Limulus amoebocyte lysate and direct sampling methods for surveillance of operating nebulizers.

The Limulus amoebocyte lysate test for detection of endotoxin (Pyrogent; Mallinckrodt Chemical Co.) and the Easicult method (Orion Diagnostica) for detection of bacteria were compared with direct dilution sampling, a standardized technique for respiratory therapy surveillance previously developed in our laboratory. Tests of 206 reservoirs of nebulizers were done in three hospitals in Georgia. Forty-five percent of all reservoirs sampled were contaminated. Gram-negative, nonfermentative bacilli were the predominant contaminants. The results of the Limulus test and the Easicult system were in agreement with those of the direct dilution sampling tests approximately 84 and 90% of the time, respectively. Direct dilution of water samples onto blood agar plates was the most sensitive, reliable, and informative method for detecting viable bacteria. The Easicult and Limulus systems were sensitive enough to detect greater than or equal to 10(3) colony-forming units per ml. Positive Limulus tests and negative culture tests, reflecting detection of endotoxin but not of viable gram-negative bacteria, occurred in 20 of 206 (9.7%) instances. Positive cultures and negative Limulus tests were noted in 13 of 206 (6.8%) samplings. The Limulus test is a valuable procedure, for it can detect moderate-to-heavy microbial contamination within 1 h of testing and affords the opportunity to remove contaminated equipment from patients within minutes of a positive test result. These results demonstrate the potential value of the Easicult and Limulus tests for selective surveillance of operating nebulizers.     

气道内微量雾化机对质粒DNA完整性和基因转染效率的影响

目的:探索新型气道内微量雾化机对质粒DNA(pDNA)完整性和整体动物基因转染效率的影响。方法:首先,研究新型气道内微量雾化机对pDNA破坏程度。分别向新型气道内微量雾化机和目前临床普遍使用的机械喷射式雾化机的加药池内加入2ml pDNA(20μg/ml),在开始雾化后第1min、3min、5min分别收集两种雾化机嘴处的雾化液滴,用琼脂糖凝胶电泳观察比较质粒的完整性。其次,研究整新型气道内微量雾化机在整体动物上对基因转染效率的影响。分别用新型气道内微量雾化机和目前临床普遍使用的机械喷射式雾化机,给大鼠雾化经多聚乙烯亚胺(polyethylenimine,PEI)修饰的相同量的绿色荧光蛋白质粒(plasmid DNA of green fluorescent protein gene, pEGFP)3.3μg(PEI/pEGFP),24h后提取动物肺组织进行反转录,用real time PCR及琼脂糖凝胶电泳观察分析绿色荧光蛋白(green fluorescent protein, GFP)的mRNA表达情况。结果:新型气道内微量雾化机在雾化第1min、第3min、第5min后完整的质粒比例分别为(99.6±0.7)%、(100±1.2)%、(99.6±0.7)%,与未雾化对照相比(P>0.05,n=3),无统计学差异,新型雾化机对质粒破坏性可以忽略。临床常用的喷射式雾化机在相同时间段内完整的质粒比例分别为(70.3±1.5)%、(49.3±1.5)%、(32.7±0.6)%。与未雾化对照相比(P<0.05,n=3)有统计学差异,并且随雾化时间增加临床常用的喷射式雾化机对质粒的完整性破坏程度逐渐增加;新型气道内微量雾化机的基因转染效率高于临床普遍使用的机械喷射式雾化机转染效率的(382.1±101.1)倍(P<0.01,n=3)。结论:新型气道内微量雾化机对质粒没有破坏性,能显著增加雾化吸入基因转染效率。为雾化基因治疗提供了一种合适的工具。     

A Comparison of Methods to Measure Operator Protection Factors in Open-fronted Microbiological Safety Cabinets

Comparative tests to measure operator protection factors in microbiological safety cabinets in accordance with British Standard 5726 have demonstrated good agreement in the results obtained by a microbiological method using a Collison nebulizer and the technique producing an aerosol of potassium iodide. Either method is suitable for testing for operator protection factors in Class I and Class II safety cabinets.
The Collison nebulizer should be considered as the standard aerosol generator for the microbiological method; alternative nebulizers meeting the general requirements of BS 5726 should be compared in performance with this nebulizer if they are to be used for containment tests.
Any microbiological safety cabinet specified for a new installation should have been 'type' tested to ensure compliance with BS 5726. However, in order to ensure adequate performance, on-site commissioning tests (and routine maintenance checks thereafter) are necessary to verify that air velocity, filtration and operator protection factor requirements are met.     

Prediction of Pneumonia in Acute Stroke Patients Using Tongue Pressure Measurements

Swallowing dysfunction caused by stroke is a risk factor for aspiration pneumonia. Tongue pressure measurement is a simple and noninvasive method for evaluating swallowing dysfunction. We have hypothesized that low tongue pressure may be able to predict pneumonia occurrence in acute stroke patients. Tongue pressure was measured using balloon-type equipment in 220 acute stroke patients. The modified Mann Assessment of Swallowing Ability (MASA) score was evaluated independently on the same day. Tongue pressure was measured every week thereafter. An improvement in tongue pressure was observed within the first 2 weeks. Receiver operating curve analysis was performed to determine the ability of tongue pressure to predict modified MASA score <95, which suggests swallowing dysfunction. The optimal cutoff for tongue pressure was 21.6 kPa (χ² = 45.82, p<0.001, sensitivity 95.9%, specificity 91.8%, area under the curve = 0.97). The tongue pressure was significantly lower in patients with pneumonia than in those without pneumonia. Using a Cox proportional hazard model for pneumonia onset with a cutoff tongue pressure value of 21.6 kPa and adjustment for age, sex, and National Institutes of Health Stroke Scale score at admission, the tongue pressure had additional predictive power for pneumonia onset (hazard ratio, 7.95; 95% confidence interval, 2.09 to 52.11; p = 0.0013). In the group with low tongue pressure, 27 of 95 patients showed improvement of tongue pressure within 2 weeks. Pneumonia occurred frequently in patients without improvement of tongue pressure, but not in patients with improvement (31/68 and 2/27, p<0.001). Tongue pressure is a sensitive indicator for predicting pneumonia occurrence in acute stroke patients.     

Effect of change in particle number on pulmonary clearance of aerosolized 99mTc-DTPA

Pulmonary clearance (PCl) of inhaled aerosolized 99mTc-diethylenetriamine pentaacetic acid (DTPA) across the alveolocapillary membrane is diffusion limited. Therefore, if the mixing of the 99mTc-DTPA in the aqueous hypophase underlying surfactant is slow or incomplete or if there were no hypophase, an increase in the alveolar surface area occupied by 99mTc-DTPA particles would increase the absorption rate. The aim of this study was to examine whether there is an effect on PCl of changing the number of inhaled particles. The change in particle number was accomplished by a setup of four parallel jet nebulizers feeding a central delivery chamber of 400 cm3. We performed two kinds of experiments in eight healthy nonsmokers between 28 and 52 yr of age. In the first experiment, 99mTc-DTPA in saline was nebulized in one nebulizer, while saline was nebulized in the other three. In the second experiment the number of inhaled particles containing 99mTc-DTPA was increased by a factor of four by nebulizing 99mTc-DTPA in saline in all four nebulizers simultaneously. Increasing the number of inhaled 99mTc-DTPA particles caused an increase in PCl of 24.2% (P less than 0.01). We conclude that there is a slight but significant effect of changing the number of DTPA particles on PCl and that this is probably due to an uneven mixing of the 99mTc-DTPA in the aqueous hypophase underlying the surfactant lining and the alveoli.     

Effects of Temperature and Humidity on Laser Diffraction Measurements to Jet Nebulizer and Comparison with NGI

Laser diffraction (LD) and next generation impactor (NGI) are commonly used for the evaluation of inhaled drug formulations. In this study, the effect of temperature and humidity on the assessment of the nebulizer particle size distribution (PSD) by LD was investigated, and the consistency between NGI and LD measurements was evaluated. There was an increase in particle size with higher temperature or lower humidity. The particle population with a diameter less than 1 μm was significant at a temperature of 5°C or at relative humidity >90%; however, the same particle population became undetectable when temperature increased to 39°C or at relative humidity of 30–45%. The results of the NGI and LD measurements of aerosol generated from three types of jet nebulizers were compared. A poor correlation between the NGI and LD measurements was observed for PARI LC (2.2 μm) (R ²?=?0.893) and PARI LC (2.9 μm) (R ²?=?0.878), while a relatively good correlation (R ²?=?0.977) was observed for the largest particle size nebulizer (PARI TIA (8.6 μm)). We conclude that the ambient environment and the nebulizer have significant impacts on the performance and consistency between these instruments. These factors should be controlled in the evaluation of inhaled aerosol drug formulations when these instruments are used individually or in combination.     

Nonbacteremic Pseudomonas Pneumonia in Immunosuppressed Guinea Pigs

An experimental model of nonbacteremic pneumonia with a virulent strain of Pseudomonas aeruginosa was successfully established in guinea pigs immunosuppressed with cortisone acetate although the organisms were eliminated rapidly from the lungs without cortisone treatment. Using a pocket nebulizer, almost all the animals which received 10⁶ organisms/g-lung developed bronchopneumonia without any septic findings as long as 10 days after challenge. The lesions produced in such animals were characterized by dissemination of multiple purulogranulomatous changes. In the early stage of infection, infiltration of polymorphonuclear cells (PMNs) in the bronchiolar and alveolar spaces was diffuse, later showing multifocal accumulation with the formation of central spherical grains enclosing bacterial colonies. In the later stage, granulation tissue consisting of large mononuclear cells, fibroblasts and collagen fibers developed around the PMN accumulation. The animals which received 10⁷ organisms/g-lung, on the other hand, developed severe pulmonary hemorrhages and necrosis followed by septic death.     

Community-acquired pneumonia in adults.

Although the frequency of community-acquired pneumonia caused by Streptococcus pneumoniae continues to be high, studies show that Mycoplasma pneumoniae, Chlamydia pneumoniae, or Legionella pneumophila are the etiologic agents in 20% to 40% of community-acquired pneumonia in adults. The clinical presentation of pneumonia caused by these organisms may be indistinguishable from pneumonia due to S pneumoniae. Separation of cases of pneumonia due to S pneumoniae as typical and that caused by M pneumoniae, C pneumoniae, or L pneumophila as atypical is unwarranted and unhelpful in planning therapy. As many as 35% to 50% of patients do not have an etiologic agent identified. Community-acquired pneumonia can have high morbidity and mortality in patients who are older, have underlying lung disease, diabetes mellitus, or other comorbid conditions, or who have decreased immune function regardless of the specific etiologic agent. In choosing appropriate empiric antimicrobial therapy in hosts who are not immunocompromised, erythromycin and other macrolide antibiotics have the advantage of being effective against a wide range of pathogens likely to be encountered, including S pneumoniae, M pneumoniae, and L pneumophila, and of having some benefit against C pneumoniae. In other patients, the selection of antibiotic therapy can be based on age, clinical suspicion, epidemiologic data, and laboratory test results. Antimicrobial therapy can be directed at specific organisms when and if they are identified.     

Initial Characterization of Micafungin Pulmonary Delivery via Two Different Nebulizers and Multivariate Data Analysis of Aerosol Mass Distribution Profiles

Pharmaceutical aerosols have been targeted to the lungs for the treatment of asthma and pulmonary infectious diseases successfully. Micafungin (Astellas Pharma US, Deerfield, IL, USA) has been shown to be an effective antifungal agent when administrated intravenously. Pulmonary delivery of micafungin has not previously been reported. In the present pilot study, we characterize the performance of two nebulizers and their potential for delivering micafungin to the lungs as well as the use of multivariate data analysis for mass distribution profile comparison. The concentration of micafungin sodium increased by 21% when delivered by the Acorn II nebulizer and by 20% when delivered by the LC Plus nebulizer, respectively, from the first to the second sampling period. The Acorn II nebulizer delivered a fine particle fraction FPF_5.8 (%<5.8 μm) of 92.5 ± 0.8 and FPF_3.3 (%<3.3 μm) of 82.3 ± 2.1 during the first sampling period. For the LC Plus nebulizer, FPF_5.8 was 92.3 ± 0.1 and FPF_3.3 was 67.0 ± 0.7 during the first sampling period. The mass median aerodynamic diameter (MMAD) increased from 1.67 ± 0.05 to 1.77 ± 0.04 μm (Acorn II nebulizer) and from 2.09 ± 0.01 to 2.20 ± 0.01 μm (Pari LC Plus nebulizer) from the first to the second sampling periods. These changes in MMAD were statistically significant by paired t test. Multivariate data analysis showed that this could be explained systematically by greater drug deposition on stages with larger cutoff sizes and reduced drug deposition on stages with smaller cutoff sizes rather than multimodal deposition or other anomalies in size distribution.     

Evaluation of bacterial contamination on objects d'art by membrane filtration and epifluorescence microscopy

Bacterial contimination on proteinaceous objets d'art such as parchment and mummies exposed to low relative humidity and low oxygen levels has been assessed using a polycarbonate NUCLEPORE filter and incedent light (epifluorescence) microscopy. The technique described is a rapid and sensitive means for observing and evaluating bacterial populations including anaerobic organisms involved in the depolymerization of collagen. Using contaminated 17th century parchment treated to microenvironmental control and modified atmospheres a positive relationship has been found between fluorescence counts and plate colony counts.     

Laboratory Study Comparing Pharmacopeial Testing of Nebulizers with Evaluation Based on Nephele Mixing Inlet Methodology

Determination of fine droplet dose with preparations for nebulization, currently deemed to be the metric most indicative of lung deposition and thus in vivo responses, involves combining two procedures following practice as described in the United States Pharmacopeia and the European Pharmacopeia. Delivered dose (DD) is established by simulating tidal breathing at the nebulizer, collecting the medication on a filter downstream of the nebulizer mouthpiece/facemask. Fine droplet fraction (FDF_<x μm) is determined separately using a cooled cascade impactor operated at 15 L/min. FDD_<x μm is subsequently calculated as the product of DD and FDF_<x μm. Development of the Nephele mixing inlet has allowed cascade impactor-based assessments to be made at a constant flow rate while simultaneously subjecting the nebulizer to the continuously varying flow profile associated with breath simulation. The study purpose was to investigate the feasibility of this approach, termed mixing inlet lung simulation (MILS), for direct determination of FDD_<x μm. An optimal upper size limit for FDF is not given for nebulizers, but 5 μm was chosen since this limit is the European norm when testing other inhalation products. Vibrating membrane nebulizers (eFlow® Rapid) were used to deliver aqueous salbutamol sulfate, simulating an adult tidal-breathing pattern (inspiratory to expiratory ratio = 1:1, tidal volume = 500 mL, 15 breaths per minute, peak inspiratory flow rate = 24 L/min). The two procedures were inequivalent, as FDD_<5 μm by the MILS approach was 72% of that obtained using the compendial “combination” method. Since the MILS methodology more closely mimics clinical use, we infer that the compendial approach likely overestimates the dose reaching the human lung.     

Contaminated Heparin and Outcomes after Cardiac Surgery: A Retrospective Propensity-Matched Cohort Study

Background

During 2007 and 2008 it is likely that millions of patients in the US received heparin contaminated (CH) with oversulfated chondroitin sulfate, which was associated with anaphylactoid reactions. We tested the hypothesis that CH was associated with serious morbidity, mortality, intensive care unit (ICU) stay and heparin-induced thrombocytopenia following adult cardiac surgery.

Methods and Findings

We conducted a single center, retrospective, propensity-matched cohort study during the period of CH and the equivalent time frame in the three preceding or the two following years. Perioperative data were obtained from the institutional record of the Society of Thoracic Surgeons National Database, for which the data collection is prospective, standardized and performed by independent investigators. After matching, logistic regression was performed to evaluate the independent effect of CH on the composite adverse outcome (myocardial infarction, stroke, pneumonia, dialysis, cardiac arrest) and on mortality. Cox regression was used to determine the association between CH and ICU length of stay. The 1∶5 matched groups included 220 patients potentially exposed to CH and 918 controls. There were more adverse outcomes in the exposed cohort (20.9% versus 12.0%; difference = 8.9%; 95% CI 3.6% to 15.1%, P<0.001) with an odds ratio for CH of 2.0 (95% CI, 1.4 to 3.0, P<0.001). In the exposed group there was a non-significant increase in mortality (5.9% versus 3.5%, difference = 2.4%; 95% CI, −0.4 to 3.5%, P = 0.1), the median ICU stay was longer by 14.1 hours (interquartile range −26.6 to 79.8, S = 3299, P = 0.0004) with an estimated hazard ratio for CH of 1.2 (95% CI, 1.0 to 1.4, P = 0.04). There was no difference in nadir platelet counts between cohorts.

Conclusions

The results from this single center study suggest the possibility that contaminated heparin might have contributed to serious morbidity following cardiac surgery.     

Contamination of Sputum Induction Equipment During Patient Usage

Sputum induction equipment was evaluated for its capacity to become contaminated by patients harboring Mycobacterium tuberculosis. The mouthpiece, goose-neck, and 10% NaCl solution were found capable of being contaminated by tuberculous patients. Two per cent glutaraldehyde was shown to be an effective means of decontamination. This study indicates that the entire induction apparatus must be cleaned and decontaminated between patients.     

Studies on aerosol delivery of plasmid DNA using a mesh nebulizer

Aerosol delivery of plasmid DNA therapeutic solutions is promising for the treatment of respiratory diseases. However, it poses challenges, most significantly the need to protect the delicate supercoiled (sc) structure of plasmid during aerosolization. Nebulizers for liquid aerosolization using meshes appear a better method for delivery than conventional jet and ultrasonic nebulizers. This paper explores their application to the delivery of plasmid DNA. A computational fluid dynamics model of the dynamics of fluid flow through the nozzle of the MicroAIR mesh nebulizer indicated high strain rates (>10(5) s(-1)) near the nozzle exit capable of causing damage to the shear-sensitive plasmid DNA. Knowledge of the strain rates predicted using CFD and molecule size determined using atomic force microscopy (AFM) enabled estimation of the hydrodynamic force and whether damage of shear-sensitive therapeutics was likely. Plasmids of size 5.7 and 20 kb were aerosolized in the mesh nebulizer. The sc structure of the 5.7-kb plasmid was successfully delivered without damage, while aerosolization of the 20-kb plasmid led to disintegration of the pDNA sc structure as observed in AFM. Subsequent formulation of the sc 20-kb plasmid with PEI resulted in successful aerosol delivery. The maximum hydrodynamic forces computed for the aerosolization of structures of the size of 5.7-kb and PEI formulated 20-kb plasmids were less than the forces reported to damage the structure of double-stranded DNA. A combination of CFD analysis and structure analysis may be used to predict successful aerosol delivery in such a mesh nebulizer.     

Effect of formulation on the stability and aerosol performance of a nebulized antibody

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.     

Effect of formulation on the stability and aerosol performance of a nebulized antibody

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.     

Detection of Staphylococcus aureus (MRSA/MSSA) in surfaces of dental medicine equipment

Methicillin-Resistant Staphylococcus aureus (MRSA) represents one of the major causes of nosocomial infections, leading to high mortality. Surfaces in clinics, as well as the attending uniform and the hands of the dental doctor can be MRSA reservoirs. Having this in mind, the purpose of this study was to evaluate the presence of Methicillin-Sensitive Staphylococcus aureus (MSSA) and MRSA on dental medicine equipment surfaces. 354 Samples were collected from six equipment surfaces in six attendance areas before and after patient consultation and cultured in a selective medium. Polymerase Chain Reaction (PCR) was used to confirm the identity of bacterial strains as MRSA or MSSA. Data analysis was performed with chi-square tests with Bonferroni correction. It was observed 55.6% of uncontaminated samples. Contamination was: 17.5% MRSA (5.9% of samples collected before patient attendance and 11.6% after); 39.3% MSSA (14.1% collected before and 25.2% after). The prevalence of MRSA and MSSA was significantly higher after patient care. Integrated Clinic represented the most contaminated attendance area (MRSA − 41.7%, MSSA − 51.2%), the chair arm rest was the most contaminated surface for MRSA (29.7%) and the dental spittoon the most contaminated surface for MSSA (23.5%). Although a low level of contamination was observed, dental clinics, through patients possibly carrying bacteria, may be reservoirs for MRSA and MSSA transmission, and might contribute to potential nosocomial infections.     

Medical Assistance to the Aged

Aerosol therapy has three principal objectives: Mobilization of bronchial secretions, relief of bronchospasm and topical chemotherapy. It has become an important tool in the treatment of bronchopulmonary diseases. The equipment for inhalation therapy, however, should be adequate. Both large-capacity and small-capacity nebulizers must be available, and they must be the kind that will produce a mist with most of its particles only 0.5 to 2.5 micra in diameter. These nebulizers may be used alone or in conjunction with a variety of appliances that will deliver the aerosols to the respiratory tract. The use of humidifying agents as aerosols is extremely helpful in patients with retained bronchopulmonary secretions. In some patients who have particularly thick or gelatinous secretions and in patients with mucoviscidosis, ordinary water or saline solution is often not enough. Hypertonic saline may be of value in these cases, and it is suggested that half-molar (2.9 per cent) saline be administered in 10 per cent propylene glycol. In these cases, preparations containing detergents (tyloxypal) or other preparations containing enzymes (desoxyribonuclease or trypsin) may be given by the aerosol technique, with care not to cause irritation.The bronchodilator aerosol agents are of proved benefit in the treatment of bronchospastic disorders and are indicated in most cases of asthma and in those cases of emphysema in which there is definite evidence of associated bronchospasm.The value of the aerosol method of administering chemotherapeutic and antibiotic drugs has probably been overrated, and it is suspected that much of the benefit previously attributed to the therapeutic agent was actually a result of humidification and liquefaction.     

Prediction of the pathogens that are the cause of pneumonia by the battlefield hypothesis

Commensal organisms are frequent causes of pneumonia. However, the detection of these organisms in the airway does not mean that they are the causative pathogens; they may exist merely as colonizers. In up to 50% cases of pneumonia, the causative pathogens remain unidentified, thereby hampering targeting therapies. In speculating on the role of a commensal organism in pneumonia, we devised the battlefield hypothesis. In the "pneumonia battlefield," the organism-to-human cell number ratio may be an index for the pathogenic role of the organism. Using real-time PCR reactions for sputum samples, we tested whether the hypothesis predicts the results of bacteriological clinical tests for 4 representative commensal organisms: Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas spp., and Moraxella catarrhalis. The cutoff value for the organism-to-human cell number ratio, above which the pathogenic role of the organism was suspected, was set up for each organism using 224 sputum samples. The validity of the cutoff value was then tested in a prospective study that included 153 samples; the samples were classified into 3 groups, and each group contained 93%, 7%, and 0% of the samples from pneumonia, in which the pathogenic role of Streptococcus pneumoniae was suggested by the clinical tests. The results for Haemophilus influenzae, Pseudomonas spp., and Moraxella catarrhalis were 100%, 0%, and 0%, respectively. The battlefield hypothesis enabled legitimate interpretation of the PCR results and predicted pneumonia in which the pathogenic role of the organism was suggested by the clinical test. The PCR reactions based on the battlefield hypothesis may help to promote targeted therapies for pneumonia. The prospective observatory study described in the current report had been registered to the University Hospital Medical Information Network (UMIN) registry before its initiation, where the UMIN is a registry approved by the International Committee of Medical Journal Editors (ICMJE). The UMIN registry number was UMIN000001118: A prospective study for the investigation of the validity of cutoff values established for the HIRA-TAN system (April 9, 2008).     

Propagation and Purification of Rat Pneumocystis carinii in Short-Term Cell

A short-term cell culture is used to propagate and purify rat-derived Pneumocystis carinii (Pc). An aliquot of pelleted material washed out of the lungs of rats with moderate to severe Pc pneumonia is cultured for 7 to 10 days on the adherent mink lung cell line Mv 1 Lu, and the rest of the material is frozen down in medium with 10% glycerol. Although it has not been established that substantial multiplication of Pc occurs in culture, the Pc organisms harvested from the supernatant at the end of the culture period arc relatively free of both host and feeder cells. This is in marked contrast with the lung wash inoculum in which the Pc organisms arc heavily contaminated with rat cells and enmeshed in a highly sticky material. Lung wash preparations frozen down in glycerol and stored at −70.°C for as long as 6 months or more can be successfully cultured upon thawing with no apparent loss of viability of the Pc organisms.