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What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined.

What was the purpose of this research?

The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently , and their structures see Table 1 were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis .

The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower. Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

The first imported case of pH1N1/2009v was identified on 5 th July 2009 week 29 in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July week 30 . pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 . . . . .

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity . Hence, the threshold of 1/40 may underestimate the number of infected individuals.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the pH1N1/2009v .

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States .

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 . Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

As mentioned earlier, cumulative incidence rates peaked in phase C W40-44 , and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant P,0.0001 . To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample sample 1 was obtained just after the epidemic wave phase C, W40-44 , and the corresponding second sample was collected at the end of the survey phase D, W45-52 .

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples , or non representative adult cohorts military, health care workers, long-stay patients . Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

the same day when inclusion started in our study cohort; ii 7 to 15 days are required to develop an antibody response after viral infection; iii On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses H. Pascalis, manuscript in preparation .

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011. Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.

When did WHO declare a pandemic of pH1N1/2009v influenza?

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season July-December 2009 : prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research INSERM , the Institute of Research for Development IRD and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health EHESP .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera.

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples , or non representative adult cohorts military, health care workers, long-stay patients . Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended.

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively P<0.0001 . A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively P<0.0001 .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold i.e. 1/40 .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July week 30 , i.e. the same day when inclusion started in our study cohort; ii 7 to 15 days are required to develop an antibody response after viral infection; iii On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32.

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

= 1/40 . The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly $60 yrs and weaker in children and adolescents ,20 yrs and adults 20-59 yrs , with highly significant differences between age groups P,0.0001 .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample sample 1 was obtained just after the epidemic wave phase C, W40-44 , and the corresponding second sample was collected at the end of the survey phase D, W45-52 . Seronegation rates were 27.0% 61/226 for all age groups, 17.4% 12/69 in children and adolescents ,20 yrs , 32.3% 41/127 in adults 20-59 yrs and 26.7% 8/30 in the elderly $60 yrs . Differences between the seronegation rates according to age were statistically weakly significant P = 0.0671 .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

We then tested in this particular group, the impact of baseline pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 Table 4 . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents ,20 yrs .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

weeks 40-44 was 20.4%. Similar results of early antibody decay were recently reported . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level.

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

As mentioned earlier, cumulative incidence rates peaked in phase C W40-44 , and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant P,0.0001 . To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample sample 1 was obtained just after the epidemic wave phase C, W40-44 , and the corresponding second sample was collected at the end of the survey phase D, W45-52 .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity.

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive i.e. HI titers $1/40 in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults $60 yrs in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 weeks 45-52 .

What is the classical cutoff value for antibody titers?

Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island.

immunity to neuraminidase and conserved T cells epitopes might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive i.e.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. The results reported in this study were based only on serological analysis of paired sera.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases S2pi and the age-specific baseline-proxy seroprevalence rate S1pA s2 pi -s1 pA .

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

Hemagglutination inhibition assay HIA . A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009 . Briefly, the virus was propagated onto MDCK cells under standard conditions.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

Initial HIA antibody titers in this group were ,1/40, The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples , or non representative adult cohorts military, health care workers, long-stay patients . Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection qRT-PCR positive in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 as in the elderly , but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics as shown in younger persons .

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age P,0.0001 Figure 4 . The multivariate adjusted odds ratio for seroconversion were 0.15 95%CI: 0.06-0.37, P,0.0001 per two-fold increase in baseline titer, 1.79 95%CI: 1.23-2.59, P,0.003 per other household members who seroconverted, 5.33 95%CI: 1.56-19.27, P,0.008 Figure 1 . The cohort profile and major outcomes.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

HIA titer $1/40 . for age ,20 years vs age $60 years and 11.35 95%CI: 0.41-4.47, P = 0.62 for age 20-60 years vs age $60 years . The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic due to social contacts and mixing patterns . It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA i.e. immunity to neuraminidase and conserved T cells epitopes might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly.

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents ,20 yrs . The highest seroconversion rate .56% was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age P,0.0001 Figure 4 .

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

Seroconversion was defined as a shift from seronegative at inclusion i.e. HIA titer ,1/40 to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion i.e. HIA titer $1/40 .

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

Baseline-proxy ,pre-epidemic HIA titers to the pH1N1/ 2009v were measured on sample 1 Table 2 , obtained from 249 subjects 103 households recruited at the very beginning of the investigation during weeks 30 and 31 phase A, Figure 2 , when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort data not shown . The overall, the baseline-proxy seroprevalence rate HIA $1/40 , over all ages, was 43.4% 95%CI: 37.4%-49.6% .

What is meant by a protective HIA titer?

Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e.

In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive i.e. HI titers $1/40 in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults $60 yrs in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 weeks 45-52 .

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

The cohort profile and major outcomes. Figure 1 details the three phases of the protocol: i inclusion weeks 30-44 and serum samples S1 collection; ii follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii end of the study weeks 45-52 and samples S2 collection. HIA on paired sera S1+S2 allowed estimating seroconversion rates.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents ,20 yrs , young adults 20-39 yrs , middle-age adults 40-59 yrs , and elderly adults $60 yrs . As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group 20-59 yrs .

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay HIA with titers ≥1/40 being considered positive.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

Sera were aliquoted and stored at 280uC. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research Nu ID RCB AFSSAPS: 2009-A00689-48 and was approved by the local Ethics Committee Comité de Protection des Personnes of Bordeaux 2 University . Every eligible person for participation was asked for giving their written informed consent.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model ALR with an exchangeable log Odds Ratio OR to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. Data were analysed with respect to subject age.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate. The sample size was calculated for identifying risk factors in the prospective cohort study.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 The Epidata Association, Odense, Denmark . SAS version 9.1 SAS Inc., Cary, NC, USA was used for statistical analysis.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

In all tests, a P value,0.05 was considered significant. We estimated 95% confidence intervals CIs of proportions by using a cluster bootstrap technique with 1000 re-samples . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. .1371/journal.pone.0025738.t002 Data are numbers, percentages 95% confidence intervals and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample S1 collection at inclusion, in each age group, after controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

Differences between the seronegation rates according to age were statistically weakly significant P = 0.0671 . We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection phase A, B or C . Criteria of seroconversion were defined in the method section.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

SAS version 9.1 SAS Inc., Cary, NC, USA was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test or Fisher's exact test when non applicable was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection i.e.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A weeks 30-31 corresponded to early epidemic time, phase B W32-39 to the epidemic unfolding, phase C W40-44 to the immediate post-epidemic stage and phase D W45-52 to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more.

What are the results of the study?

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study.

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

To interpret these data, one should remember that antibodies at seroprotective levels HIA $1/40 , in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase A W30-31 . The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave.

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July week 30 , i.e. the same day when inclusion started in our study cohort; ii 7 to 15 days are required to develop an antibody response after viral infection; iii On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32.

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection qRT-PCR positive in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 as in the elderly , but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics as shown in younger persons .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature . We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity.

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold i.e. 1/40 .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 as in the elderly , but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics as shown in younger persons . The observed seroconversion rates depend on age, after adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic due to social contacts and mixing patterns .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly 5, 6, 8, , though large variations do exist between countries . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C P,0.0001 . To interpret these data, one should remember that antibodies at seroprotective levels HIA $1/40 , in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection.

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly $60 yrs and weaker in children and adolescents ,20 yrs and adults 20-59 yrs , with highly significant differences between age groups P,0.0001 . The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera HI $1/40 steadily increased during the epidemic unfolding phase B, W32-39 and in immediate post epidemic period phase C, W40-44 when it reached its maximum level, then declined in the late post epidemic period phase D, W45-52 .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

We then tested in this particular group, the impact of baseline pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 Table 4 . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents ,20 yrs .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

= 1/40 . The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly $60 yrs and weaker in children and adolescents ,20 yrs and adults 20-59 yrs , with highly significant differences between age groups P,0.0001 .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States .

What was the interpretation for the crossreactive antibodies?

These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 . Baseline seropositivity rates that we report in children and in younger adults i.e. 30%-35% were notably higher than those reported from other parts of the world 6, 8, 22, 23, .

the same day when inclusion started in our study cohort; ii 7 to 15 days are required to develop an antibody response after viral infection; iii On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses H. Pascalis, manuscript in preparation .

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

The first imported case of pH1N1/2009v was identified on 5 th July 2009 week 29 in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July week 30 . pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 . . . . .

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In England, Baguelin et al. estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States . The rapid spread of infection to other continents led the World Health Organization WHO to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated .

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

Sixty subjects among the 464 individuals 12.9%, belonging to 33 households 24.3% were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample sample 2 was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave weeks 45-52 which allowed the constitution of a serobank of 1,687 paired-sera.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

weeks 40-44 was 20.4%. Similar results of early antibody decay were recently reported . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted . In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season July-December 2009 : prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research INSERM , the Institute of Research for Development IRD and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health EHESP .

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

The second follow up serum sample sample 2 was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave weeks 45-52 which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are provided in figure 2 .

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

As mentioned earlier, cumulative incidence rates peaked in phase C W40-44 , and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant P,0.0001 . To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample sample 1 was obtained just after the epidemic wave phase C, W40-44 , and the corresponding second sample was collected at the end of the survey phase D, W45-52 .

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

Report of ILI fever $37.8uC associated with any respiratory or systemic symptom led to three consecutive visits of a nurse to the incident case-dwelling on day 0, +3 and +8 post-report to record symptoms and collect nasal swabs from all family members for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second post-epidemic serum sample sample 2 was obtained weeks 45-52 to determine seroconversion rates. Sera were aliquoted and stored at 280uC.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island and accumulating reports from other countries and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. Lower infection rates were found in adults and the lowest rates were recorded in the elderly. Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%.

How long did the pH1N1/2009 viral outbreak last?

Based on clinical cases reported to the epidemiological surveillance services , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 attack rate: 12.85% . In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response i.e., weeks 40-44 , a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction almost two thirds of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level.

The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community . The cumulative incidence rates were much higher in children and adolescents ,20 yrs , indicating very active transmission of infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C W40-44 , and then declined indicating some lability of the humoral immune response against the pH1N1/2009v.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA .

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI .

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents.

What was the focus of this study?

In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population.

The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min.