Rejection criteria for endotracheal aspirates from pediatric patients.

Endotracheal aspirates (ETAs) from mechanically ventilated pediatric patients frequently are cultured as part of an evaluation for suspected sepsis. There are now well-defined criteria for rejecting low-yield ETAs from adults, but it is uncertain whether the same criteria can be applied to ETAs from children. Therefore, we compared the Gram stain and culture results for 361 consecutive ETA specimens collected from pediatric patients over a 1-year period. Results for patients for whom a blood culture was performed within 48 h of the time that a culture of ETA was performed were also reviewed. Gram stains were examined under x100 magnification to quantitate the number of polymorphonuclear neutrophils and squamous epithelial cells (SECs) per low-power field and under x1,000 magnification for the presence of organisms. No organisms were seen by Gram staining in 225 (62%) of the ETAs. Culture of these specimens rarely yielded useful information: 52% were sterile, 32% grew rare to 1+ quantities of expected respiratory flora only, 12% grew rare to 1+ quantities of gram-negative rods mixed with expected respiratory flora, and only 10 (4%) yielded a pure or predominant growth of a potential respiratory pathogen. Unlike adult patients, we did not find the number of SECs to be a useful screening criterion. Only 17 (5%) of the ETAs had greater than 10 SECs per low-power field, and 5 (29%) of these yielded pure growth of a gram-negative rod. When blood culture results were positive, they correlated with ETA culture results in only 6 of 10 cases. On the basis of our findings, the absence of organisms on Gram staining is a useful criterion for rejecting ETAs from pediatric patients for culture and would have excluded 62% of the specimens from further processing.     

Campylobacter pylori infection in biopsy specimens of gastric antrum: laboratory diagnosis and estimation of sampling error.

Campylobacter pylori infection was sought in 382 consecutive patients referred for upper gastrointestinal endoscopy. Five antral biopsy specimens were taken from each patient: one was inserted into a CLO-test to detect the urease activity of C pylori, two were sent for histological analysis where multiple sections were stained by the Warthin-Starry silver method, and two were sent for microbiological evaluation by Gram stain and culture. A patient was deemed to be infected when C pylori was cultured or seen in either the histological sections or the Gram stain of the biopsy smear. One hundred and seventy four (46%) patients were infected. Culture, Gram stain, histological examination and the CLO-test showed sensitivities of 92%, 87%, 93% and 90%, respectively. In 27 (15%) infected patients an uneven distribution of C pylori was seen between samples in the biopsy pair sent for histology. Examination of multiple sections stained with Warthin-Starry silver was more sensitive at detecting infection (93%) than examination of multiple sections from only one biopsy specimen (84%). Fifty seven of 80 patients, biopsied a median seven days (range 5 to 55) after completing colloidal bismuth subcitrate treatment, were still infected with C pylori. There was no decrease in the sensitivities of the above tests to detect infection after treatment. It is concluded that at least two antral biopsy specimens should be examined when attempting to diagnose C pylori infection by histological methods.     

Antibiotic sensitivity of bacteria isolated from pathological specimens and utilization of beta-lactams in an orthopedic surgery service

From 1980 to 1984, computerized data on the sensitivity to the main antibiotics of 1991 strains isolated from clinical specimens were evaluated in relation to beta-lactam use and hospital activity in a unit of orthopedic surgery. No major variations were found in distribution of species throughout the study period, whereas sensitivity to antimicrobial agents changed. From 1980 to 1982, patients had postoperative prophylactic treatment with cephalosporin (cefazolin) for two days; during the same period, 59% of 557 Gram negative organisms were resistant to cefazolin and 31% of Staphylococci were resistant to methicillin (and to other antibiotics). In 1983 and 1984, cefazolin was replaced by intraoperative flash therapy with a penicillin-M (cloxacillin); concomitantly, sensitivity to cefazolin increased among Gram negative organisms (38% of 485 isolates were cefazolin-resistant; p less than 0.001) and Staphylococci (16% of 342 isolates were methicillin-resistant; p less than 0.001). Phage typing of S. aureus failed to disclose any epidemic outbreak. Since hospital activity remained the same throughout the period under study, it seems justified to correlate the increase in bacterial sensitivity observed to the decrease in use of cephalosporin, although other factors (microepidemic, isolation techniques) may be involved.     

Lack of evidence for Nocardia asteroides in brain specimens from Lewy body-containing disorders

Previous studies have suggested that Nocardia asteroides may play a role in the pathogenesis of Parkinson's disease (PD), including the production of Lewy bodies, the inclusion bodies present in this disorder. This study explored the possible connection between Nocardia and two Lewy body-containing disorders, PD and dementia with Lewy bodies (DLB). Substantia nigra specimens from individuals with PD, DLB, other neurodegenerative disorders, and normal subjects were evaluated for nocardial infection by in situ hybridization, PCR, and Gram staining. Brain specimens from a cynomolgus monkey experimentally infected with N. asteroides for 48 h served as the controls for in situ hybridization and Gram staining, and a nocardial pellet was the PCR control. The organism was detected by in situ hybridization and Gram stain in the experimentally infected monkey brain, and by PCR from the nocardial pellet. However, in situ hybridization reactivity was detected in only three of the 125 human brain specimens (2.4%; one case each of PD, DLB, and Alzheimer's disease), and none of the specimens was positive for Nocardia by PCR or Gram staining. These findings do not support an association of Nocardia with Lewy body-containing disorders.     

Performance of the BacT/Alert Virtuo Microbial Detection System for the culture of sterile body fluids: prospective multicentre study

ObjectivesContinuous monitoring blood culture systems are commonly used for sterile body fluid cultures. In this multicentre study, we evaluated the performance of the new-generation BacT/Alert Virtuo system compared to the BacT/Alert 3D and conventional culture for the recovery of microorganisms from sterile body fluids.MethodsPeritoneal, cerebrospinal, pericardial, pleural and synovial fluids from adult patients submitted for culture were collected from three different centres. Specimens were inoculated into two bottles of the same bottle type (SA, SN, FA Plus or FN Plus) in equal volumes for simultaneous incubation in the Virtuo and 3D instruments. Each specimen was also Gram stained and seeded to solid media.ResultsA total of 811 specimens were inoculated to 1257 bottle pairs. The Virtuo and 3D showed equivalent recovery of clinically significant microorganisms (127/155, 81.9%, vs. 126/155, 81.3%, respectively). Solid media cultures recovered fewer pathogens than either continuous monitoring system (95/155, 61.3%, p <0.001), including significantly fewer Enterobacteriaceae and enterococci. The Virtuo was significantly faster than the 3D in median time to detection of isolates from the same specimen (12.5 (range, 2.8–101.5) hours vs. 15.5 (range, 4.3–78.5) hours, p <0.001). Direct specimen Gram stain detected the eventual pathogen in 30 (26.1%) of 115 significant positive specimens.ConclusionsThe BacT/Alert Virtuo system was equivalent to the 3D system in organism recovery from sterile body fluid culture but showed faster detection of growth as a result of design enhancements.     

Nonvalue of sputum culture in the management of lower respiratory tract infections.

Establishment of the microbiological etiology of bacterial pneumonia by sputum culture is confounded by both lack of recovery of fastidious pathogens and contamination of specimens with oropharyngeal flora. We reviewed the clinical records from 249 patients over a 3-month period for evidence of pneumonia. Gram staining and cultures were performed on 381 specimens isolated from this population of patients. Recovery of respiratory tract pathogens was accomplished with 354 specimens from 226 patients; 27 specimens yielded normal flora in culture but were smear positive. An additional 256 specimens submitted to our microbiology laboratory did not meet smear criteria for purulence nor did they yield respiratory tract pathogens in culture. A total of 637 specimens submitted to the microbiology laboratory were evaluated for sputum purulence by the criteria of Bartlett. Of the total 354 specimens which were positive in culture for a pathogen, 182 (52%) were submitted from 150 patients with no objective evidence of pneumonia. The majority of specimens obtained from patients without pneumonia were nonpurulent. However, 71 of 182 culture-positive specimens obtained from 50 patients without pneumonia were purulent. Approximately half of these patients (31 of 50) had other pulmonary or upper respiratory tract pathology which could account for the sputum purulence. Among the 172 culture-positive specimens from 76 patients with pneumonia, only 100 (58%) were acceptable by smear criteria. An additional 23 patients provided expectorated purulent sputum from which no respiratory tract pathogen could be isolated. Of these 23, 7 had pneumonia. We conclude that sputum culture and Gram staining are neither specific nor sensitive as diagnostic tools. Objective criteria for purulence of Gram-stained specimens must be applied before their inoculation into culture media. Specimens should be sought only from patients with objective evidence of pneumonia.     

Utility of gram stain in evaluation of sputa from patients with cystic fibrosis.

The utility of sputum Gram stain in assessing salivary contamination and in predicting the presence of pathogens on the basis of morphology was investigated in 287 respiratory specimens from patients with cystic fibrosis. Where acceptability for culture was defined as a leukocyte/squamous epithelial cell ratio of > 5, 76.6% (220 of 287) of respiratory specimens received in the laboratory were considered acceptable. Unacceptable specimens were more common in younger patients. The positive predictive value of the Gram stain for growth from acceptable sputum samples was 98% for Pseudomonas aeruginosa, 84.4% for Pseudomonas cepacia, 86.3% for Staphylococcus aureus, and 100% for Haemophilus influenzae. In cystic fibrosis patients, as has been reported for respiratory specimens in general, Gram stain of respiratory specimens in helpful for interpreting culture results.     

Clinical laboratory evaluation of a urine screening device

A study was conducted to compare the Bac-T-Screen Bacterial Detection Device for Urines (BDD; Marion Laboratories, Kansas City, Mo.) with urine Gram stain as a screen for bacteriuria. We analyzed 631 urine samples with the BDD and compared the results to urine Gram stains and quantitative cultures. A total of 90 (14%) specimens could not be analyzed with the BDD due to interfering pigments (67 specimens) or clogging of the filter (23 specimens). Of the 541 specimens that were analyzed, the BDD correctly identified 67 (88.2%) of the 76 specimens with greater than or equal to 10(5) CFU/ml but only 294 (63.2%) of the 465 specimens with less than 10(5) CFU/ml. The majority of the false negative specimens had either gram-positive organisms or yeasts. The predictive value of a negative BDD reading was 97.0%. The urine Gram stain correctly identified 92.1% of all positive cultures and 77.8% of all negative cultures. The predictive value of a negative urine Gram stain was 98.4%. In summary, the BDD compares favorably with the urine Gram stain as a screen for bacteriologically negative urine specimens.     

Value of intensive diagnostic microbiological investigation in low- and high-risk patients with community-acquired pneumonia

In a prospective study to evaluate the diagnostic yield of different microbiological tests in hospitalised patients with community-acquired pneumonia, material for microbiological investigation was obtained from 262 patients. Clinical samples consisted of the following: sputum for Gram staining, culture, and detection of pneumococcal antigen; blood for culture and serological tests; urine for detection of Legionella pneumophila serogroup 1 antigen and pneumococcal antigen; and specimens obtained by fiberoptic bronchoscopy. A pathogen was identified in 158 (60%) patients, with Streptococcus pneumoniae (n=97) being the most common causative agent of community-acquired pneumonia. In 82% of the 44 patients with an adequate sputum specimen, a positive Gram stain was confirmed by positive sputum culture. S. pneumoniae infections were detected principally when adequate sputum specimens were examined by Gram stain and culture and when adequate and inadequate sputum specimens were tested for the presence of pneumococcal antigen (n=58; 60%). The urinary pneumococcal antigen test was the most valuable single test for detection of S. pneumoniae infections (n=52; 54%) when sputum pneumococcal antigen determination was not performed. Fiberoptic bronchoscopy was of additive diagnostic value in 49% of the patients who did not expectorate sputum and in 52% of those in whom treatment failed. Investigation of sputum by a combination of Gram stain, culture, and detection of pneumococcal antigen was the most useful means of establishing an aetiological diagnosis of community-acquired pneumonia, followed by testing of urine for pneumococcal antigen. Fiberoptic bronchoscopy may be of additional value when treatment failure occurs.This revised version was published online in April 2005 with a correction to the article title.     

Gram stain evaluation of the quality of sputum specimens for mycobacterial culture.

A group of 34 mycobacteria, consisting of 25 Mycobacterium tuberculosis and nine strains of three other species, was isolated from 400 expectorated sputum specimens submitted on 148 patients from county-wide sources. Eight strains (24% of the total) were isolated from specimens evaluated by Gram stain to be oropharyngeal fluids. The remaining 26 strains were isolated from ungradable specimens and those primarily of lower respiratory origin. It was concluded that the random examination of sputum by Gram stain to determine the specimen's quality for mycobacterial isolation is not necessary.     

Multicenter Assessment of Gram Stain Error Rates

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories.     

Use of DNA hybridization test for diagnosing bacterial vaginosis in women with symptoms suggestive of infection

The purpose of this study was to evaluate a DNA hybridization test (Affirm VPIII) as an alternative to Gram stain for the rapid diagnosis of bacterial vaginosis in women with clinical signs of vaginal infection. Vaginal specimens were collected from 321 symptomatic women, and analyzed for bacterial vaginosis by both Gram stain using Nugent criteria and DNA hybridization test. Sensitivity, specificity, positive predictive value, and negative predictive value of the DNA hybridization test were determined using the Gram staining as the standard for diagnosis of bacterial vaginosis. Of the 321 patients, 115 (35.8%) were Gram positive for bacterial vaginosis and 126 (39.2%) were negative. 80 patients (25.0%) demonstrated intermediate Gram staining that was also considered negative. The Affirm system detected G. vaginalis in 107 (93.0%) of 115 vaginal specimens positive for bacterial vaginosis diagnosed by Gram stain. Compared to the Gram stain, DNA hybridization test had a sensitivity of 87.7% and a specificity of 96.0%. Positive and negative predictive values of the DNA hybridization test were 93.0% and 92.7%, respectively. In conclusion, Affirm VPIII hybridization test correlated well with Gram stain and may be used as a rapid diagnostic tool to exclude bacterial vaginosis in women with genital complaints.     

Evaluation of Bactigen latex agglutination and Phadebact coagglutination for detection of bacterial antigens in cerebrospinal fluid.

The Bactigen latex agglutination and Phadebact coagglutination tests were evaluated for their ability to detect bacterial antigens of Haemophilus influenzae type b, Streptococcus pneumoniae (83 serotypes) and Neisseria meningitidis groups A, B, C, and Y in 214 samples of cerebrospinal fluid (CSF). Bactigen latex agglutination was more sensitive than Phadebact coagglutination: it detected 87% (59/68) of culture positive CSF specimens, whereas Phadebact detected 72% (52/72). Bactigen detected all cases of meningitis caused by S pneumoniae and H influenzae. Of the 19 specimens that were positive for N meningitidis, 74% were detected by Phadebact and only 53% by Bactigen. Gram stain results were positive for 85% of all specimens positive on culture. Bactigen was slightly more specific (97%) than Phadebact (96%). Bactigen, however, showed less specificity (81%) than Phadebact (94%) on 31 CSF specimens that were culture positive for organisms other than the test organisms. These included two CSF specimens from patients with tuberculous meningitis which gave false positive results for S pneumoniae with the Bactigen reagents. No false positive results were obtained on 104 culture negative CSF samples. Bactigen latex agglutination was superior to Phadebact coagglutination and Gram stain for the detection of S pneumoniae and H influenzae in CSF specimens from patients with bacteriologically proved meningitis.     

深圳市龙岗区患者痰培养细菌菌谱和耐药性研究

目的研究深圳市龙岗区患者痰培养细菌菌谱和耐药性,为临床合理应用抗菌药物提供依据。方法对来自深圳市龙岗区4间医院2007年1月至2008年6月患者合格痰标本中培养的细菌药敏试验进行回顾性分析。结果727株细菌中,革兰阴性杆菌595株,占81．8％,以鲍曼不动杆菌、铜绿假单胞菌为主;革兰阳性球菌132株,占18．2％,以凝固酶阴性葡萄球菌、金黄色葡萄球菌为主,而且凝固酶阴性葡萄球菌株数超过金黄色葡萄球菌。药敏结果提示革兰阴性杆菌及革兰阳性球菌对相当多数的抗生素耐药率较高。结论深圳市龙岗区患者痰培养细菌耐药情况较严重,条件致病菌菌株数比例较高。应加强药物尤其是抗菌药物的使用管理,规范应用抗生素。     

Direct identification and susceptibility testing by the AutoMicrobic system of gram-negative bacilli from urine specimens.

A total of 1,800 urine specimens were screened by Gram stain to detect bacteriuria. Pellets of bacteria were obtained by centrifuging specimens containing greater than or equal to 1 gram-negative bacillus of a single morphological type per oil immersion field. Direct susceptibility tests and identifications were performed from pellets by using the AutoMicrobic system (AMS). Results were compared with culture results by routine AMS methods. Of the 145 specimens showing only gram-negative bacilli on Gram stain, 113 grew greater than or equal to 10(5) CFU of a single species per ml. Compared with routine AMS identifications, the direct method correctly identified 105 (92.9%) of the isolates. Identifications were available within 8 h for 77% of the isolates. When compared by MICs, 93.2% of the direct susceptibility test results agreed with routine AMS results within one twofold dilution. Comparisons by category call indicated that overall complete and essential agreements were 89.9 and 97.8%, respectively, with 1.0% very major, 1.0% major, and 8.1% minor errors. Cefamandole and cephalothin had the lowest correlations by both comparisons. Within 8 h, susceptibility results were available for 94.3% of the isolates. This method offers the advantage of rapid detection, prompt processing, and earlier reporting of complete results for positive urine specimens.     

Comparative evaluation of Vitek 2 identification and susceptibility testing of urinary tract pathogens directly and isolated from chromogenic media

We evaluated the use of urine specimens for direct identification and antibiotic testing of urinary tract pathogens using the Vitek system. A total of 343 urine specimens from patients with suspected UTI were selected by pyuria and screened by Gram staining to detect bacteriuria. Of those, 132 were analysed after Gram staining, showing a high number of micro-organisms of a single morphological type. Direct susceptibility testing and identification were performed by using the Vitek system. Results were compared using the standard inoculation method based on the incubation of solid media. After sub-culture, 107 specimens grew a significant count of a single species and were used for the comparative analysis. The direct method correctly identified 88 isolates (82.3 %). When compared according to antibiotic susceptibility testing, the error rate was 2.4 % overall with 0.2 % very major, 0.4 % major and 1.8 % minor errors. 84.7 % of the Gram-negative bacilli had a complete susceptibility report in ≤8 h. This method offers the advantage of prompt processing and earlier reporting of complete results for positive urine specimens.     

Polymicrobial enteric infections in African infants with diarrhoea—results from a longitudinal prospective case–control study

ObjectivesThis longitudinal case–control study aimed to determine the frequency of polymicrobial enteric detections in Ghanaian infants with and without diarrhoea.MethodsInfants aged 1–12 months with and without diarrhoea attending the outpatient department of a peri-urban Ghanaian hospital were prospectively assessed and stool samples were collected on days 0, 6 and 28 and analysed for 18 enteric pathogens with PCR.ResultsAt least one enteric pathogen was detected in 100 of 107 cases with diarrhoea (93%) and in 82 of 97 controls (85%). The number of pathogens was higher in cases than in controls (median three versus two pathogens, p 0.001). The adjusted attributable fraction (AF) for diarrhoea was highest for enterotoxigenic Escherichia coli (7.2%, 95% CI –2.0% to 16.3%), rotavirus (4.1%, 95% CI 0.6%–7.5%), Giardia lamblia (2.3%, 95% CI –0.7 to 5.3%) and astrovirus (2.3%, 95% CI –2.9 to 7.5%). In cases, a higher pathogen number was significantly associated with watery stool consistency (median 3, interquartile range (IQR) 2–5 versus median 2.5, IQR 1–4, p 0.014), stool frequency five or more per day (median 4, IQR 3–5 versus median 3, IQR 2–4, p 0.048) and vomiting (median 4, IQR 3–5 versus median 3, IQR 2–4, p 0.025). During follow-up, 94% (78/83) of cases and 85% (67/79) of controls had acquired at least one new pathogen without developing a new episode of diarrhoea.ConclusionEnteric pathogens could be identified in the stool of the vast majority of Ghanaian infants, whereby pathogens were very frequently acquired without resulting in new episodes of diarrhoea during follow-up. A higher number of co-occurring pathogens may increase the risk of diarrhoea and disease severity.     

Determination of bacterial meningitis: a retrospective study of 80 cerebrospinal fluid specimens evaluated by four in vitro methods

A total of 80 cerebrospinal fluid specimens were analyzed for bacterial meningitis by four procedures readily available to most laboratories. These tests included routine culturing. Gram staining, countercurrent immunoelectrophoresis, staphylococcal coagglutination (CoA) with laboratory-prepared reagents, and CoA with Pharmacia Diagnostics reagents. A total of 56 specimens were positive for bacterial agents by routine culturing: Gram stain results were positive for 64% of all specimens positive by culturing. For 36 specimens from patients with suspected meningitis due to either Haemophilus influenzae type b, Streptococcus pneumoniae, or group B streptococci, detection was 97% with Pharmacia CoA reagents, 94% with laboratory-prepared CoA reagents, 89% with routine culturing, 78% with countercurrent immunoelectrophoresis, and 75% with Gram staining. One specimen which contained Klebsiella pneumoniae was false positive for S. pneumoniae in tests with both of the CoA reagents and in countercurrent immunoelectrophoresis. A Gram stain of this specimen clearly showed gram-negative bacilli, which were confirmed by culturing. Although a positive culture and a positive Gram stain are definitive evidence of bacterial meningitis, rapid immunological tests can provide valuable clinical information as an adjunct to culture and Gram stain results. Serological tests with Pharmacia CoA reagents produced more positive results than either laboratory-prepared CoA reagents or countercurrent immunoelectrophoresis.     

Helicobacter pylori in duodenal ulcer disease and its eradication

Antral biopsy specimens were processed for Helicobacter pylori by Gram staining, rapid urease test (RUT) and culture from 25 patients with symptoms of duodenal ulcer, amongst whom the positivity rate was 84%. Follow up of 16 patients after appropriate therapy showed complete regression of the disease in 87.5% of cases whereas in 12.5% of cases a decrease in the extent of duodenal ulceration was noted.     

Antibiotic-associated diarrhoea: an overlooked aetiology?

Sixty-three faeces samples from hospital in-patients with probable antibiotic-associated diarrhoea (AAD) are investigated. All samples are examined for routine bacterial enteric pathogens, pus cells, red blood cells and parasites. The samples are also screened for Clostridium difficile cytotoxin B (CDT), C. perfringens enterotoxin and Candida spp. (by microscopy and quantitative culture). Faecal samples from two control groups (healthy volunteers and community samples from GP patients) are also screened. A possible pathogen was found in 71% of AAD cases. Candida spp. overgrowth was the most common (44.4%), followed by CDT (34.9%) and Clostridium perfringens enterotoxin (9.5%). There was good agreement between the significant Gram films and quantitative Candida spp. culture (kappa=0.683). Clinical information revealed the majority of patients were on multiple antibiotic regimes, receiving 'high risk' antibiotics. Of community samples, 21% were positive for Clostridium perfringens enterotoxin, indicating that C. perfringens is a problem in the community, not necessarily associated with antibiotic use. The results suggest that quantitative Candida spp. culture should be performed on all specimens requesting AAD investigations.