25 Oct 2014

#Ebola #medical #worker quarantined in #NewJersey tests negative for virus (@Reuters, October 25 2014)

[Source: Reuters, full page: (LINK).]

Ebola medical worker quarantined in New Jersey tests negative for virus [   !   ]

NEW YORK (Reuters) - A medical worker in New Jersey quarantined after arriving at an airport after treating Ebola victims in West Africa has tested negative for the virus, the state's health department said on Saturday.




#Serologic #screenings for #H7N9 from three sources among high-risk groups in the early stage of #H7N9 circulation in #Guangdong Province, #China (Virol J., abstract, edited)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Virol J. 2014 Oct 23;11(1):184. [Epub ahead of print]

Serologic screenings for H7N9 from three sources among high-risk groups in the early stage of H7N9 circulation in Guangdong Province, China. [      ][      ]

Wu J, Zou L, Ni H, Pei L, Zeng X, Liang L, Zhong H, He J, Song Y, Kang M, Zhang X, Lin J, Ke C.




The aim of this study was to assess the prevalence of the novel avian influenza A virus (H7N9) in three high risk groups. The groups were divided into those exposed through infected individuals, those exposed through poultry and those individuals exposed through the external environment, in the early stage of the epidemic in Guangdong Province, which is located in the southern region of China.


Serologic studies were conducted among samples collected from individuals who had close contact with the first H7N9 infected patient reported in Guangdong Province, those who were most likely exposed to the first group of H7N9 infected poultry, and those who might have been exposed to H7N9 in the environmental settings, using hemagglutinin inhibition (HI) and microneutralization(MN) with 3 viruses as antigens.


The alignment results indicated that the similarity of the HA gene sequence among viruses from exposure to infected poultry, infected humans and contaminated environments were highly conserved. Seven samples of individuals exposed to contaminated environments were positive using HI and one sample among them was positive using MN using poultry H7N9 virus as the antigen. One sample was positive against human H7N9 virus and 3 samples were positive against environmental H7N9 among those that were in contact with infected patients with HI. None of these were positive with MN. All HI titers of the 240 samples from those individuals in contact with infected poultry were less than 40 with 3 viruses as antigens.


The results suggest that when the H7N9 virus was in the early stages of circulation in Guangdong Province, the antigenic sites of the HA proteins of the H7N9 strains from different hosts were highly conserved. The risk of new infection is low in individuals who have contact with infected patients, poultry or a contaminated environment in the early stages of the circulation of the H7N9 virus.

PMID: 25342002 [PubMed - as supplied by publisher]



#Ebola, #epidemics, and #ethics - what we have learned (Philos Ethics Humanit Med., abstract, edited)

[Source: US National Lilbrary of Medicine, full page: (LINK). Abstract, edited.]

Philos Ethics Humanit Med. 2014 Oct 24;9(1):15. [Epub ahead of print]

Ebola, epidemics, and ethics - what we have learned. [      ]

Donovan GK.



The current Ebola epidemic has presented challenges both medical and ethical. Although we have known epidemics of untreatable diseases in the past, this particular one may be unique in the intensity and rapidity of its spread, as well as ethical challenges that it has created, exacerbated by its geographic location. We will look at the infectious agent and the epidemic it is causing, in order to understand the ethical problems that have arisen.

PMID: 25342227 [PubMed - as supplied by publisher]



Sign Me Up: #Rules of the Road for #Humanitarian #Volunteers During the #Ebola #Outbreak (Disaster Med Public Health Prep., abstract, edited)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Disaster Med Public Health Prep. 2014 Oct 24:1-2. [Epub ahead of print]

Sign Me Up: Rules of the Road for Humanitarian Volunteers During the Ebola Outbreak. [      ]

Wildes R1, Kayden S2, Goralnick E3, Niescierenko M4, Aschkenasy M5, Kemen KM6, Vanrooyen M7, Biddinger P8, Cranmer H5.

Author information: 11Partners Healthcare Risk and Insurance Services,Boston,Massachusetts. 22Division of International Emergency Medicine and Humanitarian Programs,Department of Emergency Medicine,Brigham and Women's Hospital,Boston,Massachusetts. 33Brigham and Women's Healthcare,Boston,Massachusetts. 44Global Health Program,Boston Children's Hospital,Boston,Massachusetts. 55Center for Global Health,Massachusetts General Hospital,Boston,Massachusetts. 66Partners HealthCare Emergency Preparedness,Boston,Massachusetts. 77Harvard Humanitarian Initiative,Boston,Massachusetts. 88Partners Healthcare and Massachusetts General Hospital,Boston,Massachusetts.



The current Ebola outbreak is the worst global public health emergency of our generation, and our global health care community must and will rise to serve those affected. Aid organizations participating in the Ebola response must carefully plan to carry out their responsibility to ensure the health, safety, and security of their responders. At the same time, individual health care workers and their employers must evaluate the ability of an aid organization to protect its workers in the complex environment of this unheralded Ebola outbreak. We present a minimum set of operational standards developed by a consortium of Boston-based hospitals that a professional organization should have in place to ensure the health, safety, and security of its staff in response to the Ebola virus disease outbreak. (Disaster Med Public Health Preparedness. 2014;0:1-2).

PMID: 25343427 [PubMed - as supplied by publisher]



#Triage #Management, #Survival, and the #Law in the Age of #Ebola (Disaster Med Public Health Prep., abstract, edited)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Disaster Med Public Health Prep. 2014 Oct 24:1-6. [Epub ahead of print]

Triage Management, Survival, and the Law in the Age of Ebola. [      ]

Burkle FM1, Burkle CM2.

Author information: 11Harvard Humanitarian Initiative,Harvard University,Cambridge,Massachusetts, andWoodrow Wilson International Center for Scholars,Washington,DC. 22Mayo Graduate School of Medicine,Mayo Clinic,Rochester,Minnesota.



Liberia, Sierra Leone, and Guinea lack the public health infrastructure, economic stability, and overall governance to stem the spread of Ebola. Even with robust outside assistance, the epidemiological data have not improved. Vital resource management is haphazard and left to the discretion of individual Ebola treatment units. Only recently has the International Health Regulations (IHR) and World Health Organization (WHO) declared Ebola a Public Health Emergency of International Concern, making this crisis their fifth ongoing level 3 emergency. In particular, the WHO has been severely compromised by post-2003 severe acute respiratory syndrome (SARS) staffing, budget cuts, a weakened IHR treaty, and no unambiguous legal mandate. Population-based triage management under a central authority is indicated to control the transmission and ensure fair and decisive resource allocation across all triage categories. The shared responsibilities critical to global health solutions must be realized and the rightful attention, sustained resources, and properly placed legal authority be assured within the WHO, the IHR, and the vulnerable nations. (Disaster Med Public Health Preparedness. 2014;0:1-6).

PMID: 25343493 [PubMed - as supplied by publisher]



#Saudi Arabia reported a new #MERS-CoV case in Taif in the last 24 hours (@SaudiMOH, October 25 2014, edited)

[Source: Saudi Arabia Ministry of Health, full page: (LINK). Edited.]

#Saudi Arabia reported a new #MERS-CoV case in Taif in the last 24 hours [      ][      ]



New Cases:

  1. man, 75 years old, Saudi national, resident in Taif, currently hospitalized, with pre-existing medical condition; history of contacts with confirmed/suspected cases in hospital settings.

Earlier reported cases discharged from hospital:

  1. woman, 35 years old, foreign citizen, resident in Taif, an HCW.

Deaths in previously announced cases:

  • No reports







[Source: World Health Organization, full PDF document: (LINK). Edited.]




A total of 10 141 confirmed, probable, and suspected cases of Ebola virus disease (EVD) have been reported in six affected countries (Guinea, Liberia, Mali, Sierra Leone, Spain, and the United States of America) and two previously affected countries (Nigeria, Senegal) up to the end of 23 October. There have been 4922 reported deaths.

Following the WHO Ebola Response Roadmap structure, country reports fall into two categories:

  • 1) those with widespread and intense transmission (Guinea, Liberia, and Sierra Leone); and
  • 2) those with or that have had an initial case or cases, or with localized transmission (Mali, Nigeria, Senegal, Spain, and the United States of America).

An overview of the situation in the Democratic Republic of the Congo, where a separate, unrelated outbreak of EVD is occurring, is also provided (see Annex 1).



A total of 10 114 confirmed, probable, and suspected cases of EVD and 4912 deaths have been reported up to the end of 18 October 2014 by the Ministry of Health of Liberia, 21 October by the Ministry of Health of Guinea, and 22 October by the Ministry of Health of Sierra Leone (table 1).

All but one district in Liberia and all districts in Sierra Leone have now reported at least one case of EVD since the start of the outbreak (figure 1).

Of the eight Guinean and Liberian districts that share a border with Cote d’Ivoire, only two are yet to report a confirmed or probable case of EVD.

A total of 450 health-care workers (HCWs) are known to have been infected with EVD up to the end of 23 October: 80 in Guinea; 228 in Liberia; 11 in Nigeria; 127 in Sierra Leone; one in Spain; and three in the United States of America. A total of 244 HCWs have died.

WHO is undertaking extensive investigations to determine the cause of infection in each case.

Early indications are that a substantial proportion of infections occurred outside the context of Ebola treatment and care.

Infection prevention and control quality assurance checks are now underway at every Ebola treatment unit in the three intense-transmission countries.

At the same time, exhaustive efforts are ongoing to ensure an ample supply of optimal personal protective equipment to all Ebola treatment facilities, along with the provision of training and relevant guidelines to ensure that all HCWs are exposed to the minimum possible level of risk.


Table 1: Confirmed, probable, and suspected cases in Guinea, Liberia, and Sierra Leone

[Country - Case definition - Cumulative Cases – Deaths]

  • Guinea
    • Confirmed – 1312 – 732
    • Probable 194 – 194
    • Suspected – 47 – 0
      • All – 1553 – 926
  • Liberia*
    • Confirmed – 965 – 1241
    • Probable – 2106 – 803
    • Suspected – 1594 – 661
      • All – 4665 – 2705
  • Sierra Leone**
    • Confirmed – 3389 – 1008
    • Probable – 37 – 164
    • Suspected – 470  - 109
      • All – 3896 – 1281
  • Total - 10 114 – 4912


* For Liberia, 276 more confirmed deaths have been reported than have confirmed cases.

**For Sierra Leone, 127 more probable deaths have been reported than have probable cases.

Data are based on official information reported by Ministries of Health. These numbers are subject to change due to ongoing reclassification, retrospective investigation and availability of laboratory results.



Five countries (Mali, Nigeria, Senegal, Spain, and the United States of America) have now reported a case or cases imported from a country with widespread and intense transmission.

In Nigeria, there were 20 cases and eight deaths.

In Senegal, there was one case and no deaths. However, following a successful response in both countries, the outbreaks of EVD in Senegal and Nigeria were declared over on 17 October and 19 October 2014, respectively.

On 23 October, Mali reported its first confirmed case of EVD (table 2). The patient was a 2-year old girl who travelled from the Guinean district of Kissidougou with her grandmother to the city of Kayes in western Mali, which is approximately 600 km from the Malian capital Bamako and lies close to the border with Senegal. The patient was symptomatic for much of the journey. On 22 October the patient was taken to Fousseyni Daou hospital in Kayes, where she died on on 24 October. At present, 43 contacts, of whom 10 are HCWs, are being monitored; efforts to trace further contacts are ongoing. A WHO team was already in Mali to assess the country state of readiness for an initial case. A rapid-response team will also arrive in the coming days.

In Spain, the single case tested negative for EVD on 19 October. A second negative test was obtained on 21 October. Spain will therefore be declared free of EVD 42 days after the date of the second negative test if no new cases are reported. A total of 83 contacts are being monitored.


There have now been four cases and one death (table 2) in the United States of America. The most recent case is a medical aid worker who volunteered in Guinea and returned to New York City on 17 October. The patient was screened and was asymptomatic on arrival, but reported a fever on 23 October, and tested positive for EVD. The patient is currently in isolation at Bellevue Hospital in New York City, one of eight New York State hospitals that have been designated to treat patients with EVD. Possible contacts are being identified and followed up.

Two HCWs who became infected after treating an EVD-positive patient at the Texas Presbyterian Hospital of Dallas, Texas, have now tested negative for EVD.

Of a total of 176 possible contacts linked with these cases, 109 are currently being monitored; 67 have completed 21-day follow-up.

In Ohio, 153 crew and passengers who shared a flight with one of the infected HCWs (prior to the patient developing symptoms) are being followed-up, though they are considered low-risk and are not considered to be contacts.


Table 2: Ebola virus disease cases and deaths in Mali, Spain, and the United States of America

[Country - Case definition – Cases – Deaths]

  • Mali
    • Confirmed – 1 – 1
    • Probable – * – *
    • Suspected – * – *
      • All – 1 – 1
  • Spain
    • Confirmed – 1 – 0
    • Probable – * – *
    • Suspected – * – *
      • All – 1 – 0
  • United States of America
    • Confirmed – 4 – 1
    • Probable – * – *
    • Suspected – * – *
      • All – 4 – 1
  • Total – 6 – 2


*No available data.

Data are based on official information reported by Ministries of Health. These numbers are subject to change due to ongoing reclassification, retrospective investigation and availability of laboratory results.




As at 21 October 2014 there have been 67 cases (38 confirmed, 28 probable, 1 suspected) of Ebola virus disease (EVD) reported in the Democratic Republic of the Congo, including eight among health-care workers (HCWs). In total, 49 deaths have been reported, including eight among HCWs.

Of 1121 total contacts, 1116 have now completed 21-day follow-up. Of five contacts currently being monitored, all were seen on 21 October, the last date for which data has been reported. On 10 October, the last reported case tested negative for the second time and was discharged. The Democratic Republic of the Congo will therefore be declared free of EVD 42 days after the date of the second negative test if no new cases are reported. This outbreak is unrelated to the outbreak that originated in West Africa.




#Research Articles #Abstracts on #Influenza & Other Respiratory #Viruses–October 25 2014 Issue

[Source: AMEDEO, homepage: (LINK).]

#Research Articles #Abstracts on #Influenza & Other Respiratory #Viruses–October 25 2014 Issue [      ]


This Week’s Abstracts:


  • Ann Intern Med
      • Review: Neuraminidase inhibitors reduce symptomatic influenza; oseltamivir does not reduce hospitalizations.
  • Epidemiol Infect
    • VAIDYA NK, Morgan M, Jones T, Miller L, et al
      • Modelling the epidemic spread of an H1N1 influenza outbreak in a rural university town.
  • J Clin Microbiol
    • YI L, Guan D, Kang M, Wu J, et al
      • Family Clusters of Avian Influenza A H7N9 Infection in Guangdong Province, China.
    • PECI A, Winter AL, King EC, Blair J, et al
      • Performance of Rapid Influenza Diagnostic Test in an outbreak setting.
  • J Gen Virol
    • KONG W, Liu L, Wang Y, He Q, et al
      • The C-terminal elongation of NS1 of H9N2 influenza virus induces a high level of inflammatory cytokines and increases transmission.
  • J Infect Dis
    • VIBOUD C, Eisenstein J, Reid AH, Janczewski TA, et al
    • CHEN Q, Griffin MR, Nian H, Zhu Y, et al
      • Influenza Vaccine Prevents Medically-Attended Influenza-Associated Acute Respiratory Illness in Adults 50 Years or Older.
    • HE XS, Holmes TH, Sanyal M, Albrecht RA, et al
      • Distinct patterns of B-cell activation and priming by natural influenza infection versus inactivated influenza vaccination.
  • J Virol
    • BI Y, Xie Q, Zhang S, Li Y, et al
      • Assessment of the internal genes of influenza A (H7N9) virus contributing to the high pathogenicity in mice.
    • COSTELLO DA, Whittaker GR, Daniel S
      • Variation of pH sensitivity, acid stability, and fusogenicity of three influenza H3 subtypes.
    • GAO S, Wu J, Liu RY, Li J, et al
      • Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells.
    • MORALES DJ, Monte K, Sun L, Struckhoff JJ, et al
      • A novel mode of ISG15 mediated protection against influenza A virus and Sendai virus in mice.
    • BAEK YH, Song MS, Lee EY, Kim YI, et al
      • Profiling and characterization of potentially multidrug-resistant influenza neuraminidase 1 (N1) strains against neuraminidase inhibitors.
    • LANZ C, Yanguez E, Andenmatten D, Stertz S, et al
  • MMWR Morb Mortal Wkly Rep
    • AQUINO TL, Brice GT, Hayes S, Myers CA, et al
      • Influenza Outbreak in a Vaccinated Population - USS Ardent, February 2014.
  • PLoS One
    • KUCK LR, Sorensen M, Matthews E, Srivastava I, et al
    • POWELL JD, Dlugolenski D, Nagy T, Gabbard J, et al
      • Polymerase Discordance in Novel Swine Influenza H3N2v Constellations Is Tolerated in Swine but Not Human Respiratory Epithelial Cells.
    • HADIFAR F, Ignjatovic J, Tarigan S, Indriani R, et al
      • Multimeric Recombinant M2e Protein-Based ELISA: A Significant Improvement in Differentiating Avian Influenza Infected Chickens from Vaccinated Ones.
    • MAROIS I, Cloutier A, Meunier I, Weingartl HM, et al
    • YANG X, Steukers L, Forier K, Xiong R, et al
      • A Beneficiary Role for Neuraminidase in Influenza Virus Penetration through the Respiratory Mucus.
    • OU C, Shi N, Yang Q, Zhang Y, et al
      • Protocatechuic Acid, a Novel Active Substance against Avian Influenza Virus H9N2 Infection.
    • HORM SV, Mardy S, Rith S, Ly S, et al
      • Epidemiological and Virological Characteristics of Influenza Viruses Circulating in Cambodia from 2009 to 2011.
    • DE ARAUJO J, de Azevedo Junior SM, Gaidet N, Hurtado RF, et al
      • Avian Influenza Virus (H11N9) in Migratory Shorebirds Wintering in the Amazon Region, Brazil.
  • Proc Natl Acad Sci U S A
    • LINDERMAN SL, Chambers BS, Zost SJ, Parkhouse K, et al
      • Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013-2014 influenza season.



#Dynamics and #control of #Ebola virus #transmission in Montserrado, #Liberia: a mathematical modelling analysis (The Lancet Infect Dis., abstract, edited)

[Source: The Lancet Infectious Diseases, full page: (LINK). Abstract, edited.]

The Lancet Infectious Diseases, Early Online Publication, 24 October 2014 / doi:10.1016/S1473-3099(14)70995-8

Copyright © 2014 Elsevier Ltd All rights reserved.

Dynamics and control of Ebola virus transmission in Montserrado, Liberia: a mathematical modelling analysis [      ]

Original Text

Joseph A Lewnard BA a b †, Martial L Ndeffo Mbah PhD a b †, Jorge A Alfaro-Murillo PhD a b, Prof Frederick L Altice MD a c, Luke Bawo MPH d, Tolbert G Nyenswah MPH d, Prof Alison P Galvani PhD a b




A substantial scale-up in public health response is needed to control the unprecedented Ebola virus disease (EVD) epidemic in west Africa. Current international commitments seek to expand intervention capacity in three areas: new EVD treatment centres, case ascertainment through contact tracing, and household protective kit allocation. We aimed to assess how these interventions could be applied individually and in combination to avert future EVD cases and deaths.


We developed a transmission model of Ebola virus that we fitted to reported EVD cases and deaths in Montserrado County, Liberia. We used this model to assess the effectiveness of expanding EVD treatment centres, increasing case ascertainment, and allocating protective kits for controlling the outbreak in Montserrado. We varied the efficacy of protective kits from 10% to 50%. We compared intervention initiation on Oct 15, 2014, Oct 31, 2014, and Nov 15, 2014. The status quo intervention was defined in terms of case ascertainment and capacity of EVD treatment centres on Sept 23, 2014, and all behaviour and contact patterns relevant to transmission as they were occurring at that time. The primary outcome measure was the expected number of cases averted by Dec 15, 2014.


We estimated the basic reproductive number for EVD in Montserrado to be 2·49 (95% CI 2·38—2·60). We expect that allocating 4800 additional beds at EVD treatment centres and increasing case ascertainment five-fold in November, 2014, can avert 77 312 (95% CI 68 400—85 870) cases of EVD relative to the status quo by Dec 15, 2014. Complementing these measures with protective kit allocation raises the expectation as high as 97 940 (90 096—105 606) EVD cases. If deployed by Oct 15, 2014, equivalent interventions would have been expected to avert 137 432 (129 736—145 874) cases of EVD. If delayed to Nov 15, 2014, we expect the interventions will at best avert 53 957 (46 963—60 490) EVD cases.


The number of beds at EVD treatment centres needed to effectively control EVD in Montserrado substantially exceeds the 1700 pledged by the USA to west Africa. Accelerated case ascertainment is needed to maximise effectiveness of expanding the capacity of EVD treatment centres. Distributing protective kits can further augment prevention of EVD, but it is not an adequate stand-alone measure for controlling the outbreak. Our findings highlight the rapidly closing window of opportunity for controlling the outbreak and averting a catastrophic toll of EVD cases and deaths.


US National Institutes of Health.


a Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA; b Center for Infectious Disease Modeling and Analysis, Yale School of Public Health, New Haven, CT, USA; c Infectious Diseases Section, Yale University School of Medicine, New Haven, CT, USA; d Ministry of Health and Social Welfare, Monrovia, Liberia

Correspondence to: Prof Alison P Galvani, Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA

† Joint first authors



#Phylodynamic #Analysis of #Ebola Virus in the 2014 #SierraLeone #Epidemic (PLOS Currents Outbreaks, abstract, edited)

[Source: PLoS Currents Outbreaks, full page: (LINK). Abstract, edited.]

Phylodynamic Analysis of Ebola Virus in the 2014 Sierra Leone Epidemic [      ]

October 24, 2014 · Research

Citation: Volz E, Pond S. Phylodynamic Analysis of Ebola Virus in the 2014 Sierra Leone Epidemic. PLOS Currents Outbreaks. 2014 Oct 24. Edition 1. doi: 10.1371/currents.outbreaks.6f7025f1271821d4c815385b08f5f80e.


Authors: Erik Volz Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom. Sergei Pond Medicine, University of California San Diego, San Diego, CA, USA.




The Ebola virus (EBOV) epidemic in Western Africa is the largest in recorded history and control efforts have so far failed to stem the rapid growth in the number of infections. Mathematical models serve a key role in estimating epidemic growth rates and the reproduction number (R0) from surveillance data and, recently, molecular sequence data. Phylodynamic analysis of existing EBOV time-stamped sequence data may provide independent estimates of the unobserved number of infections, reveal recent epidemiological history, and provide insight into selective pressures acting upon viral genes.


We fit a series mathematical models of infectious disease dynamics to phylogenies estimated from 78 whole EBOV genomes collected from distinct patients in May and June of 2014 in Sierra Leone, and perform evolutionary analysis on these genomes combined with closely related EBOV genomes from previous outbreaks. Two analyses are conducted with values of the latent period that have been used in recent modelling efforts. We also examined the EBOV sequences for evidence of possible episodic adaptive molecular evolution during the 2014 outbreak.


We find evidence for adaptive evolution affecting L and GP protein coding regions of the EBOV genome, which is unlikely to bias molecular clock and phylodynamic analyses. We estimate R0=2.40 (95% HPD:1.54-3.87 ) if the mean latent period is 5.3 days, and R0=3.81, (95% HPD:2.47-6.3) if the mean latent period is 12.7 days. The estimated coefficient of variation (CV) of the number of transmissions per infected host is very high, and a large proportion of infections yield no transmissions.


Estimates of R0 are sensitive to the unknown latent infectious period which can not be reliably estimated from genetic data alone. EBOV phylogenies show significant evidence for superspreading and extreme variance in the number of transmissions per infected individual during the early epidemic in Sierra Leone.

Funding Statement

EMV and SLKP are supported by NIH Models of Infectious Disease Agent Study (MIDAS) grant U01GM110749. Computational work was supported by the UCSD Center for AIDS Research (BIT Core, P30 AI036214). EMV was partly supported by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.