The continued rise of respiratory viruses

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Diagnostics

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. explain how viruses affect populations based on age;
2. recognize the primary arguments for and against viral testing;
3. identify appropriate collection sites, collection methods, and transport media used for viral testing; and
4. differentiate between viral testing methodologies and their respective results and interpretations.

A gym in Los Angeles put up an ad billboard featuring a strange-looking alien with the warning: “When they come, they’ll eat the fat ones first!” Despite howls of protest, the warning proved prescient. When a novel influenza hit in 2009, it did preferentially kill the obese.¹ Absent natural predators to eat us, the natural predator to which we will most likely succumb is a microbe. The microbes for which we have the fewest treatments are viruses. In the large surface area of the lungs, their thinness means a single cell separates the blood supply from the environment at the alveolar level, and the sea of particle-laden air in which they bathe makes the lungs especially vulnerable to infection. When “they” do come, they will probably be respiratory viruses.

Conceptually, viruses are rather simple. The prototype contains an “F” fusion protein, a “G” protein to facilitate passage of genetic material into the human cell, some structural proteins to create a virion wall, and then either RNA or DNA. Looking beyond this cartoon of a virus reveals layers of evolution and, ultimately, perhaps two broad threats. The first leads to the pattern with which we are familiar, those viruses we diagnose and treat year in year out, usually with a specific season in mind. The second threat is that sudden changes in the virus leads to a large proportion of the population being vulnerable.

The role of and the type of testing needed for these scenarios is different, but clinical labs will have to respond to both. The clinical outcome of a respiratory virus infection is dependent on both virus and host factors. Respiratory viruses may be inconsequential, may cause substantial morbidity, or may kill.

Respiratory viruses may be inconsequential: Most upper-respiratory viral infections are so mild as to warrant no concern, and many are asymptomatic. Most are rhinoviruses,² most will never cause the patient to seek medical attention, and most will never be identified.

Respiratory viruses can cause substantial morbidity: While the potential for adenovirus to cause hemorrhagic pneumonitis and bronchiolitis has long been recognized, there is an increasing recognition of the importance of other viruses. Rhinovirus and respiratory syncytial virus (RSV), long associated with childhood morbidity, are increasingly being recognized as the causative pathogen of pneumonia in the elderly. A recent study demonstrated a viral etiology for pneumonia in 29% of adult cases,³ Consequently, laboratory staff in community hospitals can anticipate receiving RSV specimens from patients much older⁴ than they would traditionally expect. The impact of viruses in pediatrics is well known; bronchiolitis, a viral infection of the lower airways, accounts for up to 31/1000 infant hospitalizations a year.⁵ Even rhinoviruses can cause significant disease.⁴

Respiratory viruses kill: Particularly following the 2009 H1N1 epidemic, physicians realized that respiratory viruses kill their patients rather than just patients in the abstract sense. RSV and influenza virus (INF V) alone cause almost 54,000 deaths a year in the U.S.⁶ This may be an underestimate as some deaths attributed to presumed bacterial pneumonia are probably due to viral pneumonia with or without bacterial secondary infection. Respiratory viruses kill differently, depending on the virus and the host. Viruses can kill infants and the elderly with either respiratory failure from pneumonitis with or without secondary bacterial infection, or by a gradual sepsis response with multi-organ failure. Severe acute respiratory syndrome (SARS), a coronavirus showed a stepwise increase in mortality with age, diabetes, COPD, and other comorbidity.^7,8

Viruses can kill younger people in the same manner or, as was seen in some cases of the recent novel influenza outbreak, extraordinarily rapidly as a result of a cytokine storm.⁹ Rapid, very high levels of interferon-γ and TNF-α, and other pro-inflammatory proteins lead to the systemic inflammatory response syndrome with pulmonary infiltrates and multi-organ failure.¹⁰ A characteristic feature of these patients is their extremely rapid deterioration, prolonged intensive-care unit, or ICU, admission, and high mortality often in previously healthy young adults.

Finally, viruses can kill vulnerable infants by causing central apnea, which occurs when the infant’s normal central respiratory drive fails. Young infants frequently have irregular breathing patterns with pauses. Normally, if the respiratory pause is prolonged, the respiratory centers in the brain respond to the rising carbon dioxide and, if this mechanism fails, by the falling oxygen level in the blood. Infection with RSV, INF V, human metapneumovirus (hMV), and possibly others can cause these mechanisms to fail.^11,12 If not detected by caregivers, for example, at night when they are sleeping, death ensues. Premature infants and those with underlying co-morbidity are especially at risk.¹³

Despite the accepted pathogenic role of viruses, the role for actual viral testing is debated. The position taken by treating clinicians tends to reflect their patients’ severity of illness. The sicker the patient, the more likely testing will be ordered. And doctors who see numerous sicker patients are likely to be more aggressive in testing for viral illness. Even within this broad generalization, physicians disagree about the value of viral testing.

The case for virus testing

The primary arguments for viral testing are improving diagnosis, preventing unnecessary testing for bacterial infection, preventing inappropriate antibiotic use, directing antiviral therapy, and facilitating protocol-driven care.

Improving diagnosis: While diagnosing respiratory infections is generally straightforward, diagnosing the etiology is more difficult.¹⁴ The number of possible pathogens is daunting, and the respiratory tract can only respond in a limited number of ways to an infection. Even applying clinical case definitions is of limited help; at least half of adults with confirmed INF V in one study did not meet the Centers for Disease Control and Prevention’s case definition for an “influenza-like illness.”¹⁵ This is especially true for elderly patients and young infants^16,17 in whom making the correct diagnosis requires viral testing performed liberally, based on physician judgment, rather than insisting on a classical presentation or testing criteria.¹⁶

Preventing unnecessary testing for bacterial infection in the ER: A related argument is the realization that many prolonged and intensive evaluations for bacterial illness can be avoided if a viral cause can be proven. This has been demonstrated for INF V testing in infants where emergency physicians ordered fewer additional studies and prescribed fewer antibiotics when a child had a positive rapid influenza test.¹⁸ More importantly, at least for infants over two months of age,¹⁹ and, possibly, even younger,²⁰ the approach seems to be safe. This is also true for RSV,²¹ although caution is warranted in those less than eight weeks of age.²² Even in bronchiolitis, a clear-cut viral illness, the prevalence of bacterial co-infection is lower when RSV is shown to be present by antigen testing.²³ The value of testing for this reason, however, is age dependent. Figure 1 quantifies how, as infants get older, the risk of occult bacterial infection in the presence of a viral illness (in this case, bronchiolitis) falls to virtually zero; whereas early in infancy the risk is non-trivial.²³ Urine catheterizations and lumbar punctures, thus, can be used more judiciously based on viral testing. This prevents unnecessary antibiotic use. Similar arguments for parinfluenza and some other viruses can also be made but have less supporting evidence.

Research in adults has had mixed results.²⁴ While one study that used PCR to distinguish viral from bacterial infection did show appropriate antibiotic cessation, the number of patients enrolled was too low to reach statistical significance. The increase in cost of care was significant.²⁴ Another adult study using rapid antigen testing had more favorable results.²⁵

Diagnostic uncertainty in the primary-care setting can lead to pre-emptive antibiotic use.²⁶ Even in the absence of diagnostic uncertainty, the pressure to give antibiotics for viral illness can be intense and is often influenced by non-medical considerations.²⁷ Parents need to go to work rather than stay home with children.²⁸ Physicians are allowed only a limited time per patient. Writing a prescription for an antibiotic can act to close a consultation far more quickly than attempting to educate a patient who has attended seeking antibiotics for a viral illness. The physician who does not maximize both patient throughput and satisfaction metrics will be portrayed as “slow and unpopular” and not merely unemployed but unemployable. Confirming the presence of a specific virus allows the physician to say “No.”

Cohorting: A traditional argument for respiratory viral testing has been that it can be used to cohort inpatients as part of infection control.²⁹ The argument is limited by the reality that many viruses and strains of each virus co-circulate during bronchiolitis season when such measures are sometimes implemented. Resource-poor settings that might benefit typically cannot afford viral testing, and better equipped settings generally will isolate each child individually. Those intermediate settings (e.g., emergency rooms) usually have insufficient space or staff time to effectively cohort during seasonal outbreaks.

Protocol-driven care: Clinical pathways and other variants of protocol-driven care, offer efficiency advantages in busy clinical settings (e.g., emergency departments). Problems can arise when a patient is placed on the wrong clinical pathway, sometimes with waste and delay — or disastrous results. Obtaining objective virology data early in a patient’s course can reduce the risk of an infant with bacterial meningitis being managed on an upper respiratory-tract infection or viral illness clinical pathway.

Directing antiviral therapy: Antiviral therapeutic options for respiratory viruses are limited. Amantidine and Rimantidine are effective against susceptible strains of INF A when started early, ideally within 12 hours³⁰ but certainly within two days of disease onset.³¹ Resistance has become a problem, rendering these drugs ineffective against novel 2009 H1N1,³² H3N2, and other seasonal strains.³³ Oseltamivir and Zanamavir remain effective against most strains, but resistance is rising.³⁴ Again, these need to be started within two days of symptoms to be effective.³⁵ Antiviral resistance patterns can be tested by PCR assays, thereby directly guiding therapy. Novel RSV treatments targeting the F protein are inherently virus-specific and likely to be expensive; diagnostic certainty prior to their use will be necessary.

Arguments against viral testing. Primary arguments against viral testing are 1) knowledge of the offending virus does not often change patient management, 2) viral testing adds time and expense to patient care, and 3) clinicians misinterpret the results. Opponents point out that knowledge of the specific virus responsible for a patient’s illness does little to alter patient management — that only clinical manifestations matter. They correctly point out that most patients with INF V present too late for treatment to be effective. Even then, benefits of antiviral medication for influenza (the only respiratory virus for which treatment is currently available) are modest. The remainder of treatment, even for the sickest patients, is largely symptomatic and supportive.

Expense: Viral testing is expensive relative to physician fees. A rapid antigen testing kit’s cost easily exceeds the professional fee Medicaid programs pay primary-care physicians, and nucleic-acid-based testing may even cost more. Because viral respiratory illness is so common, even testing a small fraction of infected patients adds enormous costs to healthcare systems. Under-testing for viral disease risks diagnostic nihilism, antibiotic overuse, and, ultimately, a failure to appreciate the role of viruses in serious pathology. Conversely, testing every case of viral respiratory illness is neither feasible nor justifiable medically or economically. In the context of day-to-day practice, a highly selective approach based on individual patient need is best. In the context of an outbreak of severe respiratory illness, a much broader approach to testing is justified to accurately define the threat.

Performing respiratory-virus testing

When the decision to test is made, the clinician must consider the specimen site he wishes to test, the method of sampling, collection and transport system, and the testing method.

Sampling sites: Typical sample sites include the anterior nares and mid-turbinate region, which seem to work well for RSV, hMV, and influenza recovery,³⁶ nasopharynx, and throat. Sometimes, more than one site may be sampled. Sputum and occasionally bronchoalveolar-lavage washings can also be used.

Sample-collection and transport methods: The sample-collection method chosen determines to some extent the transport-collection system. Sample-collection methods fall into two categories: saline and swab-based. Saline-based methods include nasal washes — typically used in infants. Saline is introduced into the nares and suctioned out, along with virus containing nasal secretions. More aggressive methods extend this to nasopharyngeal instillation and subsequent aspiration of saline. Swab-based methods use a swab and transport medium. Various approaches target the anterior nares, mid-turbinate region, or the posterior nasopharynx. Paired comparisons of sample-collection methods have been performed by investigators. Finally, some work suggests that a key component is the transport medium used, particularly for polymerase chain reaction- (PCR-) based testing, which appears susceptible to inhibitors in nasal secretions.

Nasopharyngeal aspirates are labor intensive, difficult to obtain, and also operator dependent. Early comparisons showed nasopharyngeal nylon-flocked swabs and aspirates to be essentially equivalent for culture and immunoflourescence.³⁷ Nasopharyngeal swabs are easier to obtain, and less resource- and operator-dependent. Demonstrations that adequate results could be obtained using nasal washes (for RSV and INF), led to its popularity, particularly as it dovetailed with the need to clear nasal secretions in infants.³⁸ Saliva samples are inferior.³⁹

Comparison of saline washes to saline washes preserved with a modified Hanks solution (universal transport medium room temperature, or UTM-RT) found that, despite the dilution of the sample, detection rates were significantly higher in the preserved specimens. Sensitivity was 71% (95% CI 59% to 81%) and specificity 88% (95% CI 81% to 94%) for saline washes, and for UTM-RT preserved saline washes sensitivity 99% (93% to 100%) and specificity 95% (88% to 98%). Mid-turbinate nylon-flocked swabs performed similarly to the preserved nasal washes. The swabs had a slightly higher virus detection rate (45% vs. 42% for UTM-RT preserved saline and 35% for unpreserved saline) but lower sensitivity 92% (95% CI 83% to 97%) and specificity 85% (95% CI 77% to 91%) because the gold standard was defined as the virus being detected by two of the three methods. Clinical practice should favor the collection system with the highest detection rate, provided its other test characteristics are reasonable.⁴⁰ Similar work comparing nasopharyngeal flocked swabs to mid-turbinate swabs has found excellent agreement between these swab types for viral direct immunofluorescence antibody (DFA) testing.⁴¹ In actual use, adults prefer swabs for nasal washes or nasopharyngeal aspirates. Swabs seem to work best for infants.⁴²

Swab materials have also been compared. Cotton and alginate swabs may inhibit PCR and have generally been superseded by synthetic materials. Although swab composition may be less a consideration for culture and immunonchromatographic tests for nucleic-acid-amplification techniques, even the physical design of the swab may be relevant. One study, albeit small, showed a trend to increased viral-detection rates in infants when a specifically designed swab based on age-related anatomical considerations was compared with a generic “Q-tip” design.⁴⁰ One study found better detection rates using a polyurethane foam sponge rather than nylon flocked swabs for one rapid antigen testing kit.⁴³

Transport media: In the past, many labs produced and, in some cases, devised their own unique culture media.^44,45 Such an approach may reduce materials cost — always a concern to researchers — but outside of academic settings, it has largely given way to the use of commercial pre-made solutions (typically UTM-RT or M4-RT) already placed in the collection vial, sometimes with beads to increase surface area. The ideal transport medium would 1) allow the sample be tested using any method without loss of virus-detection rates, 2) be stable across a wide range of environmental conditions, 3) never expire, and 4) be cheap. While the ideal does not exist, commercially available transport media come close, generally performing well and being compatible across diagnostic platforms.^46,47

Although it is self-evident that samples should be processed as quickly as possible, there is relatively little independent evidence measuring the effect of leaving a sample sitting on a bench before processing it. INF viruses will survive for 24 to 48 hours on non-porous and eight to 12 hours on porous substances.⁴⁸ The implication is that ordinary lab delays should not adversely impact virus detection, at least when testing is performed on the day of collection. Unpreserved saline washes contain leukocytes and cytokines that may adversely affect virus survival in the sample. It certainly leads to decreased virus detection by PCR when compared with preserved samples.⁴⁰ If delays are expected, then the sample should be preserved. Saline washes are typically preserved by freezing to -70^0C. This is an expensive proposition and adding UTM-RT may be a more cost-effective option. One study showed that leaving saline washes diluted 1:1 with UTM-RT in an uncontrolled environment results in only modest loss of testing performance, even over many days (see Figure 2).⁴⁹

Choice of testing technology

The most commonly described methods of virus testing are immunochromatographic rapid antigen tests, nucleic-acid-amplification-based tests, immunoflourescence-based tests, and virus culture.

Rapid antigen tests are easily and quickly performed. Despite manufacturers’ assertions, however, independent assessments of these tests typically demonstrate only modest test characteristics.^50,51 This is particularly the case with adults in whom these tests should probably not be used.^52,16,53,54

Nucleic-acid-amplification-based tests can be thought of broadly as reverse transcriptase PCR and microarray methods.

PCR-based testing works well if the patient is likely to have a recognized pathogen. Typical clinical platforms strive for sensitive and specific uniplex or multiplex testing for a defined list of likely recognized pathogens. Multiplex testing is more efficient, but it must be validated against uniplex testing before deployment.⁵⁵ Both commercial and in-house PCR solutions have been developed. Generally, these work well, although care must be taken to ensure the validity of in-house assays.

This strategy does not work as well when the infecting virus is unusual. When there is some suspicion as to which family the likely culprit belongs, solutions using degenerate primers, which will amplify all members of a particular virus family, can be used. This approach has been developed in response to coronovidiae-induced SARS epidemics. The result is an assay that will screen for all known (and, possibly, unknown) coronaviruses.⁵⁶

When the clinician is unable to provide any insight into potential culprits, or clinical suspicions have been proven incorrect, microarrays allow simultaneous testing for hundreds of different viruses.⁵⁷ The principle involved is to bind DNA probes for important genetic material onto a silicon or glass sheet referred to as a chip. Non-specific amplification of the genetic material present in the sample with a luminescent label is performed, which is then allowed to pass over the chip those sequences that are present, which bind to the probes present on the chip. Different combinations of different sequences can be used to estimate the probability of a given known virus being present. If the combination of sequences found cannot be accounted for by simultaneous infection with known viruses, the presence of a previously unknown virus may be postulated.⁵⁷ Although widely used in research, day-to-day clinical use for the diagnosis of respiratory viruses is rare.

Despite forming the original basis for virology research, viral culture and immunoflourescence are infrequently used in clinical practice. Culture is time consuming, often difficult, and sometimes impossible. Immunoflourescence is microscopist dependent. Neither technique, therefore, is readily available in many centers in a time-sensitive manner.

In the context of a sudden epidemic of a virus to which the population has little immunity, use of microarrays during the discovery phase, followed by PCR testing will be required. Even if immunochromatographic methods could be developed quickly, their performance is likely to be too poor to be relied upon during the initial phase where specific virus diagnosis is needed. Once an epidemic is established within a community, there is much less need for testing.

A factor which cannot be underestimated is the importance of timely reporting of results. One study demonstrated this dramatically, a rapidly reported enzyme-linked immunoassay RSV test, for all its imperfections, had far more impact on clinical decision making and antibiotic use than a panel, which tested multiple viruses, but sometimes did not report until after the patient was discharged.⁵⁸ Current technology allows the testing to be performed quickly,⁵⁹ future efforts must focus on decreasing the delay from obtaining the sample to getting the results to the beside.

Interpreting the results

Unlike many other lab tests, viral tests are typically reported as positive or negative. Clinicians have little interest in anything else; after all, a “maybe” answer from the lab would leave the clinicians precisely where they started when they first obtained the test. Quite apart from whether a PCR “creeper” deserves such certitude, there is the more vexing problem of interpretation of results in the context of prevalence. Even results from a test with good receiver, operating curve characteristics need to be interpreted in light of the pre-test probability of the patient for whom the virus is being tested.

The testing technology used is determined by local clinical practice patterns and local resources. For most patients, this will mean rapid antigen tests. When prevalence is low, as it could occur when diagnosing undifferentiated fever in infants or performing surveillance in the context of an approaching epidemic, other tests are preferred.

Figure 3 demonstrates the importance of prevalence in interpreting these results. It shows why when prevalence is low say 5%, a positive test will be a true-positive in only 61% of the cases. Conversely, when prevalence is high, for example, 50%, a negative test will be a false-negative in 24% of the cases.⁶⁰ It also shows how testing in older children is more likely to yield the correct result, partly because disease presents differently at different ages and partly because preverbal toddlers can contribute less to their own diagnosis than older children.⁶⁰
Similarly, a positive RSV antigen test in June should be regarded as less certain a true-positive than one in the middle of bronchiolitis season, and a negative RSV test in bronchiolitis season is less certain a true-negative.

Clinical laboratories typically report the methodology and the result. Specifics of which testing kit or platform was used may not be provided and, as yet, it is unclear that the clinical community has the time or inclination to ponder these subtleties. Incorporating individual level characteristics relevant to the patient being tested may also be difficult given the limited clinical information provided with samples. Incorporation of local prevalence information relevant to the patient is similarly difficult. In the future, such individualized reporting may become possible with highly integrated electronic medical record systems that incorporate clinical findings and up to date local population prevalence; for now, they are an aspiration.

Summary

Respiratory viral illness ranges from trivial to fatal. Testing is indicated only in a minority. The decision to test is based on clinical presentation, available resources, and the clinician’s philosophy. Flocked swabs are easier to obtain than aspirates and perform similarly. Commercially available room-temperature transport media perform well and should be used with both swabs and aspirates sent for PCR testing. Nucleic-acid-amplification techniques are preferred to immunochromatographic antigen tests but are less widely available and take longer. Immunochromatographic antigen tests should not be used if prevalence is low. The development of novel antiviral agents will tend to increase viral testing and cost containment measures to decrease it. Overall, testing for respiratory viruses is likely to increase.

Paul Walsh, MD, BCh, BAO, MSc, DME, DipHI, F(ACEP), is an associate clinical professor of Health Sciences at the David Geffen School of Medicine, University of California-Los Angeles, and research director in the Department of Emergency Medicine at Kern Medical Center in Bakersfield, CA.

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