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Tuesday 18 December 2018
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Point of care testing in virology

The role of the diagnostic virology laboratory in patient care has changed markedly in recent years. Twenty years ago, the work involved growing viruses from patient specimens or detecting evidence of a change in concentration in serum antibodies in response to a viral infection. In most cases, it would take several weeks to achieve a final result. However, in the 21st century, virologists have the technology and scientific expertise to be much more responsive both in terms of diagnosing commonly encountered viral infections and in dealing with new and emerging viruses.1
 
Culture of viruses from respiratory and faecal samples in cell lines has largely been replaced by detection of the viral genome by molecular methods, particularly polymerase chain reaction (PCR). Instead of collecting a blood sample during the acute phase of the infection and another sample two weeks later, in order to compare antibody titres, enzyme immunoassay (EIA) is now used to identify specific antibodies when the patient presents with the symptoms. Both of these approaches allow the laboratory to provide a result in days; where it is clinically relevant, it can be possible for the sample to be processed, tested and the results interpreted in less than 24 hours.
 
However there is an increasing repertoire of point of care tests (POCTs) for viral diagnosis on the market. These offer the prospect of an answer in a matter of hours without the need to transport the sample from the hospital ward or primary care centre to the virology department and wait for the laboratory to communicate their findings. These POCTs – also known as near patient tests (NPTs) or rapid diagnostic tests (RDTs) – can be useful in patient care and management because they are easy to use and can often give a result in a relatively fast time. 
 
However, it should be borne in mind that they are not always as accurate as the equivalent test performed by scientists in the main laboratory and they are usually more expensive. Therefore the decision to introduce a POCT into a particular situation needs careful consideration.2 There are some interesting examples of their implementation into diagnostic virology. The types of POCT formats currently available to detect evidence of viral infection fall into two broad formats – lateral flow assays (LFAs) and desk top molecular analysers. 
 

LFAs 

LFAs comprise test strips made of a material such as cellulose and embedded with biological markers (specific antibodies or antigens). The strip is held in a plastic case to keep it dry and clean and there is an opening for the patient’s sample to be added. The most familiar example is home pregnancy testing kits. In virology, formats are available to detect either the presence of the virus itself, in specimens such as faeces or throat swabs, or specific serum antibodies against particular pathogens. The principle is similar to the EIA. 
 
The sample flows across the test strip and activates the reagents. If a reaction between occurs between the virus (or antibody) from the patient and a marker in the test strip, a visible colour change will occur.3 The devices require small volumes of a type of sample, which can be collected relatively easily from a patient.  The sample is treated with a reagent to release the virus particles or antibodies from cellular material and enhance their binding reaction to the relevant marker(s) in the test. Figure 1 shows an LFA for detection of norovirus in faecal samples. 
 
 Lateral flow device for detection of norovirus in a faecal sample (virus not detected)
Figure 1: Lateral flow device for detection of norovirus in a faecal sample (virus not detected)
 
In this case, no virus was detected, but note the activation of the control line in the strip, which indicates that the test was carried out correctly. All the necessary reagents and equipment are included in the kits so that anyone who has been trained properly can perform and interpret an LFA. The time it takes to get a result varies slightly depending on the particular assay, but including the specimen preparation step and the time taken to read the results, it usually takes less than 30 minutes.
  

Desk top molecular analysers 

A large proportion of the work in a modern diagnostic virology laboratory involves detection and sequencing of viral genomes. The assays use the principle of nucleic acid amplification (for example, PCR), which in theory can recognise a single molecule of viral RNA or DNA and copy it multiple times to increase its concentration to a level that is possible to measure. The technique involves a number of steps, including extraction of the nucleic acid from the cellular material in the patient’s sample, running the amplification steps and then detecting the resulting RNA or DNA copies. It requires scientific understanding, technical expertise and dedicated laboratory space. 
 
This is needed not only to accommodate the various analysers, but also to maintain an environment free of stray pieces of nucleic acid (for example, from the scientists’ skin or hair). Figure 2 shows a clean area, a designated cabinet, sterile equipment and laboratory coats that are only used in that room as required. In recent years it has proved possible to scale down the amplification systems into a small desk top analyser. The person operating the test simply has to load the sample into the machine and all the necessary steps are carried out inside it. Thus, as with LFA, all that is needed is suitable training; scientific understanding of the principle or technical ability to interpret the data are not required. The time it takes to achieve a result varies, but is usually one to three hours.
 
Figure 2: Scientist preparing sample prior to nucleic acid amplification analysis
Figure 2: Scientist preparing sample prior to nucleic acid amplification analysis 
 

Use of POCT devices in diagnostic virology 

The LFA format has proved quite useful in the main virology laboratory. For example, to confirm a new diagnosis of human immunodeficiency virus (HIV) infection, the blood sample needs to be re-tested using a number of different types of assay, which could include a POCT kit. Before PCR was widely available, it was common to test respiratory samples for respiratory syncytial virus (RSV) or influenza virus by LFA before cell culture, in case it could provide a preliminary result. More recently, they have found an application in reconfigured laboratory services, where a main laboratory at one site serves several other hospitals.
 
Using POCT (LFAs and molecular analysers) in the smaller ‘satellite’ microbiology departments has proved very successful. The turnaround time for many specimens will be relatively fast and the number of specimens transported to the main laboratory will be reduced.4 With full training, this function could be performed by laboratory staff who do not usually work in virology.  Another situation where POCT could be useful is for unusual infections for which a main laboratory assay is not routinely available, but a rapid result is desirable (for example testing for Ebola virus).5
 
One obvious application of POCT in the ‘near patient’ setting would be screening of patients in hospitals during outbreaks of norovirus or RSV, prior to cohort nursing. Laboratory scientific staff can be involved in the testing, but the idea would be for other healthcare workers to do the tests and take responsibility for recording the results. While LFAs have been widely evaluated for this purpose, it should be noted that performance can be quite variable. Although most kits are found to have high specificity (meaning a positive result can be taken as accurate), sensitivities are often less than 90% (which means that one in ten tests could give a false negative result).
 
When it is important to identify exactly which paediatric patients do actually have RSV (and which have a different respiratory infection), the main laboratory PCR test can be a more clinically acceptable and cost effective option.6 It should be feasible to obtain a result within one working day and commercial PCR kits that allow testing for several pathogens in one assay (multiplex PCR) are becoming widely available. In addition, viruses are prone to mutation and newly emerging strain variants may not be detected by LFA kits.7
 
Another situation where POCT might be valuable is in the genitourinary medicine setting. A patient who might not return a week later to be given a test result regarding a sexually transmitted infection might be prepared to wait for an hour. Lateral flow kits are available to screen for viruses such as hepatitis B (HBV), hepatitis C (HCV) and HIV, and some formats use oral fluid instead of blood, which can be more acceptable.8 However, there are issues with sensitivity and this might lead to a protocol that requires individuals to be screened more regularly than when using the main laboratory assays. Also, any patient who is found to be positive in the POCT test will require further tests to determine the stage of infection, viral load, sequence and type of virus in order to inform treatment and management.9 Thus it might be more cost effective to do the initial screening test and all the follow up work in the main laboratory.
   
Desk top molecular analysers should be more accurate than LFAs because they are detecting the viral genome directly. The range of viruses which can be detected in molecular POCT is currently quite limited compared to the multiplex main laboratory tests and they are more expensive. Also as mentioned above for the blood borne viruses, positive results might still require follow up sequencing and typing for patient management and epidemiological purposes. However an interesting innovation is the development of a low cost analyser capable of monitoring HIV viral load for use in situations where demand is high, but resources are limited.10 Another potential application of a respiratory POCT is to identify which patients admitted to hospital with acute respiratory symptoms have virus infections and therefore do not require antibiotics,11 but could benefit from treatment with an antiviral drug.  This could be an important tool in the work to reduce antimicrobial resistance.
    

Conclusions

Virology POCT has the potential to supplement the work of the main diagnostic laboratory, but it seems unlikely that they will be a replacement. Although the cost per test is greater at the moment, this could start to reduce if more healthcare organisations use them more widely. Evaluation of POCT in virology involves comparison of the test with the main laboratory EIA or multiplex PCR. These studies tend to include laboratory staff doing the tests, either in the laboratory itself or supervising others in the primary or secondary care setting. More work needs to be carried out to ensure that results are consistent when non-scientists implement the full process of sample collection, testing and reporting. 
 
Key areas to consider are training of colleagues and quality assurance of the reagents and equipment. Another issue is how to make sure that any POCT results are recorded accurately in the patient’s notes and are communicated to the laboratory promptly. This is particularly important when follow-up laboratory tests are required. It is clear that POCT devices could be used in more settings and they are being refined using methods such as microfluidic technology (which uses paper instead of cellulose and a smartphone application to analyse the results). However, it is important to note that scientific advances in the main laboratory continue and have recently brought the powerful tool of next generation sequencing. This reveals information about the whole genome of each pathogen in the patient’s sample rather than just detecting a small section. Thus it provides data that will enhance patient care and management in different ways. 
 

References

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3 Koczula KM, Gallotta A. Lateral flow assays. Essays Biochem 2016;60:111–20.
4 Drancourt M et al. The point-of-care laboratory in clinical microbiology. Clin Microbiol Rev 2016;29:429–47.
5 Broadhurst MJ et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet, 2015;386(9996):867–74.
6 Moesker FM et al. Diagnostic performance of influenza viruses and RSV rapid antigen detection tests in children in tertiary care. J Clin Virol 2016;79:12–7.
7 Théry L et al. Evaluation of immunochromatographic tests for the rapid detection of the emerging GII. 17 norovirus in stool samples, January 2016. Eurosurveillance. 2016;21(4).
8 Chevaliez S et al. Prospective assessment of rapid diagnostic tests for the detection of antibodies to hepatitis C virus, 
a tool for improving access to care. Clin Microbiol Infec 2016;22: 459.e1–e6.
9 Bottero J et al. Effectiveness of hepatitis B rapid tests toward linkage-to-care: results of a randomized, multicenter study. Eur J Gastroen Hepat 2016;28: 633–9.
10 Goel N et al. Performance of the SAMBA I and II HIV-1 Semi-Q Tests for viral load monitoring at the point-of-care. J Virol Methods 2017; 244:39–45.
11 Brendish NJ et al. Routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (ResPOC): a pragmatic, open-label, randomised controlled trial.  Lancet Resp Med 2017; 5:401–11.

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