Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus.

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 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus.

Health Beliefs and Patient Perspectives of Febrile Illness in Kilombero, Tanzania.

This qualitative study assessed the knowledge and beliefs surrounding fever syndrome among adult febrile patients seeking health care in Kilombero, Tanzania. From June 11 to July 13, 2014, 10% of all adult (≥ 15 years) febrile patients enrolled in the larger syndromic study, who presented with an axillary temperature ≥ 37.5°C and symptom onset ≤ 5 days prior, were randomly selected to participate in an in-depth physician-patient interview, informed by Health Belief Model constructs. Interviews were audio recorded, translated, and transcribed. Transcripts were coded using NVivo Version 11.1, and the thematic content was analyzed by two separate researchers. Blood and nasopharyngeal/oralpharyngeal specimens were collected and analyzed using both acute febrile illness and respiratory TaqMan Array Cards for multipathogen detection of 56 potential causative agents. A total of 18 participants provided 188 discrete comments. When asked to speculate the causative agent of febrile illness, 33.3% cited malaria and the other 66.6% offered nonbiomedical responses, such as "mosquitoes" and "weather." Major themes emerging related to severity and susceptibility to health hazards included lack of bed net use, misconceptions about bed nets, and mosquito infestation. Certain barriers to treatment were cited, including dependence on traditional healers, high cost of drugs, and poor dispensary services. Overall, we demonstrate low concurrence in speculations of fever etiology according to patients, clinicians, and laboratory testing. Our findings contribute to the important, yet limited, base of knowledge surrounding patient risk perceptions of febrile illness and underscore the potential utility of community-based participatory research to inform disease control programs.

A Combined Syndromic Approach to Examine Viral, Bacterial, and Parasitic Agents among Febrile Patients: A Pilot Study in Kilombero, Tanzania.

The use of fever syndromic surveillance in sub-Saharan Africa is an effective approach to determine the prevalence of both malarial and nonmalarial infectious agents. We collected both blood and naso/oro-pharyngeal (NP/OP) swabs from consecutive consenting patients ≥ 1 year of age, with an axillary temperature ≥ 37.5°C, and symptom onset of ≤ 5 days. Specimens were analyzed using both acute febrile illness (AFI) and respiratory TaqMan array cards (Resp TAC) for multiagent detection of 56 different bloodstream and respiratory agents. In addition, we collected epidemiologic data to further characterize our patient population. We enrolled 205 febrile patients, including 70 children (1 < 15 years of age; 34%) and 135 adults (≥ 15 years of age; 66%). AFI TAC and Resp TAC were performed on 191 whole blood specimens and 115 NP/OP specimens, respectively. We detected nucleic acid for Plasmodium (57%), Leptospira (2%), and dengue virus (1%) among blood specimens. In addition, we detected 17 different respiratory agents, most notably, Haemophilus influenzae (64%), Streptococcus pneumonia (56%), Moraxella catarrhalis (39%), and respiratory syncytial virus (11%) among NP/OP specimens. Overall median cycle threshold was measured at 26.5. This study provides a proof-of-concept for the use of a multiagent diagnostic approach for exploratory research on febrile illness and underscores the utility of quantitative molecular diagnostics in complex epidemiologic settings of sub-Saharan Africa.

A diagnostic and epidemiologic investigation of acute febrile illness (AFI) in Kilombero, Tanzania.

INTRODUCTION: In low-resource settings, empiric case management of febrile illness is routine as a result of limited access to laboratory diagnostics. The use of comprehensive fever syndromic surveillance, with enhanced clinical microbiology, advanced diagnostics and more robust epidemiologic investigation, could enable healthcare providers to offer a differential diagnosis of fever syndrome and more appropriate care and treatment. METHODS: We conducted a year-long exploratory study of fever syndrome among patients ≥ 1 year if age, presenting to clinical settings with an axillary temperature of ≥37.5°C and symptomatic onset of ≤5 days. Blood and naso-pharyngeal/oral-pharyngeal (NP/OP) specimens were collected and analyzed, respectively, using AFI and respiratory TaqMan Array Cards (TAC) for multi-pathogen detection of 57 potential causative agents. Furthermore, we examined numerous epidemiologic correlates of febrile illness, and conducted demographic, clinical, and behavioral domain-specific multivariate regression to statistically establish associations with agent detection. RESULTS: From 15 September 2014-13 September 2015, 1007 febrile patients were enrolled, and 997 contributed an epidemiologic survey, including: 14% (n = 139) 1<5yrs, 19% (n = 186) 5-14yrs, and 67% (n = 672) ≥15yrs. AFI TAC and respiratory TAC were performed on 842 whole blood specimens and 385 NP/OP specimens, respectively. Of the 57 agents surveyed, Plasmodium was the most common agent detected. AFI TAC detected nucleic acid for one or more of seven microbial agents in 49% of AFI blood samples, including: Plasmodium (47%), Leptospira (3%), Bartonella (1%), Salmonella enterica (1%), Coxiella burnetii (1%), Rickettsia (1%), and West Nile virus (1%). Respiratory TAC detected nucleic acid for 24 different microbial agents, including 12 viruses and 12 bacteria. The most common agents detected among our surveyed population were: Haemophilus influenzae (67%), Streptococcus pneumoniae (55%), Moraxella catarrhalis (39%), Staphylococcus aureus (37%), Pseudomonas aeruginosa (36%), Human Rhinovirus (25%), influenza A (24%), Klebsiella pneumoniae (14%), Enterovirus (15%) and group A Streptococcus (12%). Our epidemiologic investigation demonstrated both age and symptomatic presentation to be associated with a number of detected agents, including, but not limited to, influenza A and Plasmodium. Linear regression of fully-adjusted mean cycle threshold (Ct) values for Plasmodium also identified statistically significant lower mean Ct values for older children (20.8), patients presenting with severe fever (21.1) and headache (21.5), as well as patients admitted for in-patient care and treatment (22.4). CONCLUSIONS: This study is the first to employ two syndromic TaqMan Array Cards for the simultaneous survey of 57 different organisms to better characterize the type and prevalence of detected agents among febrile patients. Additionally, we provide an analysis of the association between adjusted mean Ct values for Plasmodium and key clinical and demographic variables, which may further inform clinical decision-making based upon intensity of infection, as observed across endemic settings of sub-Saharan Africa.