Multicenter Evaluation of the BD Max GBS Assay for Detection of Group B Streptococci in Prenatal Vaginal and Rectal Screening Swab Specimens from Pregnant Women

A new integrated extraction and real-time PCR-based system for the detection of group B streptococci in antepartum screening samples enriched in Lim broth was compared to the CDC-recommended culture method. The BD Max GBS assay exhibited acceptable sensitivity (95%) and specificity (96.7%) compared to those of the culture method in this multisite evaluation.The number of neonatal group B streptococcus (GBS) infections has decreased significantly over the past 4 decades; however, GBS still remains one of the most common causes of neonatal sepsis in the United States (3). Current management guidelines recommend that all pregnant women be screened for vaginal/rectal GBS colonization at 35 to 37 weeks of gestation, with those found to be colonized receiving intrapartum antibiotic prophylaxis.While culture-based methods have historically been the gold standard for demonstrating GBS colonization, several recent studies have demonstrated the utility of PCR-based detection as a sensitive and specific alternative (1, 2, 4-6, 8, 9). The BD Max GBS assay (BDM) implemented using the BD Max system (previously known as the HandyLab Jaguar system; BD-HandyLab, Ann Arbor, MI) is one such PCR-based alternative. The BD Max system is a benchtop molecular diagnostic system, which provides fully automated clinical sample preparation, cell lysis, nucleic acid extraction, and mixing of nucleic acid with master mix reagents. With no user intervention, the system then dispenses the sample into a microfluidic chamber where real-time PCR amplification and detection are performed.The goal of this three-site investigational study was to compare the results obtained by BDM to those obtained by the CDC-recommended culture procedure, which served as the reference method (3). This study was designed to generate the data necessary for 510(k) submission to the FDA, so the study design, reference method, and evaluation criteria were performed as required by the FDA. Performance characteristics of the assay were derived from the results of 601 compliant specimens collected from antepartum women presenting for routine prenatal screenings at clinical locations within the United States (site 1, University of Michigan Health System [UM], Ann Arbor, MI, collected 184 specimens; site 2, DCL Medical Laboratories [DCL], Indianapolis, IN, collected 198 specimens; and site 3, TriCore Reference Laboratories [TRL], Albuquerque, NM, collected 219 specimens). The vaginal/rectal swab specimens were inoculated in Lim broth (DCL and UM obtained broth from Thermo Fisher-Remel, Lenexa, KS; TRL obtained broth from Becton-Dickinson, Sparks, MD) and incubated for 18 to 24 h (as per protocol) prior to testing by either method. The growth obtained with each Lim broth was subcultured onto a sheep blood agar plate and incubated for up to 48 h. Colonies with morphology and color suggestive of GBS (both hemolytic and nonhemolytic) were Gram stained, tested for catalase production, and confirmed as GBS using latex agglutination (DCL and TRL used PathoDX Strep Grouping reagents, Thermo Fisher-Remel; UM used Slidex, bioMérieux, Durham, NC) and/or CAMP testing. BDM (cfb gene target and limit of detection of 200 CFU of GBS/ml of sample preparation reagent) (data not shown) was performed using a residual 15-μl aliquot of Lim broth, and when possible, an alternate, validated PCR method was performed at each site (at DCL, the IDI-Strep B assay was performed on the Cepheid SmartCycler system with a cfb gene target; at TRL, the Roche analyte-specific reagent was used on the Roche LightCycler system with a pstI gene target; and at UM, the Cepheid Smart GBS assay was performed on the Cepheid SmartCycler system, with a proprietary GBS-specific target) and used to resolve discrepancies between BDM and the culture method. This alternate PCR method was performed only using the discrepant specimens. Lim broths were stored at 4°C for up to 7 days prior to BDM testing. Stability studies demonstrated no loss of analytic performance in samples stored in this manner (data not shown). In addition to the alternate PCR assay, “false-negative” specimens were retested at each site using BDM, and “false-positive” specimens were resubcultured from the original Lim broth at each site. The repeat PCR and culture testing was performed for informational purposes only and was not used to adjust the observed performance characteristics. This study was approved by Institutional Review Boards, as appropriate, of the performance sites.Table ​Table11 shows a summary of the combined site results obtained using BDM and the culture-based method. Overall agreement was 96.3%. A total of 13 of the 15 BDM-positive, culture-negative specimens (“false positives”) were available for testing by the alternate PCR assay, and 8 tested positive. A total of 12 of the 15 “false positives” were also recultured, and 10 of the 12 again yielded negative results. The two culture-positive specimens in this subset were also positive when tested by the alternate PCR assay. These results are consistent with those from previous studies that have demonstrated an increased rate of GBS detection with broth-enriched specimens using PCR compared to that with culture (7), likely reflecting the detection of bacteria present at levels below the limits of detection for culture. Furthermore, there is a low probability that “false positives” were due to nonspecific amplification, as no significant cross-reactivity was demonstrated against a panel of 127 nontarget pathogens in which 119 viable bacteria, fungi, and viruses and 8 genomic DNA samples were tested (see the supplemental material for a complete list of organisms tested). Five of the seven culture-positive, BDM-negative specimens (“false negatives”) were retested by the alternate PCR assay, and three tested negative. Four of the seven “false negatives” were also retested using BDM, and three yielded a negative result for a second time. It is unlikely that these “false-negative” BDM results are the result of sample degradation or overgrowth of competing organisms during storage, as the stability studies mentioned above demonstrated the maintenance of integrity of positive signals for specimens stored under these conditions. The “false negatives” are most likely a result of sampling error due to low levels of the organism in the sample, as evidenced by the fluctuation in results following repeat testing by both culture and PCR. Although the BDM “false-negative” rate might be reduced by increasing the volume of the input sample from 15 μl, this volume of sample was found by BD-HandyLab through internal development studies to be optimal for BDM processing in the context of the biological amplification of the target from Lim broth enrichment (data not shown).

TABLE 1.

Summary comparison of BDM and culture from all performance sites

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.     

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.     

Evaluation of Trans-Vag Broth,Colistin-Nalidixic Agar,and CHROMagar StrepB for Detection of Group B Streptococcus in Vaginal and Rectal Swabs from Pregnant Women in South Africa

Maternal vaginal colonization with group B streptococcus (GBS) is a major risk factor for invasive GBS infection in newborns. The CDC-recommended method for detecting GBS colonization is to culture vaginal and rectal swabs in a selective broth followed by subculture on blood agar or a selective medium. A high incidence of antimicrobial resistance in the fecal microflora can compromise the recovery of GBS from the selective broth. Here, we compared CHROMagar StrepB (CA), Columbia colistin-nalidixic agar (CNA), and Trans-Vag selective broth enrichment for the isolation of GBS from 130 vaginal and 130 rectal swabs from pregnant women. The swabs were randomized for plating first on either CA or CNA, and they then were inoculated in Trans-Vag broth. GBS was cultured from 37.7% of the vaginal swabs and 33.1% of the rectal swabs. There were no differences in the detection rates for the vaginal swabs between CA (31.5%), CNA (26.2%), and the selective broth (30.0%). The sensitivities in relation to a composite score were 83.7%, 69.4%, and 79.6%, respectively. However, recovery of GBS from the rectal swabs was significantly higher from CA (29.2%; P < 0.0001) and CNA (23.8%; P = 0.002) than from the selective broth (9.2%). The sensitivities were 88.4%, 72.1%, and 27.9%, respectively. The order of plating on the solid medium was significant (P = 0.003), with GBS detection rates of 30.8% and 24.6% when swabs were plated first and second, respectively. These findings show that a selective broth is not suitable for the recovery of GBS from rectal swabs in settings such as ours, due to masking of the GBS colonies by persistent microflora.     

Method for Rapid Detection of Group B Streptococci by Coagglutination

A quick and reliable technique for the identification of group B streptococci has been developed. The method requires no elaborate equipment or expensive reagents and can be used to detect the group B organisms in mixed broth cultures or to identify suspect colonies selected from agar plates. The method is a coagglutination technique in which 1 drop of specifically sensitized protein A-containing Staphylococcus aureus is mixed with 1 drop of supernatant of an actively growing culture. The soluble group-specific carbohydrate substance of the group B streptococci reacts with the staph particles to produce agglutination that is macroscopically readable. One colony of group B streptococci taken from an agar plate and inoculated into Todd-Hewitt broth will give a positive reaction within 6 h of incubation; with a larger inoculum, the positive reaction occurs within a shorter period. The method was applied for detection of group B streptococci in mixed broth cultures. In laboratory studies involving random mixtures of organisms, 59.3% of positive cultures were detected within the first 8 h of incubation, and 71.7% were found within 24 h. In clinical studies with mixed broth cultures grown directly from vaginal swabs, 78.6% of the positive cultures were detected within the first 8 h of incubation, and 92.9% were found within 24 h.     

Xpert GBS Assay for Rapid Detection of Group B Streptococcus in Gastric Fluid Samples from Newborns

The Xpert GBS real-time PCR assay was applied to gastric fluid samples from 143 newborns, and it detected group B streptococcus (GBS) within 1 h for 16 (11.2%) cases, while microscopic examination detected only 2 cases. The sensitivity and specificity of the Xpert GBS were 80% and 100%, respectively, with regard to 20 cases of GBS colonization or infection. Concordance of Xpert GBS results versus culture was 92.3%. This test detects in a timely manner newborns at risk for invasive GBS disease.     

Rapid Hippurate Hydrolysis Method for Presumptive Identification of Group B Streptococci

A rapid test to detect the hydrolysis of sodium hippurate by beta-hemolytic streptococci within 2 h was developed. All group B streptococci tested were positive using this method and all other groups were negative.     

Comparative Evaluation of the AccuProbe Group B Streptococcus Culture Test,the BD GeneOhm Strep B Assay,and Culture for Detection of Group B Streptococci in Pregnant Women

We compared a rigorous culture method with the Gen-Probe AccuProbe Group B Streptococcus Culture Test (APGB) and the BD GeneOhm StrepB assay (GOSB) for the detection of group B streptococci (GBS) from an 18- to 24-h LIM broth. Culture (95.3%) and GOSB (95.3%) were more sensitive than APGB (86.5%) for the detection of GBS.Prenatal screening for group B streptococci (GBS) is routinely performed on women at 35 to 37 weeks of gestation (10, 11, 12). We and others have previously presented data supporting the use of the AccuProbe Group B Streptococcus Culture Test (APGB; Gen-Probe, San Diego, CA) for the detection of GBS from LIM broths (3, 13).The advent of PCR-based technology for the detection of GBS offers the potential for increased sensitivity and a shorter turnaround time than culture (2, 4, 11). APGB is a nonamplified nucleic acid hybridization assay, while the BD GeneOhm StrepB assay (GOSB; BD Diagnostics, Ste-Foy, Quebec, Canada) is a real-time PCR nucleic acid amplification assay. Moreover, since the time when we performed our initial evaluation of APGB, improved culture media for the detection of GBS have been developed (5, 6). The purpose of this study was to compare the performance of APGB with a commercially available PCR assay, GOSB, and a rigorous culture method.Vaginal and vaginal/rectal specimens submitted to the Geisinger Medical Center microbiology laboratory for prenatal testing for GBS colonization were included in this study. All of the specimens were collected with Copan Venturi Transystem double swabs and liquid Stuart''s medium (Copan Diagnostics, Marietta, CA).Swabs were first used to inoculate a neomycin-nalidixic acid blood agar plate (NNA; Becton-Dickinson Microbiology, Cockeysville, MD) and then placed into a 3-ml tube of LIM broth (Remel, Lenexa, KS). Following 18 to 24 h of incubation, LIM broths were vortexed and subcultured to an NNA plate. Primary plates and LIM broth subculture plates were incubated at 35°C in a 5 to 10% CO₂ incubator and examined after overnight incubation and a second time 24 h later if the first reading of the plates yielded no GBS. Organisms were identified by the routine bacteriology procedures used by the Geisinger Medical Center microbiology laboratory, including Lancefield identification of GBS with the PathoDX kit (Remel).After 18 to 24 h of incubation, a 50-μl aliquot of each LIM broth was tested with APGB. The directions in the product insert were followed, with one exception. The product insert specifies that when an enrichment broth such as LIM broth is used, the swab should be dipped into the LIM broth and then discarded. We followed the standard procedure that is used in our laboratory for APGB and did not remove the swab from the LIM broth after inoculation.The product insert for GOSB provides instructions for direct testing of swab specimens for GBS. We utilized a protocol that tested a sample from a LIM broth rather than a swab (9). Briefly, 0.2 ml (200 μl) of the LIM broth growth was transferred to sample buffer tubes (blue cap; contains 1 ml of sample buffer) with extended, filtered pipettes. The sample buffer tube stood for 5 min (in a cold block) and was then vortexed at high speed for 15 s. Fifty microliters of each cell suspension was transferred to a corresponding lysis tube (yellow cap), and the cap was closed tightly. The standard GOSB protocol was followed to complete the performance of the assay.For this study, a true positive result was defined as one of the following: GBS from the primary plate culture, GBS from the LIM broth subculture, or GBS from both the primary culture plate and the LIM broth subculture; GBS culture negative but GOSB and APGB positive; GOSB positive, primary culture and APGB negative, but culture positive for GBS following additional culture testing; or APGB positive, primary culture and GOSB negative, but culture positive for GBS following additional culture testing. For any specimen that did not yield the same result with APGB, GOSB, and culture, the result was classified as discrepant. For any specimen with discrepant results, the following protocol was followed: APGB was repeated from the LIM broth; the GOSB was repeated from the reserved lysis tube, as well as from the LIM broth; the LIM broth was subcultured to NNA, and plates were examined for GBS as with the primary culture; and primary plates and initial LIM broth subcultures were reexamined for GBS. Importantly, none of the results were changed based upon the use of the discrepant protocol. The discrepant analysis protocol was used only to correctly classify the initial result. For example, two isolates were initially GOSB positive and culture and APGB negative. Repeat LIM broth subcultures yielded GBS, so these two specimens were classified as GOSB true positive and culture false negative. Statistical analysis was performed as described by Ilstrup by using McNemar''s asymptomatic chi-square test (7).A total of 498 specimens were tested in this study. One hundred twenty-three specimens yielded positive results by all of the methods, APGB, GOSB, primary culture, and/or LIM broth subculture. Three hundred forty-seven specimens were negative by all of the methods. Of the 28 specimens that yielded discrepant results, 25 were determined to be true positives by the above-described criteria. Among these 25 specimens, 3 were culture negative but positive by APGB and GOSB and thus were classified as true positive results. Three specimens were GOSB positive only and were considered false positives. The overall performance characteristics are summarized in Table ​Table1.1. The comparative performance characteristics of the three methods are summarized in Table ​Table2.2. Culture was more sensitive than APGB (P = 0.025). GOSB was more sensitive than APGB (P = 0.002). Culture and GOSB were equivalent in sensitivity.

TABLE 1.

Performance characteristics of culture, APGB, and GOSB

Multisite Evaluation of Cepheid Xpert Carba-R Assay for Detection of Carbapenemase-Producing Organisms in Rectal Swabs

Rapid identification of patients who are colonized with carbapenemase-producing organisms (CPO) is included in multiple national guidelines for containment of these organisms. In a multisite study, we evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative diagnostic test that was designed for the rapid detection and differentiation of the bla_KPC, bla_NDM, bla_VIM, bla_OXA-48, and bla_IMP-1 genes from rectal swab specimens. A double rectal swab set was collected from 383 patients admitted at four institutions (2 in the United States, 1 in the United Kingdom, 1 in Spain). One swab was used for reference culture (MacConkey broth containing 1 mg/liter of meropenem and subcultured to a MacConkey agar plate with a 10-μg meropenem disk) and for sequencing of DNA obtained from carbapenem-nonsusceptible isolates for carbapenemase identification. The other swab was used for the Xpert Carba-R assay. In addition to the clinical rectal swabs, 250 contrived specimens (108 well-characterized CPO and 142 negative controls spiked onto negative rectal swabs) were tested. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay. In 6 samples, multiple targets were detected (4 VIM/OXA-48, 1 IMP-1/NDM, and 1 NDM/KPC). The Xpert Carba-R assay detected 155 targets (26 IMP-1, 30 VIM, 27 NDM, 33 KPC, 39 OXA-48) within a time range of 32 to 48 min. The sensitivity, specificity, and positive and negative predictive values of the Xpert Carba-R assay compared to those of the reference culture and sequencing results were 96.6% (95% confidence interval [CI], 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Cepheid Xpert Carba-R assay is an accurate and rapid test to identify rectal colonization with CPO, which can guide infection control programs to limit the spread of these organisms.     

Multiplex Detection of Bacteria Associated with Normal Microbiota and with Bacterial Vaginosis in Vaginal Swabs by Use of Oligonucleotide-Coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.Bacterial vaginosis (BV), which is defined by a reduction in the vaginal Lactobacillus populations and an increase in the number of microbial species present, including Gardnerella spp., Atopobium spp., and others, is increasingly recognized as an important risk factor for adverse reproductive health outcomes, such as miscarriage and premature birth, and sexually transmitted diseases, including human immunodeficiency virus infection (25). Despite intense investigation, the etiology and clinical course of BV have not been well defined, probably because the normal variation of the vaginal microbial communities between individuals and their dynamics over time are complex and poorly understood. Consequently, the accurate diagnosis of BV, as well as the elaboration of effective prevention and treatment strategies, remains a major challenge (26, 44).The current “gold standard” for the diagnosis of BV in the laboratory setting relies on microscopic profiling of microbial types in Gram-stained vaginal swab smears by using the criteria of Nugent et al. (29) or Ison and Hay (20). These scoring systems are based on the relative abundance of Lactobacillus morphotypes (large gram-positive rods) compared to the abundance of the bacterial morphotypes suggestive of BV (gram-negative or gram-variable coccobacilli and curved rods and gram-positive cocci). Although this method provides reliable information and is particularly well suited for use in resource-poor settings, it requires well-trained, highly experienced individuals to interpret the results and is simplistic in its reflection of the variety of organisms present in the microbial communities present in healthy individuals with a normal vaginal flora and patients with BV. Potential problems with the subjective interpretation of the Gram stain result, along with an increasing appreciation of the fact that the complexity of the vaginal microbiota can be missed by these classical methods, have resulted in a search for other methods for the diagnosis of BV that can also identify the different species present (5, 25).In-depth studies of the makeup of vaginal microbial communities are quite common, including studies that use the older culture-based approaches (31) and more recent molecular studies that involve the construction of a clone library on the basis of the 16S rRNA gene (30) or the chaperonin-60 (cpn60) (16) target. While the information generated by these studies is critical to obtaining a greater understanding of the variability and ecological dynamics of vaginal microbial communities, they remain far too labor-intensive at present for the screening of large numbers of samples or for the routine laboratory diagnosis of BV.Most recently described molecular approaches for the diagnosis of BV are based on quantitative PCR (qPCR) of the 16S rRNA gene or other targets in bacteria extracted from vaginal samples. For example, one recent study found that the simultaneous quantification of the Gardnerella cpn60 target and the Atopobium 16S rRNA gene target resulted in the sensitive and specific diagnosis of BV in comparison to the sensitivity and specificity of the diagnosis by use of the scoring system of Nugent et al. (29). The advantages of the cpn60 universal target (UT) as an alternative to the 16S rRNA gene for the identification of bacterial species are increasingly recognized and include the increased resolution available at the species level and its presence at a single copy per genome, improving the reliability of quantitation (7).Multiplexed, bead-based flow cytometric detection and quantification of bacterial targets by use of the Luminex platform have been described previously (3, 9, 14, 38). The main advantage of this approach is its ability to detect a large number of molecular targets simultaneously after a single PCR, making high-throughput analysis of multiple samples from large study groups or longitudinal studies technically feasible. In the context of BV, the use of this multiplex approach, coupled with highly discriminatory cpn60-specific probes, facilitates screening for multiple species of Lactobacillus and a number of BV-associated bacteria simultaneously, providing an unprecedented level of information about the variability of vaginal bacterial communities, defined as those associated with a normal vaginal flora and those associated with BV, in a single, rapid assay.The goal of the present study was the design of a flow cytometric bead array for the rapid profiling of microbial communities extracted from vaginal samples and its evaluation as a potential technique for the rapid diagnosis of BV in the laboratory setting.     

三种方法筛查孕妇B族链球菌的效果评价

目的 比较三种不同的方法检测B族链球菌的结果,对三种B族链球菌筛查方法进行效果评价.方法 收集我院2014年1月至2015年12月产科门诊孕35 ～ 37周孕妇1374例,采集孕妇的阴道拭子和肛周拭子标本,分别用血琼脂培养法,LIM肉汤增菌培养法和PCR方法检测B族链球菌.结果 1374例孕妇标本中,血琼脂培养法阳性60例,阳性检出率为4.4％,PCR方法检测阳性66例,阳性检出率为4.8％,LIM肉汤增菌培养法阳性128例,阳性检出率9.3％.三种筛查方法比较,LIM肉汤增菌培养法阳性率与PCR法,血琼脂培养法比较差异具有统计学意义.血琼脂培养法和PCR法筛查阳性率差异不具有统计学意义.以LIM肉汤增菌法为金标准,血琼脂培养法的灵敏度、特异性、阳性预测值、阴性预测值分别为46.9％,100％,100％,94.8％,PCR法的灵敏度、特异性、阳性预测值、阴性预测值分别为50％,99.6％,96.9％,95.1％.结论 LIM肉汤增菌培养法筛查B族链球菌的阳性率最高,血琼脂培养法和PCR法检测灵敏度较低,只能筛查出50％左右的GBS定植孕妇.建议临床筛查B族链球菌使用LIM肉汤增菌培养法筛查GBS,灵敏度高,特异性好.     

Comparison of Perirectal versus Rectal Swabs for Detection of Asymptomatic Carriers of Toxigenic Clostridium difficile

For long-term care and spinal cord injury patients, the sensitivity, specificity, and positive and negative predictive values of perirectal versus rectal cultures for detection of asymptomatic carriers of Clostridium difficile were 95%, 100%, 100%, and 97%, respectively. Perirectal cultures provide an accurate method to detect asymptomatic carriers of C. difficile.     

Multicenter Clinical Evaluation of the Xpert GBS LB Assay for Detection of Group B Streptococcus in Prenatal Screening Specimens

Neonatal infection with Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis and meningitis in newborns. Recent guidelines have recommended universal screening of all pregnant women to identify those colonized with GBS and administration of peripartum prophylaxis to those identified as carriers to reduce the risk of early-onset GBS disease in neonates. Enriched culture methods are the current standard for prenatal GBS screening; however, the implementation of more sensitive molecular diagnostic tests may be able to further reduce the risk of early-onset GBS infection. We report a clinical evaluation of the Xpert GBS LB assay, a molecular diagnostic test for the identification of GBS from broth-enriched vaginal/rectal specimens obtained during routine prenatal screening. A total of 826 specimens were collected from women undergoing prenatal screening (35 to 37 weeks'' gestation) and tested at one of three clinical centers. Each swab specimen was tested directly prior to enrichment using the Xpert GBS assay. Following 18 to 24 h of broth enrichment, each specimen was tested using the Xpert GBS LB assay and the FDA-cleared Smart GBS assay as a molecular diagnostic comparator. Results obtained using all three molecular tests were compared to those for broth-enriched culture as the gold standard. The sensitivity and specificity of the Xpert GBS LB assay were 99.0% and 92.4%, respectively, compared to those for the gold standard culture. The Smart GBS molecular test demonstrated sensitivity and specificity of 96.8% and 95.5%, respectively. The sensitivities of the two broth-enriched molecular methods were superior to those for direct testing of specimens using the Xpert GBS assay, which demonstrated sensitivity and specificity of 85.7% and 96.2%, respectively.     

孕妇B族链球菌的检测及其临床价值

目的 探讨免疫层析法在检测孕妇B族链球菌(GBS)感染中的临床价值.方法 选取2014年12月至2015年12月间我院待产孕妇320例,各取2份阴道分泌物标本.1份用免疫层析法快速检测GBS;另1份用于接种THB培养基,第2天转至血平板,并对可疑菌落进行生化鉴定.同时对320例孕妇的妊娠结局进行随访.结果 免疫层析法检测出GBS感染41(12.81％)例,培养法检测出GBS感染36(11.25％)例,以细菌培养法为参考标准,免疫层析法的符合率为98.4％.通过临床跟踪随访,320例孕妇中有16例发生早产、胎膜早破等异常情况,其中有13例GBS阳性.结论 GBS与早产、胎膜早破等异常妊娠关系密切;免疫层析法较传统细菌培养法检出率无统计差异,且有步骤简单、省时等优点,适合应用于临床筛查GBS感染.     

Evaluation of Remel Spectra CRE Agar for Detection of Carbapenem-Resistant Bacteria from Rectal Swabs Obtained from Residents of a Long-Term-Care Facility

We compared the Remel Spectra CRE agar plate to CDC standard methodology for the isolation of carbapenem-resistant Enterobacteriaceae (CRE) from 300 rectal swab specimens obtained from patients residing in a long-term-care facility (LTCF). Multiplex PCR experiments were performed on isolates to identify specific Klebsiella pneumoniae carbapenemases (KPC) and additional β-lactamases. Of the 300 patients, 72 (24%) harbored CRE and were PCR positive for KPC enzymes. The Remel Spectra CRE plates detected KPC-type CRE in isolates from 70 of 72 patients (97.2%), while the CDC method detected CRE in 56 of 72 (77.8%). CRE identification results were available in 18 h compared to 36 h for the CDC method. Remel Spectra CRE agar plates can provide useful means for a fast and reliable method for detecting KPC-type CRE and for accelerated institution of appropriate infection control precautions.     

Evaluation of the New Brilliance GBS Chromogenic Medium for Screening of Streptococcus agalactiae Vaginal Colonization in Pregnant Women

Three commercial chromogenic agar media were evaluated for Streptococcus agalactiae screening in 200 vaginal swabs from pregnant women. The sensitivity and specificity were 94.3% and 100% for Granada medium (bioMérieux), 100% and 90.3% for Brilliance GBS medium (Thermo Fisher Scientific), and 100% and 98.8% for ChromID STRB medium (bioMérieux), respectively.     

Evaluation of a Chromogenic Agar for Detection of Group B Streptococcus in Pregnant Women

We compared ChromID Strepto B agar (STRB; bioMérieux, Inc.), a selective and differential medium for group B streptococcus, with culture using neomycin-nalidixic acid agar (NNA) and LIM broth. STRB alone was more sensitive (87.7%) than NNA alone (79.0%), while each had a sensitivity of 100% when used in conjunction with LIM broth.Meningitis, pneumonia, and septicemia due to group B Streptococcus (GBS) infection are leading causes of infant mortality and morbidity (7). In 1996 the American College of Obstetricians and Gynecologists (ACOG) and the CDC issued recommendations for intrapartum antibiotic prophylaxis according to either a risk-based or a screening-based approach to prevent GBS transmission to neonates. The risk-based guidelines called for intrapartum prophylaxis in the event of delivery at <37 weeks of gestation, a temperature of >100.4°F, or the rupture of membranes for ≥18 h, while the culture-based approach called for the screening of all pregnant women between 35 and 37 weeks of gestation for vaginal or rectal colonization with GBS and subsequent intrapartum prophylaxis of those who were culture positive. In 2002 these guidelines were revised to recommend that culture-based prenatal GBS screening at 35 to 37 weeks of gestation be universally adopted (8). This time point was chosen as an appropriate predictor of intrapartum colonization (11). U.S. national estimates of early-onset (<7 days of life) neonatal invasive GBS disease using data collected by the CDC Active Bacterial Core Surveillance system (ABCs) indicate a decrease in incidence since the institution of GBS screening and prophylaxis guidelines, from 0.7 case/1,000 live births in 1997 to 0.34 case/1,000 live births in 2007 (3).The CDC and ACOG recommend that vaginal/rectal swabs be inoculated into a selective broth medium to aid in the recovery of GBS. The broth is then to be subcultured on sheep blood agar plates after 18 to 24 h of incubation (1, 8). The addition of a solid agar medium inoculated upon receipt of the swab in the laboratory can allow for the identification of GBS after 24 h of incubation, instead of the 48 h required when swabs are inoculated only into selective broth. Neomycin-nalidixic acid agar (NNA) medium is a GBS-selective solid agar that can be used for primary plating, since it is significantly more sensitive than colistin-nalidixic acid agar (CNA) for this purpose (4). However, NNA should never be used as a substitute for a selective broth medium, because the combination of a selective broth medium and NNA improves the detection of GBS by 15% over that with either medium used alone (5).ChromID Strepto B agar (STRB; bioMérieux, Inc.) is a newly developed selective and differential medium for the presumptive identification of GBS. STRB contains a chromogenic substrate that, in the presence of actively growing GBS, generates pink to red colonies that are easily identified during routine inspection. The growth of bacteria of other species either is inhibited or appears in other colors (violet, blue, or colorless). We evaluated the performance of STRB in our laboratory compared to that of NNA, with or without the addition of a selective broth medium, as a suitable screening method for the detection of GBS colonization among pregnant women.Our established protocol calls for the inoculation of vaginal/rectal swabs onto NNA, which is incubated at 35°C under 5% CO₂, and into LIM broth, which is incubated at 35°C in room air. The NNA plate is examined at 24 h for the presence of characteristic beta-hemolytic colonies, which are further evaluated by Gram staining and by catalase, PYR (pyrrolidonyl-β-naphthylamide), and rapid latex agglutination tests to identify GBS. If beta-hemolytic colonies are not present after 24 h of incubation, then (i) the NNA plate is held for an additional 24 h and is examined the following day, and (ii) the LIM broth is subcultured onto 5% sheep blood agar, which is incubated for 48 h and is examined daily for the presence of beta-hemolytic colonies. To evaluate the performance of the STRB agar, primary swabs were processed by our routine protocol and were also inoculated onto the STRB plate, which was incubated at 35°C for 48 h in the dark. A single swab was collected from each patient, and the order of inoculation onto STRB or NNA plates was randomized. Plates were examined daily for the presence of pink colonies, which were then subjected to latex agglutination testing to confirm the identification of GBS. Gram staining and catalase and latex agglutination testing can be performed directly from STRB agar, while PYR testing cannot. However, according to the manufacturer, the appearance of pink to red colonies that type as group B by latex agglutination is sufficient for the identification of GBS.A total of 250 prenatal screening swabs were included in the study. Eighty-one of these samples (32.4%) were positive for GBS by our reference method (i.e., they were recovered from either NNA or LIM broth). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of either combination (NNA plus LIM broth subcultured onto 5% sheep blood agar or STRB plus LIM broth subcultured onto 5% sheep blood agar) were all 100%. A direct comparison of NNA to STRB without broth amplification as a screening medium revealed sensitivities of 79.0% (95% confidence interval [95% CI], 68.3% to 86.9%) for NNA and 87.7% (95% CI, 78.0% to 93.6%) for STRB (Table ​(Table1).1). For both NNA and STRB, the specificity was 97.6% (95% CI, 93.8% to 99.3%), with four isolates being incorrectly identified as group B Streptococcus. The four isolates from NNA were identified as Streptococcus porcinus, while three of the four S. porcinus isolates from NNA were also incorrectly identified on STRB, along with a fourth isolate that was identified as Streptococcus thoraltensis. There is some potential for misidentification of other streptococcal species as GBS by using STRB. S. porcinus isolated from NNA would likely be evaluated further if a wide zone of beta-hemolysis, which is not characteristic of GBS, were recognized. This would not be the case, however, if isolates were identified from STRB alone, since there would be no potential to observe the hemolysis pattern. Conversely, GBS can exhibit a nonhemolytic phenotype that may be overlooked on NNA, but these isolates would be identified using chromogenic agar (10).

TABLE 1.

Performance characteristics of STRB, NNA, and LIM broth

Serotype Identification of Group B Streptococci by PCR and Sequencing

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (cps) gene clusters to further define partial cps gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.