GeneXpert Testing: Applications for Clinical

Clinical Microbiology Newsletter Vol. 30, No. 23

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$88 December 1, 2008

GeneXpert Testing: Applications for Clinical Microbiology, Part I* Elizabeth M. Marlowe, Ph.D., D(ABMM),1 and Donna M. Wolk, Ph.D., D(ABMM),2 1Southern California Permanente Medical Group, Regional Reference Laboratories, North Hollywood, California, 2Department of Pathology, University of Arizona, College of Medicine, BIO5 Institute, Tucson, Arizona

Abstract The impact of rapid polymerase chain reaction (PCR) technology on infectious disease testing is continuing to evolve outside the realm of a centralized laboratory. The GeneXpert Dx system is the first unit dose, near-point-of-care molecular device commercially available. To date, there are five FDA-cleared assays available for the GeneXpert System: group B Streptococcus (GBS) from vaginal-rectal swabs, enterovirus from cerebrospinal fluid, methicillin-resistant Staphylococcus aureus (MRSA) from nares swabs, MRSA and methicillin-susceptible S. aureus (MSSA) from skin and soft tissue infection swabs, and MRSA and MSSA from positive blood cultures. Advantages of the GeneXpert assays include ease of use, random access, rapid results, and the ability to run the assay without the need for pre- and post-analytical rooms. Limitations include a currently limited menu with few specimen types and potential delay of results due to indeterminate results. Part I of this article presents a review of this technology and its application for the detection of GBS, enterovirus, and MRSA from various clinical specimens.

Introduction When Kary Mullis first tested the concept of polymerase chain reaction (PCR), the initial experiments were performed using water baths monitored by a simple laboratory timer. This manual cycling of temperatures quickly evolved to automated cycling in thermocyclers, followed by detection of PCR amplicons with gel electrophoresis. Real-time PCR is now used in many clinical laboratories because it allows the rapid detection of PCR products by the use of specific fluorescent chemistries in a closed system. It has become a mainstay of core molecular laboratory technologies. While molecular diagnostics has revolutionized the way medicine is *Editor’s Note: Part II of this article will be published in the December 15, 2008 issue of CMN (Vol. 30, No. 24). Mailing Address: Elizabeth M. Marlowe, Ph.D., D(ABMM), Southern California Permanente Medical Group, Regional Reference Laboratories, North Hollywood, CA 91605. Tel.: 818-503-7067. Fax: 818-5036713. E-mail: [email protected]

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practiced, after 2 decades, the practice of molecular diagnostics remains tied to a highly skilled laboratory staff and batch processing. Emerging technology has the potential to change the reference laboratory-centric model of molecular diagnostics. The future of molecular diagnostics is changing and is focused on producing rapid results in a general hospital setting. The concept of pointof-care molecular diagnostics is evolving and is closer than was ever thought possible. The technology that has transformed molecular testing from “batch” to “STAT” is Cepheid’s GeneXpert system (Sunnyvale, CA), which can offer nearpoint-of-care results in 1 to 2.5 h. The system consists of an instrument and a personal computer with preloaded software for running the tests and interpreting the results. The assays are simple to run and utilize a single-unit disposable cartridge. The cartridge automates and combines specimen processing, nucleic acid extraction, amplification, and detection. The PCR reagents are lyophilized and held in various chambers in © 2008 Elsevier

the cartridge. Once the sample and accompanying dispensable reagents are added to the cartridge, no additional manual intervention is required. Controls include probe checks, pressure checks, and specimen-processing controls. Internal controls are incorporated into each run to ensure that the cartridge and chemistry are functioning properly. The cartridges are placed in an independently controlled GeneXpert module, which is a configuration of the independent heating and cooling optical reaction (ICORE) module found in the Cepheid SmartCycler instrument. Accompany-

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ing software automates data interpretation. The GeneXpert instrument is available in random-access 1-bay, 4-bay, and 16-bay module, and more automated 48-bay configurations. A more automated 72-bay configuration is due to be released soon. Cepheid has received FDA clearance for their GeneXpert device for a variety of pathogens, including group B Streptococcus (GBS) from vaginal-rectal swabs (1,2), enterovirus (EV) from cerebrospinal fluid (CSF) (3,4), and methicillin-resistant Staphylococcus aureus (MRSA) from nares swabs (5). The Xpert methods are rapid and relatively easy to perform; the GBS and MRSA methods are rated as “moderate complexity,” and the EV method is rated as a “high complexity” test according to Clinical Laboratory Improvement Act (CLIA) categorization. Recently, a GeneXpert assay that simultaneously detects MRSA and S. aureus (SA) from skin and soft tissue specimens was FDA cleared. A similar MRSA-S. aureus assay has also been FDA approved for use in blood culture bottles.

Applications of the Technology GBS GBS emerged as a leading cause of neonatal morbidity and mortality in the 1970s. As GBS is an inhabitant of the gastrointestinal tract, transmission from mother to infants can occur at the time of delivery. Early-onset disease (EOD) occurs within 7 days of birth and accounts for about 80% of GBS infections in infants. Late-onset disease appears after 7 days of age (6,7). Colonization of pregnant women is approximately 10 to 30% depending on the patient population (8,9). Higher rates of colonization have been noted among African Americans, non-smokers, and those with a high body mass index (10).

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In 2002, the CDC issued revised guidelines in collaboration with the American College of Obstetrics and Gynecology and the American Academy of Pediatrics recommending universal prenatal screening of all pregnant women between 35 and 37 weeks of gestations to determine their vaginal-rectal GBS colonization status. Since GBS is easily treated with antibiotics, intrapartum antibiotic prophylaxis (IAP) can be provided during labor and delivery if a woman is GBS positive (8). In 2005, the CDC examined the impact of these guidelines and found that overall EOD had decreased more than 30% (11). Despite these preventative measures, disparities still remain among certain ethnic groups and those with a lower socioeconomic status (11). A 2007 report from the CDC highlighted that among African Americans the incidence of EOD had risen 70% to pre-recommendation levels (12). Furthermore, the majority of EOD cases arise from women with negative GBS cultures and no risk factors (13). Maternal GBS colonization may be transient, chronic, or intermittent (14). Thus, antepartum screening may not provide the most accurate determination of GBS colonization status at the time of delivery (15). Preterm deliveries can also miss the 35- to 37-week GBS culture. These births make up 7 to 11% of deliveries but comprise 32 to 38% of EOD (13). The solution to these problems would be to screen these women at delivery, since IAP given early enough (≥2 to 4 h) to GBS positive mothers at the time of delivery significantly diminishes GBS transmission to newborns (16). The CDC recommends the use of antepartum GBS screening, primarily with culture from vaginal-rectal swabs. Swabs are placed in selective media, typically Todd-Hewitt or LIM broth,

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incubated for 18 to 24 h, and then plated onto sheep blood agar. After 24 h, the plates are examined for GBS; if negative, the plates are incubated another 24 h and re-examined. Suspicious colonies can be confirmed as GBS by latex agglutination methods (8). For an assay to have maximum impact when used for intrapartum testing, the CDC suggested that it would have to be rapid, sensitive, a minimum of 85% accurate compared to culture, and fit easily into the clinical laboratory’s routine (8). Routine PCR meets some of these requirements; however, the simplicity of the GeneXpert makes it a good fit for around-the-clock testing scenarios. GBS testing could potentially occur in STAT laboratories to support labor and delivery suites as testing is needed. PCR for GBS from vaginal-rectal swabs is available. Only FDA-cleared PCR methods will be discussed, since full discussion of alternate PCR methods is not within the scope of this review. GBS-PCR has the ability to both decrease turnaround time (TAT) and increase sensitivity (17). The BD GeneOhm Strep B PCR (BDGO) assay (BD Diagnostics, San Diego, CA) was the first FDA-cleared test on the market for testing on the Cepheid SmartCycler DX system (Cepheid, Sunnyvale, CA), with results available within 2 h. A multi-center study demonstrated that the BDGO Strep B PCR had a sensitivity and a specificity of 94% and 95.5%, respectively (17). Cepheid also has two FDA-cleared GBS assays: the Smart GBS, which is run on their SmartCycler platform, and the Xpert GBS assay that is run on the GeneXpert Dx System. The Smart GBS is FDA cleared for testing directly from swabs or from LIM broth-enriched specimens. The direct method demonstrated


a sensitivity of 85% and a specificity of 97% compared to culture among intrapartum specimens tested in a clinical trial, while the enriched method demonstrated a sensitivity and specificity of 98.7% and 90.4%, respectively, compared to culture among anetpartum specimens (18). One limitation of the PCR assays performed on the SmartCycler is that they require a laboratory with sufficient space to house the instrument and a medical technologist to perform the test. These tests are rated high complexity by CLIA. Clinical-trial data demonstrated that the Xpert GBS assay has a sensitivity and a specificity of 88.6% and 96.7%, respectively, for both antepartum and intrapartum specimens compared to culture (1). In the intrapartum arm of the study, the sensitivity and specificity were 91.3% and 95.6%, respectively. The mean TAT was 1.84 h. During the clinical trials, the FDA required that nurses perform the testing on the GeneXpert System. The assay was FDA cleared in July 2006 as the first molecular test given a moderate-complexity rating by CLIA. In a prospective study, Gavino and Wang (2) reported the Xpert GBS assay to have a sensitivity and a specificity of 95.5% and 64.5%, respectively. Because of its sensitivity and performance simplicity, the Xpert GBS assay has the potential to decrease EOD, particularly among populations for which there are marked disparities. However, culture techniques will remain, as PCRbased tests, as yet, do not provide susceptibility results for the pencillinallergic populations. Antepartum cultures would still need to be performed to ensure GBS remains susceptible to first line antibiotics. Another potential limitation of the Xpert GBS assay is that unresolved results, due to failed controls or inhibition, require repeat testing, thus delaying results and IAP, as well as adding to the cost of testing. Enterovirus EV is composed of positive-sense, single-stranded RNA viruses. The genus is in the family Picornaviridae and currently consists of 68 distinct serotypes (19). In the United States, an estimated 75,000 cases of aseptic meningitis occur annually, and approximately 80 to 90% of these are caused by EVs (2022). Typically, most infants and young Clinical Microbiology Newsletter 30:23,2008

children recover from EV meningitis without sequelae. However, the clinical diagnosis of EV is often one of exclusion, so that a bacterial etiology cannot be excluded. Without actual EV diagnostic confirmation, patients may be hospitalized for further diagnostic testing and the administration of antibiotics. This practice leads to extended length of stay and increased hospital costs. Isolation of EV from CSF in cell culture requires multiple cell lines and 3 to 8 days of incubation before cytopathic effect appears. This time frame is typically not rapid enough to affect therapy or the duration of hospitalization. Additionally, the sensitivity of cell culture for recovery of EVs from CSF is approximately 50 to 75% depending on the serotype causing infection and the concentration of virus in the CSF (23,24). It is no surprise that nucleic acid amplification methods have replaced traditional culture-based methods as the gold standard for detecting EV in CSF, as PCR supports a considerable increase in sensitivity and a shorter time to result (23,25-31). Depending on when results are available and how they are used in the discharge plan, significant health care-associated cost savings have been coupled with PCR diagnosis of EV meningitis (21,32-37). While many laboratories have employed laboratory-developed assays (LDAs) for the detection of EV for years, LDAs require that each laboratory independently verify its assays. In 2001, a multi-center study found only 66% of laboratories performed adequately on EV proficiency testing, highlighting the need for more standardized testing (38). The Xpert EV cartridge is currently the only FDA-cleared molecular assay for the detection of EV from CSF. The Xpert EV assay is able to detect 63 different serotypes of EV with a limit of detection range of 0.0002 to 200 tissue culture infective doses/ml (3,39). A multi-center beta trial study demonstrated that the performance characteristics of the Xpert EV assay were comparable to those of assays currently being used in clinical laboratories (3). Marlowe et al. (4) compared it to realtime NASBA and a Taq-Man reverse transcription-PCR and found similar results. Assays were evaluated using a © 2008 Elsevier

12-member proficiency panel and up to 138 CSF specimens. The Xpert EV, NASBA, and TaqMan assays correctly identified 10/12, 8/12, and 7/12 proficiency panel members, respectively. For detection of EV RNA in CSF, the sensitivities of the Xpert EV, NASBA, and TaqMan were 100%, 87.5%, and 96%, respectively. In a prospective, unblinded comparative study of the Xpert EV to the Argene EV consensus kit (Argene SA, Varilhes, France) and an in-house real-time PCR, the initial and resolved sensitivities of the Xpert EV assay were 90.45% and 98.8%, respectively (40). QCMD proficiency panel results were comparable to those of Marlowe et al. (4). A limitation of the Xpert EV assay is the ability to test alternative specimen types, as the assay is FDA cleared only for CSF. While customers are independently examining the off-label ability of the cartridge to detect EV from alternative specimens, data are limited and have yet to be validated in multiple laboratories (41). The ease of use and software interpretation make the Xpert EV assay a very attractive alternative for molecular EV testing from CSF. Random-access testing makes it particularly useful for a low-volume assay and a short-staffed laboratory. Testing can be performed as specimens are received with less than 5 minutes hands-on time. Random access and decreased TAT, compared to the batch mode of testing have the potential to significantly decrease hospital costs. A cost analysis study demonstrating the use of the Xpert EV assay in emergency rooms and laboratories would be of interest. MRSA The spread of drug-resistant bacteria, such as MRSA, is a major concern for healthcare and communities in which aggressive infections have caused deaths in both hospitalized patients and otherwise healthy individuals (42). A known colonizer, MRSA is often harbored in the human nares, skin, throat, and mucosa of the vagina and rectum. Human “carriers” can spread MRSA to others in health care settings, who can fall victim to potential infections. Since 2003, MRSA has accounted for more than 60% of all S. aureus health care-associated infections (HAIs) reported in U.S. hospitals (43). At last report, the National 0196-4399/00 (see frontmatter)

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Health and Safety Network estimated that in the U.S., hospitalized patients acquire 2 million HAIs each year, causing 90,000 deaths and $4.5 billion in excess health care costs. A large percentage of HAIs are due to MRSA (44). Specific guidance continues to accumulate for MRSA-related practices in health care and the community. In 2003, active surveillance was recommended by national guidelines put forth by the Society for Healthcare Epidemiology of America (45). In 2005 and 2006, the CDC’s Healthcare Infection Control Practices Advisory Committee issued recommendations focused on reporting and management of multidrug-resistant organisms in health care settings (46,47). These recommendations detail approaches for the reduction of MRSA infections in health care facilities (48). As of September 2008, the Association for Professionals in Infection Control reported 35 states that require some form of mandatory MRSA reporting or had study laws in place; 2 additional states had voluntary reporting. On 26 September 2008, the state of California passed legislation that will require hospitals to increase their infection prevention efforts and to report their infection rates for posting to the public by 2011.

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