Special precautions reduce oropharyngeal contamination in bronchoalveolar lavage for bacteriologic studies

Despite the use of quantitative culture, oropharyngeal contamination of bronchoalveolar lavage (BAL) specimens is still a factor that limits the usefulness of this technique in the diagnosis of lower respiratory tract infection. To investigate whether special precautions could reduce contamination, 20 noninfected patients undergoing diagnostic bronchoscopy were randomized into 2 groups of 10 patients: BAL was performed routinely in group R and with special precautions in group P. These precautions consisted of giving topical lidocaine by inhalation rather than by bolus injection, and passing the bronchoscope used for BAL through a previously inserted endotracheal tube. Quantitative culture of BAL specimens showed that 5 patients in group R (50%), but none of the patients in group P (0%), had at least 1 organism recovered in concentrations greater than or equal to 10(4) colony-forming units CFU/ml (p = 0.016). Fifteen of 39 isolates (38.5%) in group R and none of 18 isolates in group P (0%) were present in concentration greater than or equal to 10(4) CFU/ml (p = 0.001). We conclude that oropharyngeal contamination of BAL specimens can be minimized by adopting special precautions during the procedure and by using quantitative culture with 10(4) CFU/ml as the cut-off point. This may increase the specificity of the technique in the diagnosis of lower respiratory tract infection without reducing its sensitivity.     

Bronchoalveolar lavage or oropharyngeal cultures to identify lower respiratory pathogens in infants with cystic fibrosis

As collections of lower respiratory tract specimens from young children with cystic fibrosis (CF) are difficult, we determined whether oropharyngeal cultures predicted lower airway pathogens. During 1992–1994, 75 of 90 (83%) infants with CF diagnosed by neonatal screening had 150 simultaneous bronchoalveolar lavage (BAL) and oropharyngeal specimens collected for quantitative bacterial culture at a mean age of 17 months (range, 1–52). Ten children undergoing bronchoscopy for stridor served as controls. Total and differential cell counts and interleukin-8 concentrations were measured in BAL fluid. A subset of bacterial pathogens were typed by pulsed field gel electrophoresis. A non-linear relationship with inflammatory markers supported a diagnosis of lower airway infection when ≥10⁵ colony-forming units/ml were detected. This criterion was met in 47 (31%) BAL cultures from 37 (49%) children. Staphylococcus aureus (19%), Pseudomonas aeruginosa (11%), and Hemophilus influenzae (8%) were the major lower airway pathogens. In oropharyngeal cultures, S. aureus (47%), Escherichia coli (23%), H. influenzae (15%), and P. aeruginosa (13%) predominated. The sensitivity, specificity, and positive and negative predictive values of oropharyngeal cultures for pathogens causing lower respiratory infections were 82%, 83%, 41%, and 97%, respectively. When there was agreement between paired oropharyngeal and BAL cultures, genetic fingerprinting showed some strains of the same organism were unrelated. We conclude that oropharyngeal cultures do not reliably predict the presence of bacterial pathogens in the lower airways of young CF children. Pediatr Pulmonol. 1996; 21:267–275. © 1996 Wiley-Liss, Inc.     

Lower airway bacterial colonization in asymptomatic smokers and smokers with chronic bronchitis and recurrent exacerbations

Bacterial colonization of the lower airways in patients with chronic bronchitis (CB) has been described mainly in patients with co-existing chronic obstructive pulmonary disease (COPD). Although smoking has been identified as a risk factor for bacterial colonization it is not known whether asymptomatic smokers (AS) can be colonized. The aim of this study was to study lower airway bacterial colonization in smokers with stable CB and recurrent exacerbations and compare with AS and healthy never-smokers (NS). Thirty-nine smokers with CB and recurrent exacerbations (median FEV1 85% of predicted normal), 10 AS and 10 NS, underwent bronchoscopy and a two-step bronchoalveolar lavage (BAL) procedure where the first portion (20 ml, 'pre-BAL') was recovered separately from the rest (140 ml, 'BAL'). The degree of oropharyngeal contamination of pre-BAL and BAL samples was evaluated by cytology. Semiquantitative bacterial cultures were performed on all samples. Higher bacterial numbers than 10(3) colony-forming units (cfu) x ml(-1) in BAL were found only in the two smoking groups. Using 10(3) cfu x ml(-1) as cut-off, 6/10 (60%) in the AS-, and 7/35 (20%) in the CB-group were colonized in the lower airways. In all, 29% of all smokers had bacterial colonization. Only bacteria belonging to the normal oropharyngeal flora were found. The proportion of samples with oropharyngeal contamination was significantly lower in BAL than in pre-BAL (5% vs. 21%, P=0.039). The proportion of sterile samples was significantly higher in BAL than in pre-BAL (49% vs. 26%, P=0.002). Lower airway bacterial colonization was found both in asymptomatic smokers and in patients with CB. Colonization with potential respiratory pathogens is uncommon in patients with CB and recurrent exacerbations without severe airflow obstruction. The two-step BAL procedure seems to decrease oropharyngeal contamination.     

The bacteriology of bronchiectasis in Hong Kong investigated by protected catheter brush and bronchoalveolar lavage

Bacteria often colonize the lower respiratory tract of patients with bronchiectasis. Although the role of these bacteria in the pathogenesis of the disease is uncertain, their accurate identification is important for epidemiologic and treatment purposes. Therefore, the aims of this study were: (1) to identify these bacteria in patients with bronchiectasis without cystic fibrosis using the protected catheter brush (PCB) in order to avoid oropharyngeal contamination, and (2) to compare the results of bronchoalveolar lavage (BAL) with PCB. Quantitative culture was performed on PCB and BAL specimens obtained from the most severely affected lobes of 23 patients with bronchiectasis. Results of PCB showed no significant growth (less than 10(3) colony-forming units [cfu]/ml) in nine patients and 17 significant isolates (greater than 10(3) cfu/ml) in the rest: H. influenzae, 5; P. aeruginosa, 4; K. ozaenae, 2; S. aureus, 2; P. fluorescens, 1; S. pneumoniae, 1; Veillonella, 1; and coag.-ve Staph., 1. For BAL, the results were the same (20 isolates) regardless of whether 10(4) or 10(5) cfu/ml was chosen as the cutoff point. More organisms were cultured from BAL specimens, and these included all but one of the organisms cultured from PCB. We conclude that the bacteriology of bronchiectasis in Hong Kong is different from that reported in sputum studies in the West (mainly H. influenzae, S. pneumoniae, and S. aureus), and with 10(4) cfu/ml as the cutoff point, BAL gives comparable results to PCB.     

Spontaneous interleukin 2 release of bronchoalveolar lavage cells in sarcoidosis is a codeterminator of prognosis

There is mounting evidence that activated interleukin 2 (IL-2)-releasing lymphocytes play a central role in the immunopathogenesis of sarcoidosis by directing inflammatory reactions and granuloma formation. In the context that a significant proportion of these cells accumulates in the lung and releases mediators, we hypothesized that different immunologically defined stages of sarcoidosis can be identified. A cohort of 89 sarcoidosis patients was allocated to four groups according to the following criteria: stage A, a low number of bronchoalveolar lavage (BAL) lymphocytes (<20%) without IL-2 release (<1 unit/ml in BAL cell culture supernatant); stage B, BAL lymphocytes <20%, with IL-2 release (1 unit/ml); stage C, BAL lymphocytes 20% with IL-2 release; and stage D, 20% BAL lymphocytes without IL-2 release. Although patients of stages C and D (n = 49) exhibited lymphocytic inflammation, only 20/49 of these patients had activated IL-2-releasing alveolar lymphocytes. BAL of groups A and B showed a low number of lymphocytes, but the lymphocytes were activated in 20/40 patients. Forty-four patients not receiving therapy were reevaluated by pulmonary function tests 8 ± 1 months after BAL. Progressive disease was found in 9/12 patients of group C and stable or regressing disease in 13/13 patients of group A. These results demonstrate that a combination of BAL parameters can yield prognostic information. Offprint requests to: Joachim Müller-Quernheim     

Quantitative bacterial cultures of bronchoalveolar lavage fluids and protected brush catheter specimens from normal subjects

Bronchoalveolar lavage (BAL) is quite useful in the diagnosis of nonbacterial lung infections, especially in immunocompromised patients, and recent studies have suggested that BAL may be useful in the diagnosis of bacterial pneumonia as well. Because previous studies indicated that bronchoscopic aspirates are usually contaminated by oropharyngeal flora, we anticipated that BAL fluid would also likely be contaminated. Therefore, the purpose of this study was to perform quantitative bacterial cultures on BAL fluids obtained from eight normal subjects. Prior to each procedure, saline was aspirated through the bronchoscope and submitted for culture. A protected brush catheter (PBC) specimen was obtained from each subject's right middle lobe, and then a BAL specimen was obtained from the same location. All specimens were quantitatively cultured for aerobic and anaerobic organisms. In addition, lidocaine concentrations were measured in the BAL fluids and the PBC specimens. Six of the eight bronchoscope cultures were sterile. Seven of the eight PBC specimens were sterile and one yielded less than 10(3) cfu/ml of normal oropharyngeal flora. One BAL fluid specimen was sterile and seven yielded from one to four bacterial strains each; however, quantitation revealed less than 10(4) cfu/ml in all specimens. Lidocaine concentrations (mean +/- 1 SD) were as follows: PBC specimen, 0.81 microgram/ml (+/- 0.62); BAL fluid specimen, 62.6 micrograms/ml (+/- 43). We conclude that BAL fluid obtained from normal subjects is frequently contaminated by oropharyngeal bacterial flora.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnostic value of bronchoalveolar lavage in patients with opportunistic and nonopportunistic bacterial pneumonia

Summary In 29 patients with community-acquired pneumonia, 24 patients with hospital-acquired pneumonia and 35 patients with pneumonia in the immunocompromised host the diagnostic value of bronchoalveolar lavage (BAL) with quantitative bacterial and fungal cultures was studied; 32 patients with noninfectious pulmonary diseases and 14 healthy volunteers served as controls. An infectious etiology could be established in 81% of the pneumonia patients without differences between the three groups; significant infection was associated with colony counts of 10⁴ cfu/ml. Prior antibiotic therapy lowered the yield of BAL culture only in community-acquired pneumonia (94%vs 55% positive cultures in untreatedvs pretreated patients, p<0.02). Furthermore the culture results were related to the radiographic extension of pulmonary infiltrates (92% positive cultures in multilobarvs 54% in lobar or segmental infiltrates, p<0.001). Therapeutic consequences of BAL were shown by resistance of the isolated organisms to predefined empiric treatment regimens in 41% community-acquired pneumonia, 43% pneumonia in the immunocompromised host and 67% hospital-acquired pneumonia patients.

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Diagnostischer Wert der bronchoalveolären Lavage bei Patienten mit opportunistischer und nicht opportunistischer bakterieller Pneumonie

Zusammenfassung Bei 29 Patienten mit ambulant erworbener Pneumonie, 24 Patienten mit hospital-erworbener Pneumonie und 35 Patienten mit Pneumonie unter Immunsuppression wurde der diagnostische Wert der bronchoalveolären Lavage mit quantitativem Erregernachweis untersucht; 32 Patienten mit nichtinfektiösen Lungenerkrankungen und 14 gesunde Probanden dienten als Kontrollen. Die Diagnose konnte in 81% der Pneumoniepatienten gesichert werden, ohne signifikante Unterschiede zwischen den Gruppen. Eine manifeste Infektion war mit Keimzahlen 10⁴ cfu/ml korreliert. Eine vorausgegangene antibiotische Therapie verminderte die Ausbeute der Lavagekultur nur bei ambulant erworbener Pneumonie (94%vs. 55% positive Kulturen in unbehandeltenvs. vorbehandelten Patienten, p<0,02). Darüber hinaus waren die Kulturergebnisse von der röntgenologischen Ausdehnung der Infiltrate abhängig (92% positive Kulturen bei lappenübergreifendenvs. 54% bei lappen- oder segmentbegrenzten Infiltraten, p<0,001). Therapeutische Konsequenzen der BAL ergaben sich durch Resistenz der kultivierten Erreger gegenüber der vorab definierten empirischen Chemotherapie in 41% (ambulant erworben) bzw. 43% (Immunsuppression) und 67% (hospitalerworbene Pneumonie).

     

Serum HCV RNA levels correlate with histological liver damage and concur with steatosis in progression of chronic hepatitis C

The role of HCV RNA levels and host factors in the severity of liver injury was studied. Enrolled were 298 consecutive liver biopsy-proven chronic hepatitis (CH) C patients (179 men; median age: 52 years, range 19–68; CH, 198; cirrhosis, 100) and 18 chronic hepatitis C with normal ALT. HCV genotypes were: 1a, 4.3%; 1b, 53%; 2a/c, 28%; 3a, 7%; 4, 1.3%, and mixed 6.4%. Serum HCV RNA levels were similar for all genotypes (median: 2.8 × 10⁶ eq/ml; range <0.2–69). In patients with chronic hepatitis without cirrhosis, the serum HCV RNA levels reflected the grade of liver necroinflammatory activity (R = 0.45; P < 0.001) and the stage of fibrosis (R = 0.51; P < 0.001), regardless of age, gender, HCV genotype, hepatic steatosis, and hepatic iron overload. Patients with high serum HCV RNA levels (3 × 10⁶ eq/ml) had higher ALT values (P < 0.002) than those with lower HCV RNA levels. Patients with normal ALT showed low HCV RNA levels (median: 0.82 × 10⁶ eq/ml) and histological features of minimal or mild chronic hepatitis. Cirrhotic patients showed significantly lower levels of viremia than those with chronic hepatitis with a similar HAI. The data of a subgroup of 62 patients with an established time of infection showed that for a similar duration of disease, patients with serum HCV RNA levels 3 × 10⁶ eq/ml had a significantly higher fibrosis score than those with lower levels. HAI and fibrosis score were significantly higher in patients with HCV RNA levels 3 × 10⁶ eq/ml and grade 3–4 steatosis than those with lower HCV RNA levels and steatosis grades. The data indicate that the liver damage is correlated with the HCV RNA levels and that a high viral load acts together with steatosis in accelerating the progression of liver injury.     

Diagnosing bacterial respiratory infection by bronchoalveolar lavage

We prospectively evaluated 75 patients by fiber-optic bronchoscopy and bronchoalveolar lavage (BAL) for the presence of bacterial lower-respiratory-tract infection. BAL specimens were cultured quantitatively for aerobic bacteria, and a cell differential was obtained of the BAL cell population. In 18 "control" patients without evidence of respiratory infection, the presence of greater than 1% squamous epithelial cells (SECs) in the BAL sample accurately predicted the presence of heavy contamination of the sample by oropharyngeal flora. In the remaining "study" patients with potential infection, polymorphonuclear leukocytes were readily identified, and potential lower-respiratory-tract pathogens were recovered in concentrations greater than 10(5) colony-forming units (cfu) per milliliter in 16 of 18 patients with bacterial infection (none had greater than 1% SECs in their BAL sample). No patients without evidence of bacterial infection and with less than or equal to 1% SECs had greater than 10(5) cfu/ml in BAL cultures. These studies establish the ability of BAL techniques to diagnose bacterial respiratory infection.     

Defining lower airway bacterial infection in children with chronic endobronchial disorders

Background

Differentiating lower airway bacterial infection from possible upper airway contamination in children with endobronchial disorders undergoing bronchoalveolar lavage (BAL) is important for guiding management. A diagnostic bacterial load threshold based on inflammatory markers has been determined to differentiate infection from upper airway contamination in infants with cystic fibrosis, but not for children with protracted bacterial bronchitis (PBB), chronic suppurative lung disease (CSLD), or bronchiectasis.

Methods

BAL samples from children undergoing bronchoscopy underwent quantitative bacterial culture, cytologic examination, and respiratory virus testing; a subset also had interleukin‐8 examined. Geometric means (GMs) of total cell counts (TCCs) and neutrophil counts were plotted by respiratory pathogen bacterial load. Logistic regression determined associations between age, sex, Indigenous status, antibiotic exposure, virus detection and bacterial load, and elevated TCCs (>400 × 10³ cells/mL) and airway neutrophilia (neutrophils >15% BAL leukocytes).

Results

From 2007 to 2016, 655 children with PBB, CSLD, or bronchiectasis were enrolled. In univariate analyses, Indigenous status and bacterial load ≥10⁵ colony‐forming units (CFU)/mL were positively associated with high TCCs. Viruses and bacterial load ≥10⁴ CFU/mL were positively associated with neutrophilia; negative associations were seen for Indigenous status and macrolides. In children who had not received macrolide antibiotics, bacterial load was positively associated in multivariable analyses with high TCCs at ≥10⁴ CFU/mL and with neutrophilia at ≥10⁵ CFU/mL; GMs of TCCs and neutrophil counts were significantly elevated at 10⁴ and 10⁵ CFU/mL compared to negative cultures.

Conclusions

Our findings support a BAL threshold ≥10⁴ CFU/mL to define lower airway infection in children with chronic endobronchial disorders.

     

Bacterial colonisation of the respiratory tract in chronic bronchitis

One hundred and nine subjects with chronic bronchitis were studied prior to winter and without clinical infection, to determine baseline patterns of bacterial colonisation. Qualitative analysis of cultures of oropharyngeal swabs showed little difference from age matched normal controls (17) except for growth of small numbers of Gram negative coliforms in the chronic bronchitic group. Quantitation of bacteria colonising the oropharynx showed small numbers (mean of 10⁵ cfu/ml), with no particular bacteria dominating. Haemophilus influenzae was present in 7.3% of throat swabs from chronic bronchitic patients, but the organism was always less than 10% of the total count. Quantitation of bacteria in sputum showed significantly higher numbers (mean 10⁷ cfu/ml). H. influenzae was detected in 25.7% of available specimens, and when present constituted > 90% of the total count. Biotyping of H. influenzae isolates demonstrated a separate colonisation of the upper and lower respiratory tracts.     

Bronchoalveolar lavage quality influences the T4/T8 ratio in sarcoidosis

BACKGROUND: Sarcoidosis is characterised by a T-lymphocytic alveolitis with a typically increased T4/T8 ratio. The diagnostic value of this ratio is under debate. AIM OF THE WORK: We prospectively evaluated the influence of BAL pre-lavage and the impact of bronchial contamination on BAL differential cell count in 108 BAL specimens obtained from patients with histologically confirmed sarcoidosis. METHODS: BAL was performed by instilling 150-300 ml normal saline either in the middle lobe or the lingula. Fifty-one patients (47%) underwent additional pre-lavage with 50 ml normal saline. Bronchial contamination was assessed by semi-quantitative analysis of mucus, ciliated and squamous cells in the untreated BAL recovery. RESULTS: Pre-lavage did neither influence the lavage cellularity nor extend of contamination of the BAL. Content of mucus and ciliated cells, indicating bronchial contamination, showed a high correlation (Kendal's tau=0.61). Presence of either mucus or ciliated cells in the BAL recovery was associated with a significant lower T4/T8 ratio (mucus: 4.9 vs. 8.0, p=0.009; ciliated cells: 4.1 vs. 7.4, p=0.001). Squamous cells in the BAL recovery representing oropharyngeal contamination did not significantly influence the T4/T8 ratio (7.7 vs. 5.6, p=0.10). CONCLUSION: Bronchial contamination of BAL as determined by the presence of mucus and ciliated cells in the recovery decreases the T4/T8 ratio of BAL in sarcoidosis.     

The Normal Cultivable Microflora in Upper Jejunal Fluid in Healthy Adults

Bacteriological studies of uncontaminated upper jejunal fluid were performed in 85 normal subjects. Fifty-three per cent of the samples were sterile (<10¹ CFU/ml). In 10% of the cases the total number of microorganisms exceeded 10⁵ CFU/ml. The main groups of microorganisms isolated were Streptococcus sp (‘Viridans group’), Lactobacillus sp., Veillonella parvula, Actinomyces sp., Haemophilus sp., Corynebacterium sp., and Candida albicans, each found in more than 10% of the subjects. Only the Streptococcus sp. exceeded 10⁵ CFU/ml, and enterobacteria were found in 5% of the subjects, the number not exceeding 10³ CFU/ml. No other typical members of the lower gastrointestinal tract were isolated. The number of subjects harbouring bacteria and the distribution of bacterial species were the same in both sexes and in different age groups.     

Quantitative culture of bronchoalveolar lavage fluid in community-acquired lower respiratory tract infections.

To evaluate the diagnostic value of quantitative bacterial culture of bronchoalveolar lavage (BAL) fluid obtained by fibreoptic bronchoscopy, 67 consecutive immunocompetent adult patients admitted to hospital with community-acquired lower respiratory tract infections from September 1997 to May 1998 were investigated. Results were compared to the findings in eight healthy control persons investigated in February 1998. There was no difference between study patients and control persons when quantitative culture of total cumulative bacterial findings or bacteria categorized as members of the oropharyngeal normal flora were compared. The culture of normal flora in bronchial washings probably reflects contamination of the lower airways with secretions from upper arways by the fibreoptic procedure itself, as fractionated sampling showed a 10-fold reduct on in quantitative culture results when a primary bronchial washing was compared to a secondary sampling from the same bronchus in the control group. Twenty-four (36%) of 67 patients were cultured as positive in the study group while all control persons were cultured as negative for bacteria categorized as potential pathogens. With a threshold value for positive culture of 10(4) cfu ml(-1) the specificity of lavage culture of potential pathogenic bacteria in relation to actual lower airway infection was 100%. Therefore, quantitative bacterial culture of potential pathogenic bacteria in BAL fluid is very specific but only positive in about one-third of unselected immunocompetent adult patients with a lower respiratory tract infection.     

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria

Background Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion‐Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion‐Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. Material and Methods Four Bacteria References (Staphylococcus epidermidis PEI‐B‐06, Streptococcus pyogenes PEI‐B‐20, Klebsiella pneumoniae PEI‐B‐08 and Escherichia coli PEI‐B‐19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. Results Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19–1·32 × 10⁷ CFU/ml, S. pyogenes: 0·58–0·69 × 10⁷ CFU/ml, K. pneumoniae: 18·71–20·26 × 10⁷ CFU/ml and E. coli: 1·78–2·10 × 10⁷ CFU/ml. Conclusion The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low‐titre spiking of blood components, (ii) the property of donor‐independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion‐Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.     

Isoniazid Levels in the Bronchoalveolar Lavage Fluid of Patients with Pulmonary Tuberculosis

Isoniazid (INH) is one of the most important first line drugs in the treatment of tuberculosis. We utilized high performance liquid chromatography with a hydrazone extraction technique to measure INH in bronchoalveolar lavage (BAL) fluid specimens from six patients with active pulmonary tuberculosis. We found BAL fluid INH levels to be similar to 2-h peak serum levels. The concentration of INH in BAL fluid from lobes with infiltrate was similar to the concentration of INH in BAL fluid from lobes without infiltrate (0.062 μg/ml and 0.073 μg/ml, respectively). After adjusting for protein concentration in the BAL fluid, INH levels in lobes with infiltrate were threefold lower than in lobes without infiltrate. The correlation between the concentration of INH in serum and BAL fluid approached significance after correcting for protein (lobes with infiltrate, r ²= 0.60 (p= 0.07); lobes without infiltrate, r ²= 0.50 (p= 0.12)). INH penetrates into bronchoalveolar fluid, and concentrations of INH in the BAL fluid suggest that assessment of the INH serum concentration is adequate to evaluate bioavailability of the drug in patients with pulmonary tuberculosis. Accepted for publication: 26 June 1997     

Distribution and transmission of Pseudomonas aeruginosa and Burkholderia cepacia in a hospital ward

Genotyping and antibiotic susceptibility testing were used to analyze Pseudomonas aeruginosa and Burkholderia cepacia strains from sink drain from 14 pediatric patients with cystic fibrosis (CF) and from hospital personnel as part of a 4 week prospective study of strain transmission in a pediatric ward. A total of 87.5% of all washbasin drains were contaminated with P. aeruginosa [10² to 10⁵ colony forming units (CFU)/ml sink fluid], whereas B. cepacia was found only once in a sink drain. From the eight CF patients already infected with P. aeruginosa upon entering the ward, we isolated six genotypes that were identical with strains found in sink drains of the ward. Four of the 16 members of the personnel had one positive P. aeruginosa hand culture. B. cepacia was never found in patients or on personnel hands. Hand washing in contaminated sinks (≥ 10³ CFU/ml) led to positive P. aeruginosa or B. cepacia hand cultures. P. aeruginosa or B. cepacia embedded in sputum were transmissable by hand shaking for up to 180 min, whereas both pathogens suspended in physiological saline were transmissable to other hands only up to 30 min. Genotyping of P. aeruginosa revealed strain transmission from CF patients or the environment to other patients or the personnel, as well as one transmission from the environment to a CF patient. The ability of CF sputum to prolong survival of P. aeruginosa and B. cepacia may be important for strain transmission. The results suggest that improved hygienic measures are required to prevent routes of bacterial transmission via the hands and sink drains. Pediatr Pulmonol. 1996; 21:90–100. © 1996 Wiley-Liss, Inc.     

Detection of Pneumocystis jirovecii by Two Staining Methods and Two Quantitative PCR Assays

Abstract Background:    Pneumocystis jirovecii is an opportunistic pathogen that causes pneumonia, particularly in immunodeficient hosts. Materials and Methods:   We retrospectively compared the results obtained by two staining methods (toluidine blue and calcofluor white) and two quantitative (q) real time PCR assays for the detection of P. jirovecii in bronchoalveolar lavage (BAL) specimens. For the qPCR assays, we used newly selected probes and primers targeting the Kex-1 gene, which codes for a serine endoprotease, and compared the results to those from the published assay targeting the β-tubulin gene. Results:   A total of 1,843 BAL specimens were analyzed microscopically in parallel, and 74 (4.0%) were found to be positive with both stains, 23 (1.2%) were positive only with the toluidine blue stain, and six (0.3%) only with the calcofluor stain (p = 0.003). Of these, a selection of 186 consecutive BAL fluid samples were tested by qPCR using the respective different primer pairs. 21 of the 186 samples (11.3%) were microscopically positive with both stains as well as qPCR positive after 18–31 cycles (corresponding to 5.24 × 10⁶ copies/ml to 640 copies/ml of native BAL) using the Kex-1 primer pair and between 21–33 cycles using the β-tubulin assay. A good correlation between semi-quantitative microscopy and the number of PCR cycles needed for a positive signal was noted. Of the remaining 165 samples, 153 (82%) were both microscopically and PCR negative (PCR with the two sets of primers); the remaining 12 samples (7%) were Kex-1-based PCR positive (from cycles 33 to 41, corresponding to 160 copies/ml of BAL or less) but microscopically negative. Of these latter samples, ten (6%) were also positive (from cycles 34 to 38) with the primers targeting the β-tubulin gene. Taking microscopy as a reference, the sensitivity of qPCR targeting the Kex-1 gene was 100%, and the specificity was 92.4%. Conclusion:   The sensitive qPCR analysis proved to be a rapid and reliable method to detect P. jirovecii in BAL.     

Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates

Background Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT‐PCR) was evaluated in an inter‐laboratory comparison in three different German blood services. Methods Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10³–4·5 × 10⁸ CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. Results The inter‐laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT‐PCR‐screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non‐detected positive samples were below the assay’s detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10⁵ and 2·10 × 10⁷ CFU/ml, respectively, K. pneumoniae: 4·79 × 10⁶ CFU/ml, S. aureus: 6·03 × 10⁵ CFU/ml). All rapid screening methods revealed no false‐positive results. Conclusions Both BactiFlow and 23S rRNA RT‐PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram‐negative strains.     

Pigment vs cholesterol cholelithiasis

Although pigment (calcium bilirubinate) gallstones in Japanese subjects are associated with bacterial infection, the role of infection in Americans with pigment gallstones has not been assessed. Anaerobic and aerobic cultures of gallbladder bile, stone, and wall were obtained at cholecystectomy from nine patients with pigment stones and 25 with cholesterol stones. Among pigment-stone subjects, only 1 of 9 grew organisms in greater than 10⁵ colony-forming units (CFU)/ml or g in gallbladder bile or wall. Likewise, growth greater than 10⁵ CFU/ml or g was present in only 1 of 26 biles and 2 of 26 walls from cholesterol-stone patients.Propionibacterium acnes was found in less than 10⁵ CFU/g or ml in at least 1 specimen from 6 of 9 pigment- and 12 of 26 cholesterol-stone patients. This organism was considered a contaminant because propionic acid concentrations in bile, an index of active bacterial metabolism, were similar in specimens with or without low-titer growth. The concentrations of bile salts, phospholipids, cholesterol, and bilirubin in gall-bladder bile was unaffected by the type of bacteria in low-titer growth. But the lipid concentrations were markedly depressed in two biles with bacterial growth greater than 10⁵ CFU/ml. The molar ratio of bile salts and phospholipids to cholesterol was significantly higher in biles surrounding pigment stones than those surrounding cholesterol stones (P<0.01). We conclude that significant bacterial infection (>10⁵ CFU/ml) is not associated with pigment or cholesterol stones in asymptomatic American subjects at cholecystectomy. These data suggest that pigment-stone formation in the United States is not primarily related to bacterial alteration of bile composition, as the experience with Japanese patients would suggest.Supported by NIH Grant AM-16549 and institutional funds of the Veterans Administration.Presented in part at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, May 8–13, 1977.