Yuji Kouzaki

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

PURE-LAMP Procedure for the Diagnosis of Extrapulmonary Tuberculosis: A Case Series.

Although the polymerase chain reaction is effective for the diagnosis of extrapulmonary tuberculosis (EPTB), it is typically unavailable in resource-limited situations. In contrast, the loop-mediated isothermal amplification (LAMP) assay is a relatively cost-effective and accessible method. Additionally, when combined with the procedure for ultra-rapid extraction (PURE) kit, which enables simple DNA extraction, LAMP can detect Mycobacterium tuberculosis in sputum within 1.5 hours using a simple procedure. In this study, we investigated the utility of the PURE-LAMP technique to diagnose three cases of EPTB and showed that it may potentially be a valuable tool for the diagnosis of EPTB.

Mortality and severity evaluation by routine pneumonia prediction models among Japanese patients with 2009 pandemic influenza A (H1N1) pneumonia.

BACKGROUND Influenza-related pneumonia, referred to as influenza pneumonia, was reported relatively more frequently during a recent influenza pandemic in 2009. The validity of adapting routine pneumonia severity prediction models for various types of pneumonia is unclear. METHODS We conducted a nationwide survey to evaluate influenza pneumonia among adult patients in Japan. Questionnaires were sent to physicians working in departments of respiratory medicine at 2491 hospitals. Both the outcome and pneumonia severity, using invasive positive pressure ventilation (IPPV) as an indicator, were evaluated by routine pneumonia severity index (PSI), CURB-65 (confusion, urea, respiratory rate, blood pressure, and age ≥ 65 years), and A-DROP (age, dehydration, respiration, disorientation, and blood pressure). RESULTS Data collected from 320 patients with influenza pneumonia, including 25 cases (7.8%) of death and 43 (13.4%) of IPPV, were analyzed. Although all routine prediction models showed that higher mortality tended to be associated with a higher risk class/grade, the actual mortality rates were higher than predicted. The risk class of mortality calculated by the PSI was influenced by pneumonia patterns. Although pneumonia severity was similarly predicted, the types of pneumonia also affected severity in all prediction models. A-DROP showed the highest accuracy on receiver operating characteristic analysis for both mortality and severity. CONCLUSIONS CURB-65 and A-DROP are fair predictors of mortality regardless of pneumonia patterns. However, the current pneumonia prediction models may underestimate the severity and appropriate site of care for patients with influenza pneumonia.

Single nucleotide polymorphisms in genes encoding penicillin-binding proteins in β-lactamase-negative ampicillin-resistant Haemophilus influenzae in Japan

Objectiveβ-Lactamase-negative ampicillin-resistant Haemophilus influenzae is a common opportunistic pathogen of hospital- and community-acquired infections, harboring multiple single nucleotide polymorphisms in the ftsI gene, which codes for penicillin-binding protein-3. The objectives of this study were to perform comprehensive genetic analyses of whole regions of the penicillin-binding proteins in H. influenzae and to identify additional single nucleotide polymorphisms related to antibiotic resistance, especially to ampicillin and other cephalosporins.ResultsIn this genome analysis of the ftsI gene in 27 strains of H. influenzae, 10 of 23 (43.5%) specimens of group III genotype β-lactamase-negative ampicillin-resistant H. influenzae were paradoxically classified as ampicillin-sensitive phenotypes. Unfortunately, we could not identify any novel mutations that were significantly associated with ampicillin minimum inhibitory concentrations in other regions of the penicillin-binding proteins, and we reconfirmed that susceptibility to β-lactam antibiotics was mainly defined by previously reported SNPs in the ftsI gene. We should also consider detailed changes in expression that lead to antibiotic resistance in the future because the acquisition of resistance to antimicrobials can be predicted by the expression levels of a small number of genes.

A Case of Bacillus Calmette-Guérin Cystitis Diagnosed with a Novel Loop-mediated Isothermal Amplification Method

Intravesical bacillus Calmette–Guérin (BCG) instillation is broadly used to prevent bladder cancer recurrence or to treat carcinoma in situ. BCG infection is rare but can cause serious problems because this strain has intrinsic resistance to pyrazinamide, a first-line anti-tuberculosis drug. Furthermore, there had been no specific and easy procedure accurately diagnosing BCG infection. In this case report we present the first case of BCG cystitis diagnosed with a newly developed easy-to-use diagnostic procedure using the loop-mediated isothermal amplification method.

Mucus de alta atenuación en aspergilosis broncopulmonar alérgica

Please cite this article as: Kouzaki Y, Kanoh S, Kawana A. Mucus de alta atenuación en aspergilosis broncopulmonar alérgica. Arch Bronconeumol. 2016;52:46–47. ∗ Corresponding author. E-mail address: kanoh@ndmc.ac.jp (S. Kanoh). endobronchial metastasis from renal cell carcinoma and performed repeated fiberoptic bronchoscopy to collect biopsy specimens. Although there were no malignant cells, mucus plugs showing Charcot–Leyden crystals and Aspergillus were found. Furthermore, total and Aspergillus-specific IgE levels were elevated. Aspergillus