Rapid diagnostics — An urgencyApril 4, 2020
Rapid diagnostics and therapeutics are important interventions for the management of 2019 novel coronavirus (2019-nCoV) outbreak, especially in India. For prompt and accurate public health control measures, prevention and surveillance, it is important to have fast and accurate specific viral detection systems. This kind of diagnostics can identify cases, and move their close contacts to quarantine so that local transmission and cluster cases can be prevented.
Identification of high-risk groups and facilities can lead to decontamination by authorities at the right time. Laboratory diagnosis can be performed by: (i) detecting the genetic material of the virus, (ii) detecting the antibodies that neutralise the viral particles of interest, (iii) detecting the viral epitopes of interest with antibodies (serological testing), or (iv) culture and isolation of viable virus particles.
All these techniques come with specific limitations, such as cross-reactivity of non-specific genetic regions for acute-phase infections for genetic tests. In serological testing, there is a need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses.
In case of detection of neutralising antibodies and culture methods, there is a long wait time for confirmation results, thus rendering it not useful in present conditions. The origin of the biospecimen also plays a major role in the sensitivity of the tests. In the case of SARS-CoV and MERS-CoV, biospecimens were collected from lower respiratory tract such as sputum and tracheal aspirates where there is higher viral load and prolonged presence of viral RNA due to tropism of the virus. The samples collected from upper respiratory tract, such as nasopharyngeal or oropharyngeal swabs, could potentially have lower viral loads and have a higher risk of false-negatives, which can prove to be catastrophic in the present 2019-nCoV case.
The nucleic acid-based tests include reverse transcription polymerase chain reaction (PCR), real-time PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays. The Chinese health authorities have recently shared the full genome of 2019-nCoV in the GenBank and in GISAID portals. This resulted in several laboratories developing assays and the World Health Organization (WHO) developing interim guidelines for 2019-nCoV testing. Corman et al from Germany was first to develop RT-PCR assay using SARS or SARS-related coronavirus as it was suggested earlier that it was SARS-like. However, once the sequence of 2019-nCoV was released, two assays were developed. Two genes, namely RNA dependent RNA polymerase (RdRp) gene and E gene sequences, were used for assay development. E gene-based assay was a first-line screening tool and RdRp gene assay is a confirmatory test.
The Centre for Disease Control, United States, shared an RT-PCR assay for detection of 2019-nCoV with complete information about probes and primers. However, the closed nature of the protocol proves to be difficult, unless validated in different platforms and chemistries. Along with this, some of the limitations, mainly false negatives, could be due to insufficient organism in the specimen, improper collection, transport/handling or the degradation of RNA due to temperature variation in the logistics step. Another important variable is that RNA viruses show substantial amounts of genetic variability, which can result in primer mismatch to target sequence, resulting in false-negative results. The use of sample collection systems, which can denature the pathogen but not the genetic component, and point-of-care test kits can potentially minimise these limitations, while helping with the identification of specific regions in viruses like non-coding regions. Research must focus on such urgent medical needs and develop required tools in the next few months.
The author is a medical scientist and former director of SGRF, Bangalore