Sensitive amplification methods to detect and sequence human- parechoviruses, enteroviruses and adenoviruses in cerebrospinal fluid in sudden unexpected death in infancy cases
Introduction: In 2023, approximately three million children under the age of five died worldwide. Sudden Unexpected Death in Infancy (SUDI) includes all unexplained deaths, aged 7 days to one year, before any investigation has taken place. Infant mortality rate (IMR) is an important global indicator...
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| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-03-01
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| Series: | International Journal of Infectious Diseases |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1201971224007951 |
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| Summary: | Introduction: In 2023, approximately three million children under the age of five died worldwide. Sudden Unexpected Death in Infancy (SUDI) includes all unexplained deaths, aged 7 days to one year, before any investigation has taken place. Infant mortality rate (IMR) is an important global indicator of child-health and well-being, and South Africa (SA) is currently among the top 10 countries with the highest IMR with an IMR between 23.13 (2024) and 23.57 (2023) (Macrotrends.net, 2024).According to the Triple Risk Hypothesis (Filiano and Kinney, 1994), SUDI is most likely to occur when vulnerable infants enter a critical developmental period and experience outside stressors simultaneously. Infection of the Central Nervous System (CNS), particularly with meningitis, meningo-encephalitis and encephalitis in SUDI cases has largely remained unexplored and underreported in SA.Cerebrospinal fluid (CSF) is present in the intracranial and spinal compartments and the composition normally remains constant (Hrishi and Sethuraman, 2019). Detection of any pathogen in the CSF is highly indicative of infection. The role of human parechoviruses (HPeVs), -enteroviruses (HeVs) or -adenoviruses (HAdVs) in SUDI is largely unknown, and these viruses are seldom tested in CSF of SUDI cases. Additionally, these assays sometimes have a lower limit of detection (LOD) at only a 100 copies/µL for CSF specimens. Therefore, a sensitive targeted approach aimed at binding to conserved viral genome regions would increase detection of these potentially underreported viruses. Methods: HPeV and HeV RNA were reverse transcribed and DNA from all three targets were amplified by respective nested touch-down PCR reactions. PCR products were cloned with CloneJET PCR cloning kit and HPeV and HeV were in vitro transcribed. HAdV DNA and HPeV and HeV RNA were respectively purified and quantified by fluorometry. Results: Panels utilised currently for screening in diagnostics has a lower LOD at a 100 copies/µL in CSF specimens. The lowest LOD achieved for the respective assays were determined to be as follows: HPeV 3 copies/µL, HeV 30 copies/µL and 80 copies/µL for HAdV. Discussion: Investigation into the presence of HPeV, HeV and HAdV in CSF specimens associated with SUDI cases remain unexplored and underreported despite recent research implicating these viruses in CNS infections. A sensitive screening assay targeting conserved viral genome regions would increase the detection of these potentially underreported viruses in CSF.Assays targeting more hypervariable regions of the viral genome for HPeV and HeV are also being developed. This would allow for the determination of molecular epidemiology. Conclusion: Ideally, two thirds of replicates need to be detected at a specific concentration. A probit regression has not yet been carried out. Assays have displayed detection at low copy numbers and successful amplicons were produced that can be sequenced on Oxford Nanopore. |
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| ISSN: | 1201-9712 |