Multi-assay investigation of viral etiology in pediatric central nervous system infections.

INTRODUCTION: In an attempt to identify a wide spectrum of viral infections, cerebrospinal fluid (CSF) specimens were collected from pediatric cases with the preliminary diagnosis of viral encephalitis/meningoencephalitis in two reference hospitals, from October 2011 to December 2015. METHODOLOGY: A combination of nucleic acid-based assays, including in house generic polymerase chain reaction (PCR) assays for enteroviruses, flaviviruses and phleboviruses, a commercial real-time PCR assay for herpesviruses and a commercial real time multiplex PCR, enabling detection of frequently-observed viral, bacterial and fungal agents were employed for screening. RESULTS: The microbial agent could be characterized in 10 (10%) of the 100 specimens. Viral etiology could be demonstrated in 7 (70%) specimens, which comprises Human Herpesvirus 6 (4/7), Herpes Simplex virus type1 (2/7) and Enteroviruses (1/7). In 3 specimens (30%), Streptococcus pneumoniae, Listeria monocytogenes and Staphylococcus aureus were detected via the multiplex PCR, which were also isolated in bacteriological media. All specimens with detectable viral nucleic acids, as well as unreactive specimens via nucleic acid testing remained negative in bacteriological cultures. CONCLUSIONS: Herpes and enteroviruses were identified as the primary causative agents of central nervous system infections in children. Enterovirus testing must be included in the diagnostic work-up of relevant cases.

Molecular markers for the classification of cytologically indeterminate thyroid nodules.

BACKGROUND: The diagnosis of indeterminate lesions of the thyroid is a challenge in cytopathology practice. Indeed, up to 30% of cases lack the morphological features needed to provide definitive classification. Molecular tests have been developed to assist in the diagnosis of these indeterminate cases. The first studies dealing with the preoperative molecular evaluation of FNA samples focused on the analysis of BRAFV600E or on the combined evaluation of two or three genetic alterations. The sensitivity of molecular testing was then improved through the introduction of gene panels, which became available for clinical use in the late 2000s. Two different categories of molecular tests have been developed, the 'rule-out' methods, which aim to reduce the avoidable treatment of benign nodules, and the 'rule-in' tests that have the purpose to optimize surgical management. The genetic evaluation of indeterminate thyroid nodules is predicted to improve patient care, particularly if molecular tests are used appropriately and with the awareness of their advantages and weaknesses. The main disadvantage of these tests is the cost, which makes them rarely used in Europe. To overcome this limitation, customized panels have been set up, which are able to detect the most frequent genetic alterations of thyroid cancer. CONCLUSIONS: In the present review, the most recent available versions of commercial molecular tests and of custom, non-commercial panels are described. Their characteristics and accuracy in the differential diagnosis of indeterminate nodules, namely Bethesda classes III (Atypical follicular lesion of undetermined significance, AUS/FLUS) and IV (Suspicious for follicular neoplasm, FN/SFN) are fully analyzed and discussed.

The Mono-Prep system increases the detection rate of sputum smear microscopy for diagnosing tuberculosis.

OBJECTIVE: Direct sputum smear microscopy (DSSM) has a low detection rate. This study investigated whether an alternative method called Mono-Prep smear microscopy (MPSM) can enhance the diagnosis of tuberculosis in tuberculosis laboratories that perform direct smear microscopy in China. METHODS: A total of 117 sputum samples were collected from outpatients who attended Beijing Chest Hospital. DSSM, MPSM, solid culture, and Xpert MTB/RIF were performed on the samples. RESULTS: The positive rates of DSSM, MPSM, solid culture, and Xpert MTB/RIF were 27.4% (32/117), 40.2% (47/117), 35.9% (42/117), and 52.1% (61/117), respectively. MPSM could detect 15 more cases of tuberculosis compared with DSSM (47 vs 32) among 117 sputum samples. This represented a significantly higher positive rate and sensitivity of MPSM compared with DSSM. However, MPSM appeared to have a lower specificity (81.3%) compared with DSSM (90.7%) with the solid culture used as a standard. CONCLUSION: Use of MPSM can increase the number of positive sputum samples, but it still needs improvement.

Usage trends and performance characteristics of a "gene expression classifier" in the management of thyroid nodules: An institutional experience.

BACKGROUND: The gene expression classifier (GEC; Afirma-Veracyte) has proven to be an effective triage modality in the management of thyroid nodules. We evaluate our institutional experience with GEC, specifically examining performance as a first line testing strategy versus in conjunction with repeat fine needle aspiration (FNA), usage trends based on clinical setting, and performance related to diagnostic categories of The Bethesda System for Reporting Thyroid Cytology (TBSRTC). METHODS: All nodules undergoing GEC analysis from 1/2011 to 12/2015 at the Hospital of the University of Pennsylvania were identified using electronic database search methods. Corresponding cytologic diagnoses, GEC results, origin of the sample (in-house vs. satellite site), number and diagnosis of prior FNA's, and clinical and histologic follow-up were collected. RESULTS: The cohort included 294 nodules. Of these, 145 (49%) were classified as benign, 136 (46%) as suspicious, and 13 (5%) as quantity insufficient by GEC. Surgical resection was performed in 130 (130/294-44%) cases (107, 82% "suspicious" by GEC); final histopathologic diagnosis was benign in 85 (65%) and malignant in 45 (35%) cases. Three false negative diagnoses were identified in the setting of GEC analysis as a first line testing strategy. Most cases with GEC as a first line testing strategy came from satellite clinical sites (112, 66%). CONCLUSIONS: The GEC showed improved performance characteristics when coupled with a repeat FNA. It continues to be of low specificity and positive predictive value in oncocytic follicular lesions. Diagn. Cytopathol. 2016;44:867-873. © 2016 Wiley Periodicals, Inc.

Diagnosing emerging and reemerging infectious diseases: the pivotal role of the pathologist.

CONTEXT: Molecular diagnostics continues to evolve very rapidly, and its impact in the diagnosis of infectious diseases is undeniable. Molecular tools have played a pivotal role in discovering and characterizing several emerging infectious agents and have now become the gold standard for the diagnosis of infectious diseases caused by fastidious or uncultivable agents. Multiple challenges still remain for the widespread use of cost-effective, validated, and commercially available molecular tools. Automated instruments capable of sample processing and multiplex nucleic acid amplification and postamplification analysis have already been approved by the US Food and Drug Administration (FDA) for use in the clinical setting. Nanobiotechnology is beginning to impact laboratory diagnostics in the clinical setting. OBJECTIVE: To address current nucleic acid techniques used in the clinical laboratory for diagnosis of infectious diseases. FDA-approved tests are listed, as well as molecular techniques (amplification and postamplification analysis). A comprehensive list of emerging pathogens during the last 4 decades is also presented. Biosurveillance systems are discussed in the context of molecular tools. The rapidly evolving field of nanobiotechnology is briefly addressed. DATA SOURCES: Original publications, major reviews, and book chapters were used to present a comprehensive, yet short, review of molecular diagnostics in infectious diseases. CONCLUSIONS: We will continue to witness an exponential growth of molecular techniques used for the initial diagnosis of infectious diseases. Molecular tools will also continue to have an impact on disease prognosis and response to therapeutic interventions. Automation, multiplexing, and miniaturization will continue to be driving forces in the development of new instruments.

A simple method to combine multiple molecular biomarkers for dichotomous diagnostic classification.

BACKGROUND: In spite of the recognized diagnostic potential of biomarkers, the quest for squelching noise and wringing in information from a given set of biomarkers continues. Here, we suggest a statistical algorithm that--assuming each molecular biomarker to be a diagnostic test--enriches the diagnostic performance of an optimized set of independent biomarkers employing established statistical techniques. We validated the proposed algorithm using several simulation datasets in addition to four publicly available real datasets that compared i) subjects having cancer with those without; ii) subjects with two different cancers; iii) subjects with two different types of one cancer; and iv) subjects with same cancer resulting in differential time to metastasis. RESULTS: Our algorithm comprises of three steps: estimating the area under the receiver operating characteristic curve for each biomarker, identifying a subset of biomarkers using linear regression and combining the chosen biomarkers using linear discriminant function analysis. Combining these established statistical methods that are available in most statistical packages, we observed that the diagnostic accuracy of our approach was 100%, 99.94%, 96.67% and 93.92% for the real datasets used in the study. These estimates were comparable to or better than the ones previously reported using alternative methods. In a synthetic dataset, we also observed that all the biomarkers chosen by our algorithm were indeed truly differentially expressed. CONCLUSION: The proposed algorithm can be used for accurate diagnosis in the setting of dichotomous classification of disease states.

Genetic testing coverage and reimbursement: a provider's dilemma.

The rapid growth of new molecular genetic tests stimulated by the diagnostic potential of DNA/RNA analyses has resulted in the capability of molecular genetic assay technology outpacing the American Medical Association Current Procedural Terminology (CPT) codes and Medicare reimbursement. The AMA CPT Editorial Panel is poised to change the way we report genetic testing, and this change may have the potential to stimulate a governmental review of how Medicare is paying for diagnostic genetic testing. Genetic assays are costly, and those in laboratory management need to be aware of potential changes that may influence the ability of their laboratory to provide access to genetic testing services for their physician clientele. The commercialization of genetic testing has resulted in a proliferation of commercial laboratories and university medical center laboratories, CLIA-certified to perform high complexity testing, offering some level of genetic testing. The genetic tests are offered as home brew (in-house developed) assays, most of which are using analyte-specific reagents. Because these are home brew assays, there is no standardization in how the industry tests for specific mutations. As these genetic assays are billed using the generic molecular diagnostic codes, 83890 through 83912, from the Pathology and Laboratory Chemistry subsection of the CPT, payers are not able to identify the specific mutations being tested and make payment determinations based on the mutations as they relate to the diagnosis code. This article discusses the history of molecular diagnostic coding and related reimbursement, current coverage issues, and the genetic coding proposal under consideration by the AMA CPT Editorial Panel.