effusion, hypoxemia, pulmonary atelectasis, and D-dimer levels > 2 times the reference value.

Predicting the severity of mycoplasma pneumoniae pneumonia in pediatric and adult patients: a multicenter study.

The purpose of this study is to develop a nomogram model for early prediction of the severe mycoplasma pneumoniae pneumonia (SMPP) in Pediatric and Adult Patients. A retrospective analysis was conducted on patients with MPP, classifying them into SMPP and non-severe MPP (NSMPP) groups. A total of 550 patients (NSMPP 374 and SMPP 176) were enrolled in the study and allocated to training, validation cohorts. 278 patients (NSMPP 224 and SMPP 54) were retrospectively collected from two institutions and allocated to testing cohort. The risk factors for SMPP were identified using univariate analysis. For radiomic feature selection, Spearman's correlation and the least absolute shrinkage and selection operator (LASSO) were utilized. Logistic regression was used to build different models, including clinical, imaging, radiomics, and integrated models (combining clinical, imaging, and radiomics features selected). The model's discrimination was evaluated using a receiver operating characteristic curve, its calibration with a calibration curve, and the results were visualized using the Hosmer-Lemeshow goodness-of-fit test. Thirteen clinical features and fourteen imaging features were selected for constructing the clinical and imaging models. Simultaneously, a set of twenty-five radiomics features were utilized to build the radiomics model. The integrated model demonstrated good calibration and discrimination in the training cohorts (AUC, 0.922; 95% CI: 0.900, 0.942), validation cohorts (AUC, 0.879; 95% CI: 0.806, 0.920), and testing cohorts (AUC, 0.877; 95% CI: 0.836, 0.916). The discriminatory and predictive efficacy of the clinical model in testing cohorts increased further after clinical and radiological features were incorporated (AUC, 0.849 vs. 0.922, P = 0.002). The model demonstrated exemplary predictive efficacy for SMPP by leveraging a comprehensive set of inputs, encompassing clinical data, quantitative and qualitative radiological features, along with radiomics features. The integration of these three aspects in the predictive model further enhanced the performance of the clinical model, indicating the potential for extensive clinical applications.

A randomized controlled non-inferiority trial of placebo versus macrolide antibiotics for Mycoplasma pneumoniae infection in children with community-acquired pneumonia: trial protocol for the MYTHIC Study.

BACKGROUND: Mycoplasma pneumoniae is a major cause of community-acquired pneumonia (CAP) in school-aged children. Macrolides are the first-line treatment for this infection. However, it is unclear whether macrolides are effective in treating M. pneumoniae CAP, mainly due to limitations in microbiological diagnosis of previous studies. The extensive global use of macrolides has led to increasing antimicrobial resistance. The overall objective of this trial is to produce efficacy data for macrolide treatment in children with M. pneumoniae CAP. METHODS: The MYTHIC Study is a randomized, double-blind, placebo-controlled, multicenter, non-inferiority trial in 13 Swiss pediatric centers. Previously healthy ambulatory and hospitalized children aged 3-17 years with clinically diagnosed CAP will be screened with a sensitive and commercially available M. pneumoniae-specific IgM lateral flow assay from capillary blood. Mycoplasma pneumoniae infection in screened patients will be verified retrospectively by respiratory PCR (reference test) and IgM antibody-secreting cell enzyme-linked immunospot (ELISpot) assay (confirmatory test for distinguishing between carriage and infection). Patients will be randomized 1:1 to receive a 5-day treatment of macrolides (azithromycin) or placebo. The co-primary endpoints are (1) time to normalization of all vital signs, including body temperature, respiratory rate, heart rate, and saturation of peripheral oxygen (efficacy), and (2) CAP-related change in patient care status (i.e., admission, re-admission, or intensive care unit transfer) within 28 days (safety). Secondary outcomes include adverse events (AEs), as well as antimicrobial and anti-inflammatory effects. For both co-primary endpoints, we aim to show non-inferiority of placebo compared to macrolide treatment. We expect no macrolide effect (hazard ratio of 1, absolute risk difference of 0) and set the corresponding non-inferiority margins to 0.7 and -7.5%. The "at least one" success criterion is used to handle multiplicity with the two co-primary endpoints. With a power of 80% to reject at least one null hypothesis at a one-sided significance level of 1.25%, 376 patients will be required. DISCUSSION: This trial will produce efficacy data for macrolide treatment in children with M. pneumoniae CAP that might help to reduce the prescription of antibiotics and therefore contribute to the global efforts toward reducing antimicrobial resistance. TRIAL REGISTRATION: ClinicalTrials.gov, NCT06325293. Registered on 24 April 2024.

The protective effect and immunomodulatory ability of orally administrated Lacticaseibacillus rhamnosus GG against Mycoplasma pneumoniae infection in BALB/c mice.

Mycoplasma pneumoniae represents one of the significant etiologies of community-acquired pneumonia in pediatric patients. However, clinical treatment of M. pneumoniae infection in children has encountered challenges due to the escalating resistance to quinolones. Numerous studies have highlighted the potential of probiotic lactobacillus administration in boosting immune responses to bacterial and viral respiratory infections. In this study, the protective efficacy of pre-oral administration of Lacticaseibacillus rhamnosus GG (LGG), Limosilactobacillus reuteri F275, Lactiplantibacillus plantarum NCIMB 8826, L. plantarum S1 or L. plantarum S2 was evaluated in the BALB/c mice model; it was observed that among these five strains of lactobacillus, the supplementation of LGG exhibited the most significant protective effect against M. pneumoniae infection. Moreover, when administered orally, both live LGG and heat-inactivated LGG have demonstrated efficacy in reducing the burden of M. pneumoniae in the lungs and alleviating pulmonary inflammation. Oral supplementation with LGG resulted in the inhibition of neutrophil recruitment into the lungs and increased recruitment of alveolar macrophages in M. pneumoniae-infected mice. Additionally, LGG supplementation led to increased production of IL-10 and secretory IgA (sIgA), while suppressing the levels of IL-1ß, IL-6, IL-17A, and TNF-α in the lungs of mice infected with M. pneumoniae. The data suggests that supplementation with LGG can modulate immune responses, decrease pathogen load, and alleviate inflammatory injury in the lungs of M. pneumoniae-infected mice.

Mycoplasma pneumoniae: not a typical respiratory pathogen.

Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia among school-aged children and young adults. Infections occur throughout the year but tend to surge during winter months across Europe. A characteristic epidemic cycle, where a substantial surge in the number of infections occurs, is seen approximately every 1-4 years and hypothesized to be driven by changes in immunity and a shift in circulating variants. Once thought to be an organism of low virulence, it has now been found to possess several virulence factors, including toxin production, biofilm formation and evasion of antibody-mediated immunity. The lack of a cell wall and reduced metabolic pathways limit the options for antibiotic treatment. Acquired macrolide resistance is a growing concern, with >80% of cases in China being macrolide-resistant. Although efforts have been made to develop a vaccine, there are still substantial hurdles to overcome in relation to vaccine-enhanced disease, which results from an inappropriate immune response among vaccinated individuals.

Establishment and evaluation of MIRA-qPCR assay for the rapid and sensitively detection of Mycoplasma pneumoniae.

Aim: Mycoplasma pneumoniae (MP) is a common cause of respiratory infections, and its incidence has increased post-COVID-19 due to "immune debt." Real-time quantitative polymerase chain reaction (qPCR) is the standard for detecting MP, but it has a lengthy detection time. This study aimed to establish a highly sensitive rapid detection method for MP.Materials & methods: We developed an integrated assay combining multienzyme isothermal rapid amplification (MIRA) with qPCR, referred to as MIRA-qPCR, for the rapid detection of MP, delivering results within approximately 40 min.Results: The analytic sensitivity of the MIRA-qPCR assay was 10 copies per reaction, and it exhibited no cross-reactivity with other respiratory pathogens, ensuring high specificity. Clinical sample analysis demonstrated higher sensitivity for MIRA-qPCR compared to qPCR reported in the literature, and 100% concordance with commercial qPCR kit.Conclusion: The MIRA-qPCR method established in this study is a promising tool for the clinical detection of MP, offering significant advantages for the rapid diagnosis of MP infections.

Mycoplasma pneumoniae is a bacteria that can make us sick. It mainly affects the lungs and can cause a sickness called "walking pneumonia". This is because it can make you poorly, but not so badly that you are unable to walk around. This bacteria spreads when someone that is infected sneezes or coughs. It is important that M. pneumoniae can be diagnosed quickly. This article looks at a new, fast way to identify infection called MIRA-quantitative PCR.

The epidemic characteristics of Mycoplasma pneumoniae infection among children in Anhui, China, 2015-2023.

The number of pediatric respiratory tract infection cases in China has significantly increased this year, and Mycoplasma pneumoniae is one of the main pathogens. This study aimed to investigate the epidemiological characteristics of M. pneumoniae in children in the Anhui region and to provide evidence for the prevention and control strategies of M. pneumoniae in children in this region. A total of 66,488 pediatric patients with respiratory tract infection were enrolled from January 2015 to November 2023 in this study. The results of this study exhibited that M. pneumoniae infection in the Anhui region was characterized by a high positive rate during 2021-2023, especially this year is considered a year of pandemic for M. pneumoniae infection. Moreover, the positive rate of M. pneumoniae in female children is significantly higher than in male children, and the infection rate of M. pneumoniae in children increases significantly with age, particularly in school-aged children. IMPORTANCE: The number of pediatric respiratory tract infection cases in China has significantly increased this year, and Mycoplasma pneumoniae is one of the main pathogens. This study aimed to investigate the epidemiological characteristics of M. pneumoniae in children in the Anhui region and provide evidence for the prevention and control strategies of M. pneumoniae in children in this region.

Portable and simultaneous detection of four respiratory pathogens through a microfluidic LAMP and real-time fluorescence assay.

Respiratory pathogen infections are seasonally prevalent and are likely to cause co-infections or serial infections during peak periods of infection. Since they often cause similar symptoms, simultaneous and on-site detection of respiratory pathogens is essential for accurate diagnosis and efficient treatment of these infectious diseases. However, molecular diagnostic techniques for multiple pathogens in this field are lacking. Herein, we developed a microfluidic LAMP and real-time fluorescence assay for rapid detection of multiple respiratory pathogens using a ten-channel microfluidic chip with pathogen primers pre-embedded in the chip reaction well. The microfluidic chip provided a closed reaction environment, effectively preventing aerosol contamination and improving the accuracy of the detection results. Its corresponding detection instrument could automatically collect and display the fluorescence curve in real time, which was more conducive to the interpretation of results. The results showed that the developed method could specifically recognize the nucleic acid of influenza A(H1N1), Mycoplasma pneumoniae, respiratory syncytial virus type A, and SARS-CoV-2 with low detection limits of 104 copies per mL or 103 copies per mL. The test results on clinical samples demonstrated that the developed method has high sensitivity (92.00%) and high specificity (100.00%) and even has the capability to differentiate mixed-infection samples. With simple operation and high detection efficiency, the present portable and simultaneous detection assay could significantly improve the efficiency of on-site detection of respiratory infectious diseases and promote the accurate treatment, efficient prevention and control of the diseases.

Clinical profiles of Mycoplasma pneumoniae pneumonia in children with different pleural effusion patterns: a retrospective study.

BACKGROUND: The clinical significance of the presence or absence of Mycoplasma pneumoniae (MP) in pleural effusion in Mycoplasma pneumoniae pneumonia (MPP) children has not yet been elucidated. Herein, we investigated the clinical implication of pleural fluid MP positive in children with MPP. METHODS: A total of 165 MPP children with pleural effusion requiring thoracocentesis were enrolled in this study. They were subsequently divided into two groups according to the presence or absence of MP in pleural effusion, namely positive group (n = 38) and negative group (n = 127). Information on their clinical manifestations, laboratory findings, radiological characteristics and treatment modalities was retrospectively collected from medical chart reviews. RESULTS: The length of hospitalization (15.00 (10.75-19.25) vs. 11.00 (9.00-14.00) days, p=0.001) and total course of illness (23.00 (18.00-28.00) vs. 20.00 (17.00-24.00) days, p=0.010) were significantly longer in the positive group than in the negative group. The occurrence of pericardial effusion (23.7% vs. 7.9%, p=0.017), atelectasis (73.7% vs. 53.5%, p=0.027) and necrotizing pneumonia (23.7% vs. 7.9%, p=0.017) were more frequent in the positive group compared to the negative group. The levels of neutrophil percentages (82.35% (75.40%-85.78%) vs. 72.70% (64.30%-79.90%), p<0.001), C-reactive protein (CRP) (71.12 (37.75-139.41) vs. 31.15 (13.54-65.00) mg/L, p<0.001), procalcitonin (PCT) (0.65 (0.30-3.05) vs. 0.33 (0.17-1.13) ng/ml, p=0.005), serum lactate dehydrogenase (LDH) (799.00 (589.00-1081.50) vs. 673.00 (503.00-869.00) U/L, p=0.009), D-dimer (6.21 (3.37-16.11) vs. 3.32 (2.12-6.62) mg/L, p=0.001) on admission were significantly higher in the positive group than in the negative group. These pronounced differences significantly contributed to the identification of MPP with MP positive pleural effusion, as evidenced by the ROC curve analysis. Marked elevations in adenosine deaminase (49.25 (36.20-60.18) vs. 36.20 (28.10-46.50) U/L, p<0.001) and LDH levels (2298.50 (1259.75-3287.00) vs. 1199.00 (707.00-1761.00) U/L, p<0.001) were observed in pleural fluid of the positive group when compared to the negative group. Meanwhile, the number of patients on low molecular weight heparin (LMWH) therapy (9 (23.7%) vs. 12 (9.4%), p=0.028) was higher in the positive group. Multivariate logistic regression analysis revealed that D-dimer > 7.33 mg/L was significantly associated with the incidence of MP positive pleural effusion in MPP (OR=3.517). CONCLUSIONS: The presence of MP in pleural fluid in MPP children with pleural effusion indicated a more serious clinical course. D-dimer > 7.33 mg/L was a related factor for MP positive pleural effusion in MPP. The results of the present study would help in the creation of a therapeutic plan and prediction of the clinical course of MPP in children.

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.

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.

A Case of Erythrocyte Condensation Caused by Mycoplasma Pneumoniae Infection.

BACKGROUND: Blood routine testing was the most commonly used laboratory method in clinical practice. The results are often influenced by factors such as instruments, reagents, and samples, among which, the interference of cold agglutinin is a very rare element. In our article, we reported a case of red blood cell agglutination caused by Mycoplasma pneumoniae infection. METHODS: The number of blood cells were detected by blood routine analyzer with or without treatment at 37℃ water bath. The red blood cell agglutination was observed through blood smear staining. The cold agglutination test were performed using O-type red blood cells added into patient's plasma and refrigerated overnight at 4℃. We also used luminescent immunoassay technology to detect the content of MP antibodies in patient's serum. RESULTS: The patient's results were RBC (2.69 x 1012/L), MCH (48.5 pg), MCHC (522 g/L). Through a microscope, we observed red blood cell agglutination. The concentration of MP-igM was 60.37 AU/mL. The cold agglutination test was positive. Following a 37℃ water bath, the patient's results changed: RBC (3.85 x 1012/L), MCH (31.2 pg), MCHC (352 g/L). The phenomenon of massive agglutination of red blood cells has also disappeared. CONCLUSIONS: The cold agglutinin produced by MP infection can alter the results of red blood cell. During the epidemic period of MP infection, it is important to pay attention to the phenomenon of abnormal elevation of MCHC in clinical practice.

Reemergence of Mycoplasma pneumoniae disease: Pathogenesis and new approaches.

The review discusses the recurrence of Mycoplasma pneumoniae (M. pneumoniae), a bacterium causing atypical pneumonia, primarily affecting Europe and Asia due to climate change, immunity decline, antibiotic resistance, and genetic heterogeneity. The COVID-19 pandemic initially reduced M. pneumoniae cases due to preventative measures, but its reemergence suggests different transmission dynamics and exacerbates clinical severity with co-infections with other viruses. The pathogenicity of M. pneumoniae is attributed to its intracellular changes, toxin release, and adhesion processes, which can result in a variety of symptoms and problems. Antibiotics and immunomodulators are used in treatment, and attempts are being made to create vaccines. Effective management of its reappearance necessitates surveillance and preventative measures, especially in the context of co-infections and potential outbreaks. M. pneumoniae's resurgence highlights its reliance on a polarized cytoskeletal architecture for host cell attachment and pathogenicity through cytoadherence and cytotoxic agent synthesis. M. pneumoniae has returned even though the COVID-19 pandemic originally reduced incidence; this might be because of things like declining immunity and particular pathogenic characteristics. Meteorological factors like temperature and humidity, along with air quality, including pollutants like PM2.5 and NO2, increase susceptibility to environmental hazards. During the pandemic, non-pharmaceutical measures decreased transmission but did not eradicate the infection. Epidemics typically occur three to five years apart, emphasizing the need for ongoing study and observation. Antimicrobial resistance is a serious issue, necessitating caution and alternative therapies, especially in macrolides. COVID-19 pandemic lessons, such as mask use and hand hygiene, may help limit M. pneumoniae transmission.

Critical insights from recent outbreaks of Mycoplasma pneumoniae: decoding the challenges and effective interventions strategies.

OBJECTIVES: Mycoplasma pneumoniae (M. pneumoniae) continues to pose a significant disease burden on global public health as a respiratory pathogen. The antimicrobial resistance among M. pneumoniae strains has complicated the outbreak control efforts, emphasizing the need for robust surveillance systems and effective antimicrobial stewardship programs. DESIGN: This review comprehensively investigates studies stemming from previous outbreaks to emphasize the multifaceted nature of M. pneumoniae infections, encompassing epidemiological dynamics, diagnostic innovations, antibiotic resistance, and therapeutic challenges. RESULTS: We explored the spectrum of clinical manifestations associated with M. pneumoniae infections, emphasizing the continuum of disease severity and the challenges in gradating it accurately. Artificial intelligence and machine learning have emerged as promising tools in M. pneumoniae diagnostics, offering enhanced accuracy and efficiency in identifying infections. However, their integration into clinical practice presents hurdles that need to be addressed. Further, we elucidate the pivotal role of pharmacological interventions in controlling and treating M. pneumoniae infections as the efficacy of existing therapies is jeopardized by evolving resistance mechanisms. CONCLUSION: Lessons learned from previous outbreaks underscore the importance of adaptive treatment strategies and proactive management approaches. Addressing these complexities demands a holistic approach integrating advanced technologies, genomic surveillance, and adaptive clinical strategies to effectively combat this pathogen.

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.

Comparative genomic analysis identifies potential adaptive variation in Mycoplasma ovipneumoniae.

Mycoplasma ovipneumoniae is associated with respiratory disease in wild and domestic Caprinae globally, with wide variation in disease outcomes within and between host species. To gain insight into phylogenetic structure and mechanisms of pathogenicity for this bacterial species, we compared M. ovipneumoniae genomes for 99 samples from 6 countries (Australia, Bosnia and Herzegovina, Brazil, China, France and USA) and 4 host species (domestic sheep, domestic goats, bighorn sheep and caribou). Core genome sequences of M. ovipneumoniae assemblies from domestic sheep and goats fell into two well-supported phylogenetic clades that are divergent enough to be considered different bacterial species, consistent with each of these two clades having an evolutionary origin in separate host species. Genome assemblies from bighorn sheep and caribou also fell within these two clades, indicating multiple spillover events, most commonly from domestic sheep. Pangenome analysis indicated a high percentage (91.4 %) of accessory genes (i.e. genes found only in a subset of assemblies) compared to core genes (i.e. genes found in all assemblies), potentially indicating a propensity for this pathogen to adapt to within-host conditions. In addition, many genes related to carbon metabolism, which is a virulence factor for Mycoplasmas, showed evidence for homologous recombination, a potential signature of adaptation. The presence or absence of annotated genes was very similar between sheep and goat clades, with only two annotated genes significantly clade-associated. However, three M. ovipneumoniae genome assemblies from asymptomatic caribou in Alaska formed a highly divergent subclade within the sheep clade that lacked 23 annotated genes compared to other assemblies, and many of these genes had functions related to carbon metabolism. Overall, our results suggest that adaptation of M. ovipneumoniae has involved evolution of carbon metabolism pathways and virulence mechanisms related to those pathways. The genes involved in these pathways, along with other genes identified as potentially involved in virulence in this study, are potential targets for future investigation into a possible genomic basis for the high variation observed in disease outcomes within and between wild and domestic host species.

Exploring the pathogenicity of Mycoplasma pneumoniae: Focus on community-acquired respiratory distress syndrome toxins.

Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX) is a unique exotoxin produced by Mycoplasma pneumoniae (MP) and has been confirmed to possess ADP-ribosyltransferase (ART) and vacuolating activities. CARDS TX binds to receptors on the surfaces of mammalian cells followed by entry into the cells through clathrin-mediated endocytosis, and exerts cytotoxic effects by undergoing retrograde transport and finally cleavage on endosomes and cellular organelles. In addition, CARDS TX can trigger severe inflammatory reactions resulting in airway dysfunction, producing allergic inflammation and asthma-like conditions. As a newly discovered virulence factor of MP, CARDS TX has been extensively studied in recent years. As resistance to macrolide drugs has increased significantly in recent years and there is no vaccine against MP, the development of a vaccine targeting CARDS TX is considered a potential preventive measure. This review focuses on recent studies and insights into this toxin, providing directions for a better understanding of MP pathogenesis and treatment. IMPORTANCE: A serious hazard to worldwide public health in recent years, Mycoplasma pneumoniae (MP) is a prominent bacterium that causes community-acquired pneumonia (CAP) in hospitalized children. Due to their high prevalence and fatality rates, MP infections often cause both respiratory illnesses and extensive extrapulmonary symptoms. It has recently been shown that MP produces a distinct exotoxin known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Mycoplasma pneumoniae pneumonia (MPP)-like tissue injury is caused by this toxin because it has both ADP-ribosyltransferase and vacuolating properties. A better knowledge of MP etiology and therapy is provided by this review, which focuses on latest research and insights into this toxin.

Effects of inflammatory stimuli on the development of Mycoplasma bovis pneumonia in experimentally challenged calves.

Many cattle infected with Mycoplasma bovis remain healthy while others develop severe chronic respiratory disease. We hypothesized that inflammatory stimuli such as co-pathogens worsen disease outcomes in M. bovis-infected calves. Calves (n=24) were intrabronchially inoculated with M. bovis and either killed bacterial lysate, transient M. haemolytica infection, or saline. Caseonecrotic lesions developed in 7/7 animals given M. haemolytica and M. bovis compared to 2/8 given M. bovis with no inflammatory stimulus, and 6/9 animals given bacterial lysate and M. bovis (P=0.01). Animals receiving M. haemolytica and M. bovis had more caseonecrotic foci in lungs than those receiving M. bovis with no inflammatory stimulus (median = 21 vs 0; P = 0.01), with an intermediate response (median = 5) in animals given bacterial lysate. In addition to caseonecrotic foci, infected animals developed neutrophilic bronchiolitis that appeared to develop into caseonecrotic foci, peribronchiolar lymphocytic cuffs that were not associated with the other lesions, and 4 animals with bronchiolitis obliterans. The data showed that transient lung inflammation at the time of M. bovis infection provoked the development of caseonecrotic bronchopneumonia, and the severity of inflammation influenced the number of caseonecrotic foci that developed. In contrast, caseonecrotic lesions were few or absent in M. bovis-infected calves without a concurrent inflammatory stimulus. These studies provide insight into how caseonecrotic lesions develop within the lung of M. bovis-infected calves. This and other studies suggest that controlling co-pathogens and harmful inflammatory responses in animals infected with M. bovis could potentially minimize development of M. bovis caseonecrotic bronchopneumonia.