The authors have declared that no competing interests exist.
Conceived and designed the experiments: HP ST KD. Performed the experiments: MT ST HP. Analyzed the data: ST OR XdL PG HP KD. Wrote the paper: KD ST HP. Reviewed/edited the manuscript: ST AF XdL FC PG HP KD.
The aim of the present study was to weigh up, at the community level, the respective roles played by pandemic
A population-based prospective cohort study was conducted in Reunion Island during the austral winter 2009 (weeks 30–44) that allowed identification of 125 households with at least one member who developed symptoms of
Two successive waves of viral infections were identified: a first wave (W33–37) when pH1N1 was dominant and co-circulated with NIRVs, sharply interrupted by a second wave (W38–44), almost exclusively composed of NIRVs, mainly human
Our results suggest that NIRV co-infections during
Several respiratory viruses cause
In April 2009, an epidemic caused by a novel triple-reassortant
Only few studies have investigated the full range of respiratory viruses competing for ILIs in the setting of the pH1N1 pandemic
We report herein the results of a virological investigation conducted in the frame of the population-based CoPanFlu-Run cohort study. During the study period, reports of ILIs in households triggered testing for pH1N1 and NIRVs in identified individuals and all members of the same household.
Several research questions stand behind this study that are of paramount importance to the understanding of the dynamics of pH1N1 infection: i) Which NIRVs have been co-circulating with pH1N1 in the community during the course of the pandemic wave? ii) How did NIRVs spread among the different age groups before, during, and after the pandemic wave? iii) Did these viruses have significant impact on the transmission of pH1N1 between and within households? iv) Did NIRVs impact on the ultimate seroconversion rate to pH1N1?
The prospective CoPanFlu-Run cohort study (772 households, 2,164 individuals) was conducted during the austral winter 2009 in Reunion Island (for details on study design, see
ILI was defined as documented fever (≥37.8°C) with at least one symptom of Upper Respiratory Tract Infection (URTI): sore throat, cough, running and/or stopped nose, or a systemic symptom (aching). The clinical profiles of household members were assessed during the three systematic visits aimed at collecting nasal swabs and were categorized as ILIs, URTIs or asymptomatic. Familial clusters were defined as two or more members of the same household sharing the same virus. Viral co-circulation was defined as more than one virus detected in two members of the same household. A co-infection was defined as more than one virus present in the same patient, either together in the same swab, or separately in two or three consecutive swabs obtained from this individual within an 8-day.
All nasal swab samples were spiked before nucleic acid extraction with an MS2 RNA phage as internal control
The Seeplex RV15 ACE multiplex kit (Seegene) was used according to manufacturer’s recommendations to simultaneously amplify by RT-PCR, specific genomic sequences belonging to 15 human respiratory viruses. The kit detects the following viruses: InfA and InfB, hRSV-A, and hRSV-B, hRV-A,B,C, hMPV, hBoV 1,2,3,4, hCoV-229E/NL63, hCoV-OC43/HKU1, hPIV-1, hPIV-2, hPIV-3, and hPIV-4, ADV and EV
In addition to the 15 viral targets of the Seeplex 15 viral detection kit, described above, the Seeplex product used in the study, contained a pH1N1-specific detection system proposed by the manufacturer as a separate and unique set. At the time we have undertaken this study (i.e. 2010–2011), and as noted by others
Duration of virus shedding was defined as the time between the appearance of symptoms and the last positive nasal swab. Alternatively in case of co-infection, virus shedding was directly estimated on the number of nasal swabs positive for the considered virus.
Positive RT-PCR products from the pH1N1 and from the other respiratory viruses were purified on a 2% gel electrophoresis using the QIAquick gel extraction kit (Qiagen), cloned in a pGEM-T Easy vector (Promega) according to the manufacturer’s instructions, and sequenced. Sequence data were analyzed using the Geneious Pro 5.3.4 software
Specific antibodies to pH1N1 2009 were tittered using hemagglutination inhibition assay (HIA) as described previously
Proportions were compared using Pearson Chi square test or Fisher exact test as appropriate. Age distribution was categorized in four classes (<20 yrs, 20–39 yrs, 40–59 yrs, ≥60 yrs), as done previously in the CoPanFlu-Run cohort
The prospective CoPanFlu-Run cohort study (772 households, 2,164 individuals) was conducted during the austral winter 2009 in Reunion Island in accordance to the principles expressed in the Declaration of Helsinki and French law for biomedical research (N° ID RCB AFSSAPS: 2009-A00689-48). Every eligible person for participation was asked for giving their written informed consent. We obtained informed written consent from the next of kin on the behalf of the minors/children participants involved in the study.
We identified 125 households (totalizing 443 individuals, M/F ratio = 0.86) with at least one member who developed ILI symptoms during the study period (W30–44). The distribution of the households according to ILI case definition is displayed in
Individuals tested for NIRV(a) | CoPanFlu-RUN cohort(b) | Reunion Island population(c) | |
N (%) | N (%) | N (%) | |
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182 (41.1%) | 697 (32.2%) | 281,680 (35.0%) |
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117 (26.4%) | 495 (22.9%) | 224,689 (27.9%) |
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116 (26.2%) | 614 (28.4%) | 207,738 (25.8%) |
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28 (6.3%) | 358 (16.5%) | 91,393 (11.3%) |
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28.7+/−19.8 | 36.1+/−22.5 | 31.7 |
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205 (46.3%) | 1003 (46.3%) | 390,645 (48.5%) |
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238 (53.7%) | 1161 (53.7%) | 414,855 (51.5%) |
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443 | 2,164 | 805,500 |
Data are numbers and percentages, or means and standard deviations.
Age comparisons a
Gender comparisons a vs b : P = 0.97; a vs c : P = 0.349.
A total of 1196 nasal swabs were analyzed corresponding to 335, 88 and 20 individuals who had three (75.6%), two (19.9%) or one (4.5%) nasal swabs respectively. Considering that determinants of viral transmission between households likely differed from those within households, we analyzed the data of the viro-survey at both the household and the individual levels.
Out of the 125 households, pH1N1 virus was found 36 (28.8%) times in at least one nasal swab from at least one household member. All detected InfA viruses were of the pandemic type and no InfB virus was found. In contrast, NIRVs were frequently detected, essentially hRV, hCoV, and hPIV in 37 (29.6%), 36 (28.8%) and 18 (14.4%) households, respectively. Other viruses were less frequently found and concerned a subset of 18 households: hMPV (n = 5), ADV (n = 5), hRSV (n = 5), EV (n = 2), and hBoV (n = 1) (referred as “Other NIRVs”). The number of detected viruses was 1, 2, 3, 4 or more viruses in 55, 32, 6 and 2 households, respectively. Thirty households (24.0%) tested negative for all 16 respiratory viruses.
In order to evaluate the specificity of nucleic acid amplification by the Seeplex kit, we cloned and sequenced the amplified material identified as hRV (n = 22), hCoV (n = 20), pH1N1 (n = 15), hPIV (n = 7), hMPV (n = 7), EV (n = 1), hRSV (n = 1) and hBoV (n = 1). Sequencing and Blast analysis confirmed that each amplified sequence could be correctly ascribed to its respective virus confirming the high specificity of the Seeplex Kit
Out of 443 individuals, 194 (43.8%) had at least one respiratory virus detected. pH1N1 was documented in 62 (14.0%), hRV in 60 individuals (13.5%), hCoV in 55 (12.4%) and hPIV in 26 (5.9%). “Other NIRVs” concerned a total of 22 individuals with the same distribution as reported in households except for hMPV which concerned 9 individuals living in 5 households. M/F sex ratios were 1.3, 1.0, 0.9, 1.3, 2.1 for pH1N1, hRV, hCoV, hPIV, and “otherNIRVs”, respectively, and 0.7 in the group of individuals tested negative to all viruses (non significant differences).
Incidences were negatively associated with age (
pH1N1(a) | hRV(b) | hCoV | hPIV | Other NIRVs(c) | negative(d) | |
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43 (69.4%) | 37 (61.7%) | 22 (40.0%) | 10 (38.5%) | 17 (77.3%) | 74 (29.7%) |
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11 (17.7%) | 13 (21.7%) | 17 (30.9%) | 8 (30.8%) | 1 (4.5%) | 73 (29.3%) |
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7 (11.3%) | 8 (13.3%) | 14 (25.5%) | 6 (23.0%) | 2 (9.1%) | 82 (32.9%) |
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1 (1.6%)* | 2 (3.3%)* | 2 (3.6%) | 2 (7.7%) | 2 (9.1%)* | 20 (8.0%) |
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Data are numbers and percentages.
Statistical significance was set at P = 0.05 for all analyses, except for
Familial clusters (i.e. two or more members of the same household sharing the same virus), were observed in 13 households out of 36 (36.1%) for pH1N1, in 14/37 households (37.8%) for hRV, in 12/36 households (33.3%) for hCoV, in 5/18 households (27.8%) for hPIV and in 3/18 (16.7%) for “other NIRVs”, respectively (Chi square test 4 ddl: 2.95,
We also checked whether pH1N1 and NIRVs could mutually interfere for the occurrence of familial clusters. No statistically significant differences could be demonstrated (data not shown).
The temporal dynamic of pH1N1 and NIRVs is shown in
Charts depict the total number of households (
During W33–37, at least one different NIRV co-circulated with pH1N1 in 21 households out of the 34 where the pandemic virus was detected (hRV, hCoV, hPIV, and “other NIRVs”, in 8, 8, 3 and 2 households respectively). Data were stratified according to the presence or absence of pH1N1 virus and analyzed for the presence or absence of NIRVs. Similarly, data were ranked according to the detection of hRV, hCoV or hPIV in the nasal swabs at day 0 and analyzed for the presence or absence of pH1N1 during the eight days of nasal sampling. Despite the fact that pH1N1 appeared to more readily infect households where hRV did not circulate (and
Out of the 194 individuals who were detected positive for at least one respiratory virus (pH1N1 or NIRV), 31 individuals had evidence of viral co-infection (
pH1N1 | hRV | hCoV | hPIV | ADV | EV | hRSV | hMPV | hBoV | |
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– | 1 | 3 | 1 | 0 | 0 | 0 | 0 | 0 |
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3 | – | 3 | 1 | 1 | 0 | 0 | 1 | 0 |
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2 | 1 | – | 1 | 0 | 1 | 0 | 0 | 0 |
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1 | 2 | 2 | 1 | 0 | 0 | 0 | 0 | 0 |
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0 | 2 | 1 | 0 | – | 0 | 0 | 0 | 0 |
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0 | 1 | 0 | 0 | 0 | – | 0 | 0 | 0 |
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0 | 0 | 1 | 0 | 0 | 0 | – | 0 | 0 |
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0 | 0 | 0 | 0 | 0 | 0 | 0 | – | 0 |
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0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | – |
Number of individuals co-infected with combinations of different viruses: Above the diagonal; individuals that tested positive for more than one virus in consecutive swabs (8 days apart). Below the diagonal; individuals that tested positive for more than one virus in the same swab.
Of 335 individuals for whom three nasal swabs were obtained on day 0, 3 and 8, the mean duration (+/−SD) of viral shedding was 7.6 (+/−3) days for pH1N1, 10.4 (+/−5.1) days for hRV, 7.8 (+/−3.8) days for hCoV and 9.3 (+/−3.2) days for hPIV. We then estimated the duration of viral shedding for pH1N1-NIRVs co-infections among the samples detected positive for any virus. Out of eleven individuals positive for pH1N1 that had evidence of co-infection, the pH1N1 virus was detected in one single swab for seven individuals, in two swabs for two, and in three swabs for two. With regard to viral shedding of co-infecting NIRVs, they were detected in one single swab in ten cases, and in three swabs in one case. When pooled together, these data indicate that the duration of pH1N1 and NIRVs viral shedding was significantly shorter in patients with co-infection compared to those with single infection (data not shown). Hence, viral positivity restricted to only one single swab was found in 16/20 (80.0%) episodes of viral co-infection but in only 87/157 (55.4%) episodes of single viral infection (Chi square test 1 ddl: 4.40,
The clinical picture of 367 individuals for whom data were available was correlated to virus detection at day 0, after exclusion of co-infections. Almost ninety percent (35/39) of individuals with RT-PCR documented pH1N1 infection were symptomatic (i.e. either ILI or URTI) and most of them (31/39) had fever. Patients with NIRVs reported fever less frequently (26/62) compared to those with pH1N1 infection (41.9% vs. 79.5%; RR: 0.56, 95%CI: 0.14−0.76,
During the epidemic upsurge (W33–37) almost half of patients with ILIs were pH1N1 positive, 16.5% had documented NIRVs and 35.3% were negative. Of these three categories, the proportions of patients that reported URTIs were 19.5%, 26.2% and 54.8% respectively (
pH1N1(a) | hRV(b) | hCoV(c) | hPIV(d) | Other NIRVs(e) | Negative swab(f) | |
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0 (0.0%) | 0 (0.0%) | 2 (40.0%) | 0 (0.0%) | 0 (0.0%) | 3 (60.0%) |
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0 (0.0%) | 0 (0.0%) | 2 (66.7%) | 0 (0.0%) | 0 (0.0%) | 1 (33.3%) |
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0 (0.0%) | 1 (10.0%) | 1 (10.0%) | 0 (0.0%) | 1 (10.0%) | 7 (70.0%) |
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41 (48.2%) | 7 (8.2%) | 4 (4.7%) | 2 (2.4%) | 1 (1.2%) | 30 (35.3%) |
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8 (19.0%) | 5 (11.9%) | 5 (11.9%) | 1 (2.4%) | 0 (0.0%) | 23 (54.8%) |
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1 (1.4%) | 3 (4.3%) | 3 (4.3%) | 0 (0.0%) | 2 (2.9%) | 61 (87.14%) |
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2 (3.6%) | 13 (23.2%) | 6 (10.7%) | 5 (8.9%) | 3 (5.4%) | 27 (48.2%) |
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0 (0.0%) | 8 (14.6%) | 9 (16.4%) | 3 (5.4%) | 3 (5.4%) | 32 (58.2%) |
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0 (0.0%) | 6 (7.1%) | 5 (5.9%) | 6 (7.1%) | 2 (2.4%) | 66 (77.5%) |
Number of individuals (percentages) with pandemic
Data correspond to individuals included in the cohort during weeks 30–32 (T0), weeks 33 to 37 (T1) or weeks 38–44 (T3).
Comparisons of clinical expression between T1 and T2 for each virus: pH1N1:
Seroconversion rates were deduced from antibody titration to pH1N1 on paired blood samples collected from each individual. For the present study, we considered only the 170 pairs of sera for which the first blood sample was collected during the transmission window (W33–37) and the second at the end of the study, after W45. We correlated the seroconversion rates of these pairs to results of the viro-survey. Interestingly, the seroconversion rate to pH1N1 was 67.6% (23/34) for those tested pH1N1+/NIRVs–; 39.2% (38/97) for those tested pH1N1−/NIRVs–; 46.7% (14/30) for those tested pH1N1−/NIRVs+ and 77.8% (7/9) for those tested pH1N1+/NIRVs+. Hence, serologic data indicate that the pandemic virus has largely spread among the study population during the epidemic upsurge and had induced protective immunity, regardless of the infecting virus detected by molecular amplification.
Our viro-survey confirmed the very fast passage of the pandemic
The highest rate of pH1N1 detection was recorded in young individuals, confirming our previous observations based on serologic data
Familial clustering of pH1N1 or hRV or hCoV was observed in almost one third of positive households. Most interestingly, NIRVs were not uniformly distributed over time as they were most commonly detected just after the passage of the
Viral co-infection during W33–37 concerned less than 5% of investigated individuals, a low incidence which has been similarly observed in other studies
pH1N1 and NIRVs tended to be mutually exclusive when co-circulating together within the same household: in such cases, NIRVs were more likely to infect pH1N1 negative individuals than pH1N1 positive peers (RR: 3.13). Similar trends were also observed with hRV and hPIV, though they did not reach statistical significance, possibly due to the small sample size.
The mean total duration of pH1N1 viral shedding in our population-based cohort was estimated at 7.6 (+/−3) days for pH1N1, a figure consistent with those reported in the literature among hospitalized or pediatric patients
Interestingly, when data recorded for pH1N1 and NIRVs were pooled together regardless of the specific virus, the duration of viral shedding was significantly shorter (
This study has outlined various features of the dynamics of viral co-circulation during the studied epidemic: i) pH1N1 and NIRVs (especially hRV), though largely overlapping, essentially spread during two successive waves; a pH1N1 wave followed by a hRV wave that peaked on W34 and W38, respectively. ii) During W33–37, when pH1N1 and NIRV waves overlapped, the global trend was to infect different households separately. iii) In households where pH1N1 and NIRVs co-circulated, co-infection was less likely than mutually exclusive infections of different individuals by the different viruses. iv) Viral co-infection in the same individual was rare and when it occurred, excretion of either of the co-infecting virus tended to be shorter.
All these features suggest a negative interplay between NIRVs (especially hRV) and pH1N1, and suggest that competition may have played a role in the extinction of pandemic
In our study, individuals who were positive for pH1N1 virus were also mostly symptomatic and presented the classic clinical picture of ILI, including the presence of fever. Reciprocally, symptomatic individuals (i.e. presenting with either ILI or URTI) during the upsurge of epidemic (W33–37) were virologically documented three times more as pH1N1 positive than as NIRVs positive. This information may help to assess the real burden of pH1N1 during the 2009 pandemic.
Co-circulation of
Acting as viral cofactors, NIRVs may vary in dominant viral types and frequency from one country/region to another
Our results, obtained through a prospective study conducted at the community level, confirm previous conclusions from reports of hospitalized patients
We acknowledge the contribution of Dr. F. Favier, as Principal Investigator of CoPanFlu-Run (CIC-EC), and we thank Dr. D. Wilkinson for proofreading the text.