Here is a study describing how minor populations can be missed in standard tests and what you can find if you go and look carefully enough.
http://content.nejm.org/cgi/content/full/353/25/2667EXTRACT
Influenza A (H5N1) virus infection was diagnosed in another local laboratory by RT-PCR assay of a pharyngeal swab obtained at admission. This specimen was not available for further analysis. In our laboratory, influenza A (H5N1) virus was isolated from a throat swab obtained from Patient 1 on the fourth day of oseltamivir treatment (January 25, 2005).
Sequence analysis of the neuraminidase gene revealed the substitution of tyrosine for histidine at amino acid position 274 (H274Y), associated with high-level resistance to oseltamivir in influenza (N1) viruses.10 Analysis of the raw sequencing traces revealed the presence of a minor subpopulation of wild-type 274H variants among predominating 274Y mutants (Figure 2). Virus was also isolated from a throat specimen obtained on January 28, 2005, two days after the completion of treatment.
Sequence analyses of this strain as well as of viral RNA extracted directly from the swab also revealed the H274Y change in N1. Although sequencing traces also revealed the presence of a minor wild-type 274H population in viral RNA from the swab, only 274Y variants were observed in the isolate, possibly reflecting overgrowth of the predominant mutant population during culture. Determination of influenza A (H5N1) RNA levels showed that the viral load had increased in the second specimen
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Influenza A (H5N1) virus was isolated from throat swabs obtained at admission from six of the seven patients (Patients 2, 3, 5, 6, 7, and 8).
Sequence analysis of the neuraminidase genes of these viruses revealed the wild-type 274H residue alone. Measurements of the viral RNA load in sequential throat specimens showed rapid declines to undetectable levels in four patients who survived,
whereas viral RNA was still detectable at the completion of oseltamivir treatment in two patients who died (Patients 3 and 4 in Figure 3). The remaining patient died during the second day after admission, at which time an increase in the viral RNA load was observed (Patient 2 in Figure 3).Direct sequencing revealed only wild-type 274H virus in the second specimen from this patient.All subsequent throat specimens from Patients 3 through 8 were cultured. Of these specimens, influenza A (H5N1) virus was isolated only from the last specimen from Patient 4, obtained three days after the completion of treatment (Figure 3).
Sequence analysis of this isolate revealed the H274Y substitution in N1. Although sequencing traces of the isolate revealed only mutant 274Y variants, direct sequencing of viral RNA from the same swab revealed evidence of a minor subpopulation of wild-type 274H viruses similar to that in specimens from Patient 1. Patient 4 died of respiratory failure six days after the isolation of resistant virus. Direct sequences of viral RNA from swabs obtained at admission and after two days of treatment showed wild-type 274H virus alone. The limited sensitivity of the method precluded direct sequencing of further samples from this patient. Likewise, no direct sequences could be obtained from the last specimen obtained from Patient 3.
Discussion
We report the isolation from two Vietnamese patients of influenza A (H5N1) viruses with a H274Y substitution in the neuraminidase gene, which confers high-level resistance to oseltamivir.10,11 In contrast to the recent report of a partially resistant influenza A (H5N1) virus isolated during once-daily prophylactic treatment with oseltamivir,11 the viruses in our patients were isolated
during or shortly after a course of oseltamivir at therapeutic twice-daily doses, and mutant 274Y variants predominated. Furthermore, although the patient with partially resistant virus ultimately received oseltamivir at therapeutic doses and survived,11 both of our patients died.
Patient 1 was treated with doses of oseltamivir that were relatively high for her weight, especially during the first day of treatment. Moreover, in this patient, unlike most patients with influenza A (H5N1) virus infection, treatment was started when the greatest clinical benefit could be expected: within 48 hours after the onset of symptoms. Indeed, her clinical condition remained stable during the first three days of treatment without the need for supplemental oxygen.
However, on the fourth day of treatment she became progressively dependent on oxygen, her white-cell and platelet counts fell, and there was laboratory evidence of hepatitis. At the time of her death, the viral load in her throat had increased. These observations suggest that the development of drug resistance contributed to the failure of therapy and, ultimately, the death of this patient. In the second patient, the viral RNA load declined during treatment, but not to undetectable levels. Whereas only wild-type 274H virus was detectable after two days of treatment, 274Y mutant virus was isolated shortly after treatment. Although a direct relationship between the emergence of resistance and this patient's death was less clear, the presence of replicating virus after 14 days of illness suggests an effect on the outcome.
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