Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila.

BACKGROUND: Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires' disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila. RESULTS: A set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher. CONCLUSIONS: In summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.

Feasibility and Safety of Neoadjuvant Alectinib in Pulmonary Invasive Mucinous Adenocarcinoma with ALK Rearrangement: Case Report and Literature Review.

BACKGROUND: Pulmonary invasive mucinous adenocarcinoma (IMA) is a rare variant of lung adenocarcinoma that rarely shows anaplastic lymphoma kinase (ALK) rearrangement. Alectinib (tyrosine kinase inhibitors) has been listed as category 1 recommendations for advanced ALK + NSCLC first-line therapy due to low toxicity and excellent efficacy, and its median progression-free survival is 34.8 months. Here, we report a case of a patient with ALK-rearranged lung IMA who showed favorable results to neoadjuvant alectinib. CASE: A 67-year-old man with no history of smoking was diagnosed with clinical stage as IIIB invasive mucinous adenocarcinoma based on clinical symptoms, chest CT and pathological findings. The anaplastic lymphoma kinase (ALK) fusion status was assessed by real-time PCR. After acquiring informed consent from the patient, we offered neoadjuvant alectinib at a dosage of 150 mg twice per day for three cycles (84 days), all lesions were undetectable on chest CT. Later, a thoracoscopic left lobectomy was performed. The postoperative pathological showed that a small amount of tumor cells remained, and the TNM stage was downstaged as T1aN0M0 IA. CONCLUSION: To our knowledge, this is the first case discussing the treatment of ALK-rearranged IMA of the lung with neoadjuvant alectinib. Alectinib is an effective ALK inhibitor, and in cases of lung adenocarcinoma with ALK rearrangement, alectinib treatment is a reasonable and priority option. Neoadjuvant alectinib may be clinically feasible and well tolerated in locally advanced NSCLC.

A Case of Pulmonary Embolism with Sarcomatoid Malignant Pleural Mesothelioma with Long-Term Pleural Effusion.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that originates from pleural mesothelial cells. In recent years, with the development of asbestos-related industries and the increase in air pollution, its incidence has been increased. The incidence of pulmonary embolism combined with sarcomatoid MPM is very low and the prognosis is extremely poor. We here report a case of a patient with long term of pleural effusion and finally diagnosed as pulmonary embolism with sarcomatoid MPM. CASE: A 75-year-old male with a 30-year history of asbestos exposure was admitted to our hospital due to chest pain and difficulty in breathing after exercise. Radiologic examination revealed pleural effusion, computed tomography pulmonary angiography (CTPA) suggests pulmonary embolism, and we consider pleural effusion caused by pulmonary embolism. After anticoagulant therapy for pulmonary embolism and pleural puncture to reduce pleural effusion, the patient's symptoms improved. However, after that, the patient was still admitted to the hospital several times because of recurrent chest pain and dyspnea symptoms, and radiologic examination always showed unexplained pleural effusion. Finally, pathological and immunohistochemical examinations of the pleural biopsy specimens were performed, and the diagnosis was confirmed as sarcomatoid MPM. CONCLUSION: In summary, sarcomatoid MPM with pulmonary embolism is relatively rare, and the prognosis is poor. Clinicians need to be alert to its occurrence. When the first diagnosis is confirmed and the effect of targeted treatment is still not good, the possibility of other diseases should be considered. In clinical practice, pleural biopsy guided by PET-CT is a good choice for patients with sarcomatoid MPM who cannot tolerate open pleural biopsies or thoracoscopy. And patients should undergo pleural morphology and immunohistochemistry as soon as possible, which are helpful for timely diagnosis.

Rapid Detection of Aspergillus fumigatus Using Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Lateral Flow.

Aspergillus fumigatus is an opportunistic, ubiquitous, saprophytic mold which can cause infection in the lungs, nose, eyes, brain, and bones in humans, especially in immunocompromised patients. However, it is difficult to diagnose A. fumigatus infection quickly. Here, we introduce a new detection method, namely multiple cross displacement amplification (MCDA) combined with nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB), which was proved to be fast, reliable, and simple for detecting A. fumigatus. We designed a set of 10 primers targeting the gene annexin ANXC4 of A. fumigatus. The best MCDA condition is 66 °C for 35 min. The minimum concentration that can be detected by this method was 10 fg. In the case of 100 sputum samples, 20 (20%) and 15 (15%) samples were positive by MCDA-LFB and PCR method, respectively. MCDA-LFB and traditional culture method showed the same results. Compared with the culture method, the diagnostic accuracy of MCDA-LFB can reach 100%. It showed that the MCDA-LFB method has better detection ability than the PCR method. We found that the whole process could be controlled within 60 min including the preparation of DNA (20 min), MCDA reaction (35 min) and results reporting (2 min). These results show that this assay is suitable for the rapid, sensitive and specific detection of A. fumigatus in clinical samples.

Nanoparticles-Based Biosensor Coupled with Multiplex Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus.

INTRODUCTION: Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is a common human pathogen, which can cause a variety of infections from mild to severe. In this article, a new diagnostic method called multiplex loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor (mLAMP-LFB) has been developed, which was proved to be fast, reliable, and simple for detecting S. aureus, and differentiate MRSA from methicillin-susceptible S. aureus (MSSA). MATERIALS AND METHODS: We designed a set of six primers targeting the nuc gene of S. aureus, and a set of five primers targeting the mecA gene of MRSA. The lateral flow biosensor visually reported the S. aureus-LAMP results within 2 mins. S. aureus species and non-S. aureus species were used to identify the specificity and sensitivity of the assay. RESULTS: The best conditions for LAMP were 50 mins at 63°C, and the sensitivity was 100 fg. No cross-reactivity was shown and the specificity of this assay is 100%. This assay requires 20 mins for DNA preparation, 50 mins for isothermal amplification and 2 mins for biosensor detection. The total time is within 75 mins. Among 96 sputum samples, LAMP-LFB and traditional culture method showed the same results, 8 (8.33%) samples were MRSA-positive, and 9 (9.38%) samples were MSSA-positive. Seven (7.29%) samples were MRSA-positive and 7 (7.29%) were MSSA-positive by PCR method. Compared with the culture method, diagnostic accuracy of m-LAMP-LFB assay was 100%. The results showed that the m-LAMP-LFB method has better detection ability than the PCR method. DISCUSSION: In short, this m-LAMP-LFB assay is a specific and sensitive method that can quickly identify S. aureus stains, and distinguish MRSA from MSSA, and can be used as a new molecular method for detection of S. aureus in laboratories.