Evaluation of Sulfamethoxazole- Trimethoprim Blood Agar Plates for Recovery of Group A Streptococci from Throat Cultures

We compared the selective blood agar medium of Gunn et al. (J. Clin. Microbiol. 5:650-655, 1977) which contains sulfamethoxazole plus trimethoprim (SXT-BA) to the conventional blood agar surface plate (SBA) and a modified blood agar pour plate plus broth method for the recovery of group A streptococci from throat swabs. The influence of CO(2) and ambient air incubation of the SXT-BA and SBA plates was also evaluated. A total of 696 throat swabs from symptomatic children were cultured simultaneously by the five methods and observed after overnight incubation; 204 positive cultures were detected overall. Recovery rates of each individual method were: SXT-BA (CO(2)), 90.7%; SXT-BA (air), 87.7%; pour plate plus broth, 83.3%; SBA (CO(2)), 79.4%; and SBA (air) 77%. Approximately one-half of the false-negative cultures in the SXT-BA (CO(2)) and SXT-BA (air) methods had colony counts of >/=10 to 100 colonies per plate. In contrast, for the SBA (CO(2)), SBA (air), and pour plate plus broth methods, approximately 70% of the false-negative cultures had colony counts of >/=10 to 100/plate. False-positive cultures obtained by the SXT-BA (CO(2)) and SXT-BA (air) methods were 11 and 12.7%, respectively-one-half as high as the rates obtained by the remaining methods. Beta-hemolytic streptococci, groups C, F, and G, are inhibited on the SXT-BA plates and were the primary cause of the higher false-positive rates on SBA and pour plate plus broth methods. An additional 3% positive cultures were obtained by incubating SXT-BA (CO(2)) plates up to 48 h before discarding as negative. We recommend either the SXT-BA (CO(2)) or the SXT-BA (air) method with up to 48 h of incubation for routine use in throat cultures.     

Practical Disk Diffusion Test for Detecting Group B Streptococcus with Reduced Penicillin Susceptibility

Although group B streptococcus (GBS) has been considered to be uniformly susceptible to β-lactams, the presence of GBS with reduced penicillin susceptibility (PRGBS) was recently confirmed genetically. We developed a feasible and reliable method for screening PRGBS in clinical microbiology laboratories using a combination of ceftibuten, oxacillin, and ceftizoxime disks.Streptcoccus agalactiae (group B streptococcus [GBS]) is a leading cause of neonatal sepsis and meningitis and is also an important pathogen for elderly people and those suffering from underlying medical disorders (1, 5, 7, 11). GBS results in the highest mortality and morbidity if it causes invasive infections in neonates, including very-low-birth-weight infants (6, 10, 12). About 5% of GBS-infected infants die, and if they survive, they often suffer from severe neurological sequelae, such as mental retardation and vision and/or auditory disabilities (2), but development of GBS vaccines is still under investigation (8). Penicillins are the first-line agents in the treatment of GBS infections because all clinical GBS isolates have been considered to be uniformly susceptible to β-lactams, including penicillins (2, 3). However, we have recently identified and molecularly characterized several clinical GBS isolates demonstrating reduced penicillin susceptibility (PRGBS) through acquisition of multiple mutations in the penicillin-binding protein 2X (pbp2x) gene (9), and similar PRGBS isolates were recently reported in the United States (4). PRGBS isolates were indeed confirmed to be nonsusceptible to penicillin G (PCG) by the agar dilution method, but this PCG nonsusceptibility was not apparent even if the PCG disk diffusion method was performed in accordance with the recommendations of the CLSI (Clinical and Laboratory Standards Institute) (3). Here we developed, therefore, a feasible and practical new disk test method for discriminating PRGBS from the clinically isolated GBS using three disks containing ceftibuten, oxacillin, and ceftizoxime, respectively.Forty-eight clinical isolates were identified as GBS using a streptococcus grouping kit (Slidex Strepto [bioMerieux, Marcy l''Etoile, France] and Streptex [Mitsubishi Chemical Medience Corporation, Tokyo, Japan]). Streptococcus agalactiae ATCC BAA-611 and ATCC 12403 were used as the reference strains in bacteriological identification. The MICs of PCG for two ATCC standard strains and clinical isolates were determined by the agar dilution method recommended by the CLSI. Streptococcus pneumoniae 49619 was used for validation of the MIC measurements. The disk diffusion method was performed as recommended by the CLSI in evaluation of the applicability of each β-lactam disk for discrimination of PRGBS strains from penicillin-susceptible strains.At first, the MICs of PCG for 2 ATCC strains and 48 clinical GBS isolates were measured by the CLSI standard agar dilution methods, and 34 strains, including 2 reference strains, proved susceptible to PCG (MIC, ≤0.12 μg/ml), whereas the remaining 16 clinical isolates were nonsusceptible to PCG (MIC, >0.12 μg/ml) (Fig. ​(Fig.1A1A and Table ​Table1).1). The highest PCG MIC for such PCG-nonsusceptible GBS strains was 1 μg/ml.Open in a separate windowFIG. 1.Scatter diagram of MICs and the sizes of the growth-inhibitory zone. PCG MICs determined by the CLSI agar dilution method and diameters of the growth-inhibitory zone measured by CLSI-recommended standard disk diffusion method are plotted using a PCG disk (A). MICs determined by the agar dilution method and the diameters of growth inhibitory zone measured by the CLSI-recommended standard disk diffusion method are plotted using an oxacillin disk (B), ceftizoxime disk (C), and ceftibuten disk (D). The numbers in the scatter diagram indicate the number of clinical isolates in each intersection.

TABLE 1.

MICs of PCG for 16 strains of PRGBS, amino acid substitutions in PBP2X of PRGBS, and the diameters of growth-inhibitory zones around oxacillin, ceftizoxime, and ceftibuten disks

Testing Susceptibility of Multidrug-Resistant Mycobacterium tuberculosis to Second-Line Drugs by Use of Blood Agar

In this study, the susceptibilities of 35 multidrug-resistant (MDR) Mycobacterium tuberculosis clinical isolates to second-line drugs, including kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH), were investigated on blood agar according to CLSI recommendations. Compared with the results of the Bactec 460 TB system, agreement was 100, 100, 97, 100, 100, 100, and 86% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively. Compared with the results of the proportion method, agreement was 100, 100, 97, 100, 97, 100, and 77% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively.Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are major public health problems, especially in developing countries (13, 18, 21). Rapid susceptibility testing is critical for early diagnosis of MDR- and XDR-TB and the initiation of effective regimens (14). The agar proportion method performed on Middlebrook 7H10 and 7H11 agars is the reference method for susceptibility of Mycobacterium tuberculosis according to CLSI recommendations (16). The Bactec 460 TB system (Becton Dickinson Diagnostic Systems, Sparks, MD), Bactec MGIT 960 (Becton Dickinson Diagnostic Systems, Sparks, MD), and Versa TREK system (formerly known as ESP II; Trek Diagnostic Systems, West Lake, OH) are cleared for used by the U.S. FDA for testing M. tuberculosis susceptibility to first-line drugs (16). It has recently been demonstrated that blood agar can be used for routine culture and testing of M. tuberculosis susceptibility to first-line drugs (1-4, 6, 7, 15).In this study, the susceptibilities of 35 MDR M. tuberculosis clinical isolates against the second-line drugs kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH) were investigated on blood agar, and results were compared to those obtained using the Bactec 460 TB system and the proportion method on Middlebrook 7H10 agar, performed according to CLSI recommendations.Clinical MDR-TB strains were obtained from the Istanbul Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Istanbul, Turkey. Isolates were identified by the Bactec NAP test as M. tuberculosis complex. Susceptibility to first-line drugs was determined by the radiometric method (Bactec 460 TB system) (19, 20). M. tuberculosis H37Rv was used as a control strain. All drugs were obtained from the manufacturers in a chemically pure form. Stock antibiotic solutions and subsequent dilutions were prepared. All stock solutions except CFM, which was stored in the dark at room temperature, were stored at −70°C in small aliquots.The agar proportion method using Middlebrook 7H10 and blood agar was performed with concentrations of 10, 5, 5, 1, 2, 1, and 2 μg/ml for CAP, ETH, KM, CFM, OFX, RBU, and PAS, respectively (9, 10, 12, 16, 17).Middlebrook 7H12 broth (Bactec 12B; Becton Dickinson Microbiology Systems) was used for radiometric testing (5, 11, 16, 19, 20). Second-line drugs (KM, RBU, PAS, CAP, CFM, OFX, and ETH) were tested by using single critical concentrations. Concentrations of second-line drugs tested in the Bactec 460 TB system were 1.25, 1.25, 5, 0.5, 2, 0.5, and 4 μg/ml for CAP, ETH, KM, CFM, OFX, RBU, and PAS, respectively (9, 10, 12, 16, 17).The inoculum was prepared from freshly grown colonies on Löwenstein-Jensen medium. The supernatant of each isolate was adjusted to a 1 McFarland standard. The agar proportion method was performed on both Middlebrook 7H10 agar and blood agar (infusion agar, Becton Dickinson) separately according to CLSI recommendations (16). The plates were monitored twice a week by the naked eye. Resistance was defined as growth of a colony count of >1% on drug-containing quadrants in comparison to drug-free quadrants (16).The Bactec 460 TB system revealed that all isolates were susceptible to KM and CFM. RBU, OFX, PAS, CAP, and ETH resistance was detected in 27, 1, 3, 2, and 17 isolates, respectively. All isolates were susceptible to KM and CFM on Middlebrook 7H10 agar; resistance was detected in 27, 1, 3, 1, and 14 isolates for RBU, OFX, PAS, CAP, and ETH, respectively. On blood agar, all isolates were susceptible to KM and CFM; resistance was detected in 27, 2, 3, 2, and 22 isolates for RBU, OFX, PAS, CAP, and ETH, respectively.Blood agar results were compared with the results obtained using the Bactec 460 TB system and the proportion method on Middlebrook 7H10 agar. Agreement, sensitivity, specificity, positive predictive value, and negative predictive value are shown in Tables ​Tables11 and ​and2.2. The results of susceptibility testing were obtained on the 21st day of incubation by the proportion method on blood and Middlebrook 7H10 agar as recommended by the CLSI. The results of susceptibility testing by the Bactec 460 TB system were obtained on the 5th day of incubation for 31 isolates and on the 8th day for 4 isolates.

TABLE 1.

Comparison of blood agar results and Bactec 460 TB system results^a

Penicillin-Susceptible Group B Streptococcal Clinical Isolates with Reduced Cephalosporin Susceptibility

We characterized penicillin-susceptible group B streptococcal (PSGBS) clinical isolates exhibiting no growth inhibition zone around a ceftibuten disk (CTB^r PSGBS). The CTB^r PSGBS isolates, for which augmented MICs of cefaclor and ceftizoxime were found, shared a T394A substitution in penicillin-binding protein 2X (PBP 2X) and a T567I substitution in PBP 2B, together with an additional G429S substitution in PBP 2X or a T145A substitution in PBP 1A, although the T145A substitution in the transglycosidase domain of PBP 1A would have no effect on the level of resistance to ceftibuten.     

Localization and Characterization of the Hippuricase Activity of Group B Streptococci

Studies are presented on the isolation, localization, and characterization of hippuricase activity of group B streptococci. Washed, intact cells, live or heat killed at 56 C, exhibited hydrolysis of hippuric acid, but cell-free filtrates of the organism did not. Excellent hippuricase activity was recoverable from supernatant fluids of mechanically disrupted cells, and evidence suggests that it exists largely intracellularly. Characteristics of the hippuricase preparation are consistent with the view that the biologically active principle is an enzyme. A quantitative microtiter technique has been developed which is useful in titrating enzymatic activity and antibody neutralization. Sera from rabbits immunized with filtered preparations neutralized hippuricase activity.     

Evaluation of the Granada Agar Plate for Detection of Vaginal and Rectal Group B Streptococci in Pregnant Women

Granada medium was evaluated for the detection of group B streptococci (GBS) in vaginal and rectal swabs compared with selective Columbia blood agar and selective Lim broth. From May 1996 to March 1998, 702 pregnant women (35 to 37 weeks of gestation) participated in this three-phase study; 103 (14.7%) of these women carried GBS. In the first phase of the experiment (n = 273 women), vaginorectal specimens were collected on the same swab; the sensitivities of Granada tube, selective Columbia blood agar, and Lim broth were 31.4, 94.3, and 74.3%, respectively. In the second and third phases (n = 429 women), vaginal and rectal specimens were collected separately; the sensitivities of Granada plate, selective Columbia blood agar, and Lim broth (subcultured at 4 h on selective Columbia agar in the second phase and at 18 to 24 h in Granada plate in the third phase) were 91.1, 83.9, and 75%, respectively, in the second phase and 88.5, 90.4, and 63.5%, respectively, in the third phase. There were no statistically significant differences in GBS recovery between the Granada agar plate and selective Columbia blood agar, but the Granada plate provided a clear advantage; the characteristic red-orange colonies produced overnight by GBS can be identified by the naked eye and is so specific that further identification is unnecessary. The use of the Granada tube and Lim broth did not result in increased isolation of GBS. In conclusion, the Granada agar plate is highly sensitive for detecting GBS in vaginal and rectal swabs from pregnant women and can provide results in 18 to 24 h.     

Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.     

Determination of Trimethoprim-Sulfamethoxazole Resistance in Streptococcus pneumoniae by Using the E Test with Mueller-Hinton Agar Supplemented with Sheep or Horse Blood May Be Unreliable

An international, multicenter study compared trimethoprim-sulfamethoxazole MICs for 743 Streptococcus pneumoniae isolates (107 to 244 isolates per country) by E test, using Mueller-Hinton agar supplemented with 5% defibrinated horse blood or 5% defibrinated sheep blood, with MICs determined by the National Committee for Clinical Laboratory Standards broth microdilution reference method. Agreement within 1 log₂ dilution and minor error rates were 69.3 and 15.5%, respectively, on sheep blood-supplemented agar and 76.9 and 13.6%, respectively, with horse blood as the supplement. Significant interlaboratory variability was observed. E test may not be a reliable method for determining the resistance of pneumococci to trimethoprim-sulfamethoxazole.     

Two Sensitive PCR-Based Methods for Detection of Hepatitis B Virus Variants Associated with Reduced Susceptibility to Lamivudine

Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5'-nuclease activity of Taq DNA polymerase, were developed for screening viral variants in lamivudine-treated patients' sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients' sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine(552) aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients' sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5'-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5'-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.     

受血者输血中Rh血型E抗原对患者机能免疫功能影响

目的 探讨受血者输血中Rh血型E抗原对患者机能免疫功能影响.方法 收集2014年3月至2016年7月200组受-供血者的血标本,其中100组受-供血者E抗原均呈阴性为A组,另100组受血者E抗原呈阴性、供血者E抗原呈阳性为B组.受血者输血前后分别检测血清白细胞介素-2(IL-2)、免疫球蛋白M(IgM)、IgG抗体及血中T淋巴细胞亚群(CD4+、CD8+).结果 A组输血前后血清IL-2、IgM及IgG抗体水平均无明显变化.B组输血后IL-2、IgM及IgG抗体水平均呈先逐渐升高,后逐渐降低的过程,均在输血后5d达到最高,在输血后18d基本恢复至输血前水平.B组输血后3、5、10d IL-2、IgM及IgG抗体水平均明显高于A组(P均＜0.05);A组输血前后血清CD4+、CD8+细胞数无明显变化.B组输血后CD4+细胞数均呈先逐渐升高后降低,CD8+细胞逐渐降低后升高,均在输血后3d达到最高和最低,在输血后18d基本恢复至输血前水平;B组输血后5、10d CD4+细胞数均明显高于A组,CD8+细胞低于A组(P均＜0.05).结论 Rh(E)抗原阴性的受血者在输注E抗原阳性血制品后,其机能免疫功能将出现有时间限制的改变;临床应加强对受-供血者的Rh(E)抗体的检测,配型满意后方进行输血,提高临床输血的科学性、合理性与安全性.     

Evaluation of the Cepheid Xpert GBS Assay for Rapid Detection of Group B Streptococci in Amniotic Fluids from Pregnant Women with Premature Rupture of Membranes

The Xpert GBS real-time PCR assay for the detection of group B streptococci (GBS) in antepartum screening samples was evaluated on amniotic fluid samples collected from 139 women with premature rupture of membrane at term. When any intrapartum positive result from the Xpert GBS or culture was considered a true positive, the sensitivities of the Xpert GBS and culture were 92.3% and 84.6%, respectively. This assay could enhance exact identification of candidates for intrapartum antibiotic prophylaxis.     

Cervical Secretions in Pregnant Women Colonized Rectally with Group B Streptococci have High Levels of Antibodies to Serotype III Polysaccharide Capsular Antigen and Protein R

Group B streptococci (GBS) colonizing the female genital tract will often infect newborn infants during delivery. In 200 pregnant women studied, 14% were colonized with GBS in the cervix, 12% in the rectum, and 9% in both cervix and rectum. We have previously reported that antibody levels to GBS serotypes Ia, II, and III in sera and cervical secretions were increased in women colonized in the rectum and/or cervix, when analyzed by a whole-cell ELISA. Here, we report the levels of antibodies to GBS serotype III capsular polysaccharide antigen (CPS III) and to protein antigen R₄, which are present in most GBS III strains. Compared to culture-negative women, the group of women colonized rectally had markedly elevated levels of immunoglobulin (Ig)A and IgG antibodies in cervical secretions to both CPS III and protein R₄ ( P  < 0.01 and P  < 0.001, respectively). In sera, the corresponding differences between culture-negative and culture-positive women were less pronounced, or not present. In contrast to antibody levels to whole-cell GBS, antibody levels to CPS III and protein R₄ in cervical secretions were not significantly increased in women colonized only in the cervix, except that IgA antibodies to protein R₄ were slightly elevated ( P  < 0.05). These findings suggest that capsular type-specific polysaccharides and protein R₄ in a mucosal vaccine might induce protective antibodies against GBS colonization of the uterine cervix.     

Characterization of non-type B Haemophilus influenzae strains isolated from patients with invasive disease. The HI Study Group

Forty-one non-type b Haemophilus influenzae isolates from cases of invasive disease were characterized. By PCR capsular genotyping, 33 nonencapsulated strains, 4 type f isolates, and 4 b(-) strains were identified. By pulsed-field gel electrophoresis, the nonencapsulated isolates exhibited great genetic heterogenicity, whereas the type f and the b(-) strains seemed to have a clonal spread. Occurrence of the hifA gene was found by PCR in 18% of the nonencapsulated, 50% of the b(-), and all of the type f strains. Hemagglutinating fimbriae were generally expressed by nonencapsulated isolates when fimbrial gene hifA was present. Two nonencapsulated isolates not susceptible to ampicillin were detected; no strains were positive for beta-lactamase production.     

Distribution by Serological Type of Group B Streptococci Isolated from a Variety of Clinical Material over a Five-Year Period (with Special Reference to Neonatal Sepsis and Meningitis)

A total of 898 group B streptococci isolated from a wide variety of human clinical sources from July 1967 through June 1972 were typed by the Lancefield precipitin test. Only 11% of the strains were nontypable. Twenty-six percent of the group B strains were from respiratory sources, 22% were from cerebrospinal fluid (CSF) and blood, 13% were from the female genital tract, 12% were urine specimens, and the remaining 27% were from other varied sources. The clinical conditions reported for patients from whom these organisms were isolated included neonatal meningitis and sepsis, pharyngitis, urinary tract and female genital tract infections, and various skin and wound infections. Seventy percent of the CSF and blood cultures from patients with meningitis or sepsis, or both, were type III, whereas the overall percentage of this type was 32%. All but three CSF isolates were from patients under 2 years of age; the distribution of CSF isolates appeared to be the same for both sexes. In contrast, group B streptococci were isolated more frequently from the blood of males than from the blood of females. There were twice as many blood cultures from patients under 2 years of age than from those that were older.     

Reduced expression of toll-like receptor 2 on peripheral monocytes in patients with chronic hepatitis B

Persistent infection with hepatitis B virus (HBV) likely depends on viral inhibition of host defenses. We report that chronic hepatitis B e antigen-positive HBV infection is associated with a significant reduction in peripheral blood monocyte expression of Toll-like receptor 2, a key component of innate immunity, thereby providing a mechanism by which wild-type HBV may establish persistent infection.     

应用CelliGen细胞罐高密度培养抗人B血型杂交瘤细胞条件的研究

应用CelliGen细胞培养罐,培养抗人B血型杂交瘤细胞,获得了细胞密度超过10~7/ml,产物IgM 2g/L以上,特异性血凝效价达到2~(13)～2~(15)。培养条件50～80r/min、pH7.2、溶解氧(DO)30％～50％,高密度时维持DO2％～10％是必要的,4种气体混合流量为0.2LPM,高密度时要增加氧气供应,细胞密度达到1.6×10~6/ml时开始灌注,随细胞密度增加,灌注量亦增加,每天补充新培养液由800ml逐渐增加到3000ml。13B1杂交瘤细胞生长代谢与葡萄糖、乳酸有关,与氨代谢无关。     

Rapid Antifungal Susceptibility Determination for Yeast Isolates by Use of Etest Performed Directly on Blood Samples from Patients with Fungemia

We prospectively determined the antifungal susceptibility of yeast isolates causing fungemia using the Etest on direct blood samples (195 prospectively collected and 133 laboratory prepared). We compared the Etest direct (24 h of incubation) with CLSI M27-A3 and the standard Etest methodologies for fluconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B. Strains were classified as susceptible, resistant, or nonsusceptible using CLSI breakpoints (voriconazole breakpoints were used for posaconazole and isavuconazole). Categorical errors between Etest direct and CLSI M27-A3 for azoles were mostly minor. No errors were detected for caspofungin, and high percentages of major errors were detected for amphotericin B. For the azoles, false susceptibility (very major errors) was found in only two (0.6%) isolates (Candida tropicalis and C. glabrata). False resistance (major errors) was detected in 46 (14%) isolates for the three azoles (in 23 [7%] after excluding posaconazole). Etest direct of posaconazole yielded a higher number of major errors than the remaining azoles, especially for C. glabrata, Candida spp., and other yeasts. Excluding C. glabrata, Candida spp., and other yeasts, the remaining species did not yield major errors. Etest direct for fluconazole, voriconazole, isavuconazole, and caspofungin shows potential as an alternative to the CLSI M27-A3 procedure for performing rapid antifungal susceptibility tests on yeast isolates from patients with fungemia. Etest direct is a useful tool to screen for the presence of azole-resistant and caspofungin-nonsusceptible strains.The incidence of fungemia continues to rise in many institutions throughout the world, and Candida is one of the leading pathogens isolated from blood (15, 28). Amphotericin B and fluconazole have been widely used for the treatment of fungemia. However, newly licensed antifungal agents (voriconazole, posaconazole, and the echinocandins) and other azoles currently under investigation (isavuconazole) have expanded the antifungal armamentarium.The mortality rate of fungemia remains high (30%) and is clearly correlated with delayed initiation of effective antifungal therapy (11, 17). Antifungal therapy is considered inappropriate when it is omitted, when the agent administered has no antifungal activity against the infecting organism, or when its serum concentrations are subtherapeutic.A growing proportion of Candida isolates obtained from blood samples have reduced antifungal susceptibility to fluconazole and other antifungal agents (14, 25). Patients with candidemia caused by Candida strains with high MICs for fluconazole or voriconazole and echinocandins can have a worse prognosis (22-24). Consequently, systematic use of empirical antifungal agents with broad-spectrum in vitro activity has led to considerable increases in the number of adverse events and in the cost of treatment (2).The combination of an increasing number of antifungal-resistant isolates and the cost of the new antifungal agents makes antifungal susceptibility testing a necessity. The reference antifungal susceptibility testing method for yeasts is the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) testing standard M27-A3. However, this method requires pure-culture isolates, and results of antifungal susceptibility testing are not available until 48 to 72 h after the isolation of fungi in blood.The Etest performed directly on blood samples may expedite antifungal testing and provide results in 24 h. Our group has previously demonstrated that the Etest performed directly on samples from the lower respiratory tract is a rapid and accurate procedure for antimicrobial susceptibility testing of bacteria in patients with ventilator-associated pneumonia (4, 5). We compared the results of the Etest performed directly on positive blood cultures with yeasts grown in Bactec blood bottles with the results of CLSI M27-A3 in isolates from patients with fungemia and blood samples generated from previously characterized isolates.(This study was partially presented at the 20th Conference of the European Congress of Clinical Microbiology and Infectious Diseases [ECCMID] in Vienna, Austria, 2010 [abstract no. P-838] [13a].)