Abstract
Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting.
Highlights
Identifying infectious etiologies is extremely complex for many clinical syndromes
Primers and hydrolysis probes were designed for specific detection of organisms for which existing assays were not currently available in our laboratories, including Toxoplasma gondii, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli/Shigella spp., Pseudomonas aeruginosa, Ureaplasma spp., Chlamydia trachomatis, Acinetobacter baumannii, Streptococcus agalactiae (Group B Streptococcus), and Neisseria meningitidis (Table S1 in File S1)
During development and validation of this assay, we occasionally observed sporadic amplification signal in no template control (NTC) reactions. This was determined to occur due to residual E. coli DNA present in the enzyme preparation from the manufacturers, which varied between production lots
Summary
The diversity and fastidious nature of pathogens causing infection and the lack of available diagnostic tests of sufficient quality and breadth contribute to the low proportion of cases with clearly identified etiology. The infectious etiology of community-acquired respiratory infections may remain unidentified in a large proportion of cases, even when advanced diagnostic technologies are employed [1,2,3,4,5]. In resource-poor settings, this proportion may be even higher as a result of factors which impede utilization of sophisticated testing procedures, including logistical challenges to specimen collection and handling, as well as the lack of adequate laboratory space or highly trained personnel. High potential for contamination in non-sterile collection settings is another considerable challenge to obtaining quality results in resource-poor areas. When more than one potential pathogen is identified, determining which organism(s) contributes to disease can be difficult
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