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Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Introduction: The ARIES® HSV 1&2 Assay is a new FDA cleared real-time PCR test for detection and differentiation of HSV-1 and HSV-2 DNA from cutaneous and mucocutaneous lesions. The test is performed on the ARIES® System, an automated sample to answer real-time PCR instrument that provides a closed system and simple workflow for performing molecular testing.Areas covered: This article reports the clinical performance of the ARIES® HSV 1&2 Assay assessed on 1963 prospectively collected specimens. Assay sensitivities were 91.1–95% (cutaneous) and 97–98.5% (mucocutaneous), and specificities were 88.8–94.2% (cutaneous) and 93.2–95.4% (mucocutaneous), as compared to the ELVIS® HSV test system.Expert commentary: Detection of HSV DNA by PCR is rapid and more sensitive than traditional culture and immunoassay methods and is being widely adopted in many laboratory settings. Sample to answer molecular platforms like ARIES® will enable routine and non-molecular labs to perform sensitive and rapid molecular t...

This multicenter study was designed to assess the accuracy and reproducibility of the Vitek MS v3.0 matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry system for identification of Mycobacterium and Nocardia species compared to DNA sequencing. A total of 963 clinical isolates representing 51 taxa were evaluated. In all, 663 isolates were correctly identified to the species level (69%), with another 231 (24%) correctly identified to the complex or group level. Fifty-five isolates (6%) could not be identified despite repeat testing. All of the tuberculous mycobacteria (45/45; 100%) and most of the nontuberculous mycobacteria (569/606; 94%) were correctly identified at least to the group or complex level. However, not all species or subspecies within the M. tuberculosis , M. abscessus , and M. avium complexes and within the M. fortuitum and M. mucogenicum groups could be differentiated. Among the 312 Nocardia isolates tested, 236 (76%) were correctly identified to the species level, with an additional 44 (14%) correctly identified to the complex level. Species within the N. nova and N. transvalensis complexes could not always be differentiated. Eleven percent of the isolates (103/963) underwent repeat testing in order to get a final result. Identification of a representative set of Mycobacterium and Nocardia species was highly reproducible, with 297 of 300 (99%) replicates correctly identified using multiple kit lots, instruments, analysts, and sites. These findings demonstrate that the system is robust and has utility for the routine identification of mycobacteria and Nocardia in clinical practice.

The accuracy and robustness of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system was evaluated by identifying mycobacteria from automated liquid-medium systems using patient samples. This is the first report to demonstrate that proteins within the liquid medium, its supplements, and decontamination reagents for nonsterile patient samples do not generate misidentification or false-positive results by use of the Vitek MS v3.0 system. Prior to testing with patient samples, a seeded-culture study was conducted to challenge the accuracy of the Vitek MS system at identifying mycobacteria from liquid medium by mimicking a clinical workflow. Seventy-seven Mycobacterium strains representing 21 species, seeded in simulated sputum, were decontaminated, inoculated into BacT/Alert MP liquid culture medium, incubated until positivity, and identified using the Vitek MS system. A total of 383 liquid cultures were tested, of which 379 (99%) were identified correctly to the species/complex/group level, 4 (1%) gave a "no-identification" result, and no misidentifications were observed. Following the simulated-sputum study, a total of 73 smear-positive liquid-medium cultures detected using BD BBL MGIT and VersaTREK Myco liquid media were identified by the Vitek MS system. Sixty-four cultures (87.7%) were correctly identified to the species/complex/group level; 7 (9.6%) resulted in no identification; and 2 (2.7%) were misidentified at the species level. These results indicate that the Vitek MS v3.0 system is an accurate tool for routine diagnostics of Mycobacterium species isolated from liquid cultures.

Objectives Infections due to Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most common bacterial sexually transmitted infections worldwide, most of which are asymptomatic. Detection of infection using a variety of specimen types in symptomatic and asymptomatic subjects is important to effectively combat CT/NG infections. The performance of the cobas CT/NG v2.0 test was assessed for urogenital swabs, urine and cervical cytology samples collected in PreservCyt Solution from 5266 symptomatic and asymptomatic women (including 202 who were pregnant), and urine from 738 men. Methods Sensitivity and specificity were estimated compared with a patient infected status determined using two US Food and Drug Administration–cleared nucleic acid amplification tests. Results Among 6004 participants, 487 CT (8.1%) and 159 NG (2.6%) infections were identified. Sensitivity estimates for CT for women ranged from 91.2% to 97.6% depending on specimen type, and the estimate for male urine specimens was 98.4%. Specificity for CT ranged from 99.2% to 99.7%. Sensitivity estimates for NG ranged from 95.6% to 100.0% for women, and the estimate for men was 100.0%. Specificity for NG ranged from 99.3% to 100.0%. Conclusions The cobas CT/NG v2.0 test performs well using urogenital swabs, urine and cervical samples collected in PreservCyt solution.

Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerella vaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated ( n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

Background Centers for Disease Control and Prevention guidelines recommend at least annual rectal screening of men who have receptive anal intercourse for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT). Only limited national data are available on the prevalence of rectal GC and CT infection among US men. Methods In collaboration with a large US commercial laboratory, we estimated positivity of the first rectal GC and CT test ("index" test) in men aged 15-60 years tested between January 2013 and May 2015. We estimated the frequency and positivity of pharyngeal or urine specimens tested for GC and CT on the index date, and the frequency and positivity of repeat rectal testing or any follow-up testing at any anatomic site after the index date. Results Of 52 063 tested men aged 15-60 years, approximately 6.1% were positive for GC only, 8.3% for CT only, and 2.7% for both GC and CT on their index date. On that date, 86.5% had either urine or pharyngeal specimens collected, and 56.1% had both specimens collected. Pharyngeal GC infection was highly associated with rectal GC infection. Follow-up testing after 12 months ranged from 42.4% among uninfected men to 56.7% among infected men on the index date. Positivity was at least 5.7% in rectal GC, rectal CT, or pharyngeal GC at their last test. Conclusions This analysis of a large number of male rectal specimens tested for GC and CT suggest that routine testing and timely repeat rectal GC and CT testing should be prioritized among men who report receptive rectal sex.

BACKGROUND: In the United States, chlamydia screening has been recommended for all pregnant women by the Centers for Disease Control and Prevention (CDC) but only for pregnant women who are at increased risk by the US Preventive Services Task Force (USPSTF). Very limited evidence, such as age-specific chlamydia positivity in pregnant women, has been used to develop these recommendations. METHODS: We analyzed data from a large commercial laboratory corporation in the United States in 2013. At the first prenatal visit made by women aged 15 to 44 years for whom a chlamydia test was performed between June 2008 and July 2010, we estimated positivity of chlamydia by age, insurance coverage, geographic region, and test type. RESULTS: Of 601,001 pregnant women aged 15 to 44 years who had routine prenatal care, 62.9% had private insurance and 32.9% had Medicaid coverage, 60.3% resided in the South region, and 43.2% were aged 15 to 24 years, 26.8% were aged 25 to 29 years, and 19.1% were aged 30 to 34 years. Chlamydia positivity was 3.6% overall, and significantly decreased as age increased (15-19 years: 9.6 %; 20-24 years: 5.2%; 25-29 years: 1.8%; 30-34 years: 0.9%; and 35-44 years: 0.6%; P < 0.05). CONCLUSIONS: Our findings of higher positivity among younger pregnant women suggest that the yield is likely to be greater from screening younger pregnant women than from screening older pregnant women to identify chlamydia infection. The benefits of harmonizing CDC and USPSTF recommendations for pregnant women could be explored by reviewing age-specific positivity data and estimating the frequency of prenatal adverse health outcomes caused by chlamydia to develop consensus regarding the age limit for pregnant women who should be screened.

Trichomonas vaginalis is the most prevalent nonviral sexually transmitted infection worldwide, and improved diagnostic methods are critical for controlling this pathogen. Diagnostic assays that can be used in conjunction with routine chlamydia/gonorrhea nucleic acid-based screening are likely to have the most impact on disease control. Here we describe the performance of the new BD T. vaginalis Q x (TVQ) amplified DNA assay, which can be performed on the automated BD Viper system. We focus on data from vaginal swab samples, since this is the specimen type routinely used for traditional trichomonas testing and the recommended specimen type for chlamydia/gonorrhea screening. Vaginal swabs were obtained from women attending sexually transmitted disease or family planning clinics at 7 sites. Patient-collected vaginal swabs were tested by the TVQ assay, and the Aptima T. vaginalis (ATV) assay was performed using clinician-collected vaginal swabs. Additional clinician-collected vaginal swabs were used for the wet mount and culture methods. Analyses included comparisons versus the patient infection status (PIS) defined by positive results with the wet mount method or culture, direct comparisons assessed with κ scores, and latent class analysis (LCA) as an unbiased estimator of test accuracy. Data from 838 women, 116 of whom were infected with T. vaginalis , were analyzed. The TVQ assay sensitivity and specificity estimates based on the PIS were 98.3% and 99.0%, respectively. The TVQ assay was similar to the ATV assay (κ = 0.938) in direct analysis. LCA estimated the TVQ sensitivity and specificity as 98.3 and 99.6%, respectively. The TVQ assay performed well using self-collected vaginal swabs, the optimal sample type, as recommended by the CDC for chlamydia/gonorrhea screening among women.

Background Chlamydia is prevalent among young persons in the United States. Infected persons have a high prevalence of infection several months later, most likely from reinfection. Retesting of all men and women with a positive test is recommended 3 months after treatment. A test-of-cure is recommended for pregnant women 3-4 weeks after treatment. Methods We analyzed 2008-2010 chlamydia testing data from a large US laboratory to estimate test positivity by patient demographic characteristics and diagnoses, and patterns of repeat testing of men and nonpregnant women with a positive test and tests-of-cures of pregnant women with a positive test. Results During the study period, 7.0% of 0.40 million tests performed in men and 4.0% of 2.92 million tests performed in women were positive. Among young women, positivity rates were highest among those aged 15-19 years, ranging from 8.5% to 10.0%. Retesting rates of persons with a positive test were suboptimal, with 22.3% of men and 38.0% of nonpregnant women retested. Although 60.1% of pregnant women with a positive test were retested, only 22.0% received a test-of-cure within the 4-week recommended time frame. Repeat tests were positive in 15.9% of men, 14.2% of nonpregnant women, and 15.4% of pregnant women. Conclusions Analyses of laboratory testing data provided important insights into chlamydia testing, retesting, and positivity among a diverse US population of men and women. Too few persons were retested as recommended, and interventions are needed to increase both healthcare provider and patient adherence to recommendations for retesting men and women with positive tests.

Quantitative PCR assays were developed for 4 organisms reported previously to be useful positive indicators for the diagnosis of bacterial vaginosis (BV)— Atopobium vaginae , Bacterial Vaginosis-Associated Bacterium 2 (BVAB-2), Gardnerella vaginalis , and Megasphaera- 1—and a single organism ( Lactobacillus crispatus ) that has been implicated as a negative indicator for BV. Vaginal samples ( n = 169), classified as positive ( n = 108) or negative ( n = 61) for BV based on a combination of the Nugent Gram stain score and Amsel clinical criteria, were analyzed for the presence and quantity of each of the marker organisms, and the results were used to construct a semiquantitative, multiplex PCR assay for BV based on detection of 3 positive indicator organisms ( A. vaginae , BVAB-2, and Megasphaera -1) and classification of samples using a combinatorial scoring system. The prototype BV PCR assay was then used to analyze the 169-member developmental sample set and, in a prospective, blinded manner, an additional 227 BV-classified vaginal samples (110 BV-positive samples and 117 BV-negative samples). The BV PCR assay demonstrated a sensitivity of 96.7% (202/209), a specificity of 92.2% (153/166), a positive predictive value of 94.0%, and a negative predictive value of 95.6%, with 21 samples (5.3%) classified as indeterminate for BV. This assay provides a reproducible and objective means of evaluating critical components of the vaginal microflora in women with signs and symptoms of vaginitis and is comparable in diagnostic accuracy to the conventional gold standard for diagnosis of BV.

Background: The Roche cobasCT/NG test (c4800), performed on the cobas 4800 system, is a new diagnostic assay using an automated workstation to isolate nucleic acids from clinical specimens and a real-time instrument for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). This study compared the perfor- mance characteristics of the c4800 with the Becton Dickinson Pro- beTec™ CT/GC Q x assay (Q x ) and Gen-ProbeAptima Combo 2 (AC2) assay for the detection of CT and NG in male urine using patient-infected-status (PIS). Methods: Urine and urethral swabs were obtained from men attend- ing STD, family planning, or OB/GYN clinics from 11 geographically distinct locations. Aliquot order was randomized for urine specimens between AC2, c4800, and Q x . Urethral swabs were randomized be- tween AC2 and Q x . Urethral swabs were only used to define PIS and were not tested on the c4800. A participant was considered infected if the 2 comparator assays with different molecular targets had positive results from either sample type. Results: A total of 790 men were screened, with 768 evaluable for CT and NG. Symptoms were reported in 296 (38.5%) participants. For urine, the overall sensitivity and specificity of the c4800 assay for CT were 97.6% and 99.5%, respectively, when compared with PIS. Sen- sitivity and specificity for NG were 100% and 99.7%, respectively. Conclusions: The c4800 has excellent sensitivity and specificity for male urine specimens when compared with PIS. Assay performance was similar in symptomatic and asymptomatic men and was equivalent to nucleic acid amplification tests that are currently on the market.

The next-generation amplification test for Chlamydia trachomatis and Neisseria gonorrhoeae (Roche cobas 4800), a fully automated system, was compared head to head, using female samples, to Gen-Probe Aptima Combo 2 and BD ProbeTec using Viper. Endocervical swabs, female urine, and endocervical samples in liquid-based cytology medium were run on at least two of three platforms. A total of 4,316 samples were evaluated, and 281 chlamydial and 69 gonococcal infections were identified. Estimates of sensitivity and specificity were obtained relative to the patient infection standard (PIS) and using latent class analysis (LCA). Chlamydia sensitivity estimates ranged from 86.9 to 95.6% using PIS and 97.6 to 98% using LCA. Specificity was ≥99.6% for all sample types. Sensitivity ranged from 95.6 to 100% using PIS and 96.9 to 100% using LCA for the detection of gonococcal infections. Specificity for gonococcal infections was ≥99.8%. cobas 4800 performance was equivalent to the comparator assays (all P values, >0.05), and the fully automated system provides high laboratory efficiency.

Objective We evaluated the performance characteristics of APTIMA Trichomonas vaginalis (ATV) transcription-mediated amplification (TMA) for diagnosis of T vaginalis (TV) infection from female vaginal swab, endocervical swab, and urine specimens and from male urethral swab and urine specimens. Performance of ATV TMA was compared with wet mount microscopy, culture, and polymerase chain reaction (PCR). Study Design In all, 296 female and 298 male subjects who attended the Jefferson County Health Department sexually transmitted diseases clinic were enrolled in the study and provided specimens for each test. Results were analyzed using 3 interpretative algorithms. Results For women, vaginal swab ATV TMA was significantly more sensitive than wet mount or culture. In male subjects, urethral swab ATV TMA was significantly more sensitive than culture or PCR. Conclusion ATV TMA provides a sensitive, commercially available nucleic acid amplification test for improved diagnosis of TV in male and female patients.

Systemic fungal diseases are primary pulmonary diseases caused by the dimorphic fungal pathogens, Blastomyces dermatitides, Coccidioides immitis, Histoplasma capsulatum, or Paracoccidioides brasiliensis. Infection occurs after inhalation of the infectious form of the fungus and may be acute, self-limited, or subclinical. Primary cutaneous infection occurs only after traumatic implantation of the fungus and is unusual. Erythema nodosum or erythema multiforme may accompany the acute form of the disease. Other cutaneous manifestations represent disseminated disease and, as such, require systemic antifungal therapy. Because cutaneous lesions have occurred coincidentally with other cutaneous pathologies, emphasis should be placed on a complete clinical history, physical examination, and diagnosis by histopathology and culture.

Gen-Probe culture confirmation tests (Gen-Probe, San Diego, CA) for Mycobacterium tuberculosis complex and Mycobacterium avium complex were performed on 276 mycobacterial isolates. All 138 M. tuberculosis complex isolates and 79 of 80 M. avium complex isolates were identified correctly. No falsely positive test results were obtained; 58 nontuberculous mycobacteria other than M. avium complex were negative by Gen-Probe. In a second phase of testing, Gen-Probe tests were performed using concentrates from 101 patient Bactec 12B cultures. Positive results by Gen-Probe tests were correlated with the growth index (GI) reading on the day of processing as well as the accumulated GI readings. For those 51 with high (greater than or equal to 999) final GIs, 40/40 (100%) M. tuberculosis complex isolates and 9/11 M. avium complex isolates were positive by Gen-Probe, and six other mycobacteria were negative. Of the 25 with moderate final readings (400 less than or equal to GI less than 999), 12/17 M. tuberculosis complex isolates and 1/1 M. avium complex isolates were correctly identified by Gen-Probe; seven other mycobacteria were negative. Of 25 with low readings (GI less than 400), 8/24 M. tuberculosis isolates were correctly identified by Gen-Probe, and no falsely positive test results were obtained with the other probes. All true negative tests on seven other mycobacteria (not M. tuberculosis complex or M. avium complex) had less than 2% hybridization. Of the 24 falsely negative tests on M. tuberculosis complex isolates or M. avium complex isolates, 22 had greater than 2% hybridization with their respective probes. Thus, percent hybridization greater than 2% may be a useful indicator of the need for retesting.

The clinical and pathologic features of allergic fungal sinusitis caused by Curvularia lunata, as seen in two patients, are described. The findings are identical to those of allergic aspergillus sinusitis. Patients have allergies, nasal polyposis, and, occasionally, eosinophilia. Radiographs show opacification of multiple sinuses without bone destruction. Surgical specimens consist of polyps and inspissated, mucoid material. Diagnostic microscopic features, termed "allergic mucin," include eosinophils, numerous Charcot-Leyden crystals, and hyphae embedded in pools of mucus. The recognition of allergic mucin may be impeded by extensive degranulation and fragmentation of the eosinophils. In addition, the fragments of mucin exhibit large, poorly stained central areas, probably due to incomplete penetration by the fixative. Eosinophils are easier to recognize in well-fixed areas. Electron microscopy, though not of diagnostic necessity, confirms the eosinophilic nature of the infiltrate. It is important that surgical pathologists recognize this distinctive clinicopathologic entity and recommend appropriate cultures.

A unique patient with chronic lymphocytic leukemia died with pneumonia caused by both Cunninghamella bertholletiae and Pneumocystis carinii . In tissue sections, the hyphase of C bertholletiae were twisted and ribbon-like, but were smaller than those typical for zygomycetes, displayed more than occasional septa, and exhibited Y branching, making histologic distinction from Aspergillus sp difficult.