Department of Anaesthesiology, Bicêtre Hospital, Assistance Publique-Hôpitaux de Paris, F-94275 Le Kremlin Bicêtre, France
Accepted for publication: 26 June, 2000
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Abstract |
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Br J Anaesth 2000; 85: 7359
Keywords: intensive care; infection, nosocomial; complications, ventilator-associated pneumonia
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Introduction |
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The present study was undertaken to evaluate prospectively whether the Gram stain of PSB or PTC predicts the results of quantitative cultures and can guide the choice the treatment of pneumonia. We also studied the possible improvement of the accuracy of Gram staining by analysing more fields or by having two independent observers analyse the stains.
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Patients and methods |
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One hundred and eight protected pulmonary specimens were taken from patients who had not received antimicrobial therapy during the previous 3 days, and 78 protected pulmonary specimens were taken from patients who had received antibiotics (for more than 72 h). No patient received local antimicrobial therapy or digestive decontamination with antibiotics.
Specimen collection
We obtained 126 PTC and 60 PSB samples after careful endotracheal suctioning and without topical anaesthesia.3 The choice of one of these two techniques was left to the physician in charge of the patient. During the procedures, 100% oxygen was given and the patients were sedated with midazolam; pancuronium was used to achieve neuromuscular block. Heart rate, arterial pressure and arterial oxygen saturation were monitored during the investigation.
Blinded PTC samplings were performed by the previously described standardized technique.4 The PTC unit was composed of an outer catheter sealed by a polyethylene glycol plug and an inner catheter for sampling. After blind introduction of a PTC unit into the bronchial system, the inner catheter was advanced, extruding the plug, and three brief aspirations were then applied to the inner catheter with a 10-ml syringe connected to its proximal port. After the catheter had been retracted into the outer sheath, the entire unit was removed from the patient; the distal end of the outer catheter was dried and then cut off with sterile scissors, the inner catheter was advanced, and 1 ml of saline was flushed through its proximal port and collected into a sterile vial. Finally, the distal segment of the catheter was transected and collected into the same vial. For the PSB, a bronchoscope (Olympus BF P 20D; Olympus Optical Corporation of America, New Hyde Park, NY, USA) was introduced through an adapter (Bodai Suction Safe Y; Sontek Medical, Lexington, MA, USA) and advanced to the bronchial orifice, selected according to the radiographic position of the new pulmonary infiltrate. After brushing, the tip of the PSB was cut aseptically and dropped into a sterile glass vial containing 1 ml of saline, according to a standard technique.3 All the specimens were taken to the laboratory within 15 min of collection.
Microbiological processing
Microscopic examination
From the original suspension, 0.2-ml aliquots were dropped into a Cytospin and centrifuged at 300 g for 10 min. The slides were Gram-stained and independently examined twice (by A.K. and S.L.) at high magnification (x100) without knowledge of the quantitative culture results. The presence of microorganisms was looked for on 10 and 50 fields and any microorganisms found were classified according to their Gram stain morphology.
Quantitative cultures
Each vial was mechanically vortexed for 60 s. Two successive 1:100 saline dilutions were prepared. Aliquots (0.1 ml) of the original suspension and of each dilution were plated onto different media for quantitative culture and identification: fresh blood agar, fresh blood agar to which nalidixic acid, amphotericin B and colistin had been added, Drigalski agar, chocolate agar and anaerobic blood agar (BioMérieux, La Balme-les-Grottes, France). All samples were incubated for 48 h in an appropriate atmosphere and the microorganisms recovered were identified by standard methods and their number expressed as c.f.u ml1. The cut-off value of protected pulmonary specimens for the diagnosis VAP was 103 c.f.u. ml1.1
Statistical analysis
Data are expressed as medians and ranges. To assess the degree of qualitative correlation between Gram staining and quantitative cultures, the results of Gram staining were divided into four categories. Gram staining was considered to be true-positive if each Gram stain morphotype present also grew at significant concentration (i.e. 103 c.f.u. ml1), true-negative if no organism was present on Gram staining and quantitative culture was either sterile or non-significantly positive (i.e. <103 c.f.u. ml1), false-positive if a morphotype present on Gram staining was not grown in significant concentration, and false-negative if a microorganism grew at significant concentration but was not present on Gram staining. For mixed cultures, all the bacterial isolates grown at significant concentration had to be morphologically present on Gram staining. If not, the case was classified as false-negative. Similarly, the case was classified as false-positive if all the morphotypes present on Gram staining did not grow at significant concentration. Sensitivity, specificity and predictive value were calculated.5 The Spearman rank test was used to assess the relationship between the number of bacilli and/or cocci seen on Gram staining and the number of microorganisms subsequently growing on appropriate culture medium; P<0.05 was considered to be statistically significant. The
statistic was used to measure the agreement between the two observers.6 A
value of 0 indicates no agreement beyond chance, whereas a
value of 1 indicates perfect agreement.
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Results |
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Eighty-one (44%) protected pulmonary specimens (54 PTC and 27 PSB) had a positive culture, including 17 specimens with two (11 PTC and six PSB) or three (one PSB) microorganisms with different Gram stain morphologies. Microorganisms isolated at significant concentration were 24 Staphylococcus aureus, 1 isolate of coagulase-negative staphylococci, 7 Streptococcus spp., 10 Streptococcus pneumoniae, 1 Neisseria spp., 9 Escherichia coli, 14 Haemophilus influenzae, 2 Proteus spp., 1 Klebsiella spp., 4 Enterobacter spp., 3 Serratia spp., 19 Pseudomonas aeruginosa, 1 Acinetobacter spp., 1 Stenotrophomonas maltophilia, 2 Branhamella catarrhalis, and 1 Bacteroides spp. Ten patients (12%) died rapidly and the remaining 71 recovered from their bacterial pneumonia while receiving appropriate antibiotic therapy for organisms cultured at a significant growth in protected pulmonary specimens. The causes of death were overwhelming sepsis (n=2), multiple organ failure of unknown origin (n=5), brain death (n=2) and refractory hypoxaemia (n=1). Two (6%) of the 31 patients with a non-significant positive protected pulmonary specimen developed bacterial pneumonia (defined as a protected pulmonary specimen culture significantly positive and a clinical outcome consistent with pneumonia while receiving appropriate antibiotic therapy) caused by the same organisms 4 and 5 days later. None of the patients with a sterile protected pulmonary specimen culture later developed bacterial pneumonia during the 5 days after their inclusion in the study.
When 10 fields were analysed, a strong relationship was found between the presence of bacteria on Gram staining and the final diagnosis of VAP (Table 1). The ability of PTC Gram staining to predict VAP seemed better than that of PSB Gram staining, although the difference was not significant. Increasing the number of fields read to 50 was associated with a slight decrease in specificity and in the positive predictive value of the Gram stain, but with a small increase in its sensitivity and negative predictive value (Table 2). Whatever the number of fields read, the agreement between the two observers was excellent (=0.88 and 0.82 for 10 and 50 fields respectively).
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Discussion |
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When invasive sampling is used, bacterial growth in the specimens is quantified and the presence of pneumonia and the identity of the causative pathogen(s) are defined by the recovery of bacteria at a concentration above a predetermined threshold (103 c.f.u. ml1).7 Results are delayed until the quantitative cultures are available, and initial therapy is therefore empirical. Previous studies found that Gram staining of protected specimens had low sensitivity, prompting several authors to use microscopic analysis of bronchoalveolar lavage material along with quantitative cultures of protected specimens.9 10 The use of two invasive techniques in combination increases the cost, carries an additional risk for the patient and increases the difficulty and the work-load of the laboratory.
The present study shows that Gram staining of protected pulmonary specimens correctly predicts the presence of VAP and may partially identify (using Gram stain morphology) the microorganisms subsequently growing at significant concentrations. Our results support previous studies in which Gram staining was performed on slides prepared directly from the brushes. Teague et al. used two brushes, one for Gram staining and the other for quantitative culture, and found a marked correlation between Gram staining and the presence of bacteria at a concentration of 103 c.f.u. ml1.11 Similar results were reported by Pollock et al. when the brush was smeared directly onto a glass slide before the brush was placed in the holding medium.12 In contrast, early studies using Gram staining on centrifuged specimens reported low sensitivity for the method.4 9 13 More recently, Marquette et al. reported 85% sensitivity and 94% specificity for cytocentrifuged Gram staining.14 The authors said they obtained good results because they first screened for bacteria using May-Grünwald Giemsa-stained slides at high magnification before performing (if these stains were positive) Gram staining on slides to determine the precise morphology of the bacteria detected. We believe that our results may be accounted for by several factors: cytocentrifuged preparations were made with a high, calibrated volume for the initial dilution (200 µl, i.e. 20% of the total dilution volume), and Acinetobacter spp., which may be detected either as Gram-negative coccobacilli or as Gram-positive cocci depending on its state of development, were rarely isolated. However, increasing the number of fields read or having the Gram staining analysed by two independent individuals were both time-consuming and did not usefully improve accuracy. Our results agree partially with those obtained previously when bronchoalveolar lavage was used to diagnose bacterial pneumonia. In a study reported recently in this journal, Gram staining of bronchoalveolar lavage was found to be useful for the rapid diagnosis of ventilator-associated pneumonia, but was not reliable for the early adaptation of empirical therapy.15 By contrast, the role of Gram staining of tracheal aspirates was not useful in the diagnosis of ventilator-associated pneumonia or in guiding empirical therapy.16
Our study has several limitations. First, the effects on patient outcome and antibiotic management in relation to the data collected were not evaluated because Gram staining was generally performed many days after sampling and without knowledge of the results of quantitative cultures. Secondly, the performance of Gram staining of protected pulmonary specimens was not compared with results obtained by bronchoalveolar lavage or tracheal aspirates. However, it was not our policy to use such sampling methods routinely to investigate patients with suspected nosocomial pneumonia. The use of both invasive techniques in combination is expensive and time-consuming and carries an additional risk for the patient. Finally, PSB and PTC were considered as reference methods of diagnosing VAP. Until recently, lung histology has been considered the gold standard in this situation. However, when 39 open lung biopsies obtained from dead patients were reviewed independently by four pathologists, the prevalence of histological VAP, as determined by each of the pathologists, varied from 18 to 38%.17 Histological features of VAP were found in seven of nine organ donors who had no clinical evidence of pulmonary infection and who were not receiving antibiotic therapy.18 Finally, studies have found discrepancies between histology and bacteriological cultures. Neither the bacterial densities from the four quantitative airway cultures nor the bacterial density from the quantitative culture of lung parenchyma separated the histological pneumonia and non-pneumonia groups accurately.19
In summary, Gram staining of protected pulmonary specimens performed on 10 fields predicted the presence of VAP and partially identified (using Gram stain morphology) the microorganisms growing at significant concentrations. Further studies on antibiotic management and patient outcome, based on this method, are warranted.
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Footnotes |
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References |
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