The effects of adulterants and selected ingested compounds on drugs-of-abuse testing in urine

Household chemicals such as bleach, table salt, laundry detergent, toilet bowl cleaner, vinegar, lemon juice, and eyedrops are used for adulterating urine specimens. Most of these adulterants except eyedrops can be detected by routine specimen integrity tests (creatinine, pH, temperature, and specific gravity); however, certain adulterants such as Klear, Whizzies, Urine Luck, and Stealth cannot. These adulterants can successfully mask drug testing if the concentrations of certain abused drugs are moderate. Several spot tests have been described to detect the presence of such adulterants in urine. Urine dipsticks are commercially available for detecting the presence of such adulterants, along with performance of tests for creatinine, pH, and specific gravity. Certain hair shampoo and saliva-cleaning mouthwashes are available to escape detection in hair or saliva samples, but the effectiveness of such products in masking drugs-of-abuse testing has not been demonstrated. Ingestion of poppy seed cake may result in positive screening test results for opiates, and hemp oil exposure can cause positive results for marijuana. These would be identified as true-positive results in drugs-of-abuse testing even though they do not represent the actual drug of abuse.     

Evaluation of the Cobas-Bact system for direct and rapid identification and antimicrobial susceptibility testing of Enterobacteriaceae from positive urine specimens

A direct antimicrobial susceptibility test and a direct identification of Gram-stained urine specimens positive for Enterobacteriaceae using a new instrument (Cobas-Bact) were compared by means of the conventional Kirby-Bauer agar diffusion disk method and the spot indole test, an in-house set of identification tests or API 20E. Bacteria from 191 cases of monomicrobial bacteriuria due to members of the family Enterobacteriaceae were tested. Direct susceptibility testing was performed in 180 cases (94%), providing 1649 antibiotic-microorganism combinations. A complete agreement was reached in 82% and essential agreement in 92% of cases. Minor discrepancies were found in 163 (9.9%) major ones in 125 (7.6%) and very major ones in 10 (0.6%) of examinations. 72% of all minor and 71% of all major discrepancies were caused by two antibiotics: cephalothin and nitrofurantoin. Of the very major discrepancies, 50% were due to amoxicillin. Of 171 direct identifications obtained, 130 (76%) were "high-confidence" correct identifications (percentage of likelihood p greater than or equal to 80%), 25 (14.6%) "low-confidence" identifications (percentage of likelihood p less than 80%) and 16 (9.4%) misidentifications. On the whole, this direct and rapid Cobas-Bact identification and susceptibility testing procedure provided satisfying information within 5-6 h after collection of urine specimens positive for members of the Enterobacteriaceae family.     

Comparison of the APTIMA CT and GC assays with the APTIMA combo 2 assay, the Abbott LCx assay, and direct fluorescent-antibody and culture assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

The Gen-Probe APTIMA Combo 2 (AC2) is a Food and Drug Administration-cleared nucleic acid amplification test (NAAT) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae from urine and urogenital swab specimens. The Centers for Disease Control and Prevention have recommended confirmation of positive NAAT results in low-prevalence populations. APTIMA CT (ACT) and APTIMA GC (AGC) are two discrete NAATs for C. trachomatis and N. gonorrhoeae detection that are suitable for confirming AC2-positive results because they target different nucleic acid sequences. Our objective was to determine if ACT and AGC could be used as confirmatory tests for AC2 and to correlate the APTIMA assays with culture, direct fluorescent-antibody (DFA), and LCx CT and GC assays. Urine and swab specimens (1,304) were initially tested with either culture, DFA, or LCx, followed by AC2. A subset (675) was then tested with ACT and AGC. There was absolute concordance between ACT-AGC and AC2. LCx did not detect 1 of 14 AC2-ACT- and 1 of 6 AC2-AGC-positive urine samples, and it yielded one C. trachomatis- and one N. gonorrhoeae-positive swab result that were not detected by AC2 and ACT-AGC. Culture failed to detect 5 of 20 AC2-ACT and 3 of 4 AC2-AGC positives, and DFA missed 4 of 4 AC2-ACT positives. Thus, ACT and AGC relative sensitivity compared to that of AC2 was 100%. All APTIMA assays detected more confirmed positive results than culture, DFA, and LCx. The performance of APTIMA assays was not altered by the use of various swab types and by long-term storage of specimens. All APTIMA assays are highly sensitive and rapid. ACT and AGC can be recommended for confirmation of positive results from other NAATs, such as AC2 and LCx.     

Enhanced enzyme immunoassay with negative-gray-zone testing compared to a single nucleic Acid amplification technique for community-based chlamydial screening of men

We evaluated a low-cost diagnostic strategy for detecting Chlamydia trachomatis in a low-prevalence population. We used an amplified enzyme immunoassay (EIA) with a reduced-cutoff "negative gray zone" to identify reactive specimens for confirmation by a nucleic acid amplification test. As part of the Chlamydia Screening Studies project, men provided a first-pass urine specimen, which they returned by post for testing. We tested 1,003 specimens by IDEIA PCE EIA (Dako) and Cobas PCR (Roche). There were 32 (3.2%) true positive specimens according to a combined standard using an algorithm requiring concordant results from at least two independent tests. All of these were positive by Cobas PCR and 24 were confirmed to be positive by PCE EIA, including 2 that gave results in the negative gray zone. There were 971 true negative specimens, 2 of which were positive by Cobas PCR and 19 of which were initially inhibitory for PCR. The relative sensitivity, specificity, positive predictive value, and negative predictive value of PCE EIA with PCR confirmation were 75.0% (95% confidence interval [CI], 56.6 to 88.5%), 100% (95% CI, 99.7 to 100%), 100% (95% CI, 88.3 to 100%), and 99.2% (95% CI, 98.4 to 99.6%), respectively. The corresponding values for Cobas PCR were 100% (95% CI, 89.1 to 100%), 99.8% (95% CI, 99.3 to 100%), 94.1% (95% CI, 76.9 to 98.2%), and 100% (95% CI, 99.6 to 100%), respectively, with 1.9% (19/1003) of the samples being initially indeterminate. When the prevalence of C. trachomatis is low, the use of an amplified EIA on urine specimens, with confirmation of results in the negative gray zone by use of a nucleic acid amplification technique, is not suitable for screening asymptomatic men. In addition, positive nucleic acid amplification test results should be confirmed and an inhibition control should be used.     

Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis

The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries.Due to its activity against semidormant bacilli sequestered within macrophages, pyrazinamide (PZA) is one of the most effect frontline drugs used in the short-course chemotherapy of tuberculosis (TB) and also in retreatment regimens for multidrug-resistant TB patients (6, 36). However, the detection of PZA resistance is difficult and often unreliable because the drug is active only at acid pH (5.5), which also affects the growth of Mycobacterium tuberculosis (5, 37). For this reason, many laboratories do not perform PZA susceptibility testing, and therefore the true extent of global PZA resistance is largely unknown. The radiometric Bactec 460 system (Becton Dickinson, Sparks, MD), using a special acid liquid medium, has been considered the reference method for detection of PZA resistance, but it requires the use of costly and problematic radioisotopes (20, 30). Other commercial tests, such the nonradiometric Bactec MGIT 960 method (Becton Dickinson, Sparks, MD), utilize protocols adapted for PZA susceptibility testing, but they are also expensive and impractical for routine use in resource-poor settings (14, 27).PZA is a prodrug that requires activation by the M. tuberculosis pyrazinamidase (PZase), and most PZA-resistant strains have mutations in pncA, the gene encoding this enzyme, that result in the loss or reduction of PZase activity (7). Thus, an indirect approach to detecting resistance is to assess PZase activity (22). The classic way to detect PZase activity is with the Wayne assay (12, 35), which monitors the hydrolysis of PZA to the active acid form, pyrazinoic acid (POA), through the color change of a ferrous ammonium phosphate solution added to the medium.Nicotinamide (NIC), a structural analogue of PZA with some activity against M. tuberculosis, is also converted to its active acid form, nicotinic acid, by the M. tuberculosis PZase (12, 24). Strains of M. tuberculosis that are resistant to PZA are also resistant to NIC (18, 20, 28), and the conversion of NIC into nicotinic acid by PZase occurs at a physiological pH that does not hinder bacterial growth. In recent studies, PZA resistance was rapidly and accurately detected with the inexpensive resazurin microtiter assay (REMA) and nitrate reductase assay (NRA), using NIC as a surrogate for PZA to avoid the need for acidification of the medium (18, 20). Another alternative colorimetric method, reported by Farnia et al., uses malachite green to test for susceptibility to first- and second-line anti-TB drugs (4). Malachite green is a triphenylmethane dye with a dark green color that becomes colorless during M. tuberculosis metabolism (9).In this study we evaluated the use of NIC in the NRA and malachite green microtube (MGMT) assay for the detection of PZA resistance and compared the results with those obtained by the Wayne assay, which served as a gold standard. We also sequenced the pncA genes from strains determined to be resistant by any of the three methods.     

Blood Cultures Positive for Coagulase-Negative Staphylococci: Antisepsis,Pseudobacteremia, and Therapy of Patients

A blood culture cohort study investigating issues related to isolation of coagulase-negative staphylococci (CoNS) and other skin microflora is reported. Data were collected over 12 weeks to determine the incidence of significant CoNS bacteremia versus that of pseudobacteremia (contaminants) and to evaluate drug therapy in patients with cultures positive for CoNS. In addition, the effectiveness of 0.2% chlorine peroxide as a bactericidal disinfectant was compared to that of 10% providone iodine. A total of 3,276 cultures of blood from 1,433 patients were evaluated in the study. Eighty-nine cultures were positive for skin flora, with 81 of 89 (91%) involving CoNS. The incidence of significant CoNS bacteremia was 20 of 81 (24.7%), that of indeterminate bacteremia was 10 of 81 (12.3%), and that of contamination was 59 of 81 (72.8%). The incidence of significant bacteremia involving CoNS was double the 10 to 12% rate based on previous estimations at our institutions. In tests with the two bactericidal disinfectants, 22 of 1,639 cultures (1.3%) in the chlorine peroxide group versus 37 of 1,637 (2.3%) in the providone iodine group were considered contaminated (P = 0.065). Rates of contamination for venipuncture versus catheter collection were not significantly different (P = 0.46). The overall contamination rate was 59 of 3,276 (1.8%), which is consistent with the lower end of published quality assurance benchmark standards. The low rate was believed to be due to the professional phlebotomy staff in our institutions. There was excellent agreement between retrospective analysis by reviewers, when formal criteria were used, and the attending physicians’ intuitive clinical impressions in the classification of significant bloodstream infections (100% agreement) or contamination (95% agreement). However, physicians still used antimicrobial agents to treat nearly one-half of the patients with contaminated blood cultures, with vancomycin being misused in 34% of patients. In addition, 10% of patients with significant bacteremia were treated with inappropriate agents. There were no significant adverse events or prolonged hospital stays due to the unnecessary use of vancomycin; however, the additional costs of treating patients whose cultures contained CoNS contaminants was estimated to be $1,000 per patient. Measures to limit the unnecessary use of vancomycin (and other agents) are important.Coagulase-negative staphylococci (CoNS), the most frequent blood culture isolates, are predominantly blood culture contaminants, but they are also a significant cause of bacteremia (2–5, 7, 9, 13). Institution-specific contamination rates vary from 2 to more than 6% (3, 5, 23, 26, 27). In the past 5 years, estimated contamination rates at our hospitals ranged from 2.5 to 3.5%. During this period, CoNS accounted for 45 to 60% of total blood isolates, and we estimated, using laboratory criteria, that 10 to 12% of CoNS isolates from blood were implicated in significant bloodstream infections. A relatively large proportion of the patient population with presumed false-positive blood cultures due to contaminants (pseudobacteremia) were treated with antimicrobial agents, in particular, vancomycin.Clinical and microbiologic guidelines for the differentiation of true bacteremia from pseudobacteremia or contamination have been published (5, 13, 15). Suggested laboratory criteria for true bacteremia include growth within 48 h and multiple blood cultures positive for the same organism. In contrast, increased duration of time before positivity, polymicrobial growth of skin organisms, or growth during antibiotic treatment suggest contamination. Others recommended that the addition of clinical guidelines is essential for the appropriate classification of bacteremia (4, 8, 9, 15, 18).We conducted a cohort study to evaluate clinical and laboratory data for adult patients with blood cultures positive for CoNS. The study was done at two tertiary-care teaching centers, Deaconess Medical Center (DMC) and Sacred Heart Medical Center (SHMC), with a combined capacity of 900 beds. We examined problems associated with false-positive bacteremia and determined the incidence of significant bacteremia. Our goal was to make recommendations to improve clinicians’ ability to recognize the significance of potentially contaminating organisms and to evaluate treatment given to patients with CoNS-positive blood cultures. To attempt to minimize contamination, we evaluated the nontoxic, antiseptic and disinfectant chlorine peroxide in comparison to a standard disinfectant.(This work was previously presented in abstract form at the 96th General Meeting of the American Society for Microbiology, New Orleans, La., 19 to 23 May 1996 [24a].)     

Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor.     

Evaluation of a Rapid Immunochromatographic ODK0501 Assay for Detecting Streptococcus pneumoniae Antigen in Sputum Samples from Patients with Lower Respiratory Tract Infection

A novel, rapid, and noninvasive test (ODK0501) to detect Streptococcus pneumoniae antigen was evaluated in a Japanese multicenter study. ODK0501 uses polyclonal antibodies to detect C polysaccharide of S. pneumoniae from sputum samples by an immunochromatographic assay. The utility of ODK0501 was evaluated for 161 adult patients with lower respiratory tract infection between March 2006 and March 2007. Bacterial culture and identification, real-time PCR, and ODK0501 assays were performed on sputum samples, and the Binax Now Streptococcus pneumoniae antigen test was performed using urine samples obtained from the same patients. The performances of all tests were compared based on the results of bacterial culture and identification. The sensitivity and specificity of ODK0501 were 89.1% (49/55 samples) and 95.3% (101/106 samples), respectively. We then compared the Binax Now Streptococcus pneumoniae antigen test with ODK0501 using samples from 142 patients. The sensitivities of ODK0501 and the Binax Now S. pneumoniae antigen test were 90.0% (45/50 samples) and 62.0% (31/50 samples), respectively (P = 0.002). The relative quantity of S. pneumoniae in expectorated sputum was calculated using real-time PCR and indicated that the possibility of false-positive results for ODK0501 due to indigenous S. pneumoniae was low. The positive and negative concordance rates of ODK0501 and Binax Now were 96.8% (30/31 samples) and 21.1% (4/19 samples), respectively. Binax Now was less capable of detecting S. pneumoniae antigen among patients with underlying chronic obstructive pulmonary disease. In conclusion, ODK0501 is noninvasive, rapid, and an accurate tool for diagnosing respiratory infection caused by S. pneumoniae.Streptococcus pneumoniae is a frequent cause of community-acquired bacterial pneumonia and lower respiratory tract infection (10, 11, 19, 21) and a major cause of significant morbidity and mortality in Japan and the rest of the world (5).Since pneumococcal infections are common and can be severe, appropriate initial selection of antimicrobial agents is crucial to an optimal outcome. Rapid and precise identification of the causative agents of infectious diseases is critical but challenging. Pathogen-oriented, prompt selection and application of antimicrobial agents improve prognosis, reduce medical costs, and prevent the development of drug-resistant bacteria due to their inappropriate use. The “gold standard,” bacterial culture to identify causative microorganisms, requires several days to yield a result and is thus unhelpful for the initial selection of appropriate antibacterial drugs. Although Gleckman et al. previously reported that classic Gram staining of sputum, which is simple and inexpensive, is useful for the diagnosis of bacterial infections (7), Garcia-Vazquez et al. and Reed et al. found that it is ineffective for rapid diagnoses (6, 18). In addition, staining results rely on several factors such as the quality of the sputum samples and the skill of laboratory personnel in processing samples (7). Thus, Gram staining is not recommended for all patients with community-acquired pneumonia, according to the most recent “Infectious Diseases Society of America/American Thoracic Society Consensus Guidelines on the Management of Community-Acquired Pneumonia in Adults” (12).Urinary antigen detection is an alternative rapid diagnostic technique for detecting antigens of S. pneumoniae and Legionella pneumophila as respiratory pathogens. The Binax Now Streptococcus pneumoniae urinary antigen test (Binax, Inc., Portland, ME) detects cell wall antigens secreted in urine using an immunochromatographic method to separate capsular polysaccharide (C-ps) of S. pneumoniae. The test is noninvasive and rapid (the entire assay can be completed in about 15 min), and the specificity and sensitivity of detecting pneumococcal infections in adult patients are >90% and 50% to 80%, respectively, which allows early diagnosis (3, 8, 16). Moreover, even when S. pneumoniae cannot be identified by bacterial culture tests after the start of antibiotic therapy, the assay can still detect this organism. However, Binax Now has significant problems, including false-positive results due to indigenous S. pneumoniae in children (17) and antigens that can be detected even 1 to 3 months after treatment (13, 15).ODK0501 (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) is a diagnostic kit that uses polyclonal antibodies to detect antipneumococcal C-ps, a cell wall antigen of S. pneumoniae (4). This immunochromatographic method is rapid and noninvasive and analyzes sputum derived directly from a local infection site. Here, we evaluated the ability ODK0501 to detect S. pneumoniae among patients with lower respiratory tract infection at several Japanese medical institutions and compared its performance with that of the Binax Now assay. The goal of this study is to prove the usefulness of ODK0501 and its performance in clinical settings.     

Testing for established viruses: from the screening to the confirmation

Enzyme Immunoassays (EIAs) detecting antibodies (Ab) or antigen (Ag) used to avoid viral transmissions by transfusion, are the most suitable methods for blood screening because they are economical and adaptable to high-throughput testing. The continuing need to improve screening for HIV and HCV, led to develop Ag/Ab combination assays. The benefit of such assays has been widely demonstrated, since they are able to detect infected individuals in viremic pre-seroconversion phase, making them particularly attractive for ensuring blood safety when nucleic acid testing (NAT) cannot be implemented. In resource-limited countries, especially in remote-areas, blood safety is often based upon rapid tests (RT) which are easy to handle and can be used with whole blood. Nevertheless, the poor sensitivity of RT compared to EIAs could significantly impair blood safety. Because NAT could efficiently detect serologically negative donors, many developed countries have implemented this method in blood screening. NAT yields vary depending on epidemiological situations. NAT is subjected to false-negative results due to viral diversity or to extremely low viral loads, but also to false-positive due to sample cross-contamination. A repeatedly NAT reactive sample with multiplexed NAT is submitted to a virus-specific amplification testing to discriminate between the viruses, creating some problematic situations consisting of samples reactive with the screening assay but non-reactive with the discriminatory assay. Although the primary purpose of viral screening testing is to prevent pathogen transmission to recipients, confirmatory testing intends to manage donors with adverse test results and to guarantee an optimal blood supply. As EIAs show a low positive predictive value in low-prevalence countries due to frequent non-specific reactions, the use of confirmatory assays is justified. Nevertheless confirmatory tests are known to be less sensitive than EIAs in the early phase of infection and show also nonspecific reactivity leading to indeterminate results which create difficulties in managing donors. As in limited-resource countries, specific confirmatory assay cannot be used, alternate strategies for confirmatory testing have been proposed, as to simultaneously test blood with two assays and to exclude donors reactive with both, or to use screening test signal values to support confirmation. Although the direct detection of the virus through its genome might be considered as the best solution to ensure blood safety, the choice of blood screening strategy should be adapted to local epidemiology and organization constraints.     

PCR-Based Pooling of Dried Blood Spots for Detection of Malaria Parasites: Optimization and Application to a Cohort of Ugandan Children

Sensitive, high-throughput methods to detect malaria parasites in low-transmission settings are needed. PCR-based pooling strategies may offer a solution. We first used laboratory-prepared samples to compare 2 DNA extraction and 4 PCR detection methods across a range of pool sizes and parasite densities. Pooled Chelex extraction of DNA, followed by nested PCR of cytochrome b, was the optimal strategy, allowing reliable detection of a single low-parasitemic sample (100 parasites/μl) in pool sizes up to 50. This PCR-based pooling strategy was then compared with microscopy using 891 dried blood spots from a cohort of 77 Ugandan children followed for 2 years in an urban setting of low endemicity. Among 419 febrile episodes, 35 cases of malaria were detected using the PCR-based pooling strategy and 40 cases using microscopy. All five cases of malaria not detected by PCR were from samples stored for >2 years with parasitemia of <6,000/μl, highlighting the issue of possible DNA degradation with long-term storage of samples. Among 472 samples collected from asymptomatic children as part of routine surveillance, 15 (3.2%) were positive by PCR-based pooling compared to 4 (0.8%) by microscopy (P = 0.01). Thus, this PCR-based pooling strategy for detection of malaria parasites using dried blood spots offers a sensitive and efficient approach for malaria surveillance in low-transmission settings, enabling improved detection of asymptomatic submicroscopic infections and dramatic savings in labor and costs.Due to large-scale implementation of effective control measures, many countries where malaria is endemic are experiencing dramatic declines in disease burden. With this success has come a shift in the end goal from control to elimination (9, 10). However, when the goal is elimination, accurate detection of persons infected with malaria parasites becomes essential (11). Standard surveillance systems depend on diagnosis by microscopy, a method that is technically challenging, labor-intensive, and often inaccurate in operational settings. More recently available rapid diagnostic tests (RDTs) provide convenience and ease of use, but they have limitations in specificity, sensitivity, species identification, and cost (22).PCR-based methods for malaria parasite detection are relatively simple and provide improved sensitivity compared to microscopy and RDTs, especially in settings where infections have low parasitemia or contain mixed species (22). PCR can also be performed on dried blood spots, which are convenient for collection, storage, and transport. Nonetheless, PCR has a long turnaround time, making it an impractical tool for clinical care of individual patients. One exception would be real-time PCR, which has a short turnaround time but carries with it cost and capacity constraints that make it unavailable for rapid diagnosis in most settings. However, there is a potential role for PCR in situations where large numbers of samples are being screened: a high-throughput system could allow accurate, rapid, and cost-effective assessments to quantify prevalence and identify species.The need for a high-throughput method to detect malaria parasites is especially important for surveillance in low-transmission and elimination settings, where low prevalence renders diagnostic testing at the individual sample level impractical and inefficient. Detection methods based on pooled nucleic acid could provide a simple solution. The pooling of sera or plasma specimens has long been used to improve the accuracy, cost-effectiveness, and throughput of screening for many infections (e.g., HIV-1, hepatitis B virus, hepatitis C virus, and West Nile virus) in low-prevalence settings, such as blood banks (4, 26). We recently reported methods for pooled analysis of dried blood spots for HIV-1 nucleic acids (1, 18).The goal of this study was to develop and apply a novel PCR-based pooling strategy for the detection of malaria parasites using dried blood spots. We first established reference standards by using laboratory strains to create dried blood spots with known Plasmodium falciparum parasite densities. After extracting DNA from pools of dried blood spots, we compared the sensitivities and specificities of different nucleic acid amplification tests. The most robust pooling strategy was identified and then applied to 891 field samples collected using passive and active surveillance from a cohort of 77 children living in Kampala, Uganda—a city with relatively low malaria endemicity—who were followed for up to 2 years. The results were compared to those obtained by microscopy.     

Ammonia vapour in the mouth as a diagnostic marker for Helicobacter pylori infection: preliminary 'proof of principle' pharmacological investigations.

Most current non-invasive tests for Helicobacter pylori depend on the conversion of labelled (13C or 14C) urea to labelled carbon dioxide (13CO2 or 14CO2) and ammonium (NH4+) by the enzyme urease, with the labelled CO2 detected in exhaled air. Despite suggestions going back over a number of years, the alternative possibility of using NH4+ (in the form of gaseous ammonia [NH3]) as the test parameter has received little or no attention. However, this approach is now being explored using a chemiresistive sensor detecting sub-parts per million concentrations of NH3. An in vitro 'glass stomach' (containing various volumes of hydrochloric acid [HCl] and ammonium chloride [NH4Cl]) was used to evaluate the means of increasing 'gastric' pH to that of the NH4+-->NH3 transition that occurs significantly at pH 9.24. This 'stomach' also was used to study mechanisms by which NH3 may be expelled in a pulse (as a surrogate belch), either by the in situ production of CO2 or through an exogenous source. On the basis of the protocols developed, H. pylori-negative subjects were tested before and after ingestion of 10 mg NH4Cl (as a surrogate for bacteria-produced NH4,), and H. pylori-positive subjects were tested without taking urea or NH4Cl. 'Intragastric' pH in the in vitro 'glass stomach' could be increased above pH 9.24 by adding a mixture of 15-30 mL magnesium hydroxide mixture (or the proprietary equivalent) and 50 mL water, and the resulting NH3 expelled by adding 100 mL CO2-saturated cold water (sparkling water). In vivo, NH3 levels in the oral cavity of H. pylori-negative subjects were increased after ingestion of 10 mg NH4Cl; however, levels in the oral cavity of a small number of H. pylori-positive subjects were two- to threefold higher after magnesium hydroxide and sparkling water. On the basis of in vitro studies, an in vivo protocol was developed to increase gastric pH above that required for the NH4+-->NH3 transition, and a mechanism established to release the NH3 into the oral cavity. Preliminary in vivo data confirm the chemiresistive sensor is sufficiently sensitive to NH3 to distinguish H. pylori-negative subjects who have taken 10 mg NH4Cl from those who have not, and clearly distinguish H. pylori-negative subjects from H. pylori-positive subjects. Ingestion of urea or other labelled tracers is not required, nor is belching; and the sensor takes less than two minutes to reach a maximum response. The data provide good evidence that the chemiresistive detection of NH3 has considerable potential as a rapid, point-of-care diagnostic test for H. pylori infection.     

PCR Diagnosis of Opisthorchis viverrini and Haplorchis taichui Infections in a Lao Community in an Area of Endemicity and Comparison of Diagnostic Methods for Parasitological Field Surveys

Opisthorchiasis is a major public health problem in Southeast Asia. Affected individuals often have mixed infections with the liver fluke Opisthorchis viverrini and minute intestinal flukes such as Haplorchis taichui. The usual methods of diagnosing these infections involve the demonstration of fluke eggs in stool samples under light microscopy, but sensitivity and specificity are low. We developed two PCR tests that detect and discriminate between O. viverrini and H. taichui infections. PCR tests were validated by stool samples from purged individuals. We then applied the PCR tests to estimate the prevalence of O. viverrini and H. taichui infections from a random sample of individuals selected from a community in an area of endemicity in Khong District, Laos. PCR results were compared with those from the Kato-Katz (KK) method and the formalin-ether concentration technique (FECT). When validated with purge results, PCR tests of O. viverrini and H. taichui had sensitivities of 93.7% (95% confidence interval [CI], 85.8 to 97.9%) and 73.3% (95% CI, 60.3 to 83.9%) and could detect as little as 0.75 pg DNA and 1.32 ng DNA, respectively. The PCR-determined community prevalences of O. viverrini and H. taichui infections were 63.9% (95% CI, 54.1 to 72.9%) and 30.6% (95% CI, 22.1 to 40.2%), respectively. Using PCR as the gold standard to detect O. viverrini, three KK thick smears performed comparably well, whereas one KK smear and FECT were poorer (sensitivities of 91.4% [95% CI, 81.0 to 97.1%,], 62.3% [95% CI, 49.8 to 73.7%], and 49.3% [95% CI, 37.0 to 61.6%], respectively). PCR may be a valuable and sensitive diagnostic tool, particularly for low-intensity O. viverrini and H. taichui infections.Food-borne trematodiases are an emerging but neglected public health issue (12). One of the food-borne trematodes, the liver fluke Opisthorchis viverrini is endemic in Cambodia, Lao People''s Democratic Republic (PDR), Thailand and Viet Nam (8). It has been estimated that the number of infections caused by O. viverrini amounts to 9 million cases, and more than 67 million people are at risk of infection (11). Chronic O. viverrini infection is an important risk factor (class 1 carcinogen) for developing cholangiocarcinoma, or biliary duct cancer, which is associated with a poor prognosis (17, 24-26).O. viverrini infection often coexists with other food-borne trematodes, including several species of flukes called the minute intestinal flukes (MIF) owing to their small size (families Heterophyidae and Lecithodendriidae). In recent studies in Lao PDR (2, 4, 23), different species of MIF were detected after purging individuals infected with O. viverrini. Of these MIF, Haplorchis taichui was the predominant species in concurrent infection with O. viverrini. The coexistence of O. viverrini with H. taichui infections has been described previously (7, 21, 29).Fecal examination under light microscopy is currently the standard method to diagnose O. viverrini infections. The Kato-Katz technique (KK) (10) is the recommended field method (34). This technique permits estimation of infection intensity expressed in eggs per gram feces (epg). Unfortunately, a single stool examination read under light microscopy has low sensitivity (24), particularly for low-intensity infections, and examination of repeated specimens per individual may be needed to improve detection rates. Furthermore, KK does not permit the differentiation of O. viverrini eggs from MIF eggs such as those of H. taichui, because the eggs are similar in size and are both oval and operculated (30). Detection by light microscopy is also operator dependent, and infection may simply be missed.When screening for O. viverrini by KK under light microscopy, identified eggs can be characterized only as “Opisthorchis-like” (21, 29). Distinction of O. viverrini eggs from MIF eggs can be achieved if the specimen is preserved and examined by the formalin-ether concentration technique (FECT) and if the eggs are later stained with iodine (9), potassium permanganate (28), or methylene blue (20). One method of confirming O. viverrini infections in the presence or absence of MIF with certainty is by identification of adult flukes, which may be obtained by treating infected individuals with praziquantel, the current drug of choice, offering them bowel purging with an oral laxative and then examining their diarrhetic stools.As an alternative to microscopy and its limitation of potential diagnostic misclassification, molecular methods have recently been developed. A PCR method for the detection of O. viverrini eggs in human stool specimens was developed (37) and then evaluated as a diagnostic tool with human stool samples collected in Lao PDR (27). A rapid cleanup procedure for human stool samples was developed using inexpensive chemicals (19). This test recognizes all opisthorchiids by a single assay and differentiates them from MIF. Most recently, PCR tests were developed to discriminate between O. viverrini, H. taichui, and another food-borne trematode, Clonorchis sinensis (22, 31, 32). Some studies using PCR tests on human stool samples were validated with light microscopy (27, 32, 33). A more accurate method such as the identification of adult flukes would be valuable to validate PCR tests.In this paper, we present the development of PCR diagnostic tests using sensitive and specific primers to differentiate O. viverrini from H. taichui with PCR tests validated by stool samples from purged individuals. We then used these PCR methods as the gold standard to estimate the infection prevalences of O. viverrini and H. taichui in a random sample drawn from a cross-sectional community survey in Khong District, Lao PDR. Finally, we compared the results of our PCR method with those from two parasitological diagnostic tests, the KK and FECT.     

Biochip System for Rapid and Accurate Identification of Mycobacterial Species from Isolates and Sputum

The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.Mycobacterium tuberculosis is an important pathogen; it is responsible for 1.3 to 1.7 million mortalities worldwide in 2007 (26). However, the incidence of opportunistic infections by nontuberculous mycobacteria (NTM) has gradually increased, causing a number of different NTM diseases (4, 5, 14). This apparent increase is thought to be the consequence of several factors; the increased use of diagnostic methods that can identify these agents has contributed to this number, while the AIDS epidemic is a principal cause (18). A report on a nationwide random survey in China on the epidemiology of tuberculosis in 2000 revealed that 11.1% of the 441 bacterial strains isolated from patient sputum were NTM (15). The low level of identification of NTM is partially due to the use of conventional methods, which rely on growth characteristics, colony morphology, pigment production, and biochemical tests. Because of the slow growth of mycobacteria, the methods are time-consuming, taking 4 to 8 weeks to complete. Extensive experience in the interpretation of the results of biochemical tests is also required, and the identity of specific species from cultures is often difficult to determine, resulting in possible misidentification. As a result of these numerous limitations, conventional methods are not widely used by the majority of clinical laboratories in China. However, the correct identification of NTM is clinically important because most NTM are naturally resistant to first-line antituberculosis drugs, and different species of NTM are sensitive to different antibiotics drugs. Thus, there is an urgent need for the development of a rapid, simple, and accurate method for mycobacterium species identification (21).Recently, different nucleic acid based molecular assays such as DNA sequencing (1), microarray assay (6, 16, 25), PCR-restriction fragment length polymorphism (RFLP) assays (23), and commercial kits such as Accuprobe (Gen-Probe, San Diego, CA) (2), GenoType (Hain, Germany), (19, 20), and INNO-LiPA (Innogenetics, Belgium) (24), have emerged as rapid tools for species identification. The molecular identification methods are based on the polymorphisms in the 16S rRNA (1) or 16S-23S rRNA spacer regions (24) or 23S rRNA (20) or hsp65 genes (12). The 16S rRNA gene sequences of most mycobacterial species are well known and can be found in online databases. Although the molecular methods mentioned above have greatly improved the diagnosis of NTM, there is still a need for a quick, semiautomated or fully automated, total solution-based system to meet the high-throughput demands of busy clinics, particularly for large centers with numerous patients.We report here a rapid diagnosis solution that includes a biochip, apparatus for sample preparation, chip hybridization, washing and data acquisition, and dedicated software for automated diagnosis. The biochip is designed to detect differences in 16S rRNA sequences for mycobacteria species identification. The entire process is semiautomatic and high throughput. The automated determination of species could also eliminate some elements of operator error.     

Evaluation of the BD GeneOhm StaphSR Assay for Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Spiked Positive Blood Culture Bottles

To improve the clinical outcome of Staphylococcus aureus septicemia, the early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represents an attractive approach for the rapid identification of S. aureus and the determination of its methicillin (meticillin) resistance. In direct comparison to other molecular assays (sa442 and mecA real-time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm StaphSR assay for its use in the detection of S. aureus and methicillin-resistant S. aureus (MRSA) from spiked blood culture bottles (n = 134). In the case of detecting S. aureus (n = 90; for methicillin-susceptible S. aureus, n = 45; for MRSA, n = 45), the BD GeneOhm StaphSR assay had a sensitivity and a specificity of 100% each (95% confidence intervals [CIs], 96.0 to 100% and 82.4 to 100%, respectively). For MRSA (n = 45), the test was 95.6% (95% CI, 84.9 to 99.5%) sensitive and 95.3% (95% CI, 86.9 to 99.0%) specific. Overall, five discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains that were not detected by the BD GeneOhm StaphSR assay (2/45). Compared to other real-time PCR-based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm StaphSR turned out to be an appropriate diagnostic tool for a rapid (∼1.5 h), preliminary identification of S. aureus and MRSA from blood cultures.Staphylococcus aureus septicemia is associated with high mortality rates, prolonged hospitalization, and increased costs (3, 5). The prevalence of S. aureus septicemia is increasing, primarily due to infections caused by methicillin (meticillin)-resistant S. aureus (MRSA) (20). Several studies have shown that mortality rates among patients suffering from MRSA septicemia is significantly higher than those of patients suffering from infections caused by methicillin-susceptible S. aureus (MSSA) (5, 18, 19).The early selection of an appropriate antibiotic regime for the treatment of MSSA or MRSA is crucial for the patient''s outcome (4, 14, 15). However, bacterial identification and preliminary antibiotic susceptibility testing by standard microbiological procedures still requires 24 to 48 h after growth detection by automated incubation systems. In contrast, new real-time PCR-based methods that use samples directly from positive blood culture bottles allows differentiation of MSSA, MRSA, and coagulase-negative staphylococci (CoNS) within 1.5 to 3 h (7, 12, 13, 16). Such tests promote an early appropriate antibiotic selection, thus avoiding the unnecessary use of vancomycin, and they reduce mortality, the length of hospitalization, and costs associated with bloodstream infections caused by these bacteria (3).We described recently a real-time PCR method for the detection of MSSA, MRSA, and CoNS directly from positive blood cultures; it turned out to have 100% sensitivity and 100% specificity for the detection of MSSA and MRSA (7). In this study, the differentiation between MSSA and MRSA directly from signal-positive blood cultures was achieved by the separate detection of the S. aureus-specific chromosomal fragment sa442 and the mecA gene (encoding methicillin resistance). However, since this test is not a commercialized system and does not run on a common platform like, e.g., the SmartCycler (Cepheid, Sunnyvale, CA), its widespread application is limited. Moreover, in blood cultures containing a mixture of MSSA (sa442⁺ but mecA negative) and methicillin-resistant CoNS (MR-CoNS; sa442 negative but mecA⁺), the test is prone to lead to the incorrect identification of MRSA (sa442⁺ mecA⁺).The BD GeneOhm StaphSR assay (BD Diagnostics GeneOhm, Québec, Canada) provides a rapid, simple, commercially available diagnostic test that runs on the SmartCycler for the detection of S. aureus and MRSA from nasal swabs, wounds, and blood cultures. This multiplex real-time PCR amplifies an S. aureus-specific target sequence and a specific target near the staphylococcal cassette chromosome mec (SCCmec) insertion site and the orfX junction in MRSA, thereby differentiating between MSSA and MRSA (9, 17).Using the herein-described setting, we evaluated the BD GeneOhm StaphSR assay and the PCR that detects sa442 and mecA (designated sa442-mecA-PCR) for the detection of MSSA and MRSA directly from spiked blood cultures.     

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Isolates

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position −15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.The rates of mortality and morbidity from tuberculosis (TB) remain high, despite intense worldwide efforts. One of the major factors sustaining the current TB epidemic is the increasing drug resistance of Mycobacterium tuberculosis strains (2). In the early 1990s, multidrug-resistant (MDR) TB cases that were resistant to at least rifampin (RIF) plus isoniazid (INH) arose (6). When the frequency and distribution of extensively drug-resistant (XDR) TB cases were assessed in 2004 by the U.S. Centers for Disease Control and Prevention and the World Health Organization, several cases of drug-resistant tuberculosis consistent with an XDR phenotype were found (7). This study revealed that 20% of the isolates met the MDR criteria; 2% of those were classifiable as XDR; and 4%, 15%, and 19% of the XDR TB cases were from the United States, South Korea, and Latvia, respectively (7). Thus, it is crucial that rapid drug susceptibility tests be developed to prevent the spread of MDR and XDR TB.Although drug susceptibility testing (DST) is a prerequisite for accurate results, such testing requires much time and labor (3). Therefore, several molecular techniques have been applied to detect mutations related to drug resistance (5, 10). Resistance to RIF and INH, the mainstays of antituberculosis treatment, is mainly attributable to mutations in genes encoding the drug target or drug-converting enzymes (8). Early studies demonstrated that 95% of the resistance to RIF is associated with mutation of the RIF resistance-determining region of rpoB, whereas mutations in katG and the regulatory zone of inhA are most frequently associated with INH resistance (11).The oligonucleotide chip method and real-time PCR have been used for detection of drug-resistant M. tuberculosis (17, 21, 27, 29). A novel method of high-resolution melting (HRM) curve analysis is an accurate and simple technique for analyzing the genotype without the need for specific probes. The dye LC Green, SYTO9, or Eva Green saturates amplified DNA, unlike SYBR green dye, during homogeneous melting curve analysis. Also, HRM curve analysis generates a difference plot curve, which analyzes nucleic acid sequences with high accuracy. Application of genotyping by HRM curve analysis has followed (13, 19). The aim of the study described here was to develop a useful molecular tool for the identification of drug resistance in M. tuberculosis in an accurate, rapid, and cost-effective manner.     

Development and Evaluation of a Novel Single-Nucleotide-Polymorphism Real-Time PCR Assay for Rapid Detection of Fluoroquinolone-Resistant Mycoplasma bovis

Monitoring of the susceptibility of Mycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However, in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains of M. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan single-nucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification of M. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of the parC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific for M. bovis, with a detection limit of 5 fg/μl (about 5 M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility of M. bovis to fluoroquinolones.Mycoplasma bovis is an important and emerging cause of bovine respiratory disease (BRD), calf pneumonia, mastitis, arthritis, and otitis media, as well as various less common presentations (11). Clinical disease associated with M. bovis is often chronic, debilitating, and poorly responsive to antimicrobial therapy, resulting in significant economic loss, the full extent of which is sometimes difficult to estimate (5, 11). Moreover, since there is no effective vaccine for M. bovis, use of antibiotics is the main control method for this infection.Fluoroquinolones are broad-spectrum antimicrobials highly effective for the treatment of a variety of clinical and veterinary infections and frequently used for the treatment of M. bovis infection in cattle. Their antibacterial activity is due mainly to inhibition of DNA replication. A major mechanism of fluoroquinolone resistance in prokaryotes (including Mollicutes) involves alterations in the target enzymes DNA gyrase and DNA topoisomerase IV, thereby altering affinity for the drug (2, 8). The mutations associated with resistance have been localized within the quinolone resistance-determining regions (QRDRs) of DNA gyrase subunits GyrA and GyrB and/or topoisomerase IV subunits ParC and ParE.Recently, the QRDRs of these genes in 42 M. bovis clinical isolates exhibiting various levels of susceptibility to fluoroquinolones were characterized (10). The data showed that 10/11 enrofloxacin-resistant isolates harbored amino acid substitutions in the QRDRs of each of two proteins (GyrA and ParC). However, the main difference between susceptible and resistant M. bovis strains was the change of nucleotide G to A at position 265 of the parC amplicon (corresponding to position 250 of the Escherichia coli parC QRDR gene), resulting in the substitution of Asn for Asp-84 (10).In routine practice, the detection of fluoroquinolone resistance in mycoplasmas is detected by in vitro susceptibility testing (agar or broth microdilution or Etest) after isolation in pure culture. These methods, however, are time-consuming and can take as long as 3 to 4 weeks. In addition, there is no standard operating procedure and no agreed reference strains for the in vitro susceptibility testing of animal-pathogenic mycoplasmas. A parC PCR-restriction fragment length polymorphism (parC-PCR-RFLP) assay detecting a point mutation that confers resistance to fluoroquinolones on M. bovis strains was recently developed to shorten the detection time (10). The parC-PCR-RFLP assay results correlated well with the results by conventional broth microdilution. However, a more sensitive, faster, and more user-friendly method than parC-PCR-RFLP would be advantageous.Therefore, the aim of this study was to develop a rapid and reliable single-step method to differentiate between M. bovis fluoroquinolone-susceptible and -resistant field isolates.     

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