Conceived and designed the experiments: TMLS JIFS FAB MTP VPM LVG AW SR VPM EV MMS. Performed the experiments: TMLS JIFS MM MS FCM MTP MdLO VPM LVG AW SR VPM EV. Analyzed the data: TMLS JIFS FAB MM MS FCM MTP MdLO VPM LVG AW SR VMCG VPM EV MMS. Wrote the paper: TMLS JIFS FAB MTP MMS.
¶ These authors also contributed equally to this work.
The authors have declared that no competing interests exist.
The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 in Brazil.
We performed a prospective single-center cohort study in a cancer center in Rio de Janeiro, Brazil. Hospitalized patients with cancer and a confirmed diagnosis of influenza A H1N1pdm were evaluated. The main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of H1N1pdm susceptibility to oseltamivir.
A total of 44 hospitalized patients with suspected influenza-like illness were screened. A total of 24 had diagnosed H1N1pdm infections. The overall hospital mortality in our cohort was 21%. Thirteen (54%) patients required intensive care. The median age of the studied cohort was 14.5 years (3–69 years). Eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. A total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant H1N1pdm virus. Viral evolution was observed in sequentially collected samples.
Prolonged influenza A H1N1pdm shedding was observed in cancer patients. However, oseltamivir resistance was not detected. Taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation.
The emergence of the novel influenza A/H1N1 pandemic virus (H1N1pdm) significantly affected the utilization of healthcare resources and increased morbidity and mortality in children and young adults
Emerging data on the clinical course of severe H1N1pdm infection have allowed the identification of high-risk groups, which include pregnant women and patients with morbid obesity
Because the analysis of this novel viral infection in cancer patients is an important component of the 2009 pandemics, we conducted a prospective cohort study aimed at evaluating the clinical course of influenza infection, the duration of viral shedding, H1N1pdm evolution and the emergence of antiviral resistance in hospitalized cancer patients with a severe H1N1pdm infection in a reference cancer center during the winter of 2009 in Brazil.
During the study period, 44 hospitalized cancer patients with a suspected influenza infection were screened, and 24 had a confirmed influenza A diagnosis using a rapid indirect immunofluorescence (IFI) test or World Health Organization (WHO)-recommended real-time RT-PCR (rRT-PCR) (
Patients diagnosed with H1N1pdm were young (median age = 14.5, range 3–69 years). In total, 14 (58.3%) were under 18 years old, and 17 (70.8%) were less than 50 years old. Hematologic cancer occurred in 75% (18) of the patients, whereas solid tumors occurred in 25% (6) patients (
Variable | All patients (n = 24) | Survivors (n = 19–79.2%) | Non-survivors (n = 5–20.8%) | |
14.5 (3–69) | 14 (3–69) | 17 (4–62) | 0.50 | |
12 (50%) | 7 (36.8%) | 5 (100%) | 0.03 | |
6 (25%) | 5 (26.3%) | 1 (20%) | 0.99 | |
18 (75%) | 14 (73.7%) | 4 (80%) | 0.99 | |
3 (12.5%) | 3 (15.8%) | 0 | 0.99 | |
9 (37.5%) | 6 (31.6%) | 3 (60%) | 0.32 | |
12 (50%) | 10 (52.6%) | 2 (40%) | 0.99 | |
6 (31.6%) | 6 (31.6%) | 0 | 0.28 | |
18 (75%) | 13 (68.4%) | 5 (100%) | 0.28 | |
18 (75%) | 13 (54.2%) | 5 (100%) | 0.28 | |
14 (58.3%) | 10 (52.6%) | 4 (80%) | 0.36 | |
14 (58.3%) | 7 (36.8%) | 3 (60%) | 0.67 | |
10 (41.6%) | 5 (26.3%) | 5 (100%) | 0.06 | |
8 (33.3%) | 4 (21.1%) | 4 (80%) | 0.03 | |
7 (0–19) | 9 (1–19) | 5 (1–18) | 0.38 |
Results are expressed as the mean ± standard deviation, median (range), n (%).
Reported
A total of 23 (95.8%) patients were treated with oseltamivir, and the median time from the initial symptoms to the initiation of therapy was three days (0–15 days;
At presentation, all patients were treated with broad-spectrum intravenous antimicrobial agents to combat community-acquired pneumonia and/or febrile neutropenia
Overall, 13 patients (five adults and eight children) were admitted to the ICU. Six patients were directly admitted from the emergency department, and the other seven patients were transferred from other hospital wards (
Of the 13 critically ill patients, 12 were treated with oseltamivir, and treatment was initiated 48 h after the first signs/symptoms of viral infection in 5 of them. Adjunct or non-conventional supportive therapies for ARDS were performed for 12 of the 13 patients that entered the ICU (92.3%). A total of 10 patients (76.9%) received systemic corticosteroids (eight due to previous use and two for shock and persistent ARDS); five (38.5%) were ventilated in a prone position, and four (30.8%) required recruitment maneuvers. No patient received extra-corporeal membrane oxygenation.
Although prolonged influenza A shedding has been observed for a cancer patient infected with the H3N2 seasonal virus
Most importantly, the maximum duration of H1N1pdm shedding in our investigation was 63 days, followed by 44 days for another patient. To our knowledge, these periods constitute the longest registered cases of H1N1pdm shedding described to date. All rRT-PCR-positive samples from these patients with the longest viral shedding durations (5645 and 5899) were culturable, meaning that these were infectious viruses (
To date, no significant variation has been detected at the amino acid level in the hemagglutinin (HA) of the 2009 pandemic virus
To evaluate the genetic characteristics of the isolates from patients with prolonged viral shedding, we sequenced two consecutive samples from a single individual (5645s2/09 and 5645s3/09) and compared their HA sequences to other H1N1pdm viruses from mild, severe and fatal cases from different countries. We observed that samples collected a month apart (5645s2/09 and 5645s3/09) clustered together and displayed a relatively large branch length from other H1N1pdm viruses (
The bootstrap probability is indicated for each interior branch, and values below 80% are hidden. The scale bar indicates the number of amino acid changes per site. The sampling number and the state of origin in Brazil are displayed. This tree is rooted by the California/04/2009 HA sequence.
Considering that influenza resistance to antivirals is likely to emerge in immunocompromised individuals and that, with respect to the H1N1pdm virus, only a few studies have detected oseltamivir resistance
Prolonged influenza shedding in cancer patients has been observed for seasonal strains
Unlike previous reports, our population was composed of hospitalized, severely immunocompromised cancer patients
Interestingly, during the influenza season, 14 patients (58.3%) with febrile neutropenia were identified as H1N1pdm cases, a condition that is not usually investigated in this scenario. However, febrile neutropenic cancer patients have an increased risk of developing respiratory distress and multi-organ failure. Therefore, screening for respiratory viruses and prompt initiation of oseltamivir treatment should be considered in these patients. Febrile neutropenia indicates a poor prognostic with respect to a patient's outcome, but neutropenia duration in our cohort of patients was less than seven days. Thus, prolonged viral shedding might not have a correlation with neutropenia.
We observed the persistence of H1N1pdm in 5 out of 10 patients studied for this purpose. In these individuals, viral shedding continued for at least 11 days, despite the use of oseltamivir. The median duration of viral shedding in our population was 23 days, and two pediatric patients with acute lymphoblastic leukemia showed even longer virus secretions (44 and 63 days;
Influenza shedding is not considered to last long, and it disappears seven days after the onset (2.4 and 4.5 days for oseltamivir- and placebo-treated groups, respectively)
General Population | Hospitalized | Immunocompromised | |||||||
Households, Hong Kong | Containment of the pandemics in Ho Chi Minh City (HCMC), Vietnam | Households, Canada | Travelers, France | Military cadets | First 426 patients hospitalized in China | No | No | No | |
ND | ND | ND | No | ND | ND | Yes | Yes | Yes | |
ND | ND | ND | No | ND | Various | Transplant recipients | Leukemia | Cancer | |
54 | 932 | 43 | 2 | 29 | 350 | 6 | 2 | 10 | |
2 children | ∼80 | 8–14 |
2 | 7 | 238 | 1 | 2 | 5 | |
8 | 11–12 |
8–11 |
14–28 |
9 | 17 | 11 | 37–44 |
63 |
– Five to six days for children under nine years old.
– Eight patients shed the virus for at least 8 days, while 14 shed for at least 11 days.
– Only two patients were evaluated.
ND – Not determined.
Our results highlight the need for closer surveillance of cancer patients with H1N1pdm infections until the detection of the first negative sample. We hypothesize that follow-up protocols aimed at monitoring the persistence of viral shedding in cancer patients may be relevant if patients are submitted to immunosuppressive therapies in the days or weeks prior to or following an H1N1pdm infection.
Next, we sought to determine viral evolution during prolonged shedding. We found that some amino acid changes persisted from the initial symptoms until 30 days thereafter, suggesting that these patients had viral persistence rather than re-infection. In addition, an extra amino acid change (D238P) was found in the viral HA sequenced a month after the onset of illness, suggesting continuing viral evolution. Although some of the mutations that we found (L52S, L70P, P100S, C153L, T214A, D238P, Q293R and I321V) in strains 5645s2/09 and 5645s3/09 have not been previously described, other amino acid residue changes that were detected in our study (P100S and T214A) have been found in H1N1pdm viruses throughout the world without a significant link to viral pathogenesis or antigenic variation
Influenza viruses resistant to antiviral drugs have been reported in immunocompromised patients,
The apparent paradox of prolonged viral shedding without antiviral resistance could be explained by either the inability of the immunocompromised host to effectively clear the H1N1pdm virus
Although our work further investigates the unique dynamics of H1N1pdm virus infection in immunocompromised hosts, some caveats must be noted. Because our investigation started during a new pandemic, the clinical evaluation and management protocols changed during the course of the study as new data emerged from the literature and from updated recommendations
In conclusion, this study provides evidence that severe H1N1pdm infection is associated with significant morbidity and mortality in cancer patients. In these patients, viral persistence without the emergence of antiviral resistance may occur during the clinical course of the disease. This result has major implications for the clinical management of H1N1pdm infections and infection control strategies. Our study may provide insights into H1N1pdm shedding and might contribute to the development of new guidelines to manage cancer patients with H1N1pdm infection.
The Ethics Committee (Comitê de Ética em Pesquisa; CEP;
This was a prospective cohort study conducted in the Hospital do Câncer-I, Instituto Nacional de Câncer (HC-I-INCa), Rio de Janeiro, Brazil, from July 8th to October 1st, 2009. The HCI-INCa is a 160-bed comprehensive cancer center primarily for the population of Rio de Janeiro and neighboring states. The present study was strictly observational, and every clinical decision was at the discretion of the attending physician.
All patients with a definite diagnosis of cancer requiring hospital admission for any reason and who displayed influenza-like illness were evaluated. Patients in complete remission from cancer for more than five years were not considered.
Data were collected using a standardized case report form that included demographic data, clinical presentation, comorbidities, cancer status, use of immunosuppressive therapies, time course of acute illness, need for intensive care, use of antivirals, adjunctive therapies, advanced life support and in-hospital mortality (supporting information; SI). Patients were included if they had a fever (>37.8°C) and/or respiratory influenza-like illness and confirmed influenza A H1N1pdm diagnosis (by at least one of three assays, IFI, rRT-PCR or cell culture, and according to case definitions from the WHO
Nasopharyngeal Dacron-swab specimens were collected from all patients and placed onto transport medium (Hanks solution with 100 U/mL penicillin and 100 µg/mL streptomycin) at the initial evaluation. Tracheal aspirates were also obtained if the patient required tracheal intubation. Patients' clinical samples were directly tested for a panel of respiratory viruses using an IFI assay for influenza A (respiratory virus panel; Biotrin, Mount Merrion, Co. Dublin, Ireland.). Specimens were also sent to the Brazilian National Influenza Center (IOC/Fiocruz) for H1N1pdm confirmation using rRT-PCR, which was performed in accordance with the current guidelines from the WHO/CDC
Blood samples were routinely sent for bacterial culturing, as were tracheal aspirates if the patient was intubated.
Standard descriptive statistics were used to describe the study population. Continuous variables were reported as the mean ± standard deviation or median (range) as appropriate. Univariate analysis was used to identify factors associated with hospital mortality. Two-sample
Clinical investigation and Influenza virus assays.
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Diagnostic tests for Influenza A virus.
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Patient's characteristics and outcomes according to age range.
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Prevalence of underlying malignancies.
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Use of Chemotherapy, corticosteroids and granulocyte colony stimulating factor previous to H1N1pdm infection.
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Patient's Neutropenia.
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Oseltamivir treatments.
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Focus of bacterial infection in cancer patients with Influenza A H1N1pdm.
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Bacteria isolates from cancer patients with Influenza A H1N1pdm.
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Organ Dysfunctions 72 h after H1N1pdm diagnosis.
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Frequency of signs and symptoms at clinical suspicion.
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Pulmonary infiltrates at Influenza diagnosis and hospital discharge, by chest radiography.
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Clinical and Viral Characteristics of the followed-up cohort.
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Phylogenetic tree of HA gene from the classical swine, Eurasian swine, American Avian and human seasonal lineages. The bootstrap probability is indicated for each interior branch, all values below 80% are hidden. The scale bar indicates the number of amino acid changes per site. Colored circles indicate the samples from our study. This tree is unrooted. Each Influenza HA lineage is displayed beside their respective clade.
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Phylogenetic tree of NA gene from the followed-up cohort. The bootstrap probability is not indicated for each interior branch since it is below 85%. The scale bar indicates the number of amino acid changes per site. The tree is rooted by California/07/2009 NA sequence.
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We are indebted to the Instituto Nacional de Câncer, Fiocruz, FAPERJ and CNPq for their support. The authors thank Marc-Alain Widdowson (CDC/CCID/NCIRD) for critical review of the manuscript. Alicia Fry (CDC Atlanta epidemiology intelligence officer) was also extremely helpful in discussions on virus shedding.