The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

We compared the performances of the Third Wave Technology Invader method and the Digene Hybrid Capture 2 assay to detect high-risk human papillomaviruses in 87 cervical brushing specimens submitted in Cytyc ThinPrep media. Two different methods for the extraction of DNA from squamous epithelial cells were also evaluated.The human papillomavirus (HPV) family consists of more than 100 types. Those shown to be causative agents of cervical cancer are referred to as "high-risk" strains. Current guidelines recommend the testing of all women with a cytological diagnosis of atypical squamous cells of undetermined significance (ASCUS) for infection by high-risk types of HPV (16,23). HPV testing has been found to have a higher sensitivity than standard or liquid-based Pap testing for detecting high-grade cervical intraepithelial neoplasia (1,(17)(18)(19)(20). Only the Digene Hybrid Capture 2 (HC2) assay (Digene, Gaithersburg, MD) has been approved by the FDA for the detection of high-risk strains of HPV as an ASCUS triage for referral to colposcopy, and, in conjunction with cervical cytology, as a primary screen for cervical cancer. The purpose of this study was to compare a laboratory-developed test utilizing analyte-specific reagents (ASRs) developed by Third Wave Technologies Inc. (TWT, Madison, WI) with the Digene HC2 assay for the detection of high-risk HPV DNA in cervical brushings. The TWT method used in this study, based on proprietary Invader technology, employed isothermal signal amplification to detect 13 high-risk HPV types, utilizing three probe pools (A5/A6 [types 51 and 56], A7 [types 18, 39, 45, 59, and 68], and A9 [types 16, 31, 33, 35, 52, and 58]) based on phylogenetic relatedness. The assay also incorporated an internal control for human alpha-actin to control for DNA quality and quantity in each reaction.The 87 specimens used in this study were cervical brushings collected in ThinPrep fluid (Cytyc Corp., Marlborough, MA) submitted to the cytology laboratory at CompuNet Clinical Laboratories for routine Pap testing. Those samples that received a cytological diagnosis of ASCUS were tested for highrisk HPV by using the Digene HC2 high-risk HPV test according to the manufacturer's instructions. Cells were collected from 2 ml of ThinPrep fluid by centrifugation at 14,000 ϫ g for 10 min and suspended in 0.4 ml of phosphate-buffered saline.DNA was extracted using the Qiagen BioRobot M48 and the MagAttract virus DNA minikit (Qiagen Inc., Valencia, CA) and eluted into 100 l of RNase-free distilled water. Duplicate samples were prepared from 2 ml of each cytology sample by using Gentra Systems Puregene spin columns (Qiagen Inc., Valencia, CA) by following the manufacturer's instructions. All extracted samples were stored at Ϫ20°C until tested. The extracted DNA was tested for the presence of high-risk HPV by a laboratory-developed assay utilizing Invader HPV ASRs. The TWT ASRs consisted of three separate oligonucleotide pools with 6-carboxyfluorescein (FAM)-labeled probes, one Redmond red (RED)-labeled oligonu...

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Plasma human immunodeficiency virus RNA and CD4 lymphocyte response to nucleoside reverse-transcriptase therapy were evaluated in a large, comparative pediatric trial. Both baseline values and changes in the two laboratory markers over time correlated well with clinical outcome and possessed independent predictive value. In comparison of RNA reduction from baseline between the dideoxyinosine (ddI) and zidovudine+ddI therapeutic arms, marginal superiority of the combination arm was not correlated with an observed clinical benefit. Despite the size of this trial and the significantly higher rate of clinical end points in the zidovudine monotherapy group, attempts to establish surrogacy for plasma RNA were difficult. Nevertheless, plasma RNA and CD4 lymphocyte count together possess strong clinical predictive power and are valuable tools for both the clinician and the evaluation of new therapies.

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In November 2001, United States citizens became acutely aware of the potential for bioterrorism within our borders. With the introduction of anthrax bacilli (Bacillus anthracis) into the U.S. Postal Service system, the threat of other forms of bioterrorism became a reality. Biological terrorism, or bioterrorism, involves the use or the threat of using diseasespreading microorganisms and/or toxins as weapons of mass destruction. The use of B. anthracis as a weapon of terrorism has resulted in a heightened review of other potential bioterrorism agents. Smallpox virus (variola virus) is considered to pose the greatest risk.In 1980 the World Health Organization Smallpox Eradication Program declared that the disease, and the need for a vaccine with possible adverse reactions, had been eliminated (1-3). However, stocks of variola virus were "officially" maintained in the United States and in the Soviet Union for experimental purposes (6). The dissolution of the Soviet Union led to concerns regarding safety control of a number of State properties, including stocks of variola virus. Other unknown illicit smallpox virus sources may exist, and these may serve as a potential for bioterrorism threats.Most adults 35 years old and older were vaccinated for smallpox at least once prior to entering school. An unknown parameter of this smallpox vaccination, however, is the duration of its protection, as determined by the presence of antibody. It is generally accepted that the original vaccination would be protective for about 5 to 10 years, although the World Health Organization Committee on International Quarantine suggested that international travelers be vaccinated within the 3 years prior to travel (5). Multiple vaccinations provide long-term protection (4); however, the duration of antibody from the single original smallpox vaccination has not been recently evaluated. Do adults who received a single vaccination as a child still have any residual antibody? Many individuals are concerned with regard to protection against smallpox infection and specifically with whether they themselves have antibodies. Each serum donor was questioned regarding the number of times he or she was vaccinated.In this study, the presence of humoral antibody in vaccinated and unvaccinated (control) individuals was investigated. Those who had received additional booster vaccinations were noted, and their results were reported separately from those for individuals who had received a single vaccination. As it is not known whether antibody detected by enzyme immunoassay (EIA) is protective, it was of interest to determine if another antibody test would yield similar results. Accordingly, for comparison, 60 of the EIA-tested sera were also examined by serum neutralization (SN). MATERIALS AND METHODSTwo hundred four sera from adults of various ages, but at least 35 years of age, were submitted to the ESOTERIX Infectious Disease Center laboratory by their physicians for the detection of vaccinia virus antibody. All claimed to have been vaccinated as childre...

We have developed a rapid, pseudohomogeneous assay for the detection of PCR amplicons, based on the use of electrochemiluminescence generated from a Tris-bipyridine ruthenium(II) label. PCR amplification of highly conserved human immunodeficiency virus type 1 (HIV-1) gag gene sequences was performed with SK38 and SK39 primers, the latter of which was 5 biotinylated. Post-PCR reaction mixtures were combined with 10 12 copies of the SK19 probe-Tris-bipyridine ruthenium(II) conjugate, denatured by heating at 100 C for 5 min, and hybridized at 55 C for an additional 15 min. Hybridization to the biotinylated strand of the amplified DNA was determined by the addition of streptavidin-conjugated magnetic particles and analyzed by using an Origen-1 electrochemiluminescence analyzer. Our results demonstrated a sensitivity of fewer than five copies of HIV-1 (pre-PCR), by using either purified plasmid DNA containing one complete copy of the HIV-1 cDNA genome or lysed, proteinase K-treated 8E5 cells as the starting material. In an evaluation of actual clinical specimens (peripheral blood monocytes from both healthy and HIV-1-infected children), the electrochemiluminescent detection assay correlated 100% with both our standard method (solution hybridization with a radiolabeled probe followed by polyacrylamide gel electrophoresis [PAGE] and autoradiography) and a commercial method (Roche Amplicor). The electrochemiluminescent method was substantially easier to perform than either the PAGE or microtiter plate assays and was considerable faster to perform than either of these alternative formats.

The coronavirus disease 2019 (COVID‐19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) diagnostics. However, emerging variants pose the risk for target dropout and false‐negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS‐CoV‐2 Panel combines reverse‐transcription polymerase chain reaction and matrix‐assisted laser desorption/ionization time‐of‐flight mass‐spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS‐CoV‐2‐positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS‐CoV‐2 variants.