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

Hercik, Christine; Cosmas, Leonard; Mogeni, Ondari D.; Wamola, Newton; Kohi, Wanze; Houpt, Eric; Liu, Jie; Ochieng, Caroline; Onyango, Clayton; Fields, Barry; Mfinanga, Sayoki; Montgomery, Joel M.

AFI TAC allows for screening of whole blood samples for the detection of 15 viruses, eight bacteria, and three protozoa in the bloodstream. 27 Total nucleic acid was extracted from 2.5 mL of whole blood specimens using High Pure Viral Nucleic Acid Large Volume Kit. MS2 and phocine herpes virus served as built-in controls to confirm success of the extraction process and amplification efficiency. We mixed 46 μL of total nucleic acid extract with AgPath One Step RT-PCR reagents (Life Technologies, Carlsbad, CA), in a 100 μL reaction, then pipetted into the inlet port of each channel. Cards were centrifuged (1 minute, 1,200 rpm twice), sealed, and the inlet ports removed as directed by the manufacturer's instructions. All AFI TACs were run on the ViiA ™ seven real-time PCR system (Life Technologies) using PCR cycling conditions comprising 10 minutes at 50°C, 20 seconds at 95°C, followed by 45 two-step cycles of 3 seconds at 95°C and 30 seconds at 60°C. 27 Resp TAC further allows for screening of NP/OP specimens for the detection of 15 viruses and 15 bacteria known to cause respiratory illness. Total nucleic acid was extracted from 100 μL of NP/OP in MagNA Pure 96 Instrument (Roche) using MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Indianapolis, IN) and eluted in 100 μL of elution buffer. Fortysix microliters of total nucleic acid extract was mixed with AgPath One Step RT-PCR reagents (Life Technologies), in a 100 μL reaction, then pipetted into the inlet port of each channel. Cards were centrifuged (1 minute, 1,200 rpm twice), sealed, and the inlet ports removed as directed by the manufacturer's instructions. Resp TACs were run on the ViiA seven real-time PCR system (Life Technologies) using PCR cycling conditions comprising 45°C for 10 minutes, 94°C for 10 minutes, and 45 cycles of 94°C for 30 seconds followed by 60°C for 1 minute. 28 Our team captured additional laboratory testing performed by clinical staff as part of routine patient care, including malaria rapid diagnostic tests and microscopy. In this regard, clinicians administered an SD Bioline malaria Ag P.f/Pan rapid diagnostic test (Alere Inc., Waltham, MA) at point-of-care for all participants. If the patient yielded a positive rapid test result, a microscopic examination of Giemsa-stained thick blood film smears was performed to determine the intensity of infection. Results were scored as 0 (no parasites), 1+ (1-9 parasites/μL), 2+ (10-19 parasites/μL), 3+ (20-29 parasites/μL), or 4+ (30 or more parasites/μL).