Aspergillus oryzae Fermentation Extract Alleviates Inflammation in Mycoplasma pneumoniae Pneumonia.

The filamentous fungus Aspergillus oryzae, also known as koji mold, has been used for centuries in the production of fermented foods in East Asia. A. oryzae fermentation can produce enzymes and metabolites with various bioactivities. In this study, we investigated whether A. oryzae fermentation extract (AOFE) has any effect on Mycoplasma pneumoniae (Mp) pneumonia. We performed solid-state fermentation of A. oryzae and obtained the ethanol extract. AOFE was analyzed by HPLC, and the major component was identified to be kojic acid. In vitro, AOFE suppressed Mp growth and invasion into A549 lung epithelial cells as determined by the gentamicin protection assay. AOFE treatment also suppressed Mp-stimulated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 at mRNA and protein levels in murine MH-S alveolar macrophages. In a mouse model of Mp pneumonia, Mp infection induced a marked pulmonary infiltration of neutrophils, which was significantly reduced in mice pre-treated orally with AOFE. AOFE administration also suppressed the production of proinflammatory cytokines and chemokines in the lungs. Collectively, our results show that AOFE has the potential to be developed into a preventive/therapeutic agent for Mp pneumonia.

Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification.

Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0 × 10-17 M (approximately 6.0 × 103 copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0 × 104 copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the "gold standard" real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens. Graphical abstract.

Assessment of serum sialic acid correlated with C3 in children with Mycoplasma pneumoniae pneumonia.

BACKGROUND: Different from the diagnosis of bacterial infections, Mycoplasma pneumoniae pneumonia (MPP) is still lacking of convenient non-specific laboratory parameters. METHOD: A total of 125 children with MPP were included in the MPP group and 89 children with Mycoplasma-negative pneumonia were included in the control group, and the sera were collected from the children at both the acute and recovery stages in the two groups. RESULTS: The sialic acid and C3 in the MPP group were significantly higher than those in the control group both at the acute and at the recovery stage. On the other hand, the sialic acid and C3 at the acute stage were significantly higher than those at the recovery stage in the MPP group. However, in the control group, the sialic acid and C3 demonstrated IgG exhibited no significant change between the acute stage and the recovery stage. Lastly, positive correlations between sialic acid level and C3 level were identified in the MPP group at both acute and recovery stages. CONCLUSION: Our study demonstrated that the serum sialic acid correlated with C3 specifically increased in children with MPP, indicating that it might be the important non-specific parameters in the diagnosis of MPP.

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia.

An air-insulated microfluidic chip was designed for the automatic centrifugal distribution of samples to 24-test cells, enabling the parallel identification of multiple clinical pneumonia-related pathogens in 1.45-µL reactions without cross-contamination in 45 min. A portable nucleic acid analyzer that integrates mechanical, confocal optical, electronic, and software functions was also developed to collect fluorescence data in a Ø3 mm imaging field near the optical diffraction limit for highly sensitive fluorescence detection of nucleic acid amplification in real time. This microfluidic chip-based portable nucleic acid analyzer could detect low abundance nucleic acids present at as few as 10 copies. In a blinded experiment, specific identification of Mycoplasma pneumoniae, Staphylococcus aureus, and methicillin-resistant S. aureus was achieved with 229 clinical patient sputum samples. The total coincidence rate of our system and traditional RT-PCR with an ABI 7500 was 99.56%. Four samples accounting for the 0.44% inconformity were retested by gene sequencing, revealing that our system reported the correct results. This novel microfluidic chip-based detection system is cost-effective, rapid, sensitive, specific, and has a relatively high throughput for parallel identification, which is especially suitable for resource-limited facilities/areas and point-of-care testing.

Antibiotic prescriptions and cycles of Mycoplasma pneumoniae infections in Norway: can a nationwide prescription register be used for surveillance?

Mycoplasma pneumoniae outbreaks cause increased use of macrolides and tetracyclines. We aimed to investigate whether drug use data, in addition to laboratory data, could improve understanding of the spread of M. pneumoniae epidemics. Number of users of Mycoplasma antibiotics (erythromycin, doxycycline, clarithromycin) per week and county of residence in an indicator age group (6-12 years) was retrieved from the Norwegian prescription database for the epidemic season 2011-2012 and compared to non-epidemic seasons. In 2011, increased use of Mycoplasma antibiotics was first observed in September on the west coast of Norway. The Norwegian laboratory-based surveillance system showed the first increase in positive tests in August 2011 and an epidemic was announced on 25 October 2011. At that time the use of Mycoplasma antibiotics had already exceeded three times the use in non-epidemic periods. Data for three counties from the regional microbiological laboratories showed that the increase in number of positive samples coincided in time with the increase in prescription data. Laboratory data cannot accurately determine the extent of an epidemic, and drug use data cannot identify the cause. Establishing a systematic interaction between the two monitoring systems will enhance surveillance and probably contribute to improved infection control and prudent antibiotic prescribing.

[Value of routine identification of respiratory infectious agents in children hospitalized with an acute asthma exacerbation]. / Intérêt de l'identification en routine des agents pathogènes respiratoires chez les enfants hospitalisés pour une exacerbation d'asthme.

BACKGROUND: Asthma is the most common chronic disease in childhood. With its high economic burden, it is considered a disease of major public health importance by the World Health Organization. The link between respiratory tract infections and acute exacerbation has been recognized for a long time. The aim of this retrospective study in routine care was to evaluate our practices concerning microbiological prescriptions in children hospitalized for asthma exacerbation. STUDY DESIGN: All children aged from 2 to 15 years hospitalized for asthma exacerbation between January 2010 and December 2011 in our unit were included in the study. Microbiological prescriptions, their indications, their results, and their cost were studied. RESULTS: One hundred ninety-seven children were included in the study. A potential causative agent was sought in 79.7% of the children (n=157) by immunofluorescence assay (IFA) and/or polymerase chain reaction (PCR). The main indications were upper airway infections, hypoxemia, and pneumonia. Viruses were detected in 23.8% of them (30/126). Mycoplasma pneumoniae was detected by PCR in only 3.2% of these patients (4/125). No other bacterial agent was identified. There was no correlation between the severity of asthma exacerbation and the microbiological diagnosis of infection. The results did not influence the therapy given. These prescriptions represented a substantial cost for each child. CONCLUSION: These analyses do not seem to have a real advantage for the patient except for epidemiology. It would be important to conduct a new study analyzing the role of rhinovirus, and of other viruses such as coronavirus, bocavirus, and enterovirus, not routinely investigated in our hospital, and to question the value of these costly microbiological tests.

Assessment of the loop-mediated isothermal amplification assay for rapid diagnosis of Mycoplasma pneumoniae in pediatric community-acquired pneumonia.

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for timely treatment with effective antibiotics; however, PCR-based methods are often too expensive and technologically intensive for general use in clinical practice. In this study, the efficacy of the loop-mediated isothermal amplification (LAMP) assay for diagnosis of M. pneumoniae pneumonia in clinical practice was prospectively evaluated. From July 2011 to March 2012, 531 children hospitalized for community-acquired pneumonia were enrolled. In all patients, throat swabs were obtained on admission for the detection of M. pneumoniae DNA, and paired serum samples were obtained to assay M. pneumoniae particle agglutination (PA) antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or an increase of 4-fold or greater in the PA titer. Overall, 271 children (51.0% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia. Among these, 258 (95.2%) and 248 (91.5%) were identified by the LAMP assay and serological tests, respectively. When the results of serological tests were considered as standard, the sensitivity, specificity, and positive and negative predictive values of the LAMP assay were 94.8%, 91.9%, and 91.1% and 95.2%, respectively. The median duration of pharyngeal carriage, as measured by the LAMP assay, was 9.5 days. Thus, the LAMP assay is useful in the rapid diagnosis of M. pneumoniae pneumonia.

Clinical features and the role of atypical pathogens in nursing and healthcare-associated pneumonia (NHCAP): differences between a teaching university hospital and a community hospital.

OBJECTIVE: The Japan Respiratory Society documented a new category of guidelines for nursing and healthcare-associated pneumonia (NHCAP), which is distinct from community acquired pneumonia (CAP). The objective of this study was to determine the epidemiological differences between NHCAP patients in a teaching university hospital and a community hospital. In addition, to clarify the strategy for treatment of NHCAP, we investigated the role of atypical pathogens. METHODS: We analyzed 250 NHCAP and 421 CAP cases in a university hospital and 349 NHCAP and 374 CAP cases in a community hospital. RESULTS: Patient age and the incidences of poor general condition were significantly higher in the community hospital compared with those in the university hospital. The distribution and frequency of pathogens, especially multidrug-resistant (MDR) pathogens, were significantly different between the two hospitals. Central nervous system disorders, dementia and poor performance status, which was possibility related to aspiration pneumonia, were significantly more frequent in patients with NHCAP compared with those with CAP in both hospitals. Atypical pathogens were detected in a few cases in patients with NHCAP. CONCLUSION: There were many differences in the clinical characteristics between NHCAP patients in a university hospital and a community hospital even for hospitals located in the same area. Aspiration pneumonia was thought to be the main characteristic of NHCAP in both hospitals. Thus, all NHCAP patients did not need the same empiric therapy with a multidrug regimen directed against MDR pathogens. In addition, physicians rarely need to consider atypical pathogens in NHCAP treatment.

[Bacteriological assessment of infectious diseases using a real-time PCR method in Tenri Hospital--focusing on diagnosis for atypical pneumonia and hemorrhagic enterocolitis].

In recent years, the detection of specific pathogens has been remarkably speedy with the development of new nucleic acid amplification methods such as real-time PCR, which is expected to be a great contribution to clinical diagnosis. Real-time PCR was introduced in our hospital, 2004 with the aim of detecting various pathogens. The system has been established and used for rapid bacteriological diagnosis for outpatient services in our hospital. This system can contribute greatly to achieving reliable and quick diagnosis, particularly for atypical pneumonia and hemorrhagic colitis caused by Escherichia coli. This system requires only 45 minutes on average for atypical pneumonia diagnosis, from receiving a specimen to the reporting of its results, which shortens the diagnosis time to about one-tenth of conventional methods. Survey on the type of initial dose in cases of Mycoplasma pneumoniae PCR positives shows that any administration of beta-lactam antibiotics, not effective to M. pneumoniae, has not been reported. Concerning the diagnosis of hemorrhagic colitis caused by Escherichia coli, it has been possible for us to report the results within one and a half hours, or within two hours including the legal notification of a third class infectious disease to a public health center. The SYBR Green Idetection system used in our hospital is superior to other detection systems comparing with versatility and cost-effectiveness. This report advocates that real-time PCR can contribute greatly to a rapid and cost-effective diagnostic system making full use of the characteristics of conventional bacteriological rapid-diagnostic methods, such as Gram staining and immuno-chromatography.

Home management of mild to moderately severe community-acquired pneumonia: a randomised controlled trial.

OBJECTIVE: To determine whether community management of mild to moderate community-acquired pneumonia (CAP) is as effective and acceptable as standard hospital management of CAP. DESIGN: Randomised controlled trial. SETTING: Christchurch, New Zealand, primary and secondary care. PARTICIPANTS: 55 patients presenting or referred to the emergency department at Christchurch Hospital with mild to moderately severe pneumonia, assessed using a validated pneumonia severity assessment score, from July 2002 to October 2003. INTERVENTIONS: Hospital treatment as usual or comprehensive care in the home delivered by primary care teams. MAIN OUTCOME MEASURES: Primary: days to discharge, days on intravenous (IV) antibiotics, patient-rated symptom scores. Secondary: health status measured using level of functioning at 2 and 6 weeks, patient satisfaction. RESULTS: The median number of days to discharge was higher in the home care group (4 days; range, 1-14) than in the hospital groups (2 days; range, 0-10; P = 0.004). There was no difference in the number of days on IV antibiotics or on subsequent oral antibiotics. Patient-rated symptom scores at 2 and 6 weeks, median change in symptom severity from baseline to 6 weeks, and general functioning at 2 and 6 weeks did not differ between the groups. Patients in both groups were satisfied with their treatment, with a clear preference for community treatment (P < 0.001). CONCLUSIONS: Mild to moderately severe CAP can be managed effectively in the community by primary care teams. This model of comprehensive care at home can be implemented by primary care teams with suitable funding structures.