Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2. Fever is a symptom common to a wide variety of infectious diseases, including some of the leading causes of death in subSaharan Africa (SSA). Many etiologic studies have been performed for respiratory infections, diarrheal illness, and meningitis (1, 2). However, the incidence and etiology of undifferentiated fever are less clear (3). Most research has examined individual agents such as Plasmodium, Salmonella, and specific zoonotic or arboviral pathogens (4-6) by utilizing blood culture (7) or a complex mixture of rapid, serologic, culture, and molecular assays and algorithms to determine an etiologic agent (8).We describe our initial development and validation of a TaqMan array card (TAC) that uses quantitative reverse transcription-PCR (qRT-PCR) for the simultaneous detection of 15 viruses, 8 bacteria, and 3 protozoa of particular relevance to SSA (5, 9-13), with the intended use for outbreak investigation and acute febrile illness (AFI) surveillance. Previous TAC assays have been developed for respiratory diseases, enteric diseases, and etiologies of neonatal sepsis (14-16), and we have shown their robust and comparable performance across several countries (17). Once developed, TaqMan array cards are stable at 4°C for 2 years, can be shipped at ambient temperature, and minimize several cumbersome steps in the field, such that they are as easy to perform as individual quantitative PCR (qPCR) assays.This work was primarily a development exercise since clinical

Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first-and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.

The current recommendation for the laboratory confirmation of Lyme disease is serology-based diagnostics. Specifically, a standardized two-tiered testing (STTT) algorithm is applied that utilizes a first-tier immunofluorescence assay or enzyme immunoassay (EIA) that, if the result is positive or equivocal, is followed by second-tier immunoblotting. Despite the standardization and performance achievements, STTT is considered technically complex and subjective, as well as insensitive for early acute infection. These issues have prompted development of novel algorithms and testing platforms. In this study, we evaluated the performance of several commonly used assays for STTT. Several modified two-tiered testing (MTTT) algorithms, including a 2-EIA algorithm and modified criteria for second-tier IgG immunoblots, were also evaluated. All tests were performed on sera from a recently available, well-defined archive of positive-and negative-control patients. Our study demonstrates differences in the results between individual first-and second-tier tests, although the overall agreement of the different STTT algorithms used was strong. In addition, the MTTT algorithm utilizing 2-EIAs was found to be equivalent to all STTT algorithms tested, with agreement ranging from 94 to 97%. The 2-EIA MTTT algorithm slightly enhanced sensitivity in early disease compared to the STTT algorithms evaluated. Furthermore, these data add to the mounting evidence that a 2-EIA-based MTTT algorithm, where immunoblotting is replaced by the C6 EIA, performs as well or better than STTT. Serologic testing has been the mainstay of laboratory diagnostics for Lyme disease for over 20 years. To date in the United States, serologic tests are the only U.S. Food and Drug Administration (FDA) 510(k)-cleared diagnostic tests for Lyme disease (1). Standardization of serologic testing for Lyme disease occurred in 1994, when attendees of the Second National Conference on Serologic Diagnosis of Lyme Disease (Dearborn, MI) determined that a two-tiered algorithm, optimized for specificity and the highest sensitivity, was most appropriate for Lyme disease laboratory diagnosis (2). The standard two-tiered testing (STTT) algorithm utilizes a first-tier immunofluorescence assay (IFA) or enzyme immunoassay (EIA) that, if the result is positive or equivocal, is followed by a second-tier immunoblot (IgM and/or IgG). The interpretation criteria for a positive immunoblot requires that at least two of three or at least five of ten bands be considered positive for IgM and IgG reactivity, respectively. Further, it was recommended that patients only be tested by IgM immunoblotting if their duration of illness is 30 days or less. STTT and interpretation criteria continue to be recommended by the Centers for Disease Control and Prevention (CDC) for laboratory confirmation of Lyme disease (2).Several dozen individual first-and second-tier diagnostic tests have been FDA 510(k) cleared and are commercially available. Most first-tier tests use either Borrelia burgdorferi whole-cel...

Training dental students is a complex process as it requires the acquisition of fine motor skills, and hand-foot and eye coordination to perform clinical work, in addition to the attainment of knowledge. Before performing invasive clinical procedures on patients, it is important that dental students reach adequate competency and this is achieved through extended use of pre-clinical simulated practice. 1,2 In Australian dental schools, students primarily receive pre-clinical training in a conventional pre-clinical laboratory for operative dentistry, prosthodontics, endodontics, and paediatric dentistry restorative procedures. Working with paediatric patients is often stressful for dental students, as child patients often lack