Cases in Medical Microbiology and Infectious Diseases. Melissa B. Miller
higher than that of DNA (10−3 to 10−5 versus 10−6 to 10−8 per base per generation), point mutations readily accumulate in influenza viruses. Although mutations occur throughout the influenza genome, the accumulation of mutations (and corresponding amino acid changes) in surface antigens, such as hemagglutinin and neuraminidase, have the greatest impact. For influenza A virus, these changes will not necessarily result in the change of the classification of a viral strain (which is based on the subtypes of the H and N antigens), but they may be sufficient to render patients with antibodies to the parent strain susceptible to the new mutant strain. This is the basis for the decision to reevaluate and potentially change the formulation of the influenza vaccine each year to include recent isolates, so that protective antibodies to the most recent isolates will be made in response to the vaccine. Both influenza A and influenza B are constantly changing by antigenic drift.
The more dramatic, and less common, antigenic shift is due to genetic reassortment of genes to form a novel human influenza virus, which typically has different hemagglutinin and/or neuraminidase proteins. Antigenic shift occurs during coinfection of a cell with two different influenza A viruses. Since the packaging of viral RNA segments occurs randomly, a coinfected cell could form a variety of different virions. The result could be a virus with a different classification (e.g., a shift from H1N1 to H5N1) or a virus of the same type but with divergent genomic sequences from nonhuman sources such as pigs or birds. The end result is a new virus that differs dramatically from parent strains.
The influenza A H1N1 pandemic of 2009 was a result of antigenic shift. Although an H1N1 influenza virus had circulated globally for years, a reassortant H1N1 virus was introduced and spread worldwide. The 2009 H1N1 virus was a result of the introduction of Eurasian swine segments (neuraminidase and matrix) into the classical swine influenza strain that previously had only caused swine-to-swine transmission and rare swine-to-human transmission. When an antigenic shift occurs, most of the world’s population has little or no protection against the new virus, resulting in large epidemics or pandemics.
3. There are a variety of ways of diagnosing influenza in the laboratory, including rapid antigen tests, direct fluorescent-antibody assay (DFA), viral culture, and molecular detection. Rapid antigen tests are immunochromatographic assays that have been used for decades and have been favored due to their fast time to result (~15 minutes). However, as diagnostic methods have improved and circulating strains have changed, studies have shown that these tests suffer from lack of sensitivity. Sensitivities down to 10% were reported during the 2009 pandemic. Typical ranges of sensitivity reported are 20 to 90% depending on the strain circulating and the method used as the reference method. A further concern is the positive predictive value of rapid antigen tests when used outside of peak influenza season. Since positive predictive value is dependent on the prevalence of disease, using a test with imperfect specificities (90 to 95%) during times of low prevalence increases the chance that a positive result may actually be false positive rather than true positive. However, the times when laboratory testing for influenza is the most helpful clinically are at the beginning and end of the epidemic season, when the differential diagnosis is much broader. Another rapid method (~2 hours) is DFA testing. DFA uses a pool of monoclonal antibodies to influenza and other common respiratory viruses to directly detect infected cells obtained from the nasopharynx of patients. Although it is more sensitive than rapid antigen tests, DFA also had decreased sensitivity (~47%) for detecting the 2009 H1N1 pandemic strain. DFA sensitivity and specificity are also dependent on the skill of the personnel performing the test. Therefore, if rapid antigen tests or DFA must be used, alternative methods should be available to confirm the results, as needed.
Viral culture sensitivity is virus specific and ranges from 80 to 95%. Specificity approaches 100%. The disadvantage to culture is its longer turnaround time (up to 7 days). Rapid shell vial cultures have decreased the time to result to 24 to 48 hours, but this is still not adequate to aid in treatment decisions for influenza, which must occur in the first 48 hours of illness for the greatest benefit. Nonetheless, it is important for public health laboratories to maintain the capability of culturing influenza so that epidemiologic typing and resistance testing can be performed to inform next year’s vaccine components and antiviral recommendations.
The increase in molecular testing for influenza has been largely due to the limitations outlined above for other methods. Several FDA-cleared assays exist for the molecular detection of influenza with turnaround times ranging from 20 minutes to 8 hours. Sensitivities of these tests are 90 to 99%, with specificities of 98 to 99%. Some of the tests can also type influenza (i.e., H1, H3, or 2009 H1N1), and others can detect other respiratory viruses simultaneously. However, the majority of these tests require significant laboratory expertise and are more expensive than the other diagnostic methods listed. Since influenza genomic sequences change rapidly, it is important to monitor the accuracy of molecular tests on an annual basis. The curves shown in Fig. 10.1 represent the increase in fluorescence during real-time detection of PCR amplification.
A fluorescent probe is incorporated into the PCR reaction to measure on a per-cycle basis the presence of amplicons. Once the level of fluorescence is higher than the background level, the sample is positive. A lower cycle number of positivity (the point at which the curve crosses the horizontal threshold line) indicates a greater amount of virus in the sample. The positive result for the patient is shown by the gray line. The cycle threshold (Ct value) for the positive result is displayed by the red vertical line (27.3) and represents the cycle at which the fluorescence from the real-time PCR detection exceeds background. An example of a negative result is depicted by the purple line. The horizontal red line represents the threshold required for positivity in the PCR.
4. Most cases of influenza in this age group are self-limited and do not require hospitalization. Influenza is a much greater threat to individuals >65 years of age and children <5 years old. During most epidemics the highest numbers of hospitalizations and deaths are in these age groups. Other individuals at risk for complications of influenza infection are those with underlying chronic pulmonary diseases, such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease; immunocompromised individuals; pregnant women, particularly in the second and third trimesters; and those with a variety of other chronic conditions such as cardiovascular disease and diabetes. This patient was a smoker and had diabetes, both of which put her at increased risk for severe influenza disease. In 2009, obesity was shown to be an independent risk factor for increased mortality due to H1N1. During the pandemic there was still significant disease and mortality in pediatric patients, with more than double the number of pediatric patients dying than in the previous three influenza seasons. Interestingly, the death rate for those 25 to 49 years of age was greatly increased as well, but little disease was seen in those >55 years of age. This suggests that influenza strains circulating prior to 1955 provided some protection against the pandemic strain, which was confirmed by serologic surveillance studies.
5. The most common complication leading to increased morbidity and mortality is pneumonia. This could be primary influenza virus pneumonia, secondary bacterial pneumonia, or a combination of the two. The majority of reported influenza-associated deaths appear to be due to influenza with accompanying bacterial pneumonia, especially pneumonia caused by Streptococcus pneumoniae and Staphylococcus aureus. For this patient, we cannot determine whether she has influenza pneumonia or bacterial pneumonia. To differentiate these, we would need a lower respiratory specimen (preferably a bronchoalveolar lavage) obtained prior to antibiotic administration to culture for bacteria and test for influenza. The sputum specimen obtained from this patient was rejected as inadequate for culture because there were no neutrophils present, suggesting a poor specimen collection. Thus, she was treated empirically for bacterial pneumonia.
6. There are currently two classes of anti-influenza drugs. The first class of agents, M2 inhibitors, blocks formation of influenza-derived ion channels. The reason these virally derived ion channels are important is that they play an important role in the “uncoating” of the virus. This is a step in viral replication in which viral RNA is released from the viral particle and enters the cytoplasm of the cell. The two drugs in this class are the oral agents amantadine and rimantadine. The drugs must be administered