Comparison of Macroscopic, Microscopic, and Radiometric Examinations of Clinical Blood Cultures in Hypertonic Media

Clinical blood cultures were collected in the Bactec 8A flask (Johnston Laboratories, Cockeysville, Md.) and examined macrosopically, microscopically, and radiometrically in an effort to determine which approach produced the fastest detection time. Of 360 blood cultures found to contain organisms by subculture, 334 were first detected by Bactec, 98 by macroscopic examination, and 68 by microscopic examination. Examination times were at 4, 8, 16, 24, 36, and 48 h after collection of the specimen. Sixteen hours after specimen collection, microscopic examination had detected 31 positive cultures, macroscopic examination had detected two positive cultures, and Bactec had detected 160 positive cultures. By the end of the first 24 h of incubation, Bactec had detected 313 (93%) of those cultures eventually found to be positive. Although Bactec produced the fastest detection time in an overwhelming majority of the cultures, it failed to detect three of three Candida spp. cultures, three of five Bacteroides spp. cultures, and six of 32 Enterococcus spp. cultures during the first 48 h of incubation.     

Detection of Mycobacterium bovis in bovine tissue samples by the Abbott LCx Mycobacterium tuberculosis assay and comparison with culture methods.

A ligase chain reaction (LCR) DNA amplification method for the molecular diagnosis of Mycobacterium tuberculosis complex (Abbott LCx MTB) was evaluated in comparison with solid (Lowenstein Jensen), liquid (7H12, Bactec 460 system) phase culture and microscopic examination (ME) on 86 tissue samples collected from 86 intradermal tuberculin positive cattle and one pool from 4 guinea pigs experimentally infected with M. bovis. Overall, 48 samples (58.81%) were culturally positive for mycobacteria, and on the basis of biochemical characters, all the isolates were identified as M. bovis. Sensitivity was 83.92% for LCx, 53.57% for LJ, 85.71% for Bactec and 41.07% for ME. In 3 out of 25 "no visible lesion" tissue samples, M. bovis was detected only by LCx and Bactec but not by LJ and ME. The concordance in the determination of positives and negatives among the methods observed in pairs was calculated according to Cohen's K concordance coefficient and showed 81.1% of concordance of LCx vs Bactec, 68.8% LCx vs LJ, 72.2% LCx vs ME, 80.0% Bactec vs LJ, 66.7% Bactec vs ME, 85.5% LJ vs ME. Despite a certain variability in concordance rates, both Cohen's K concordance coefficients or standardized (Zk) values were statistically significant. Both LCx and Bactec appear not alternative but subsidiary to the other methods traditionally applied for direct diagnosis of bovine tuberculosis on tissue samples from cattle reacting to intradermal tuberculin test.     

Comparison of automated culture systems with a CFR/USP-compliant method for sterility testing of cell-therapy products

BACKGROUND: Although widely used, commercially available automated culture methods are not US Food and Drug Administration-approved for sterility testing of cell-therapy products. For cell-therapy products regulated under Section 351 of the Public Health Service Act, sterility testing must be performed by the methods described in 21 CFR 610.12 and USP <71> (CFR/USP method), or by methods demonstrated to be equivalent. METHODS: Two automated methods, BacT/Alert (BTA; bioMerieux) and Bactec (Becton Dickinson), were compared with the CFR/USP method. Representative mononuclear cell (MNC) products were formulated using six different product media. MNC product aliquots containing 10-50 x 10(6) cells in a 0.5 mL volume were seeded with organisms, and cultured for 14 days in aerobic and anaerobic bottles of each system. Ten different organisms at target concentrations of 10 and 50 colony-forming units (CFU) per bottle were tested. RESULTS: Positives were detected in a mean (range) of 72% (7-100%) of cultures for CFR/USP, 82% (0-100%) for BTA, and 93% (57-100%) for Bactec. For nine of the 10 organisms tested, overall detection rates for BTA and Bactec were equivalent to or higher than CFR/USP. Of the six product media tested, detection of organisms was impaired only by the medium containing multiple antibiotics: this occurred in all three systems. Both BTA and Bactec had shorter times to detection than the CFR/USP method, with overall means (ranges) of 87 (24-264) h for CFR/USP, 24 (12-54) h for BTA, and 33 (12-80) h for Bactec. Detection occurred consistently within 7 days for both BTA and Bactec, but not for CFR/USP. DISCUSSION: Both BTA and Bactec are superior to the CFR/USP method for overall detection and time to detection of organisms in MNC products suspended in commonly used media. These data support general use of either BTA or Bactec for sterility testing of a variety of cell-therapy products, and suggest that a 7-day culture period is sufficient to detect clinically relevant organisms. These results confirm the need for bacteriostasis and fungistasis testing of antibiotic-containing products, even when antibiotic-binding substances are used.     

Comparison of Macroscopic Examination, Routine Gram Stains, and Routine Subcultures in the Initial Detection of Positive Blood Cultures

Blood was cultured in two vaccum bottles containing Columbia broth with sodium polyanethol sulfonate and CO₂. Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in CO₂) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture.     

Usefulness of the BACTEC MYCO/F lytic system for detection of mycobacteremia in a clinical microbiology laboratory

278 BACTEC MYCO/F lytic system blood cultures for mycobacteria were evaluated between 1997 and 1999. Sixty of them were read as positive by the system, being considered 15 of them as false positives. Twenty-seven yielded mycobacterial growth (13 Mycobacterium avium from 3 patients and 14 Mycobacterium tuberculosis from 8 patients). Other bacteria isolated were coagulase-negative Staphylococcus (13 samples), Corynebacterium sp. (5 samples), Salmonella enteritidis (2 samples) and Klebsiella pneumoniae (1 sample). Five of these isolates were considered as true episodes of bacteremia. The average time for detection of mycobacteria was 12.6 days for M. avium and 26.4 days for M. tuberculosis. BACTEC MYCO/F lytic system is useful for detection of mycobacteremia in clinical microbiology laboratories.     

The detection of positive blood cultures in children using the acridine orange, gram, and methylene blue stains

The efficacy of the acridine orange (AO), gram (G), and methylene blue (MB) microscopic procedures was analyzed using positive blood cultures monitored by a radiometric procedure (Bactec) in children. Sixty-eight positive cultures included the following isolates: Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, Enterobacteriaceae, Staphylococcus aureus, Candida spp., and seven other pathogens. The MB stain yielded the highest detection rate, 99%, in comparison with 94 and 93% for the AO and G stains, respectively. Since the MB stain yielded comparable results to the AO procedure with no requirement for a fluorescent microscope, the former method is recommended for confirming the presence and initial characterization of microorganisms in blood cultures monitored by Bactec from children.     

The rapid evaluation of bacterial growth in blood cultures by selected ion flow tube-mass spectrometry (SIFT-MS) and comparison with the BacT/ALERT automated blood culture system

We compared the performance of the BacT/ALERT automated blood culture system with real-time, quantitative volatile organic compound (VOC) detection by selected ion flow tube-mass spectrometry (SIFT-MS). Blood samples from healthy donors were artificially infected with 5 or 100 CFU of organisms commonly causing bacteremia. Positive results by SIFT-MS analysis of headspace gases were recorded for 53/60 (88.3%) bottles at 8h, and 58/60 (96.6%) bottles at 24 h. We conclude that SIFT-MS is a sensitive method for the detection of microbial VOCs. Furthermore, profiles of the VOCs detected may allow simultaneous identification of infecting organisms.     

Caught between validation of results and the case definition

A highly sensitive and timely detection system is the cornerstone of an effective response to either incursions of exotic diseases and pests or the emergence of new organisms. The trade-off is a reduction in system specificity. This presentation describes a number of investigations and responses involving MAFBNZ Investigation and Diagnostic Centre which were the result of specificity issues (false positive results). How and why did false positives occur, why are they important, the IDC approach to investigation of false positives and how this differs to traditional outbreak investigation together with some a priori decisions to reduce the impact of false positive results are explored.     

Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples

The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples. The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection. The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods. The MicroFoss system detected Listeria spp. in 12 samples, which were not detected by culture, and the culture method detected Listeria spp. in 15 samples, which were not detected by the MicroFoss method. This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods. The specificity value determined for the MicroFoss system was 92.7%. The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features. Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L. monocytogenes being the most predominant isolate detected. The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples. The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.     

Isolation of vero cytotoxin-producing Escherichia coli O157 from wild birds

In a survey of wild birds (mainly gulls), 0.9% of the bacterial isolates from faecal samples at an urban landfill site and 2.9% of bacterial isolates from faecal samples on intertidal sediments in Morecambe Bay were Vero cytotoxin-producing Escherichia coli O157. Isolation procedures employing commonly used cultural methods were hindered by the selection of a large number of false positives. The only procedure which resulted in the isolation of E. coli O157 from bird faecal samples was: enrichment (18 h) in a selective tryptone soya broth followed by filtration using hydrophobic grid membranes and growth on Chromagar O157. The majority of isolates selected as potential E. coli O157 by characteristic growth on Chromagar O157 could be eliminated by subsequent growth on CT-SMAC or CR-SMAC. This second identification (characterization) stage reduced the number of potential E. coli O157 requiring further confirmation by typing methods (serotype and Vero cytotoxin) by more than 70%.     

Rapid Identification and Susceptibility Testing of Candida spp. from Positive Blood Cultures by Combination of Direct MALDI-TOF Mass Spectrometry and Direct Inoculation of Vitek 2

Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.     

革兰阴性菌引起的血培养阳性标本直接细菌鉴定和药敏的应用

目的 评估血培养阳性标本直接细菌鉴定和药敏可行性.方法 将血培养瓶放入Bact/Alert 3D 60血培养系统进行培养筛选.选取78份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心血细胞.在收集到足量菌液后,用VITEK-32革兰阴性菌鉴定药敏卡做直接鉴定药敏试验.用标准方法及亚培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 VITEK-32直接鉴定试验,78株中的74株(94.9％)准确鉴定,直接药敏试验标准符合率95.6％.KB法血标本直接药敏试验标准符合率96.2％,但微小错误率高于VITEK-32直接药敏法.结论 Bact/Alert血培养阳性标本直接VITEK-32细菌鉴定和药敏对革兰阴性菌是切实可行的,可大幅度缩短时间,为临床及时修正用药提供依据,具有较高的应用价值.     

False positive rate in newborn screening for congenital adrenal hyperplasia (CAH)-ether extraction reveals two distinct reasons for elevated 17α-hydroxyprogesterone (17-OHP) values

Background

While the sensitivity of newborn screening for the salt wasting form of congenital adrenal hyperplasia (CAH) is good, the positive predictive value is poor due to the high false positive rate of the immunological assays for 17-OHP. Cross-reactivity with steroid sulfates is one of the main causes for false positive results. Several approaches have been described to improve CAH screening: adjusting cut-off levels to gestational age or birth weight, and second-tier molecular genetic analysis or second-tier liquid chromatography-tandem mass spectrometry (TMS).

Methods

17-OHP was extracted with diethyl ether from dried blood spots in order to separate 17-OHP from polar steroids (like steroid sulfates). The dried ether extracts of calibrators, controls, and patient samples were redissolved and measured with the 17-OHP test kit (Wallac).

Results

760 normal, 1049 false positive, and 232 samples of confirmed cases with CAH were analysed. Mean 17-OHP values were significantly lower after extraction: Normal samples: 17.5 nmol/L vs. 3.2 nmol/L; false positive samples: 97.0 nmol/L vs. 25.9 nmol/L; CAH: 275 nmol/L vs. 205 nmol/L. With a cut-off value of 11.9 nmol/L (mean + 3 SD of the normal values), 404 of the false positives turned out to be normal. Ether extraction revealed two distinct subgroups of initially false positives rather than a continuum with normal distribution of 17-OHP values.

Conclusion

Diethyl ether extraction provided evidence for two causes of false positive results in CAH screening. It reduced the rate of false positives by about 40% without loss of sensitivity.     

Culture of Ethiopian Strains of Borrelia recurrentis

A number of standard bacteriological media with supplements were tested for their ability to support in vitro growth of Ethiopian strains of Borrelia recurrentis. Propagation of 18 out of 21 strains occurred in Trypticase soy yeast broth to which bovine albumin (fraction V), N-acetyl glucosamine, and sodium pyruvate had been added. This medium supported a population of 10(7) organisms per ml and yielded a harvest of four to five times the original inoculum during the logarithmic phase of growth. Maximal yield varied from 1.4 x 10(7) to 3.4 x 10(7) organisms per ml. Generation time in optimal media was 11.3 h. Lesser multiplication of organisms occurred in other media tested. Strains from primary cultures were infective for the green monkey. Recovery of viable organisms from subculture has not been successful.     

Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome

Background  

Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem.     

Membrane filter method for enumerating Escherichia coli.

A membrane filter procedure for enumerating Escherichia coli was developed and evaluated. The method quantifies E. coli within 24 h without requiring subculture and identification of isolates. It incorporates a primary selective-differential medium for gram-negative, lactose-fermenting bacteria; resuscitation of weakened organisms by incubation for 2 h at 35 degrees C before incubation at 44.5 degrees C for 18 to 22 h; and an in situ urease test to differentiate E. coli from other thermotolerant, lactose-positive organisms. The recovery of E. coli from marine, estuarine, and freshwater samples exceeded 90%. Of the presumptively positive colonies, 91% were verified as E. coli. Less than 1% of all of the verified E. coli colonies failed to react typically.     

Implications of False Negative and False Positive Diagnosis in Lymph Node Staging of NSCLC by Means of 18F-FDG PET/CT

Background

Integrated ¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT) is widely performed in hilar and mediastinal lymph node (HMLN) staging of non-small cell lung cancer (NSCLC). However, the diagnostic efficiency of PET/CT remains controversial. This retrospective study is to evaluate the accuracy of PET/CT and the characteristics of false negatives and false positives to improve specificity and sensitivity.

Methods

219 NSCLC patients with systematic lymph node dissection or sampling underwent preoperative PET/CT scan. Nodal uptake with a maximum standardized uptake value (SUVmax) >2.5 was interpreted as PET/CT positive. The results of PET/CT were compared with the histopathological findings. The receiver operating characteristic (ROC) curve was generated to determine the diagnostic efficiency of PET/CT. Univariate and multivariate analysis were conducted to detect risk factors of false negatives and false positives.

Results

The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of PET/ CT in detecting HMLN metastases were 74.2% (49/66), 73.2% (112/153), 54.4% (49/90), 86.8% (112/129), and 73.5% (161/219). The ROC curve had an area under curve (AUC) of 0.791 (95% CI 0.723-0.860). The incidence of false negative HMLN metastases was 13.2% (17 of 129 patients). Factors that are significantly associated with false negatives are: concurrent lung disease or diabetes (p<0.001), non-adenocarcinoma (p<0.001), and SUVmax of primary tumor >4.0 (p=0.009). Postoperatively, 45.5% (41/90) patients were confirmed as false positive cases. The univariate analysis indicated age > 65 years old (p=0.009), well differentiation (p=0.002), and SUVmax of primary tumor ≦4.0 (p=0.007) as risk factors for false positive uptake.

Conclusion

The SUVmax of HMLN is a predictor of malignancy. Lymph node staging using PET/CT is far from equal to pathological staging account of some risk factors. This study may provide some aids to pre-therapy evaluation and decision-making.     

Roadmap to approval: use of an automated sterility test method as a lot release test for Carticel, autologous cultured chondrocytes

BACKGROUND: In February 2004, FDA approved a supplement to our biologics license for Carticel, autologous cultured chondrocytes, to use the BacT/ALERT microbial detection system as an alternative to the compendial sterility test for lot release. This article provides a roadmap to our approval process. The approval represents more than 4 years of development and validation studies comparing the Steritest compact system to the BacT/ALERT microbial detection system. METHODS: For this study, freshly cultured chondrocytes were prepared from a characterized cell bank. Microbial isolates were prepared from either American Type Culture Collection (ATCC) strains or from in-house contaminants. For each test condition, a suspension of chondrocyte cells and test organisms was inoculated into both aerobic media (SA standard adult culture bottles, FA FAN, tryptic soy broth) and anaerobic media (SN standard adult culture bottles, FN FAN, fluid thioglycollate media) and tested for sterility using the Steritest compact system (Millipore, Bedford, MA, USA) and the BacT/ALERT microbial detection system (bioMerieux, Durham, NC, USA). Negative control bottles were inoculated with chondrocytes and no microorganisms. All bottles were incubated for 14 days and read daily. Bacterial growth was determined by either visual examination of Steritest canisters or detection of a positive by the BacT/ALERT system. A gram stain and streak plate were used to confirm positive bottles and negative bottles after 14 days. RESULTS: The detection of a positive by either the Steritest compact system or the BacT/ALERT system was summarized for each organism in each validation study. Data generated from studies reducing the incubation temperature from 35 degrees C to 32 degrees C improved detection times in the automated method compared with the compendial method. Other improvements included the use of FAN aerobic and anaerobic media to absorb the gentamicin contained in the culture media of prepared chondrocyte samples. Chondrocytes alone did not generate positive results in either the compendial method or the automated method. DISCUSSION: Data from validation studies support the use of the BacT/ALERT microbial detection system as an alternative sterility test for Carticel.     

Distribution of Streptococcal Groups in Clinical Specimens with Evaluation of Bacitracin Screening

During a 2-year period, 4,968 strains of beta-hemolytic streptococci were examined for the clinical source distribution and bacitracin sensitivity of each group. In the upper respiratory tract, groups A (51.7%) and C (20.4%) accounted for most of the isolates, and in wounds and exudates group A (79.1%) made up most of the isolates. Group B (71.2%) was the major component of isolates from the genitorinary tract and, while composing 29.3% of the lower respiratory tract isolates, competed with group A (18.8%) and the nongroupables (22.8%) for supremacy. Bacitracin screening showed that 0.5% of group A streptococci were resistant, and sensitive non-group A isolates were group B (2.6%), group C (6.0%), group G (8.0%), and the nongroupables (2.2%). It was found that those groups which were most predominant in wounds and the upper respiratory tract gave the highest rate of false positives with bacitracin, whereas the predominant group of the genitourinary tract gave the lowest rate of false positives.