Decontamination Strategies Used for AFB Culture Significantly Reduce the Viability of Mycobacterium abscessus Complex in Sputum Samples from Patients with Cystic Fibrosis

Nontuberculous mycobacteria are important respiratory pathogens in patients with cystic fibrosis (CF). For diagnosis, international guidelines recommend culture of sputum that has been decontaminated via chemical treatment. Fifty-six sputum samples from 32 patients known to be previously colonized or infected with NTM were subdivided, and the aliquots were subjected to six different decontamination strategies, followed by quantitative culture for NTM. Thirty sputum samples contained Mycobacterium abscessus complex (MABSC) and 11 contained Mycobacterium avium complex (MAC). Decontamination strategies included treatment with N-acetyl L-cysteine with 2% sodium hydroxide (NALC-NaOH), 4% NaOH, 1% chlorhexidine, 0.5 N sulfuric acid, 5% oxalic acid, double decontamination with NALC-NaOH, followed by 5% oxalic acid, and saline (0.85%) as a control. The samples were also cultured directly with no treatment. Treatment with NALC-NaOH resulted in an average reduction in colony count of 87% for MABSC when compared with direct culture. NaOH at 4% caused a 98.3% average reduction in colony count. All treatments that included NaOH resulted in colony counts that were statistically lower than those obtained from direct culture or the saline-treated control (p < 0.05). Standard treatments using sulfuric or oxalic acids were less deleterious, but still resulted in an average reduction in colony count of at least 30%. The viability of MAC was much less affected by most decontamination treatments. In conclusion, the viability of MABSC was severely compromised by standard decontamination regimens. This supports recent evidence showing that optimal recovery of MABSC is achieved by culture on an appropriate selective agar without decontamination of sputum samples.

Evaluation of GeneXpert PA assay compared to genomic and (semi-)quantitative culture methods for direct detection of Pseudomonas aeruginosa in endotracheal aspirates

Introduction Pseudomonas aeruginosa is a common cause of ventilator-associated pneumonia (VAP). Rapid and accurate detection of lower respiratory tract colonization and/or infection with P. aeruginosa may advise targeted preventive (antibody-based) strategies and antibiotic therapy. To investigate this, we compared semi-quantitative culture results from 80 endotracheal aspirates (ETA) collected from mechanically-ventilated patients, to two culture and two non-culture-based methods for detection of P. aeruginosa. Methods P. aeruginosa-positive (n = 40) and -negative (n = 40) ETAs from mechanically ventilated patients analyzed initally by (i) routine semi-quantitative culture, were further analyzed with (ii) quantitative culture on chromogenic ChromID P. aeruginosa and blood agar; (iii) enrichment in brain heart infusion broth followed by plating on blood agar and ChromID P. aeruginosa; (iv) O-antigen acetylase gene-based TaqMan qPCR; and (v) GeneXpert PA PCR assay. Results Of the 80 ETA samples included, one sample that was negative for P. aeruginosa by semi-quantitative culture was found to be positive by the other four methods, and was included in an “extended” gold standard panel. Based on this extended gold standard, both semi-quantitative culture and the GeneXpert PA assay showed 97.6% sensitivity and 100% specificity. The quantitative culture, enrichment culture and O-antigen acetylase gene-based TaqMan qPCR had a sensitivity of 97.6%, 89.5%, 92.7%, and a specificity of 97.4%, 100%, and 71.1%, respectively. Conclusion This first evaluation of the GeneXpert PA assay with ETA samples found it to be as sensitive and specific as the routine, hospital-based semi-quantitative culture method. Additionally, the GeneXpert PA assay is easy to perform (hands-on time ≈ 5 min) and rapid (≈ 55 min assay time). The combination of the high sensitivity and high specificity together with the rapid acquisition of results makes the GeneXpert PA assay a highly recommended screening technique. Where this equipment is not available, semi-quantitative culture remains the most sensitive of the culture methods evaluated here for P. aeruginosa detection in ETA samples.

Are Semi-Quantitative Clinical Cultures Inadequate? Comparison to Quantitative Analysis of 1053 Bacterial Isolates from 350 Wounds

Early awareness and management of bacterial burden and biofilm is essential to wound healing. Semi-quantitative analysis of swab or biopsy samples is a relatively simple method for measuring wound microbial load. The accuracy of semi-quantitative culture analysis was compared to ‘gold standard’ quantitative culture analysis using 428 tissue biopsies from 350 chronic wounds. Semi-quantitative results, obtained by serial dilution of biopsy homogenates streaked onto culture plates divided into 4 quadrants representing occasional, light, moderate, and heavy growth, were compared to total bacterial load quantified as colony-forming units per gram (CFU/g). Light growth, typically considered an insignificant finding, averaged a clinically significant 2.5 × 105 CFU/g (SE = 6.3 × 104 CFU/g). Occasional growth (range: 102–106 CFU/g) and light growth (103–107 CFU/g) corresponded to quantitative values that spanned a 5-log range; moderate and heavy growth corresponded to a range of 4-log and 6-log, respectively, with a high degree of overlap in range of CFU/g per category. Since tissue biopsy and quantitative culture cannot be widely practiced and semi-quantitative analysis is unreliable, other clinically relevant approaches are required to determine wound bioburden and guide best management practices. Fluorescence imaging is a point-of-care technology that offers great potential in this field.

Evaluation of the Effectiveness of Two Automated Room Decontamination Devices Under Real-Life Conditions

To evaluate the effectiveness of automated room decontamination devices, a common aerosolized hydrogen peroxide (aHP) as well as a recent gaseous ozone-based device, which produces the disinfectant reagent without the need of consumables, were tested under real-life conditions. Twenty-two contaminated surfaces were positioned in different areas in a patient room with adjacent bathroom and anteroom. Following the decontamination process bacteria were recovered and reduction factors were calculated after performing quantitative culture. Following the manufactures instructions, the ozone-based device displayed a bactericidal effect (log10 &gt; 5), whereas the aHP system failed for a high bacterial burden and achieves only a complete elimination of a realistic bioburden (log10 2). After increasing the exposure time to 30 min, the aHP device also reached a bactericidal effect. Nevertheless, our results indicate, that further research and development is necessary, to get knowledge about toxicity, efficacy and safety by using in complex hospital conditions and achieve meaningful integration in cleaning procedures, to reach positive effects on disinfection performance.

Candida Species in Community-Acquired Pneumonia in Patients With Chronic Aspiration

BackgroundWhen Candida is found in a sputum culture, clinicians generally dismiss it as a contaminant. We sought to identify cases of community-acquired pneumonia (CAP) in which Candida might play a contributory etiologic role.MethodsIn a convenience sample of patients hospitalized for CAP, we screened for “high-quality sputum” by Gram stain (>20 WBC/epithelial cell) and performed quantitative sputum cultures. Criteria for a potential etiologic role for Candida included the observation of large numbers of yeast forms on Gram stain and the finding of >106 CFU/ml Candida in sputum. We gathered clinical information on cases that met these criteria for possible Candida infection.ResultsSputum from 6 of 154 consecutive CAP patients had large numbers of extra- and intracellular yeast forms on Gram stain, with >106 CFU/ml Candida albicans, glabrata, or tropicalis on quantitative culture. In all 6 patients, the clinical diagnoses at admission included chronic aspiration. Greater than 105 CFU/ml of a recognized bacterial pathogen (Streptococcus pneumoniae, Staphylococcus aureus, or Pseudomonas) or >106 CFU/ml of other ‘normal respiratory flora’ (Lactobacillus species) were present together with Candida in every case. Blood cultures yielded Candida in 1 case, and 1,3-beta-D glucan was >500 ng/mL in 3 of 3 cases in which it was assayed. Since all patients were treated with anti-bacterial and anti-fungal drugs, no inference about etiology can be derived from therapeutic response.ConclusionsCandida species, together with a recognized bacterial pathogen or normal respiratory flora, may contribute to the cause of CAP in patients who chronically aspirate.

Evaluation of semi-quantitative compared to quantitative cultures of tracheal aspirates for the yield of culturable respiratory pathogens – a cross-sectional study

Background Diagnosis of lower respiratory tract infections (LRTI) depends on the presence of clinical, radiological and microbiological findings. Endotracheal suction aspirate (ETSA) is the commonest respiratory sample sent for culture from intubated patients. Very few studies have compared quantitative and semi-quantitative processing of ETSA cultures for LRTI diagnosis. We determined the diagnostic accuracy of quantitative and semi-quantitative ETSA culture for LRTI diagnosis, agreement between the quantitative and semi quantitative culture techniques and the yield of respiratory pathogens with both methods. Methods This was a cross-sectional study conducted at the Aga Khan University clinical laboratory, Karachi, Pakistan. One hundred and seventy-eight ETSA samples sent for routine bacteriological cultures were processed quantitatively as part of regular specimen processing method and semi-quantitatively. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy was calculated for both methods using clinical diagnosis of pneumonia as reference standard. Agreement between the quantitative and semi quantitative methods was assessed via the kappa statistic test. Pathogen yield between the two methods was compared using Pearson’s chi-square test. Results The quantitative and semi-quantitative methods yielded pathogens in 81 (45.5%) and 85 (47.8%) cases respectively. There was complete concordance of both techniques in 155 (87.1%) ETSA samples. No growth was observed in 45 (25.3%) ETSA specimens with quantitative culture and 37 (20.8%) cases by semi-quantitative culture. The diagnostic accuracy of both techniques were comparable; 64.6% for quantitative and 64.0% for semi-quantitative culture. The kappa agreement was found to be 0.84 (95% CI, 0.77–0.91) representing almost perfect agreement between the two methods. Although semi-quantitative cultures yielded more pathogens (47.8%) as compared to quantitative ETSA cultures (45.5%), the difference was only 2.3%. However, this difference achieved statistical (chi-square p-value < 0.001) favoring semi-quantitative culture methods over quantitative culture techniques for processing ETSA. Conclusion In conclusion, there is a strong agreement between the performances of both methods of processing ETSA cultures in terms of accuracy of LRTI diagnosis. Semi-quantitative cultures of ETSA yielded more pathogens as compared to quantitative cultures. Although both techniques were comparable, we recommend processing of ETSA using semi-quantitative technique due to its ease and reduced processing time.

TLR4 Polymorphism, Nasopharyngeal Bacterial Colonization, and the Development of Childhood Asthma: A Prospective Birth-Cohort Study in Finnish Children

We aimed to explore the role of TLR4 (rs4986790) polymorphism in the nasopharyngeal (NP) bacterial colonization and its consequent impact on the development of childhood asthma. A semi-quantitative culture of NP swabs was performed on 473 children at 2 months of age and on 213 children at 13 months of age. TLR4 polymorphism was analyzed for 396 children. Children were followed from birth to the age of 7.5 years and the final outcome was physician-diagnosed asthma. The associations between TLR4 genotype, bacterial colonization, and asthma were analyzed. Children with TLR4 AG or GG genotype were more often colonized with Moraxella catarrhalis at 2 months of age (p = 0.009) and Haemophilus influenzae at 13 months of age (p = 0.018). Children who were colonized with H. influenzae at 13 months of age had a significantly higher risk of later development of asthma (p = 0.004). M. catarrhalis or H. Influenzae colonization at 2 months of age or TLR4 genotype Asp299Gly were not associated with the development of childhood asthma. TLR4 Asp299Gly polymorphism was associated with an increased risk of colonization of M. catarrhalis and H. influenzae in children. The colonization with H. influenzae at 13 months of age was associated with a higher risk of later development of childhood asthma.

Assaying Chlamydia pneumoniae Persistence in Monocyte-Derived Macrophages Identifies Dibenzocyclooctadiene Lignans as Phenotypic Switchers

Antibiotic-tolerant persister bacteria involve frequent treatment failures, relapsing infections and the need for extended antibiotic treatment. The virulence of an intracellular human pathogen C. pneumoniae is tightly linked to its propensity for persistence and means for its chemosensitization are urgently needed. In the current work, persistence of C. pneumoniae clinical isolate CV6 was studied in THP-1 macrophages using quantitative PCR and quantitative culture. A dibenzocyclooctadiene lignan schisandrin reverted C. pneumoniae persistence and promoted productive infection. The concomitant administration of schisandrin and azithromycin resulted in significantly improved bacterial eradication compared to sole azithromycin treatment. In addition, the closely related lignan schisandrin C was superior to azithromycin in eradicating the C. pneumoniae infection from the macrophages. The observed chemosensitization of C. pneumoniae was associated with the suppression of cellular glutathione pools by the lignans, implying to a previously unknown aspect of chlamydia–host interactions. These data indicate that schisandrin lignans induce a phenotypic switch in C. pneumoniae, promoting the productive and antibiotic-susceptible phenotype instead of persistence. By this means, these medicinal plant -derived compounds show potential as adjuvant therapies for intracellular bacteria resuscitation.

Real-Time PCR and Quantitative Culture for Mycoplasma pneumoniae Load in Pharyngeal Swabs from Children at Preliminary Diagnosis and Discharge

Background. Extensive studies have focused on the diagnosis and treatment of Mycoplasma pneumoniae infection; however, rare studies investigated the posttreatment conditions. We analyzed the carrying status of M. pneumoniae in the respiratory tract of children before and after treatment. Methods. Ninety-two children with M. pneumoniae pneumonia were included in this study. Clinical data were obtained from each patient, and pharyngeal swab sampling was performed at preliminary diagnosis and discharge. Real-time PCR and dilution quantitative culture were utilized to determine the DNA quantification and number of viable M. pneumoniae from samples collected upon preliminary diagnosis and discharge. Results. All the 92 cases showed DNA positivity upon preliminary diagnosis, serum IgM antibody was detected in 80 patients, and positivity of M. pneumoniae culture was observed in 82 cases. Upon discharge, the M. pneumoniae nucleotide and culture positivity were detected in 87 and 49 cases, respectively. The content of viable M. pneumoniae was 10–104 CCU/mL and 10–102 CCU/mL in the preliminary diagnosis samples and discharge samples, respectively. Conclusions. Real-time PCR was rapid and effective for the qualitative diagnosis of M. pneumoniae at the early stage, but it cannot be used to evaluate the prognosis of patients with M. pneumoniae infection. Quantitative analysis for M. pneumoniae DNA could not directly reflex the viable strain content.