CT and NG Data

CT and Neisseria gonorrhoeae (NG) are the most common cause of bacterial STDs, and both can cause urogenital tract infections ranging from acute to asymptomatic disease. CT is an obligate intracellular bacterium comprising 15 serovars, whereas NG is a fastidious intracellular diplococcus. Significant underreporting of disease can occur as the result of silent infections affecting the reproductive age group. Identification and treatment is important to prevent the sequelae of infection, such as infertility, chronic pain, and pelvic inflammatory disease.

Urogenital specimens commonly exhibit amplification inhibition. The inhibitory substances can be removed by including nucleic acid purification steps in the sample preparation. The sample preparation protocols vary among the commercially available assays, ranging from the use of crude lysates (AMPLICOR) to purified nucleic acids. The Roche AMPLICOR assay uses an amplification control in the sample that allows for detection of inhibitory substances. This control consists of a plasmid-containing CT primer binding sites and a randomized internal sequence. The BD ProbeTec (BD Diagnostics, Sparks, MD) uses 1000 copies of a linearized NG DNA containing plasmid as the internal amplification control.

Commercially available assays for CT and NG (Table 1) use target amplification methods, with the one exception being the Digene HC2 assay, which uses a signal amplification method with an RNA probe cocktail complementary to approximately 39,300 bp (4%) of the Chlamydia genomic DNA and one probe complementary to 100% of the cryptic plasmid. Nucleic acid amplification testing assays typically increase sensitivity by targeting multiple copy genes or plasmids. For CT, the targets include cryptic plasmid DNA present in nearly all serovars (5 to 10 copies), genes such as omp1, and ribosomal RNA (rRNA; 16S and 23S). For NG, targets include the cytosine methyl transferase gene (M.NgoPII), the Opa gene, Piv-1 genes, and 16S and 23S rRNA. The specimen type approved for CT and NG testing is assay specific (Table 1) and includes urethral swab and urine for males and endocervical/cervical samples, vaginal swabs, urine, and PreservCyt (Hologic, Inc.) specimens for females. A male first-void urine specimen and vaginal swabs are considered optimal specimens, according to the Association of Public Health Laboratories (http://www.aphl.org/aphlprograms/infectious/std/documents/ctgclabguidelinesmeetingreport.pdf, last accessed December 31, 2010).

The AMPLICOR NG detection kit (Roche Molecular Diagnostics, Pleasanton, CA) targets the M.NgoPII gene, whereas the BD ProbeTec ET CT/GC targets the cryptic plasmid of CT and the Piv-1 gene of NG. Both of these assays have cross-reactivity with some Neisseria species. Confirmatory testing using a different gene target is an option in such instances. Cross-reactivity has not been reported for the Real Time CT/NG (Abbott Laboratories, Des Plaines, IL), APTIMA COMBO 2 (Gen-Probe Inc., San Diego, CA), and PACE 2 (Gen-Probe Inc.) assays.20

Coinfection with CT and NG occurs in many patients. Simultaneous detection of both organisms in a single test is achieved by several assays (Table 1). The APTIMA COMBO 2 is a second-generation assay that uses target capture with transcription-mediated amplification and chemiluminescent hybridization protection. In contrast to PCR and strand displacement assays, which amplify bacterial DNA, transcription-mediated amplification amplifies specific regions of the 23S rRNA/16S rRNA. The APTIMA COMBO 2 assay does not have an internal control; however, it uses target capture technology, which removes inhibitors. The assay is a dual kinetic assay, with one signal scoring for CT and the second signal scoring for NG. Assays that target bacterial rRNA rather than plasmid DNA have a greater ability to detect lower concentrations of organisms because of the presence of up to a 1000-fold greater amount of RNA than plasmid DNA in the infected cell.

Cryptic plasmid–based detection assays could yield false-negative results because CT strains without the plasmid or with deletions in the plasmid have been described (the Swedish variant, with a 377-bp fragment deletion). False-negative results have been reported for the Roche AMPLICOR and the Abbott RealTime CT/NG assays that target the deleted region, whereas those assays that target outside of this region or the chromosomal regions detect the mutant strain.21 The BD ProbeTec assay is able to detect the CT Swedish variant because the cryptic plasmid target is outside the area of deletion. The newer Abbott RealTime CT/NG assay, FDA cleared in June 2010, includes an additional 140-bp cryptic plasmid target outside of the 377-bp deletion area. The Abbott RealTime CT/NG assay also contains a small fragment of noninfectious linearized DNA plasmid for use as an internal control throughout the sample preparation process.

A low prevalence of STDs in a specific population may reduce the positive predictive value of the molecular result. However, the test can be repeated with a separate aliquot of the same specimen or a second specimen and a different test method and/or a different target to confirm the positive result.20 The efficacy of this strategy is debatable.22

HIV-1 Assay

Qualitative assays, such as the Procleix ULTRIO and Discriminatory HIV-1/HCV/HBV assays (Gen-Probe Inc.) and the COBAS AmpliScreen HIV-1 test (Roche Molecular Systems, Pleasanton), are available for donor screening applications. The transcription-mediated amplification–based APTIMA HIV-1 RNA qualitative assay (Gen-Probe Inc.) can be used for diagnosing acute and primary infections and can detect infection before seroconversion and confirm infections in individuals when antibody test results are positive.23,24 However, in this segment, we will focus on the quantitative and genotyping assays that are the main HIV-1–related assays performed in the Molecular Diagnostics Laboratory.

HIV-1 viral load assays are important for monitoring HIV-1–infected individuals, predicting the progression of HIV disease, and monitoring antiretroviral treatment.25,26 HIV-1 is classified into three major groups (ie, M, N, and O). Group M is the most prevalent and is sub-classified into seven subtypes (ie, A–D and F–H) that are geographically distinct. Several commercial kits are available for quantitative determination of HIV viral load to assess patient prognosis during antiretroviral therapy (Table 1). Ascertaining the viral load is a prerequisite to initiating drug therapy in adherence to FDA guidelines and serves to evaluate the efficacy of antiretroviral therapy (http://www.aidsinfo.nih.gov/guidelines, last accessed December 31, 2010). In 1996, the AMPLICOR HIV-1 MONITOR test (Roche Molecular Systems) was the first FDA-approved quantitative HIV end point–based RT-PCR assay. Quantification of HIV-1 RNA copy number is determined by comparing optical density readings of the HIV-1 signal with an external quantitation standard signal, which has a known copy number input. However, a small dynamic range of 400 to 750,000 copies/mL (or 50 to 100,000 copies/mL for the ultrasensitive method) limits the assay. Recently, real-time RT-PCR HIV assays with options of automation, closed-system platform characteristics, broad dynamic range, and good specificity have become commercially available.27 By using the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Roche Molecular Systems), patient specimens are extracted on the COBAS AmpliPrep instrument and amplification/detection occurs on the COBAS TaqMan Analyzer. The assay targets the conserved region in the gag gene, which is prone to a high level of mutation and is only intended for the detection of group M subtypes of HIV-1. Calibration is not required because specific calibration values and predefined assay control ranges are included with each kit and because uracil-N-glycosylase reduces the risk of carryover contamination. Initial underestimations regarding quantification of HIV-1 group M non-B subtypes, when compared with the COBAS AMPLICOR HIV-1 MONITOR Version 1.5 and the Abbott RealTime HIV-1 assay, have been reported.28–32 A second version of the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 (Roche Molecular Systems) was recently approved. This assay improves the underquantification and subtype inclusivity issues present with the original assay. The version 2.0 assay uses a two-target approach with the combination of the new ltr primer-probe set with the original gag primer-probe set to detect the various group M HIV-1 subtypes A–D and F–H and group O. The assay has a quantitation range of 20 to 10⁷ RNA copies/mL.33,34 With the Abbott RealTime HIV-1 kit (Abbott Molecular, Inc., Abbott Park, IL), patient specimens are extracted on the m2000sp instrument and detected on the m2000rt instrument. The assay allows for flexibility in the sample input volumes and detects both group M and group O HIV-1 subtypes. In contrast to these real-time PCR-based assays, the VERSANT HIV-1 RNA 3.0 Assay (Siemens Health Care Diagnostics, Deerfield, IL) uses branched DNA chemistry, which relies on signal amplification. The assay has a lower risk of contamination because of the lack of amplicon production, and the VERSANT HIV-1 RNA 3.0 Assay has been validated for samples containing group M subtypes A–G (in 2002, subtype E was still believed to be a true subtype); however, it has decreased sensitivity compared with target amplification assays.35,36

During treatment of HIV infections, mutant HIV-1 strains emerge that are resistant to one or more drugs.37 The identification of viral resistance genotypes allows treatment strategies to be modified.38 Retrospective and prospective intervention-based studies38–41 have provided evidence supporting the clinical utility of genotype testing for resistance, and this is recommended by the International AIDS Society–USA panel for selecting new regimens after treatment failure and monitoring therapy for pregnant women. Genotype testing should also be considered before initiation of therapy for acute infections and for treatment-naïve patients with established infection.38

Two genotyping systems are commercially available: TRUGENE HIV-1 Genotyping Kit (Siemens Diagnostics, Tarrytown, NY) and ViroSeq HIV-1 Genotyping Systems (Celera Diagnostics, Alameda, CA). The ViroSeq kit provides reagents for viral RNA isolation from plasma, and both kits provide reagents for RT-PCR and sequencing.39,40,42 Typically, the entire protease and the 5′ reverse transcription coding regions of the pol gene are amplified to generate a large amplicon that is then used as a sequencing template for multiple primers that generate a consensus sequence. Software is available for comparing the consensus with a known reference, to determine any mutations present, based on which treatment options are made available. These are kept current by a panel of HIV experts and recommendations of the International AIDS Society–USA panel.37,43,44 Reportedly, genotyping assays may have limitations of sensitivity in detecting a minority variant species in a patient.45 In addition, potential mutations may be missed at positions not previously characterized as resistance mutants.

MRSA Data

Methicillin resistance is associated with increased mortality in patients with staphylococcal bacteremia.50 Approximately half of all Staphylococcus aureus pneumonias in the United States are due to methicillin-resistant S. aureus (MRSA).51 MRSA ventilator-associated pneumonias also appear to be associated with a higher mortality rate compared with methicillin-sensitive S. aureus ventilator-associated pneumonias.52 Patients colonized with MRSA (nasal carriers) are also at increased risk of developing MRSA disease and can spread the infection as well.

For direct MRSA detection from patient samples, the Xpert MRSA, Xpert SA Nasal Complete assays for the GeneXpert System (Cepheid, Sunnyvale, CA), the BD GeneOhm MRSA ACP (BD Diagnostics, La Jolla, CA), and the LightCycler MRSA Advanced test (Roche Molecular Systems) can be used. The GeneXpert System is a fully integrated and automated nucleic acid preparation, amplification, and real-time detection system. The Xpert SA Nasal Complete (Cepheid) assay can be used for surveillance of both S. aureus and MRSA carrier status, which can aid in reducing the risk of HAIs. In a recent multicenter clinical evaluation, no statistically significant performance differences were observed between the Xpert MRSA and MRSA ACP assays compared with culture.53,54 The recently approved LightCycler MRSA Advanced test was similarly evaluated compared with the BD GeneOhmMRSA ACP and had a similar sensitivity (92.2% and 93.2%, respectively) but a significantly better specificity (98.9% and 94.2%, respectively).55 All of these assays target the S. aureus orfX gene sequence incorporating the insertion site (attBssc) of the staphylococcal cassette chromosome mec (SCCmec) for the detection of MRSA. Targeting the mecA alone could result in false positivity because a large percentage of coagulase-negative staphylococcus species would also test positive. False-negative results can occur as the result of novel SCCmec elements and variants resulting from recombination. Targeting the orfX region alone can give false-negative results in those instances of mecA insertion into other sites (although this is rare).56

Both the BD GeneOhm MRSA ACP and the BD GeneOhm StaphSR assays target the MREJ (types i to vii) of the SCCmec insertion into the orfX gene and the S. aureus species-specific nuc gene, which is distinct from the SCCmec cassette. Therefore, false-positive results are reduced. However, false-positive results may still occur because of SCCmec variants with missing or nonfunctional mecA genes (empty cassette variants) and false-negative results from MREJ variants other than types i to vii.57

The LightCycler MRSA Advanced assay targets the sequence incorporating the insertion site of the SCCmec in the S. aureus orfX gene. Specifically, it targets types 2, 3, and 7 of the right extremity of the SCCmec-orfX junction. Thus, it may give false-negative results if the targeted right extremity types are not present. This assay uses the uracil-N-glycosylase enzyme before amplification to eliminate any amplicon contamination. The Xpert MRSA and Xpert SA Nasal Complete assays offer ease of use, minimal hands-on time, and a closed-tube method of testing. The assay can also be run either as a single on-demand assay or in batch mode. In addition, the Xpert SA Nasal Complete assay has high specificity because of inclusion of three targets (spa, mecA, and SCCmec), reducing false-positive results due to empty cassette variants. In this assay, all three targets must be detected for the assay to give a positive MRSA result. Therefore, coagulase-negative staphylococcus species, which may contain the mecA gene but not the S. aureus–specific spa gene, will not render a positive MRSA result. Assays may still test positive if there is mixed flora of both methicillin-sensitive S. aureus and coagulase-negative staphylococcus species in the testing sample; however, the reported incidence of such cocolonization is low.58

Cultured material can also be tested for the presence of methicillin-susceptible or resistant S. aureus causing sepsis (Table 4). Positive blood culture material may be analyzed by the nonamplified peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) method (AdvanDx, Woburn, MA). These pathogen-specific assays are based on positive blood culture and Gram stain results for detecting S. aureus and MRSA as soon as the instrument signal on continuously monitoring blood culture systems is positive.59–61 Small quantities of positive blood culture material can also be analyzed using the multiplex real-time PCR assays [BD GeneOhm StaphSR (BD Molecular Diagnostics) and Xpert MRSA/SA BC (Cepheid)]. The latter assay operates under a revised Corrective Action Letter from Cepheid that instructs laboratories not to report an MRSA-negative result when an MRSA-negative/SA-positive result is generated on the Cepheid MRSA/SA Blood Culture Assay. Instead, MRSA-indeterminate/SA-positive antimicrobial susceptibility testing pending is the recommendation, with further antimicrobial susceptibility testing performed to determine the MRSA status. The reporting of MRSA-positive/SA-positive results generated on the Cepheid MRSA/SA Blood Culture Assay is not affected.

Results can be achieved by real-time PCR, closed, walk-away systems more rapidly than by more traditional PCR assays. The commercial assays have excellent sensitivity and specificities when compared with culture. In particular, genetic excisions within the SCCmec region of MRSA strains may also yield positive PCR results in the absence of a functional mecA gene and may cause PCR-positive but phenotypically methicillin-susceptible S. aureus results (empty cassette). This prevalence has differed by geographical region and appears to be more common outside the United States.62,63 Decisions on which assay to implement will depend on laboratory capabilities, the urgency for the result, and the impact on patient care. It is recommended that the laboratory routinely perform clinical correlation of the assay results, both positive and negative, to keep abreast of diverse and evolving MRSA strains.

VRE Data

Screening for vancomycin-resistant enterococcus (VRE) directly from perianal, perirectal, rectal, or stool specimens has been recommended by the CDC, Health Care Infection Control Practices Advisory Committee (http://www.cdc.gov/hicpac/mdro/mdro_0.html, last accessed December 31, 2010) to limit the spread of antimicrobial resistance within certain high-risk populations. For surveillance of VRE, the XpertvanA (Cepheid) assay can be performed directly on rectal swab specimens from patients (Table 2). Gram-positive cocci in pairs and chains–Enterococcus faecalis/other enterococci PNA FISH (AdvanDx) is also available to identify VRE from positive blood culture results. Testing for VRE helps identify patients colonized with resistant enterococci in approximately 1 to 2 hours. Positive test results indicate the presence of either the vanA or vanB gene, or vanA alone, which confers vancomycin resistance in E. faecalis, Enterococcus faecium, and other bacteria that may colonize the human intestine. In general, although a positive result does not imply disease caused by VRE, the presence of vanA or vanB genes correlates with colonization and clinical correlation is required to determine active VRE disease. PCR testing should decrease the spread of VRE by rapid identification and isolation of colonized patients; however, conventional bacterial cultures may still be required to isolate VRE from clinical specimens (eg, blood) for the diagnosis of VRE infection. This approach allows antimicrobial susceptibility testing for selection of appropriate antimicrobial treatment and strain typing of isolates in outbreak situations.

Clostridium difficile Infection

C. difficile infection is an important cause of diarrhea in patients who are hospitalized, in long-term care facilities and receiving antibiotics, and in community settings.64 Four assays are available for the detection of toxigenic strains of C. difficile (Table 2). The Illumigene C. difficile assay (Meridian Bioscience Inc., Cincinnati, OH) uses loop-mediated isothermal amplification technology to detect the pathogenicity toxin A gene (tcdA) in the pathogenicity locus of toxigenic C. difficile. The C. difficile pathogenicity locus is a gene segment present in several known toxigenic C. difficile strains. It codes for both tcdA and the toxin B gene (tcdB). The test includes a manual extraction step but does not require costly capital equipment, and results are available in approximately 1 hour. The Xpert C. difficile (Cepheid), BD GeneOhm Cdiff (BD Diagnostics), and proGastro Cd (Gen-Probe Prodesse, Inc., Waukesha, WI) assays are based on real-time PCR and target tcdB of C. difficile. A positive test result does not necessarily indicate the presence of viable C. difficile organisms, but it does indicate the presence of tcdB. Specimen extraction and amplification for the Xpert C. difficile test is self-contained and automated, and the results are available in approximately 45 minutes. The BD GeneOhm Cdiff assay results are available in <2 hours. Mutations or polymorphisms in primer- or probe-binding regions may affect detection of C. difficile tcdA or tcdB variants, resulting in false-negative results; however, variant toxigenic C. difficile without tcdB or with a nonfunctional toxin B protein is rare. An assay may be positive for tcdB without TcdB toxin production (noncytotoxic, IX subtype), as reported in community-associated cases in Canada.65 Because of the enhanced sensitivity of these amplification methods, testing for C. difficile should be limited to patients with clinical symptoms of C. difficile infection. Testing should be limited to diarrheal or loose stools (ie, those that take the shape of the container), and the assays should also not be used for test of cure. Assay performance is unknown for asymptomatic patients.66 Tables 2 and 4 list details of assays for HAIs.

HCV Data

Hepatitis C virus (HCV) infection is the most common cause of chronic viral hepatitis. Nearly 20% of the 4 million carriers develop liver cirrhosis. Viral RNA can be detected in HCV-infected individuals as early as 1 to 4 weeks before the increase of liver enzymes, and it peaks in the first 8 to 12 weeks after infection.80 HCV infections are typically diagnosed by the detection of antibodies directed against specific HCV antigens. However, HCV serological tests demonstrate low specificity and may require confirmation of positive results. Qualitative PCR assays, such as the FDA-cleared Cobas AMPLICOR HCV Test, version 2.0 (Roche Molecular Diagnostics), the APTIMA HCV RNA qualitative assay (Gen-Probe Inc.), or the VERSANT HCV RNA Qualitative assay (Siemens Diagnostics), can be used to confirm serological findings.

Treatment for chronic HCV typically consists of a combination of pegylated interferon-α and ribavirin, and response to therapy is genotype dependent. Quantitative viral load assays are used to establish viral load at baseline, monitor viral load during therapy, and determine response to treatment. Both the rapid and complete early virologic responses have been used to predict if a patient will achieve a sustained virologic response. The early virologic response is defined as a ≥2-log₁₀ reduction in HCV RNA levels during the first 12 weeks of therapy. The rapid and complete early virologic responses are defined as no virus detected at 4 and 12 weeks after initiation of antiviral agent, respectively.81 A completely negative test result for HCV RNA at week 12 (complete early virologic response) is a better predictor of a sustained virologic response than a 2-log₁₀ reduction in HCV RNA.82 A sustained virologic response is achieved when <5 IU/mL HCV RNA is detected after 24-week treatment. After this, the treatment is discontinued for genotypes 2 and 3 and continued for an additional 24 weeks for genotypes 1 and 4. Currently, there are no FDA-approved HCV genotyping tests. Therefore, the quantitative assays used to measure HCV viral load need to be sensitive and need to generate accurate results for all genotypes and subtypes. The same quantitative test should be used throughout a patient's treatment course. The American Association for the Study of Liver Diseases has published guidelines for the use of qualitative and quantitative molecular assays for detection and quantification of HCV RNA in serum and plasma.83

There are two commercial FDA-approved assays for HCV quantitation. The COBAS AmpliPrep/COBAS TaqMan System (Roche Molecular Diagnostics) is an automated real-time RT-PCR that targets a highly conserved sequence in the HCV 5′ untranslated region.84,85 The VERSANT HCV RNA 3.0 Assay (branched DNA) (Siemens Healthcare Diagnostics) is a branched DNA signal amplification method targeting highly conserved sequences in both the 5′UTR and the core gene.86 The former method has a dynamic range of detection from 25 to 3.9 × 10⁸ IU of HCV RNA/mL of plasma or serum. The range of the latter assay is from 615 to 7.69 × 10⁶ IU of HCV RNA/mL of plasma or serum.

Group B Streptococcus

Streptococcus agalactiae [group B Streptococcus (GBS)] is a leading cause of sepsis, meningitis, and death among newborn infants in the Western world. Between 10% and 40% of healthy adult women are colonized by GBS in the genital and gastrointestinal tracts; although not associated with disease in healthy women, GBS can cause disease during pregnancy and delivery.87 The CDC, in collaboration with the American College of Obstetrics and Gynecology and the American Academy of Pediatrics, issued revised guidelines that included recommending universal prenatal screening of all pregnant women between 35 and 37 weeks of gestation to determine their vaginal/rectal GBS colonization status. The CDC recommends universal antepartum GBS screening in all pregnant women, primarily with culture from vaginal-rectal swabs, to identify candidates for intrapartum prophylaxis to decrease early-onset GBS-related complications in the newborn.87 Use of vaginal-rectal swabs improves GBS isolation by 40%, compared with use of vaginal specimens alone.88 Culture requires up to 36 hours of incubation and, therefore, rapid screening using molecular test methods may alleviate unnecessary antibiotic treatment in patients with preterm labor. A rapid and sensitive intrapartum real-time PCR assay offers the advantage of ascertaining the colonization status before delivery.

Six assays are commercially available for GBS testing (Table 4), using either direct vaginal-rectal swabs or these swabs incubated in Lim broth (culture confirmation): AccuProbe Group B Streptococcus culture confirmation test (Gen-Probe Inc.), IDI-Strep B (GeneOhm Sciences, San Diego), BD MAX GBS assay (BD Diagnostics), Smart GBS (Cepheid), Xpert GBS (Cepheid), and the GBS PNA FISH (AdvanDx) assay. The BD MAX System (BD Diagnostics) is an integrated system that uses probes (Molecular Beacons), whereas the GeneXpert (Cepheid) system uses TaqMan probes. The Xpert GBS assay is the only FDA-cleared assay for use on both intrapartum and antepartum specimens. FDA-cleared molecular methods allow for increased sensitivity and a rapid turnaround time. The GeneXpert system facilitates near-point-of-care analysis in an intrapartum scenario. The BD GeneOhm StrepB and the Smart GBS PCR assays are not yet automated and require varying degrees of hands-on specimen manipulation and sample preparation. In a high-volume laboratory setting, this may not be practical. The BD MAX system allows automation of a PCR assay that is run in a batch mode in the laboratory for antepartum testing. Although PCR tests have initially been adopted selectively in facilities with sufficient demand and resources, a general recommendation for their use by the CDC and other agencies would require the capacity for effective implementation in a wide range of hospital settings. Some disadvantages of intrapartum PCR testing include delays in the administration of antibiotics, pending test results, and lack of an isolate for susceptibility testing. Susceptibility testing is of particular concern for women who are allergic to penicillin.