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Value of washed sputum gram stain smear and culture for management of lower respiratory tract infections in children
J Infect Chemother (2004) 10:31–36 DOI 10.1007/s10156-003-0277-z
© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2004
ORIGINAL ARTICLE Luong Dong Cao · Naruhiko Ishiwada · Nobue Takeda Yukiko Nigo · Jirou Aizawa · Haruo Kuroki Yoichi Kohno
Value of washed sputum gram stain smear and culture for management of lower respiratory tract infections in children
Received: February 20, 2003 / Accepted: September 3, 2003
Abstract To date, the technique of washed sputum examinations has not been widely used in the clinical management of lower respiratory tract infections in children. A total of 224 sputum samples from 125 pediatric patients with lower respiratory tract infections were collected for washed sputum Gram stain smears and cultures. The results with these methods were compared to find correlation rates. The value of washed sputum cultures was assessed by examining the clinical responses of the patients who received antibiotic therapies instituted on the basis of the sputum culture results. Isolation rates of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus were 22.4%, 9.4%, 4.9%, and 0.4%, respectively. For the prediction of H. influenzae, S. pneumoniae, and M. catarrhalis, the sensitivities of the washed sputum Gram stain smears compared with the culture method were 86.0%, 81.0%, and 90.9%, respectively. The specificities of the washed sputum Gram stain smear technique were 94.8%, 97.5%, and 98.1%, respectively. Overall, the sensitivity and specificity of the washed sputum Gram stain smear method were 85.5% and 87.2%, respectively. S. aureus was isolated from only one specimen; and washed sputum Gram stain smear estimation was correlated with the culture result. On the basis of the washed sputum culture results, appropriate antibiotic therapies were instituted for 93.3% of the patients with acute lower respiratory tract infections. This study suggests that the techniques of washed sputum Gram stain smear and culture are valuable and should be encouraged in clinical practice for the management of lower respiratory tract infections in children.
D.C. Luong · N. Ishiwada (*) · N. Takeda · Y. Nigo · J. Aizawa · H. Kuroki · Y. Kohno Department of Pediatrics, Chiba University, Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Tel. ⫹81-43-226-2144; Fax ⫹81-43-226-2145 e-mail: [email protected]
Key words Haemophilus influenzae · Gram stain smear · Washed sputum culture · Respiratory tract infection · Children
Introduction Pediatric lower respiratory tract infections (LRTI) are common causes of morbidity and mortality worldwide. Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis, the major bacterial etiologic agents causing these infections, are responsible for cases of acute bronchitis, chronic bronchitis, otitis media, sinusitis, and pneumonia. The management of LRTI is remarkably simplified when the responsible pathogens are accurately identified. In addition, early recognition and prompt institution of initial appropriate therapy may offer the best chance to reduce mortality. Clinical signs, symptoms, and host responses (erythrocyte sedimentation rate [ESR], white blood cell count [WBC], and C-reactive protein [CRP]) may not provide reliable evidence of bacterial respiratory infections.1,2 Therefore, microbiological methods are considered to be necessary for the detection of the real bacterial pathogens. Conventionally, physicians and bacteriologists have accepted that microbiologic examinations of expectorated sputum (ES) be performed for identifying the bacterial pathogens of LRTI, because sputum is always available, and can be obtained easily and noninvasively. However, the sputum culture findings are usually not available within 18 to 24 h, and so antibiotic therapies instituted on the basis of these findings would be delayed. Therefore, ES Gram stain smears have been employed for the rapid prediction of the bacterial pathogens of LRTI, with the aim of quickly guiding the initial antibiotic choice. The advantages of this method were that it could be completed within several minutes, did not require sophisticated equipment, and was inexpensive.3–5 Unfortunately, the diagnostic yields of sputum Gram stain smear and culture were limited, because of potential contamination from the upper respiratory tract.3–8 For this reason, previous studies have con-
32
sidered an ES washing technique as one of the useful means for the improvement of sputum microbiologic examinations.9–13 However, several studies have still questioned the reliability of washed sputum Gram stain smear and culture for accurately predicting the bacterial pathogens of LRTI.3,5–8 Moreover, few studies have been carried out to evaluate the usefulness of washed sputum Gram stain smear and culture in the clinical management of pediatric patients with LRTI, because the collection of a satisfactory ES sample in children seems to be more difficult than in adults. Taking these factors into consideration, we performed the present study to evaluate the reliability of the washed sputum Gram stain smear technique for the rapid estimation of bacterial pathogens of LRTI, for the purpose of quickly guiding the initial antibiotic choice. The present study also aimed to assess the value of the washed sputum culture method in the institution of appropriate antibiotic therapy for pediatric patients with LRTI.
Patients and methods We studied 224 samples of ES obtained from 224 identical clinical episodes in 125 pediatric patients with LRTI at the Department of Pediatrics of Chiba University Hospital and other hospitals in Chiba prefecture between May 1999 and December 2000. Of the 125 patients, 115 were outpatients and 10 were hospitalized. Within 2–3 h of sampling, each sample was consecutively washed three times in three cups of sterilized saline solution to remove contaminating commensal bacteria from the saliva and upper respiratory tract.11–13 A small portion of washed sputum was homogenized and smeared on a glass slide for Gram staining, and another was cultured in sheep blood agar and horse blood chocolate agar medium and incubated at 37°C in an atmosphere containing 5% CO2 for 20–24 h. All Gram stain smears were judged to be valid according to Murray’s methods and Geckler’s classification, based on the number of leucocytes or alveolar macrophages and squamous or ciliated epithelial cells in a low-power microscopic field (100⫻).1,5 Smears with Geckler’s groups of 4–5, which contained more than 25 leucocytes or macrophages and fewer than 25 squamous or ciliated epithelial cells in a low-power microscopic field (100⫻) were considered to be satisfactory or adequate. On Gram staining slides, pathogenic bacteria were estimated on the basis of Gram stain reaction and typical morphologies. By culture methods, these bacteria were identified on the basis of the laboratory standard methodology. The H. influenzae strains were initially identified on the basis of colony morphology and the X- and V-factor requirement test (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Subsequently, all the strains were confirmed by using the ID Test HN-20 Rapid (Nissui Pharmaceutical, Tokyo, Japan). Some of the H. influenzae strains were differentiated from H. haemolyticus by subculture on rabbit blood media. All of these H. influenzae strains demonstrated nonhemolytic activity. The S. pneumoniae strains were identified on the basis of colony
morphology with α-hemolytic forming circles and positive Optochin (Becton Dickinson Microbiology Systems, Sparks, MD, USA) tests on sheep blood agar media. In further experiments, all the strains were confirmed by using bacterial cell-lysis tests, with a 10% solution of sodium desoxycholate.8 The M. catarrhalis strains were identified by observing colony morphology and by positive DNase tests. Subsequently, these strains were confirmed by using IDTest HN-20 Rapid (Nissui Pharmaceutical). The S. aureus strain was identified on the basis of colony morphology, positive catalase test, lactose fermentative activity, and positive coagulase test. Next, the strain was definitely confirmed by using the MicroScan Pos Combo 41 C test (Dade Behring, West Sacramento, CA, USA). Correlation between the results of the washed sputum Gram stain smears and cultures was evaluated by analysis of the sensitivity (100% false-negative rate) and specificity (100% false-positive rate) of the washed sputum Gram stain smear findings by considering culture results as the referent standards.9,12,13 The value of the washed sputum culture was assessed through examination of the clinical responses of the patients who received antibiotic treatments instituted on the basis of the culture results. The patients were classified into three groups on the basis of the clinical diagnoses and underlying diseases. Group 1 consisted of the patients who suffered from acute LRTI without any underlying diseases; group 2 were those patients who suffered from LRTI with a bronchial asthma attack; and group 3 consisted of the patients who suffered from LRTI with underlying immunodeficiency diseases. The clinical responses of the patients to the antibiotic treatments were evaluated after 5- to 7 day antibiotic treatment and classified as showing either “improvement” (disappearance or amelioration of acute pretreatment clinical signs and symptoms, although chest radiograph may still have shown some signs of infiltrate) or no improvement (unchanged signs and symptoms).
Results Table 1 shows the distribution of the 224 ES samples and the 125 source patients stratified by sex, age, diagnosis, and underlying diseases. Of the 125 patients, 52.8% were male and 47.2%, female. Acute bronchitis was present in 91 patients (72.8%) and chronic bronchitis was present in 17 patients (13.6%). However, 113 (50.4%) samples were collected from the patients with chronic bronchitis during various clinical episodes. Of the total number of patients, 11 (8.8%) had underlying congenital immunodeficiency diseases, and 52 (41.6%) had underlying bronchial asthma. In the 11 patients with underlying congenital immunodeficiency diseases, X-linked agammaglobulinemia (XLA) was determined in 7 patients, common variable immunodeficiency (CVID) was found in 3 patients, and WiskottAldrich syndrome (WAS) was found in 1 patient. We obtained 70 samples from patients with underlying congenital immunodeficiency diseases (31.2%) and 81 samples
33
(36.2%) from those with underlying bronchial asthma. Only 26 (20.8%) patients had had previous antibiotic therapy in 80 (35.7%) different clinical episodes. Table 2 demonstrates the distribution of the patients and ES samples in the three treatment groups stratified by patient age and underlying disease. This Table shows that the distribution of the samples collected from patients in group 1 and those collected from patients in group 3 differed significantly (P ⬍ 0.01). Most of the patients with acute LRTI were aged 5 years or less, while those with chronic bronchitis were mainly more than 5 years old (P ⬍ 0.01). In addition, most of the patients in group 1 had not received previous antibiotic treatment, while most of those in group 3 had been given previous antibiotic treatment (P ⬍ 0.05).
Table 3 shows the isolation rates of the pathogenic bacteria recovered by the washed sputum culture method. Of the 224 sputum samples, H. influenzae was isolated from 50 (22.4%) samples, S. pneumoniae was isolated from 21 (9.4%) samples, M. catarrhalis was isolated from 11 (4.9%) samples, and S. aureus was isolated from 1 (0.4%) sample. The total isolation rate was 37.1%. Correlation rates between the findings of washed sputum Gram stain smears and cultures for the prediction of H. influenzae, S. pneumoniae, and M. catarrhalis are demonstrated in Tables 4, 5, and 6, respectively. By culture, H. influenzae was predominantly isolated from 50 specimens and not isolated from 174 specimens. Of these 50 specimens, Gram-negative coccobacilli like H. influenzae were
Table 1. Distribution of patients and ES samples, by patient features
Table 3. Pathogenic bacteria dominantly isolated from washed sputum samples
Patients; n ⫽ 125 (%)
Features Sex Male Female Ages ⬍1 year 1–5 years 6–10 years ⬎10 years Diagnosis Acute bronchitis Chronic bronchitis Bronchiolitis Pneumonia Underlying diseases Immunodeficiency Bronchial asthma Other Previous antibiotics No previous antibiotics Outpatients Inpatients
ES specimens; n ⫽ 224 (%)
66 (52.8) 59 (47.2)
144 (64.3) 80 (35.7)
7 (5.6) 41 (32.8) 47 (37.6) 30 (24.0)
10 (4.5) 53 (23.7) 76 (33.9) 85 (37.9)
91 (72.8) 17 (13.6) 2 (1.6) 15 (12.0)
113 (50.4) 92 (41.1) 2 (0.9) 17 (7.6)
11 (8.8) 52 (41.6) 5 (4.0) 26 (20.8) 99 (79.2) 115 (92.0) 10 (8.0)
70 (31.2) 81 (36.2) 8 (3.6) 80 (35.7) 144 (64.3) 211 (94.2) 13 (5.8)
Isolated bacteria
Number of specimens (%)
Haemophilus influenzae Streptococcus pneumoniae Moraxella catarrhalis Staphylococcus aureus Negative, or contamination by normal flora
50 (22.4) 21 (9.4) 11 (4.9) 1 (0.4) 141 (62.9)
Total
224 (100.0)
Table 4. Correlation between findings of washed sputum Gram stain smears and cultures for prediction of H. influenzae Culture findings
Gram stain predictions Positive
Negative
Positive Negative
43 9
7 165
50 174
Total
52
172
224
False-negative, 14.0%; sensitivity, 86.0%; false-positive, 5.2%; specificity, 94.8%
ES, expectorated sputum
Table 2. Distribution of patients and ES samples in the three treatment groups, by patient features Features
Sex Male Female Age ⬍1 year 1–5 years 6–10 years ⬎10 years PAT No PAT Outpatients Inpatients
Total
Patients (n ⫽ 60)
Specimens (n ⫽ 83)
Group 1 (n ⫽ 28)
Group 2 (n ⫽ 23)
Group 3 (n ⫽ 9)
Group 1 (n ⫽ 30)
Group 2 (n ⫽ 30)
Group 3 (n ⫽ 23)
11 17
10 13
8 1
13 17
13 17
22 1
4 15 8 1 3 25 24 4
1 11 9 2 3 20 21 2
0 0 1 8 7 2 9 0
4 16 9 1 3 27 26 4
1 16 11 2 8 22 28 2
0 0 1 22 18 5 23 0
Group 1, Patients who suffered from lower respiratory tract infection (LRTI) without underlying disease; group 2, patients who suffered from LRTI and had bronchial asthma; group 3, patients who suffered from LRTI and had immunodeficiency diseases; PAT, previous antibiotic treatment
34 Table 5. Correlation between findings of washed sputum Gram stain smears and cultures for prediction of S. pneumoniae
Table 7. Overall correlation between the findings of washed sputum Gram stain smears and cultures
Culture findings
Culture findings
Gram stain predictions
Total
Positive
Negative
Positive Negative
17 5
4 198
21 203
Total
22
202
224
False-negative, 19.0%; sensitivity, 81.0%; false-positive, 2.5%; specificity, 97.5%
Gram stain predictions
Total
Positive
Negative
Positive Negative
71a 18
12 123
83a 141
Total
89
135
224
False-negative, 14.5%; sensitivity, 85.5%; false-positive, 12.8%; specificity, 87.2% a Including one specimen from which S. aureus was identified by culture, and Gram stain estimation correlated with culture result
Table 6. Correlation between findings of washed sputum Gram stain smears and cultures for prediction of M. catarrhalis Culture findings
Gram stain predictions
Total
Positive
Negative
Positive Negative
10 4
1 209
11 213
Total
14
210
224
False-negative, 9.1%; specificity, 98.1%
sensitivity,
90.9%;
false-positive,
Table 8. Clinical responses of the patients who received antibiotic treatment instituted on the basis of culture results Patients’ clinical courses
1.9%;
seen in 43 specimens and not observed in 7 specimens by Gram stain smears. Thus, the false-negative rate and the sensitivity of the washed sputum Gram stain smear were considered to be 14.0% (7/50) and 86.0% (43/50), respectively. In addition, of the 174 specimens from which H. influenzae strains were not recovered by culture, Gramnegative coccobacilli like H. influenzae were seen in 9 specimens by Gram stain smears. Therefore, the false-positive rate and the specificity of the washed sputum Gram stain smear were considered to be 5.2% (9/174) and 94.8% (165/ 174), respectively. By similar considerations, the falsenegative rate, false-positive rate, sensitivity, and specificity of the washed sputum Gram stain smear for the prediction of S. pneumoniae were 19.0%, 2.5%, 81.0%, and 97.5%, respectively. For the prediction of M. catarrhalis, the falsenegative rate, false-positive rate, sensitivity, and specificity of the washed sputum Gram stain smear technique were 9.1%, 1.9%, 90.9%, and 98.1%, respectively. In addition, the washed sputum Gram stain results were not identical to the culture results in 4 specimens. In 2 of these, H. influenzae were isolated by culture, while Gram stain smears indicated Gram-positive diplococci like S. pneumoniae in 1 and Gram-negative diplococci like M. catarrhalis in the other. In the 2 remaining specimens, S. pneumoniae and M. catarrhalis were isolated in 1 each, while the results of Gram stain smears of these specimens showed Gram-negative coccobacilli like H. influenzae and Gram-positive diplococci like S. pneumoniae, respectively. Moreover, among the total of 224 sputum samples, S. aureus was isolated from only 1 specimen. On the Gram stain smear of this sample, Gram-positive cocci in clusters like Staphylococci were dominantly observed. Overall, the values for sensitivity and specificity of the washed sputum Gram stain smear technique compared with the culture method were 85.5% and 87.2%, respectively (Table 7).
Clinical responses Improvement (%)
No improvement (%)
Patient group 1 (n ⫽ 30) Patient group 2 (n ⫽ 30) Patient group 3 (n ⫽ 23)
28 (93.3) 21 (70.0) 10 (43.5)
2 (6.7) 9 (30.0) 13 (56.5)
Total (n ⫽ 83)
59 (71.1)
24 (28.9)
Group 1, patients with acute LRTI and without underlying diseases; group 2, patients with LRTI and bronchial asthma attack; group 3, patients with LRTI and immunodeficiency diseases
The clinical responses of the patients who received antibiotic treatment on the basis of the culture results are listed in Table 8. This Table shows that the rates of clinical improvement were 93.3%, 70.0%, and 43.5% for the patients in groups 1, 2 and 3, respectively.
Discussion Several difficulties have been encountered in attempts to accurately identify the bacterial pathogens of LRTI in children. Traditionally, microbiologic examinations of respiratory tract secretions have been processed for recovering the bacterial pathogens of LRTI, but complication by upper respiratory tract contamination is always a possibility.5,7–15 Organisms that are normally resident in the oropharynx in the order of at least 106/ml markedly confound the analyzed data.3 Furthermore, if contamination is present to a significant degree, culture would yield a large number of different species that would overgrow the true etiologic agents.3 Therefore, previous authors have utilized quantitative cultures of ES as one of the methods to improve the results of bacteriologic examinations of ES.9 This method is designed to distinguish bacterial pathogens and nonpathogens on the basis of relative bacterial concentrations. Nevertheless, quantitative culture is technically more difficult for processing sputum because exudates do not permit accurate volume sampling. Thus, ES requires liquefaction and homogenization before quantitative culture.9 For this reason, the washing technique of ES is considered to be a simple
35
way of resolving contamination from upper respiratory tract secretions. This technique has been described well in several previous studies.10–13 However, variability in correlation rates between sputum Gram stain smear and culture results in previous studies has called into question the value of sputum Gram stain examination for guiding the initial antibiotic therapy choice. It was reported that the interpretation of Gram stain smears of ES was frequently inaccurate, even when interpreted by infectious diseases specialists, and, therefore, these smears may be unreliable in predicting the causative bacteria of LRTI.15 Again, sputum Gram stain smear findings correlated poorly with sputum culture results; a good correlation rate was demonstrated only for S. pneumoniae, while a poor correlation rate was demonstrated for H. influenzae and other Gram-negative rods.5,6 Moreover, the reliability of sputum culture results was impressive in a study in which more than 50% of patients with H. influenzae pneumonia, diagnosed in Gram stain smears and substantiated by positive blood culture, had negative sputum cultures.16 In contrast, other studies have revealed that a transtracheal aspiration Gram stain interpretation is not superior to sputum Gram stain interpretation in predicting pathogens involved in pneumonia.4 In particular, studies utilizing a washing technique for examining ES have revealed high correlation rates between the findings of Gram stain smears and cultures.10–13 In addition, it was reported that the washing procedure of sputum decreased the mean concentration of contaminants by approximately 100-fold for all specimens and by 1000-fold for purulent specimens.11 The results of our study were consistent with previous reports in which the washed sputum Gram stain smear technique demonstrated high sensitivity and specificity compared with washed sputum cultures. The high correlation rates obtained in our study sufficiently proved that the diagnostic value of sputum samples was enhanced by using the washing technique, thereby removing potential contaminants arising from the upper respiratory tract. It is noteworthy that the present study included only valid sputum samples selected according to Murray’s method and Geckler’s classification for examination. This practice, in part, explains the high correlation rate found in our study. On the other hand, our study still showed overall falsenegative and false-positive rates for the washed sputum Gram staining technique of 14.5% and 12.8%, respectively. The discrepancy between culture and Gram stain findings has been considered to be related to previous antibiotic administration, which can influence the growth of bacteria in the culture medium.6,10,12 In our study, there were, in total, 18 specimens from which the pathogenic bacteria were estimated by Gram stain smears but not isolated by culture. Nevertheless, the clinical records revealed that only 4 of these specimens (22.2%) were collected from the patients with previous antibiotic treatment. This finding suggests that there were some factors other than previous antibiotic treatment causing that discrepancy. Such factors could be a significant variation in the abilities of observers to correctly interpret specimens, and/or the irregular distribution of pathogens in a specimen.4,6,9
The high correlation rates between the findings of washed sputum Gram stain smears and cultures found in this study are particularly useful in the clinical management of patients with LRTI. On the basis of this technique, physicians and pediatricians can quickly institute a reliable initial antibiotic therapy for the patient, especially in some cases when antibiotic treatment should not be delayed. According to our data, most of the patients who suffered from acute LRTI and had no underlying diseases demonstrated good clinical responses to the antibiotic treatments instituted on the basis of the sputum culture results. This finding appears to substantiate the idea that the washed sputum cultures likely indicated the real causative bacteria of LRTI, and that the results of these cultures are useful for establishing appropriate antibiotic treatment. Nevertheless, in the patients who suffered from chronic bronchitis and had underlying congenital immunodeficiency diseases or bronchial asthma attacks, the effectiveness of the antibiotic treatments was limited. In fact, these poor outcomes may be due to other factors, which probably included immuneinsufficient situations, damage to the respiratory tract mucosa, and exacerbation of chronic inflammatory responses in the respiratory tracts of these patients.17–19 Moreover, in patients with asthma attack, bacterial infection mainly occurs after there is primary noninfected inflammation of the lower respiratory tract. This primary inflammation, caused by the mechanism of the asthma attack, with high concentrations of cytokines and chemokines, always damages the bronchial mucosa, and suppresses the respiratory mucous defense system.20 These conditions possibly led to the poor results of antibiotic treatment in the patients in groups 2 and 3. In conclusion, the present study suggests that the techniques of washed sputum Gram stain smear and culture are valuable and should be encouraged in clinical practice for the management of LRTI in children. The collection of valid or adequate ES samples plays an important role in the improvement of the diagnostic value of washed sputum microbiologic examinations; therefore, more attention should be paid to the training of physicians and pediatricians to obtain adequate sputum specimens and to perform washed sputum examinations correctly.
References 1. Nohynek H, Valkeila E, Leinonen M, Eskola J. Erythrocyte sedimentation rate, white blood cell count and serum Creactive protein in assessing etiologic diagnosis of acute lower respiratory infections in children. Pediatr Infect Dis J 1995;14:484– 90. 2. Ishiwada N, Kurosaki T, Toba T, Niimi H. Etiology of pediatric patients with pneumonia. Analysis of clinical symptoms, physical examination and simple laboratory findings (in Japanese). J Jpn Assoc Infect Dis 1995;3:285–90. 3. Murray PR, Washington JA. Microscopic and bacteriologic analysis of expectorated sputum. Mayo Clinic Proc 1975;50:339– 44. 4. Geckler RW, McAllister CK, Gremillion DH, Ellenbogen C. Clinical value of paired sputum and transtracheal aspirates in the initial management of pneumonia. Chest 1985;87:631–5.
36 5. Gleckman R, DeVita J, Hibert D, Pelletier C, Martin R. Sputum gram stain assessment in community-acquired bacteremic pneumonia. J Clin Microbiol 1988;26:846–9. 6. Minocha A, Moravec CL Jr, Baltimore. Gram’s stain and culture of sputum in routine management of pulmonary infection. South Med J 1993;86:1225–8. 7. Geckler RW, Gremillion DH, McAllister CK, Ellenbogen C. Microscopic and bacteriological comparison of paired sputa and transtracheal aspirates. J Clin Microbiol 1977;6:396–9. 8. Takeda Y, Yabuuchi E, Miwatani T. Biochemical tests for identification of medical bacteria (in Japanese). 2nd Ed. Tokyo: Igaku–Shoin; 1985. pp 13–17. 9. Guckian JC, Christensen WD. Quantitative culture of sputum. Am Rev Respir Dis 1978;118:997–1005. 10. Saadah HA, Nasr FL, Shagoury ME. Washed sputum gram stain and culture in pneumonia: a practical tool for the clinician. J Okla State Med Assoc 1980;73:354–9. 11. Bartlett JG, Finegold SM. Bacteriology of expectorated sputum with quantitative culture and washed technique compared to transtracheal aspirates. Am Rev Respir Dis 1972;117:1019–27. 12. Uehara S. A method of bacteriological examination of washed sputum in infants and children. Acta Paediatr Jpn 1988;30:253–60. 13. Sugioka T, Ishikawa T, Kohri Y, Suruga Y, Sugimoto K. Washed sputum gram stain smear for rapid identification of pathogenic bacteria (in Japanese). Pediatr Jpn 1999;40:1537–44.
14. Takeda N, Kuroki H, Ishikawa N, Murata A, Sugimoto K, Uehara S, et al. The usefulness of washed sputum culture in children with lower respiratory infections (in Japanese). J Jpn Pediatr Soc 1998;102:975–80. 15. Pratter MR, Irwin RS. Clinical value of the gram stain smear of respiratory secretion. Chest 1985;88:163–4. 16. Levine DC, Schwarz MI, Matthay RA, LaForce FM. Bacteremic Haemophilus influenzae pneumonia in adults. A report of 24 cases and a review of the literature. Am J Med 1977;62:219–24. 17. Groeneveld K, Alphen LV, Eijik PP, Jansen HM, Zanen HC. Changes in outer membrane proteins of nontypeable Haemophilus influenzae in patients with chronic obstructive pulmonary diseases. J Infect Dis 1988;158:360–5. 18. Groeneveld K, Alphen LV, Eijik PP, Visschers G, Jansen HM, Zanen HC. Endogenous and exogenous re-infections by Haemophilus influenzae in patients with chronic obstructive pulmonary diseases: the effect of antibiotic treatment on persistence. J Infect Dis 1990;161:512–7. 19. Cole P. The damaging role of bacteria in chronic lung infection. J Antimicrob Chemother 1997;40:5–10. 20. Nagayama Y, Tsubaki T, Toba T, Kawakami H, Ohkusu K. Role of bacterial infection in the exacerbation of acute or prolonged asthma attack in children. Allergol Int 1999;48:137–44.
© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2004
ORIGINAL ARTICLE Luong Dong Cao · Naruhiko Ishiwada · Nobue Takeda Yukiko Nigo · Jirou Aizawa · Haruo Kuroki Yoichi Kohno
Value of washed sputum gram stain smear and culture for management of lower respiratory tract infections in children
Received: February 20, 2003 / Accepted: September 3, 2003
Abstract To date, the technique of washed sputum examinations has not been widely used in the clinical management of lower respiratory tract infections in children. A total of 224 sputum samples from 125 pediatric patients with lower respiratory tract infections were collected for washed sputum Gram stain smears and cultures. The results with these methods were compared to find correlation rates. The value of washed sputum cultures was assessed by examining the clinical responses of the patients who received antibiotic therapies instituted on the basis of the sputum culture results. Isolation rates of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus were 22.4%, 9.4%, 4.9%, and 0.4%, respectively. For the prediction of H. influenzae, S. pneumoniae, and M. catarrhalis, the sensitivities of the washed sputum Gram stain smears compared with the culture method were 86.0%, 81.0%, and 90.9%, respectively. The specificities of the washed sputum Gram stain smear technique were 94.8%, 97.5%, and 98.1%, respectively. Overall, the sensitivity and specificity of the washed sputum Gram stain smear method were 85.5% and 87.2%, respectively. S. aureus was isolated from only one specimen; and washed sputum Gram stain smear estimation was correlated with the culture result. On the basis of the washed sputum culture results, appropriate antibiotic therapies were instituted for 93.3% of the patients with acute lower respiratory tract infections. This study suggests that the techniques of washed sputum Gram stain smear and culture are valuable and should be encouraged in clinical practice for the management of lower respiratory tract infections in children.
D.C. Luong · N. Ishiwada (*) · N. Takeda · Y. Nigo · J. Aizawa · H. Kuroki · Y. Kohno Department of Pediatrics, Chiba University, Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Tel. ⫹81-43-226-2144; Fax ⫹81-43-226-2145 e-mail: [email protected]
Key words Haemophilus influenzae · Gram stain smear · Washed sputum culture · Respiratory tract infection · Children
Introduction Pediatric lower respiratory tract infections (LRTI) are common causes of morbidity and mortality worldwide. Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis, the major bacterial etiologic agents causing these infections, are responsible for cases of acute bronchitis, chronic bronchitis, otitis media, sinusitis, and pneumonia. The management of LRTI is remarkably simplified when the responsible pathogens are accurately identified. In addition, early recognition and prompt institution of initial appropriate therapy may offer the best chance to reduce mortality. Clinical signs, symptoms, and host responses (erythrocyte sedimentation rate [ESR], white blood cell count [WBC], and C-reactive protein [CRP]) may not provide reliable evidence of bacterial respiratory infections.1,2 Therefore, microbiological methods are considered to be necessary for the detection of the real bacterial pathogens. Conventionally, physicians and bacteriologists have accepted that microbiologic examinations of expectorated sputum (ES) be performed for identifying the bacterial pathogens of LRTI, because sputum is always available, and can be obtained easily and noninvasively. However, the sputum culture findings are usually not available within 18 to 24 h, and so antibiotic therapies instituted on the basis of these findings would be delayed. Therefore, ES Gram stain smears have been employed for the rapid prediction of the bacterial pathogens of LRTI, with the aim of quickly guiding the initial antibiotic choice. The advantages of this method were that it could be completed within several minutes, did not require sophisticated equipment, and was inexpensive.3–5 Unfortunately, the diagnostic yields of sputum Gram stain smear and culture were limited, because of potential contamination from the upper respiratory tract.3–8 For this reason, previous studies have con-
32
sidered an ES washing technique as one of the useful means for the improvement of sputum microbiologic examinations.9–13 However, several studies have still questioned the reliability of washed sputum Gram stain smear and culture for accurately predicting the bacterial pathogens of LRTI.3,5–8 Moreover, few studies have been carried out to evaluate the usefulness of washed sputum Gram stain smear and culture in the clinical management of pediatric patients with LRTI, because the collection of a satisfactory ES sample in children seems to be more difficult than in adults. Taking these factors into consideration, we performed the present study to evaluate the reliability of the washed sputum Gram stain smear technique for the rapid estimation of bacterial pathogens of LRTI, for the purpose of quickly guiding the initial antibiotic choice. The present study also aimed to assess the value of the washed sputum culture method in the institution of appropriate antibiotic therapy for pediatric patients with LRTI.
Patients and methods We studied 224 samples of ES obtained from 224 identical clinical episodes in 125 pediatric patients with LRTI at the Department of Pediatrics of Chiba University Hospital and other hospitals in Chiba prefecture between May 1999 and December 2000. Of the 125 patients, 115 were outpatients and 10 were hospitalized. Within 2–3 h of sampling, each sample was consecutively washed three times in three cups of sterilized saline solution to remove contaminating commensal bacteria from the saliva and upper respiratory tract.11–13 A small portion of washed sputum was homogenized and smeared on a glass slide for Gram staining, and another was cultured in sheep blood agar and horse blood chocolate agar medium and incubated at 37°C in an atmosphere containing 5% CO2 for 20–24 h. All Gram stain smears were judged to be valid according to Murray’s methods and Geckler’s classification, based on the number of leucocytes or alveolar macrophages and squamous or ciliated epithelial cells in a low-power microscopic field (100⫻).1,5 Smears with Geckler’s groups of 4–5, which contained more than 25 leucocytes or macrophages and fewer than 25 squamous or ciliated epithelial cells in a low-power microscopic field (100⫻) were considered to be satisfactory or adequate. On Gram staining slides, pathogenic bacteria were estimated on the basis of Gram stain reaction and typical morphologies. By culture methods, these bacteria were identified on the basis of the laboratory standard methodology. The H. influenzae strains were initially identified on the basis of colony morphology and the X- and V-factor requirement test (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Subsequently, all the strains were confirmed by using the ID Test HN-20 Rapid (Nissui Pharmaceutical, Tokyo, Japan). Some of the H. influenzae strains were differentiated from H. haemolyticus by subculture on rabbit blood media. All of these H. influenzae strains demonstrated nonhemolytic activity. The S. pneumoniae strains were identified on the basis of colony
morphology with α-hemolytic forming circles and positive Optochin (Becton Dickinson Microbiology Systems, Sparks, MD, USA) tests on sheep blood agar media. In further experiments, all the strains were confirmed by using bacterial cell-lysis tests, with a 10% solution of sodium desoxycholate.8 The M. catarrhalis strains were identified by observing colony morphology and by positive DNase tests. Subsequently, these strains were confirmed by using IDTest HN-20 Rapid (Nissui Pharmaceutical). The S. aureus strain was identified on the basis of colony morphology, positive catalase test, lactose fermentative activity, and positive coagulase test. Next, the strain was definitely confirmed by using the MicroScan Pos Combo 41 C test (Dade Behring, West Sacramento, CA, USA). Correlation between the results of the washed sputum Gram stain smears and cultures was evaluated by analysis of the sensitivity (100% false-negative rate) and specificity (100% false-positive rate) of the washed sputum Gram stain smear findings by considering culture results as the referent standards.9,12,13 The value of the washed sputum culture was assessed through examination of the clinical responses of the patients who received antibiotic treatments instituted on the basis of the culture results. The patients were classified into three groups on the basis of the clinical diagnoses and underlying diseases. Group 1 consisted of the patients who suffered from acute LRTI without any underlying diseases; group 2 were those patients who suffered from LRTI with a bronchial asthma attack; and group 3 consisted of the patients who suffered from LRTI with underlying immunodeficiency diseases. The clinical responses of the patients to the antibiotic treatments were evaluated after 5- to 7 day antibiotic treatment and classified as showing either “improvement” (disappearance or amelioration of acute pretreatment clinical signs and symptoms, although chest radiograph may still have shown some signs of infiltrate) or no improvement (unchanged signs and symptoms).
Results Table 1 shows the distribution of the 224 ES samples and the 125 source patients stratified by sex, age, diagnosis, and underlying diseases. Of the 125 patients, 52.8% were male and 47.2%, female. Acute bronchitis was present in 91 patients (72.8%) and chronic bronchitis was present in 17 patients (13.6%). However, 113 (50.4%) samples were collected from the patients with chronic bronchitis during various clinical episodes. Of the total number of patients, 11 (8.8%) had underlying congenital immunodeficiency diseases, and 52 (41.6%) had underlying bronchial asthma. In the 11 patients with underlying congenital immunodeficiency diseases, X-linked agammaglobulinemia (XLA) was determined in 7 patients, common variable immunodeficiency (CVID) was found in 3 patients, and WiskottAldrich syndrome (WAS) was found in 1 patient. We obtained 70 samples from patients with underlying congenital immunodeficiency diseases (31.2%) and 81 samples
33
(36.2%) from those with underlying bronchial asthma. Only 26 (20.8%) patients had had previous antibiotic therapy in 80 (35.7%) different clinical episodes. Table 2 demonstrates the distribution of the patients and ES samples in the three treatment groups stratified by patient age and underlying disease. This Table shows that the distribution of the samples collected from patients in group 1 and those collected from patients in group 3 differed significantly (P ⬍ 0.01). Most of the patients with acute LRTI were aged 5 years or less, while those with chronic bronchitis were mainly more than 5 years old (P ⬍ 0.01). In addition, most of the patients in group 1 had not received previous antibiotic treatment, while most of those in group 3 had been given previous antibiotic treatment (P ⬍ 0.05).
Table 3 shows the isolation rates of the pathogenic bacteria recovered by the washed sputum culture method. Of the 224 sputum samples, H. influenzae was isolated from 50 (22.4%) samples, S. pneumoniae was isolated from 21 (9.4%) samples, M. catarrhalis was isolated from 11 (4.9%) samples, and S. aureus was isolated from 1 (0.4%) sample. The total isolation rate was 37.1%. Correlation rates between the findings of washed sputum Gram stain smears and cultures for the prediction of H. influenzae, S. pneumoniae, and M. catarrhalis are demonstrated in Tables 4, 5, and 6, respectively. By culture, H. influenzae was predominantly isolated from 50 specimens and not isolated from 174 specimens. Of these 50 specimens, Gram-negative coccobacilli like H. influenzae were
Table 1. Distribution of patients and ES samples, by patient features
Table 3. Pathogenic bacteria dominantly isolated from washed sputum samples
Patients; n ⫽ 125 (%)
Features Sex Male Female Ages ⬍1 year 1–5 years 6–10 years ⬎10 years Diagnosis Acute bronchitis Chronic bronchitis Bronchiolitis Pneumonia Underlying diseases Immunodeficiency Bronchial asthma Other Previous antibiotics No previous antibiotics Outpatients Inpatients
ES specimens; n ⫽ 224 (%)
66 (52.8) 59 (47.2)
144 (64.3) 80 (35.7)
7 (5.6) 41 (32.8) 47 (37.6) 30 (24.0)
10 (4.5) 53 (23.7) 76 (33.9) 85 (37.9)
91 (72.8) 17 (13.6) 2 (1.6) 15 (12.0)
113 (50.4) 92 (41.1) 2 (0.9) 17 (7.6)
11 (8.8) 52 (41.6) 5 (4.0) 26 (20.8) 99 (79.2) 115 (92.0) 10 (8.0)
70 (31.2) 81 (36.2) 8 (3.6) 80 (35.7) 144 (64.3) 211 (94.2) 13 (5.8)
Isolated bacteria
Number of specimens (%)
Haemophilus influenzae Streptococcus pneumoniae Moraxella catarrhalis Staphylococcus aureus Negative, or contamination by normal flora
50 (22.4) 21 (9.4) 11 (4.9) 1 (0.4) 141 (62.9)
Total
224 (100.0)
Table 4. Correlation between findings of washed sputum Gram stain smears and cultures for prediction of H. influenzae Culture findings
Gram stain predictions Positive
Negative
Positive Negative
43 9
7 165
50 174
Total
52
172
224
False-negative, 14.0%; sensitivity, 86.0%; false-positive, 5.2%; specificity, 94.8%
ES, expectorated sputum
Table 2. Distribution of patients and ES samples in the three treatment groups, by patient features Features
Sex Male Female Age ⬍1 year 1–5 years 6–10 years ⬎10 years PAT No PAT Outpatients Inpatients
Total
Patients (n ⫽ 60)
Specimens (n ⫽ 83)
Group 1 (n ⫽ 28)
Group 2 (n ⫽ 23)
Group 3 (n ⫽ 9)
Group 1 (n ⫽ 30)
Group 2 (n ⫽ 30)
Group 3 (n ⫽ 23)
11 17
10 13
8 1
13 17
13 17
22 1
4 15 8 1 3 25 24 4
1 11 9 2 3 20 21 2
0 0 1 8 7 2 9 0
4 16 9 1 3 27 26 4
1 16 11 2 8 22 28 2
0 0 1 22 18 5 23 0
Group 1, Patients who suffered from lower respiratory tract infection (LRTI) without underlying disease; group 2, patients who suffered from LRTI and had bronchial asthma; group 3, patients who suffered from LRTI and had immunodeficiency diseases; PAT, previous antibiotic treatment
34 Table 5. Correlation between findings of washed sputum Gram stain smears and cultures for prediction of S. pneumoniae
Table 7. Overall correlation between the findings of washed sputum Gram stain smears and cultures
Culture findings
Culture findings
Gram stain predictions
Total
Positive
Negative
Positive Negative
17 5
4 198
21 203
Total
22
202
224
False-negative, 19.0%; sensitivity, 81.0%; false-positive, 2.5%; specificity, 97.5%
Gram stain predictions
Total
Positive
Negative
Positive Negative
71a 18
12 123
83a 141
Total
89
135
224
False-negative, 14.5%; sensitivity, 85.5%; false-positive, 12.8%; specificity, 87.2% a Including one specimen from which S. aureus was identified by culture, and Gram stain estimation correlated with culture result
Table 6. Correlation between findings of washed sputum Gram stain smears and cultures for prediction of M. catarrhalis Culture findings
Gram stain predictions
Total
Positive
Negative
Positive Negative
10 4
1 209
11 213
Total
14
210
224
False-negative, 9.1%; specificity, 98.1%
sensitivity,
90.9%;
false-positive,
Table 8. Clinical responses of the patients who received antibiotic treatment instituted on the basis of culture results Patients’ clinical courses
1.9%;
seen in 43 specimens and not observed in 7 specimens by Gram stain smears. Thus, the false-negative rate and the sensitivity of the washed sputum Gram stain smear were considered to be 14.0% (7/50) and 86.0% (43/50), respectively. In addition, of the 174 specimens from which H. influenzae strains were not recovered by culture, Gramnegative coccobacilli like H. influenzae were seen in 9 specimens by Gram stain smears. Therefore, the false-positive rate and the specificity of the washed sputum Gram stain smear were considered to be 5.2% (9/174) and 94.8% (165/ 174), respectively. By similar considerations, the falsenegative rate, false-positive rate, sensitivity, and specificity of the washed sputum Gram stain smear for the prediction of S. pneumoniae were 19.0%, 2.5%, 81.0%, and 97.5%, respectively. For the prediction of M. catarrhalis, the falsenegative rate, false-positive rate, sensitivity, and specificity of the washed sputum Gram stain smear technique were 9.1%, 1.9%, 90.9%, and 98.1%, respectively. In addition, the washed sputum Gram stain results were not identical to the culture results in 4 specimens. In 2 of these, H. influenzae were isolated by culture, while Gram stain smears indicated Gram-positive diplococci like S. pneumoniae in 1 and Gram-negative diplococci like M. catarrhalis in the other. In the 2 remaining specimens, S. pneumoniae and M. catarrhalis were isolated in 1 each, while the results of Gram stain smears of these specimens showed Gram-negative coccobacilli like H. influenzae and Gram-positive diplococci like S. pneumoniae, respectively. Moreover, among the total of 224 sputum samples, S. aureus was isolated from only 1 specimen. On the Gram stain smear of this sample, Gram-positive cocci in clusters like Staphylococci were dominantly observed. Overall, the values for sensitivity and specificity of the washed sputum Gram stain smear technique compared with the culture method were 85.5% and 87.2%, respectively (Table 7).
Clinical responses Improvement (%)
No improvement (%)
Patient group 1 (n ⫽ 30) Patient group 2 (n ⫽ 30) Patient group 3 (n ⫽ 23)
28 (93.3) 21 (70.0) 10 (43.5)
2 (6.7) 9 (30.0) 13 (56.5)
Total (n ⫽ 83)
59 (71.1)
24 (28.9)
Group 1, patients with acute LRTI and without underlying diseases; group 2, patients with LRTI and bronchial asthma attack; group 3, patients with LRTI and immunodeficiency diseases
The clinical responses of the patients who received antibiotic treatment on the basis of the culture results are listed in Table 8. This Table shows that the rates of clinical improvement were 93.3%, 70.0%, and 43.5% for the patients in groups 1, 2 and 3, respectively.
Discussion Several difficulties have been encountered in attempts to accurately identify the bacterial pathogens of LRTI in children. Traditionally, microbiologic examinations of respiratory tract secretions have been processed for recovering the bacterial pathogens of LRTI, but complication by upper respiratory tract contamination is always a possibility.5,7–15 Organisms that are normally resident in the oropharynx in the order of at least 106/ml markedly confound the analyzed data.3 Furthermore, if contamination is present to a significant degree, culture would yield a large number of different species that would overgrow the true etiologic agents.3 Therefore, previous authors have utilized quantitative cultures of ES as one of the methods to improve the results of bacteriologic examinations of ES.9 This method is designed to distinguish bacterial pathogens and nonpathogens on the basis of relative bacterial concentrations. Nevertheless, quantitative culture is technically more difficult for processing sputum because exudates do not permit accurate volume sampling. Thus, ES requires liquefaction and homogenization before quantitative culture.9 For this reason, the washing technique of ES is considered to be a simple
35
way of resolving contamination from upper respiratory tract secretions. This technique has been described well in several previous studies.10–13 However, variability in correlation rates between sputum Gram stain smear and culture results in previous studies has called into question the value of sputum Gram stain examination for guiding the initial antibiotic therapy choice. It was reported that the interpretation of Gram stain smears of ES was frequently inaccurate, even when interpreted by infectious diseases specialists, and, therefore, these smears may be unreliable in predicting the causative bacteria of LRTI.15 Again, sputum Gram stain smear findings correlated poorly with sputum culture results; a good correlation rate was demonstrated only for S. pneumoniae, while a poor correlation rate was demonstrated for H. influenzae and other Gram-negative rods.5,6 Moreover, the reliability of sputum culture results was impressive in a study in which more than 50% of patients with H. influenzae pneumonia, diagnosed in Gram stain smears and substantiated by positive blood culture, had negative sputum cultures.16 In contrast, other studies have revealed that a transtracheal aspiration Gram stain interpretation is not superior to sputum Gram stain interpretation in predicting pathogens involved in pneumonia.4 In particular, studies utilizing a washing technique for examining ES have revealed high correlation rates between the findings of Gram stain smears and cultures.10–13 In addition, it was reported that the washing procedure of sputum decreased the mean concentration of contaminants by approximately 100-fold for all specimens and by 1000-fold for purulent specimens.11 The results of our study were consistent with previous reports in which the washed sputum Gram stain smear technique demonstrated high sensitivity and specificity compared with washed sputum cultures. The high correlation rates obtained in our study sufficiently proved that the diagnostic value of sputum samples was enhanced by using the washing technique, thereby removing potential contaminants arising from the upper respiratory tract. It is noteworthy that the present study included only valid sputum samples selected according to Murray’s method and Geckler’s classification for examination. This practice, in part, explains the high correlation rate found in our study. On the other hand, our study still showed overall falsenegative and false-positive rates for the washed sputum Gram staining technique of 14.5% and 12.8%, respectively. The discrepancy between culture and Gram stain findings has been considered to be related to previous antibiotic administration, which can influence the growth of bacteria in the culture medium.6,10,12 In our study, there were, in total, 18 specimens from which the pathogenic bacteria were estimated by Gram stain smears but not isolated by culture. Nevertheless, the clinical records revealed that only 4 of these specimens (22.2%) were collected from the patients with previous antibiotic treatment. This finding suggests that there were some factors other than previous antibiotic treatment causing that discrepancy. Such factors could be a significant variation in the abilities of observers to correctly interpret specimens, and/or the irregular distribution of pathogens in a specimen.4,6,9
The high correlation rates between the findings of washed sputum Gram stain smears and cultures found in this study are particularly useful in the clinical management of patients with LRTI. On the basis of this technique, physicians and pediatricians can quickly institute a reliable initial antibiotic therapy for the patient, especially in some cases when antibiotic treatment should not be delayed. According to our data, most of the patients who suffered from acute LRTI and had no underlying diseases demonstrated good clinical responses to the antibiotic treatments instituted on the basis of the sputum culture results. This finding appears to substantiate the idea that the washed sputum cultures likely indicated the real causative bacteria of LRTI, and that the results of these cultures are useful for establishing appropriate antibiotic treatment. Nevertheless, in the patients who suffered from chronic bronchitis and had underlying congenital immunodeficiency diseases or bronchial asthma attacks, the effectiveness of the antibiotic treatments was limited. In fact, these poor outcomes may be due to other factors, which probably included immuneinsufficient situations, damage to the respiratory tract mucosa, and exacerbation of chronic inflammatory responses in the respiratory tracts of these patients.17–19 Moreover, in patients with asthma attack, bacterial infection mainly occurs after there is primary noninfected inflammation of the lower respiratory tract. This primary inflammation, caused by the mechanism of the asthma attack, with high concentrations of cytokines and chemokines, always damages the bronchial mucosa, and suppresses the respiratory mucous defense system.20 These conditions possibly led to the poor results of antibiotic treatment in the patients in groups 2 and 3. In conclusion, the present study suggests that the techniques of washed sputum Gram stain smear and culture are valuable and should be encouraged in clinical practice for the management of LRTI in children. The collection of valid or adequate ES samples plays an important role in the improvement of the diagnostic value of washed sputum microbiologic examinations; therefore, more attention should be paid to the training of physicians and pediatricians to obtain adequate sputum specimens and to perform washed sputum examinations correctly.
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