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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

Scientists have feared that emerging infectious diseases could complicate efforts to conserve rare and endangered species, but quantifying impacts has proven difficult until now. We report unexpected impacts of West Nile virus (WNv) on radio-marked greater sage-grouse (Centrocercus urophasianus), a species that has declined 45-80% and is endangered in Canada and under current consideration for federal listing in the US. We show that WNv reduced late-summer survival an average of 25% in four radio-marked populations in the western US and Canada. Serum from 112 sage-grouse collected after the outbreak show that none had antibodies, suggesting that they lack resistance. The spread of WNv represents a significant new stressor on sage-grouse and probably other at-risk species. While managing habitat might lessen its impact on sage-grouse populations, WNv has left wildlife and public health officials scrambling to address surface water and vector control issues in western North America.

Two novel real-time PCR assays were developed for the detection of Rickettsia spp. One assay detects all tested Rickettsia spp.; the other is specific for Rickettsia rickettsii. Evaluation using DNA from human blood and tissue samples showed both assays to be more sensitive than nested PCR assays currently in use at the CDC. Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), is a tick-borne bacterial agent that is a member of the spotted fever group (SFG) rickettsiae. Symptoms of this disease may include, but are not limited to, high fever, headache, and rash, with the potential for a fatal outcome (1). National case fatality rates are Ͻ1% but can approach 7% in some regions to which it is highly endemic; a fatal outcome may be averted with timely administration of doxycycline (2, 3).In the United States, serologic tests are most often used to diagnose RMSF with seroconversion (4-fold titer increase from acute to convalescent phase) by immunofluorescence assay (IFA), the gold standard. However, serologic tests are frequently negative during the acute phase of illness, and currently available molecular tests are not reliable for use in patient management when applied to acute-phase blood samples, which may have very few organisms (3, 4). Clinical management of suspected RMSF cases based on test results is not recommended, partly because of diagnostic assay limitations, and physicians must treat suspected cases empirically (4). While empirical treatment of suspected cases will always be recommended, the development of more sensitive molecular tests that could be applied to patient specimens during the acute phase of illness will enhance the identification of nonfatal cases, improve the timeliness of public health actions following identification of a case, and aid national surveillance efforts by better defining the spectrum and burden of tick-borne rickettsial infections.At the Centers for Disease Control and Prevention (CDC), nested PCR assays have been the standard molecular diagnostic method for testing of blood and fresh tissue specimens. PCR methods for detection of R. rickettsii in clinical samples include nested assays for two SFG target genes, the 17-kDa-protein-encoding gene and the ompA outer membrane protein gene (5-7). The ompA nested amplicon may then be sequenced for species identification (8-15). However, this methodology is time-consuming (1 to 2 days, minimum) and has not proven highly sensitive for the detection of Rickettsia spp. in blood samples during acute disease, except in cases of advanced or fatal illness (4, 16). A 2000-2007 national surveillance summary noted that Ͻ0.5% of reported cases were diagnosed using nested PCR methodology (3).Several real-time PCR assays have been developed for rickettsial agent detection, including Rickettsia genus-specific (panrickettsia) and R. rickettsii-specific assays (17-20). Panrickettsia assays have utilized conserved sites in the 17-kDa, outer membrane protein (ompB), 16S rRNA, and citrate synthase (gltA) genes, while ...