Study design, population, and sampling
A cross-sectional, hospital-based study was conducted to enrol children presenting for care with febrile illness to the outpatient or inpatient departments of Dareda and Haydom hospitals in the Manyara region of north-eastern Tanzania. These hospitals provide a range of healthcare services and serve predominantly agro-pastoral communities 20 in an area of low malaria endemicity 21.
Children aged 2–59 months with an axillary temperature of ≥ 38°C at the time of presentation were eligible for inclusion in the study. Children receiving anti-retroviral therapy, tuberculosis therapy, or who reported use of antibiotic therapies for three or more days in the two weeks before screening were excluded. Whole blood samples of up to 2.5mL volume were collected into EDTA vacutainers at the time of enrolment, stored temporarily at 2–8°C for up to three days then stored at -80°C, shipped to the Kilimanjaro Clinical Research Institute Biotechnology Laboratory and stored there at -80 °C freezer until TAC testing.
Demographic and socio-economic data recorded for study participants included participant age in months, sex, the occupation of their mother and father, and if domestic animals were kept in their household. Clinical data recorded (for patients presenting to Haydom only) included the final diagnosis recorded in hospital records. Given variable information available to clinicians to inform this assessment and variable timing of field recording after initial presentation analyses of these data were limited to checks of the mention (or not) of pathogens (and corresponding disease names) detected in 10 or more individuals through TAC testing in the clinical diagnosis field.
Nucleic acid extraction and TaqMan array card testing
High pure viral nucleic acid large-volume kits (Roche, Germany) were used for nucleic acid extraction as per the manufacturer’s instructions. Briefly, 1.5 mL of EDTA blood was subjected to a lysate preparation process, followed by purification and elution of nucleic acid using spin columns, including a high pure extender assembly for an initial large volume. Extrinsic controls PhHV (Phocine Herpesvirus) for DNA targets and MS2 bacterial phage for RNA targets were added to each sample during the lysate preparation to evaluate extraction and amplification efficiency. For each batch of extractions, a blank was processed through the complete protocol and later tested on a real-time PCR to rule out contamination. The eluted nucleic acids were stored at 80 °C before testing.
A TAC system as described previously 10 was used to simultaneously detect 33 AFI-associated viral, bacterial, and parasitic pathogens. Target pathogens were Chikungunya virus, Dengue virus, Zika virus, Crimean Congo Haemorrhagic Fever (CCHF) virus, Bundibugyo virus, Ebola virus, Sudan virus, Hepatitis E virus, Lassa virus, Marburg virus, Measles virus, Nipah virus, O’nyong-nyong virus, Rift Valley fever virus, West Nile virus, Yellow fever virus, B. anthracis, Bartonella spp., Brucella spp., C. burnetii, Leptospira spp., N. meningitidis, Rickettsia spp., S. enterica Typhi, S. enterica Paratyphi A, S. aureus, S. pneumonia, Group A streptococcus, Y. pestis, Leishmania spp., Plasmodium spp., and T. brucei. Primers and probes at concentrations of 900 nM and 250 nM respectively were used 10. Test cards were loaded with a mixture of 75 µl of total nucleic acid and 25 µl of TaqMan fast virus one-step mastermix (Life Technologies; Thermo Fisher Scientific, Carlsbad, CA). No template, positive controls (for all target pathogens), and extrinsic controls (PhHV and MS2) were included in all test runs. Cards were run on the ViiA7 real-time PCR system (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA) using the following PCR cycling conditions: reverse transcription step of 10 min at 50 °C and 1 denaturation cycle of 20 s at 95 °C followed by 40 two-step cycles of 3 s at 95 °C and 30 s at 60 °C. A run was considered valid if all positive controls were amplified, extrinsic controls were amplified for all samples, and no negative controls amplified. A sample was considered positive for a given target when it showed amplification with a cycle threshold (Ct) < 35. Samples showing amplification for a given target with Ct values ≥ 35 were considered borderline and were re-tested in a singleplex qPCR on the ViiA7 instrument using the individual target primers and probes 10 and identical cycling conditions. Samples with Ct < 40 in these confirmatory singleplex reactions were considered positive. Samples where amplification was not confirmed in the singleplex reaction were classified as negative. All other samples were classified as negative. All samples classified as positive and borderline for T.brucei in the TAC assay were re-tested in singleplex reactions and classified based on these singleplex reactions.
Samples that were Brucella spp. positive were additionally tested by quantitative PCR assay to distinguish between B. melitensis and B. abortus DNA. The Brucella speciation assay applied previously published primers, probes, and procedure 22. Briefly, duplicate reactions were performed on the Rotorgene platform (Qiagen) in 25 µL volumes, containing 25 µL TaqMan Universal Master Mix, 0.2 µM of each primer, 0.1 µM of each probe and 2.5 µL of template nucleic acid. Cycling conditions were as previously described (10 min at 95 °C followed by 45 cycles at 95 °C for 15 s and 57 °C for 1 min). Positive (B. abortus 544 and B. melitensis 16 M DNA) and negative (no template) controls were included in these assays. Samples were classified as positive for B. abortus and B. melitensis if a Ct < 40 was observed in either test well.
Data analysis
All data analyses, summaries, and graphs were performed using R version 4.3.2 23. Continuous variables were summarized using median and interquartile range (IQR). Categorical variables were summarized as frequencies and percentages. Binomial confidence intervals were calculated for all proportions.
Ethics declarations
This research was performed in accordance with relevant guidelines and regulations. Written consent was obtained from the parents or guardians of all participants before enrolment in the study. Ethical clearance for the collection and storage of participant samples was obtained from the College Research Ethics Review Committee (CRERC) of Kilimanjaro Christian Medical University College, certificate number 835. Additional clearance for testing for AFI pathogens was approved by the CRERC, certificate number PG 01/2022 and by the Medical Research National Health Research Ethics Review Committee (NIMR/HQ/R.8a/V0l.1X/2079).
