Molecular assays have excellent performance char- acteristics and are usually highly sensitive and specific.
Occasionally, however, false-positive test results can occur. Laboratories should be aware of several impor- tant reasons why false-positive results occur.
Laboratories should be routinely monitoring for false- positive results by several methods so that if unex- pected results occur, they can be identified and corrected in a timely manner, before larger problems occur. Monitoring for false-positive results should include verification of primer and probe sequences, monitoring and verifying assay conditions, and the use of negative controls in their molecular assays. Some of the more common causes of false-positive test results are discussed next, with suggestions for strategies to prevent and control them.
Contamination
In general, molecular diagnostic methods, especially infectious disease assays, require very high analytical 335
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sensitivity. Because the amount of microbial DNA or RNA present in a clinical specimen may be very low, an amplification method is used to optimize sensitiv- ity. Although there are some nucleic acid detection tests based on direct hybridization, most nucleic acid detection methods now rely on some form of nucleic acid amplification, such as PCR. Theoretically, nucleic acid amplification methods have the capability to detect a single molecule of analyte, and many assays do approach this level of sensitivity, with some assays capable of detecting as few as 5 10 molecules of target analyte. However, the extremely high analytical sensi- tivity of amplification methods also increases the risk of false-positive test results due to the presence of con- taminating DNA in the reaction, where even a single molecule of DNA can serve as a template for amplifi- cation in a later reaction. Contamination of samples is a major consideration for laboratories performing amplification reactions, and procedures must be in place to prevent, monitor, and control contamination.
A false-positive PCR result can have serious implica- tions; there are numerous reports of false-positive results in the literature, many of which have significant consequences [48]. The molecular diagnostics labora- tory must ensure that methods are in place to prevent, detect, monitor, and correct for contamination events.
Sources of Contamination
Contaminating DNA may be introduced into reac- tions from a number of different sources:
Amplicon DNA: An important source of false- positive results in nucleic acid amplification testing is amplified DNA from a previous amplification reaction. If reaction tubes are opened after amplification (e.g., for gel analysis, microarray testing, and sequencing), amplified DNA can be aerosolized, contaminating the environment, and it can be carried directly into reagents and
consumables. This contaminating DNA can then be carried directly into new reactions the next time assays are set up, causing false-positive results.
Specimens: Nucleic acids from specimens may also contaminate the environment during specimen handling, for example, when pipetting specimens into tubes for nucleic acid extraction procedures.
Cross-contamination of samples containing the analyte prior to or during sample processing can lead to false-positive results. When multiple samples are processed in batches, there is potential for aerosolization, splashing, etc.
Positive control material: Incorrect handling of positive controls (e.g., plasmid DNA) may also result in contamination of patient samples, leading to false-positive results. The second potential source
of contamination is amplified material from previous reactions. If tubes are opened for post- amplification analysis (e.g., gel electrophoresis and microarray testing), amplicons can contaminate the environment and be carried over into the next reactions, causing false-positive results.
Contaminating nucleic acids can be present in the laboratory environment (on clothing, benchtop, waste containers, etc.). DNA contamination may occur in reagents, enzyme mixes, master mixes, etc.
Methods to Prevent and Control Contamination Laboratory practices to prevent contamination in the molecular diagnostics laboratory include both physical design and layout of the laboratory, workflow practices, and policies and procedures designed to pre- vent contamination. Many references are available for designing and implementing contamination control programs in the molecular diagnostic laboratory[49]:
1. Laboratory design: Optimally, a molecular diagnostic laboratory should be designed so that separate work areas for different parts of a procedure are used. Although separate rooms are optimal (e.g., specimen processing room, PCR master mix room, amplification room, and post-PCR DNA handling room), separate work areas within a large space can function to prevent contamination.
Limiting traffic in the molecular laboratory can help prevent contamination.
2. Work practices and workflow: Workflow practices should be implemented to ensure a one-way workflow from clean to amplified area. Specimens are extracted in one area and then amplified in a different area. PCR products or other amplified materials are not carried into the specimen handling or pre-amplification areas. Work practices that should be implemented include use of dedicated pipettors, use of dedicated packages of consumables such as tips and tubes, and use of dedicated lab coats. Laboratory policies and procedures should include specific methods for contamination prevention. Cleaning the work areas should be performed prior to and after every procedure.
Cleaning agents specifically designed to destroy nucleic acids should be used.
3. DNA degrading enzymes such as uracil DNA- glycosylase (UNG) can be used. Uracil is incorporated in the PCR master mix; during amplification reactions, uracil is incorporated into the amplified DNA. UNG is added to the new PCR reaction tubes; if any DNA is carried over from the amplification into the new PCR reaction tubes, the UNG will degrade the contaminating DNA prior to the start of the new PCR reaction. This method is
utilized in some commercially available PCR diagnostic assays and is quite beneficial in preventing carryover contamination.
4. Converting end-point assays to real-time assays that do not require manipulation after the amplification is an excellent method for preventing problems with environmental contamination. Much of the risk for contaminating the environment with target DNA comes from opening amplification tubes after reactions are complete. Splashing, spilling, and aerosol formation can all be avoided when tubes for real-time assays are discarded unopened.
Methods for Detection and Monitoring of Contamination
Laboratories can include in their quality assurance programs methods to ensure that any contamination issues are promptly detected and corrected:
No-template or negative controls: A no-template reaction should be included in every batch of amplified reactions. Typically, the laboratory will set up a no-template control using water or buffer without DNA. The control reaction should be handled exactly the way specimens are handled;
that is, it should be processed through the extraction and purification and then assayed.
Positive results in a negative control reaction indicate possible contamination. The laboratory should have a procedure in place to follow up and investigate a positive result in water or other negative controls.
Environmental testing for contamination: For laboratories that are at risk of contamination (e.g., when high-volume testing with open reaction conditions) or that have experienced contamination, routine environmental monitoring studies (wipe testing) can be implemented. There are no
standardized procedures for environmental testing;
the laboratory must establish its own policies and procedures for doing this type of monitoring.
Examples include using swab testing of the
environment and then processing the swab through the extraction and testing procedure, handling it as if it were a specimen. Any positive results should trigger a review of policies and procedures and implementation of cleaning and decontamination.
Environmental contamination can be difficult to control if the environment has become
contaminated with amplified DNA.
Monitoring assay positivity rates: Monitoring assay positivity rates can be a useful method for
determining if a problem is occurring with an assay.
Monitoring positivity rates is quite useful for tests such asN. gonorrhoeaeorChlamydia trachomatisPCR.
An unusual increase in the number of positive tests may indicate contamination has occurred.
Monitoring can be done as part of the laboratory’s quality assurance program.
Primer or Probe Cross-Reactivity Resulting in Nonspecific Amplification or Hybridization:
Assay Specificity
False-positive PCR results in molecular testing can occur when primers or probes exhibit cross-reactivity and nonspecifically bind to sequences that are present in the specimen or when specific sequences are present in unrelated organisms that are not pathogens.
Mispriming
Hybridization of primers to nontarget sequences (mispriming) can occur under several conditions.
Primers may bind to identical sequences present in nucleic acids that are not the specific targets of the assay. For laboratory-developed tests, primer sequences should be checked by searching against a database such as GenBank to verify that they will not cross-react to other organisms. Mispriming can also occur when assay conditions are not optimized (annealing and extension temperatures, composition of amplification mix, ion concentration, etc.).
Cross-Reactivity in Primer or Probe Binding Regions
Nucleotide sequence homology in primer or probe binding regions can give false-positive results.
Hybridization probes used to detect amplified product may exhibit cross-reactivity to closely related species.
For amplification assays that use labeled probes for detection of product, probe sequences should be checked for potential cross-reactivity to related species or genes, including pseudogenes if DNA is targeted.
False-positive results can have significant negative con- sequences. For assays that use hybridization probes (e.g., ribosomal RNA probes), if nonspecific hybridiza- tion is occurring, assay parameters should be checked.
Incorrect temperatures during the hybridization of the labeled probe are a common reason for nonspecific hybridization.
CASE REPORT: FALSE-POSITIVE REPORT OF TROPHERYMA WHIPPLEI IN CSF AND INTESTINAL BIOPSY SAMPLES DUE TO HOMOLOGY IN PRIMER BINDING REGIONS A 13-year-old male presented to the emergency room with a 2-week history of central ner- vous system symptoms. Multiple tests were performed;
a PCR-based assay forTropheryma whippleiperformed on a CSF specimen was positive. The primers used in the 337
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PCR were targeted to aT. whipplei-specific portion of the 16 S rRNA gene; product detection was by melting curve analysis and agarose gel electrophoresis.
Clinically, the diagnosis of Whipple’s disease was not consistent with the results of the PCR, and follow-up investigation was pursued. PCR testing with a differ- ent primer set was performed and was negative. The amplicons from the original PCR were sequenced.
Sequencing results revealed that the amplified mate- rial was a human gene; the original PCR test was a false positive due to primer cross-reactivity [50].
Ultimately, the child was diagnosed with chronic lym- phocytic meningitis of unknown origin.
Examples of Known Problems with Cross- Reactivity in Molecular Assays
AMPLIFICATION OF NONPATHOGENICNEISSERIA SPECIES IN PCR: FALSE-POSITIVE RESULTS FOR NEISSERIA GONORRHOEAE
Several different assays using molecular methods are available for detection of N. gonorrhoeae (GC) in urine or urogenital sites. Commercially available tests include PCR, SDA, and TMA. The molecular targets for probes and primers used in these assays varies [51];
these genes or sequences may be present in the closely related, nonpathogenic species such as N. cinerea and N. lactamica. These nonpathogenic Neisseriaspecies are common colonizers of the oropharyngeal cavity, rectal, and urogenital sites. False-positive results (due to the presence of identical DNA sequences in nonpathogenic Neisseriaspecies) can occur with some of these assays [52]. There are several recommendations for avoiding the possibility of a false-positive report of GC. Testing of specimens collected from extragenital sites such as throat or rectal specimens should be performed with culture rather than using molecular amplification assays. Laboratories should consider performing some form of confirmatory testing for specimens that test positive with molecular assays for GC and Chlamydia trachomatis(CT). Ideally, a different method or different molecular target would be used. Repeat testing of posi- tive specimens with the same method is often used for practical reasons and can be cost-effective. Laboratories performing molecular testing for GC and CT should monitor their positivity rates as part of their quality management program for molecular diagnostics. A sig- nificant increase in the positivity rate can trigger an investigation into the potential for contamination or an investigation of the performance of the assay. Screening guidelines can also help; ensuring that testing is per- formed for only those patients for whom screening is recommended will help to ensure that the positive pre- dictive value of the test is adequate.
FALSE-POSITIVE PCR RESULTS FOR METICILLIN- RESISTANTSTAPHYLOCOCCUS AUREUS
Surveillance testing for meticillin-resistant Staphylococcus aureus(MRSA) is routinely performed in many health care settings. Culture-based methods have been shown to be slightly less sensitive than PCR assays, and many laboratories have begun using PCR- based assays for detection of MRSA in screening pro- grams. Several commercially available assays are in use, most of which are based on the detection of the staphylococcal cassette chromosome mec (SCCmec) ele- ment. Resistance to meticillin is due to an altered peni- cillin binding protein, PBP2a, which is encoded by the mecA gene. The mecA gene is located on a mobile genetic element, called the SCCmec, which was origi- nally acquired from a non-S. aureus species. The SCCmec is integrated into an S. aureus-specific gene called orfX. Using primers directed to the orfX/mecA junction region allows for simultaneous identification ofS. aureus and mecA. Certain strains ofS. aureuscon- tain deletions in a portion of the mecA cassette; func- tionally, these isolates do not express mecA and are thus meticillin susceptible in phenotypic assays. These strains are phenotypically not meticillin resistant, but they give positive results in PCR assays that are designed to target the orfX/mecA region. The inci- dence of false-positive MRSA reports varies and is most likely regional. Laboratories that perform both culture- and PCR-based methods for MRSA should investigate the possibility of mecA mutants.
Laboratories may wish to monitor their positive PCR surveillance screens, monitor the rate of discordance between the PCR assay results and phenotypic results, and follow up on discordant results to determine if such strains are present in their population.