Conditions: Filamentary Keratitis
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 15
Healthy volunteers: no

Brief summary:
It is a pilot study aimed to determine if oral Mucinex® is effective in reducing the amount of corneal filaments and improving symptomology in subjects with filamentary keratitis.

Detailed description:
The study is aimed to determine if oral administration of Mucinex® is effective in reducing the amount of corneal filaments and improving symptomology in subjects with filamentary keratitis. Subjects will be instructed to take 600 mg tablets of extended release Mucinex® twice a day (total dose is 1.2 g/day) for 4 weeks.

Arms:
arm 1: 600 mg of Mucinex 2 times a day.

Interventions: intervention 1: Mucinex®
Intervention descriptions: intervention 1: Mucinex®

Protocol document:
Prot_000.pdf: 
Cover Sheet 
 
Study Title: MUCINEX for Treatment of Filamentary Keratitis 
NCT No: NCT02859246 
Date: Protocol Last updated 7/21/2016, Protocol approved by Human Research Protection Review 
Board 10/20/2016 


 
Human Studies Committee 
Tel: 617-573-3732 
Email: HSC@meei.harvard.edu 
 
 
HUMAN SUBJECTS RESEARCH PROTOCOL - RESEARCH SUMMARY (SUPP N) 
 
DATE: 7/21/2016 
TITLE OF PROJECT: MUCINEX ® for treatment of filamentary keratitis 
PRINCIPAL INVESTIGATOR: Reza Dana MD, MPH, MSc 
FUNDING SOURCE: Pending  
 
BACKGROUND
 
1)  Discuss the following in paragraph format 
 Discuss the importance of the topic (public health and/or clinical importance and impact on 
individuals/community; incidence, prevalence, mortality and morbidity) 
 Critically appraise the relevant literature and discuss the state of current knowledge on the topic (including 
deficiencies in knowledge or gaps that make the study worth doing) 
 Describe, in detail, your approach to address the research question 
 Explain how your study will contribute to existing research and benefit other individuals or the wider 
community 
 
RESPONSE: Mucolytics like n-acetylcystine have shown some success previously in the management of filamentary 
keratitis.  There is no data evaluating whether Mucinex ® is also effective at decreasing severity. Anecdotally, patients 
have reported that filamentary keratitis symptoms improved with the use of mucinex ® for upper respiratory congestion.  
Filamentary keratitis is an uncommon (chronic) eye condition frequently associated with dry eye disease.  Affected 
patients typically present with symptoms including foreign body sensation, grittiness, pain, photophobia, tearing and 
increased blink rate.  On examination there are thread-like mucus filaments adherent to the corneal surface.  Current 
management of filamentary keratitis addresses dry eye disease (lubrication, punctal plugs), inflammation (anti-
inflammatory eye drops), mucous buildup (n-acetylcystine as a mucolytic) and filament removal with forceps.   
Previous histologic evaluation has demonstrated that the filaments consist of a corneal epithelial core wrapped in mucus.  
Staining has established that there is significant overlap in the mucins produced in the eye and those from the respiratory 
tract, most notably MUC5AC and MUC16. There are several proposed mechanisms for filament formation.  The general 
process begins with an insult to the epithelium, Bowman’s membrane, or both, and is commonly associated with some 
degree of aqueous tear deficiency.   
Primary treatment modalities include lubrication, filament removal, anti-inflammatories and mucolytics.  Several past 
studies have shown limited success using n-acetylcysteine for its mucolytic properties. To date there are no trials studying  
Mucinex ®, which is well-tolerated, cost-effective and has been shown to hydrate respiratory mucus secretions, in the 
treatment of filamentary keratitis.  
 
References: 
 
Albietz, J., Sanfilippo, P., Troutbeck, R., & Lenton, L. M. (2003). Management of filamentary keratitis associated with 
aqueous-deficient dry eye. Optometry and vision science : official publication of the American Academy of Optometry, 
80(6), 420-430. doi: 10.1097/00006324-200306000-00007 


Human Studies Committee 
Tel: 617-573-6939 
Email: HSC_Submissions@meei.harvard.edu 
 
 
Updated 06/04/2012 
2
Ali, M. S., & Pearson, J. P. (2007). Upper Airway Mucin Gene Expression: A Review. The Laryngoscope, 117(5), 932-
938. doi: 10.1097/MLG.0b013e3180383651 
Diller, R., & Sant, S. (2005). A case report and review of filamentary keratitis. Optometry - Journal of the American 
Optometric Association, 76(1), 30-36. doi: 10.1016/S1529-1839(05)70252-9 
Doughty, M. J. (2012). Goblet cells of the normal human bulbar conjunctiva and their assessment by impression cytology 
sampling. The ocular surface, 10(3), 149.  
Goodman, L. S., Brunton, L. L., Blumenthal, D. K., Murri, N., & Hilal-Dandan, R. (2011). Goodman & Gilman's The 
pharmacological basis of therapeutics. New York: McGraw-Hill Medical. 
Guzman-Aranguez, A., & Argüeso, P. (2010). Structure and Biological Roles of Mucin-type O-glycans at the Ocular 
Surface. The ocular surface, 8(1), 8-17. doi: 10.1016/S1542-0124(12)70213-6 
Kim, K. C. (2012). Role of epithelial mucins during airway infection. Pulmonary pharmacology & therapeutics, 25(6), 
415-419. doi: 10.1016/j.pupt.2011.12.003 
Rose, M. C., & Voynow, J. A. (2006). Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease. 
Physiological Reviews, 86(1), 245-278. doi: 10.1152/physrev.00010.2005 
Seagrave, J. (2011). Effect of guaifenesin on mucin production, rheology, and mucociliary transport in differentiated 
human airway epithelial cells. Experimental lung research, 37(10), 606-614. doi: 10.3109/01902148.2011.623116 
Seagrave, J., Albrecht, H. H., Hill, D. B., Rogers, D. F., & Solomon, G. (2012). Effects of guaifenesin, N-acetylcysteine, 
and ambroxol on MUC5AC and mucociliary transport in primary differentiated human tracheal-bronchial cells. 
Respiratory research, 13(1), 98-98. doi: 10.1186/1465-9921-13-98 
Tanioka, H., Yokoi, N., Komuro, A., Shimamoto, T., Kawasaki, S., Matsuda, A., & Kinoshita, S. (2009). Investigation of 
the Corneal Filament in Filamentary Keratitis. Investigative ophthalmology & visual science, 50(8), 3696. doi: 
10.1167/iovs.08-2938 
Wang, G., Xu, Z., Wang, R., Al-Hijji, M., Salit, J., Strulovici-Barel, Y., . . . Crystal, R. G. (2012). Genes associated with 
MUC5AC expression in small airway epithelium of human smokers and non-smokers. BMC medical genomics, 5(1), 21-
21. doi: 10.1186/1755-8794-5-21 
 
 
AIMS
2) Your aim(s) should arise from your literature review and state what the study hopes to accomplish. 
 
RESPONSE: To determine the effectiveness of systemic mucinex ® in patients with filamentary keratitis.  
 
OBJECTIVES
3) Your focused research question needs to be further refined into one or more study objectives.  The study 
objective(s) should be single and quantifiable statement(s) that will allow you to answer your research question. 
 
RESPONSE: To determine if the mucolytic properties of Mucinex ® are effective in reducing the amount of corneal 
filaments and improving symptomatology in patients with filamentary keratitis.
 


 
 
VERSION 1.0:7/15/2013 
3
HYPOTHESES 
 
4a). Primary Hypothesis - Hypotheses are more specific than objectives and amenable to statistical evaluation.  
Your primary hypothesis is your statement of the hypothesized effect on the primary outcome measure.  A 
hypothesis is worded very simply and written as ‘testable’ statements.  Your experimental results will prove or 
disprove your hypothesis.  Hypotheses are generally stated in the null form (Ho) as they have their basis in 
inferential statistics.  Rejecting the null hypothesis increases our confidence, with a given level of probability, 
that there is a relationship between the variables being studied.  However, a classic scientific hypothesis 
includes both a null and alternative (Ha) hypothesis. 
 
*e.g. Ho: Asthma prevalence rates are not different among children from low and high socioeconomic groups in 
Istanbul.  Ha: Asthma prevalence rates are different among children from low and high socioeconomic groups 
in Istanbul. 
 
RESPONSE: H0: Oral Mucinex ® does not reduce corneal filaments or symptomatology in patients with filamentary 
keratitis.  Ha: Oral Mucinex ® twice a day reduces corneal filaments and the symptomatology in patients with filamentary 
keratitis.  
 
4b). Secondary Hypotheses - Although a study is usually based around a primary hypothesis, secondary 
hypotheses may also be pre-specified although based on outcomes of lesser importance or additional interest.  
As the primary hypothesis is usually the basis for statistical power calculations, secondary hypotheses with 
insufficient power will generally not lead to statistically robust conclusions. 
 
RESPONSE: N/A 
 
STUDY DESIGN
5) State the design of the research (e.g. randomized controlled study, cross-sectional survey, prospective or 
retrospective cohort/case-control).   
 
*Whatever the study design, you need to ensure that you provide the reader with a clear statement and 
description of your proposed design.  You may also explain why the particular study design has been chosen in 
preference to other possible designs (i.e. justification for choice of study design) 
 
RESPONSE: Prospective, non-randomized, non-controlled study.
 
ELIGIBILITY CRITERIA 
 
6a). Inclusion Criteria - Inclusion criteria are the ‘characteristics’ that clearly describe the study population 
that are required for a subject to be included in the study. The criteria may be based on factors such as age, 
gender, the type and stage of a disease, previous treatment history, and co-morbid medical conditions. They 
may state appropriate criteria for admitting special ‘at-risk’ populations such as women of reproductive age, 
children or patients with disease states or organ impairment. 


 
VERSION 1.0: 7/15/2013 
4
 
RESPONSE: 1)Patients 18 years or older who have filamentary keratitis in at least one eye. 2) Schirmers test ≥3mm in 
the eye diagnosed with filamentary keratitis (can be one eye or both the eyes). 
 
6b. Exclusion Criteria - Provide details of participants that will be considered ineligible to participate and 
justification for their exclusion. These criteria are not always clinical in nature, aiming principally to 
accommodate participants in a safe and ethical manner. Criteria may include circumstances that interfere with 
the participant’s ability to give informed consent (diminished understanding or comprehension, or a language 
other than English spoken and an interpreter unavailable), contraindications to the study 
treatment(s)/procedure(s), taking certain concomitant medication(s), or conditions that interfere with a patient's 
ability to comply with all treatment(s)/procedure(s).  
 
RESPONSE: 1) Less than 18 years or older than 80 years of age; 2) Patients unable to provide informed 
consent; 3) Patients unable to attend a follow-up visit within 4 (±1) weeks; 4) Active ocular surface infection of 
any type; 5) Recent ocular surgery (< 30 days); 6) History of nephrolithiasis as Mucinex ®  has been associated 
in rare cases to the development of kidney stones; 7) Schirmer test <3mm (in study eye; can be one eye or both 
the eyes); 8) History of hypersensitivity to  Mucinex ®; 9) Concurrent eye disease requiring immediate 
initiation of a new treatment (e.g., topical steroids); 10) Restrictions for water intake can exacerbate the risk of 
nephrolithiasis and thus patients taking oral  Mucinex ® are instructed to take each dose with a generous 
amount of water. 
STUDY OUTCOMES 
 
7a). Primary Outcome - The primary outcome should be the most important and clinically relevant outcome 
(e.g. clinical, psychological, economic, or other) of the study. This is the measure used to answer your study 
aim. However, it is also the outcome used to calculate study sample size and power and test the primary 
research hypothesis. Generally, no more than 1-2 primary outcome measures are pre-specified. Primary 
outcome measures may be measured in various ways such as: binary (e.g. caesarean/no caesarean, blood loss 
≥500mL/blood loss <500mL); continuous (e.g. weight - kg, blood loss - mL); ordinal (e.g. pain - mild, 
moderate, severe); time to event (e.g. survival), and counts (e.g. number of infections, number of events 
occurring).  
 
RESPONSE: Reduction of symptoms according to the Ocular Surface Disease Index score after treatment with 
oral  mucinex ® twice a day (total dose is 1.2 g/day) for 4 weeks 
 
7b). Secondary Outcome(s) - Secondary outcome(s) are measures of additional or less important research 
interest. They may include additional clinical, psychological, economic, or safety outcomes (e.g. treatment 
related side effects/adverse events). However, as these endpoints are not used to calculate study power and 
 sample size it is often not possible to draw robust conclusions from the results.  
 
RESPONSE: Reduction in the number of corneal filaments after treatment with oral Mucinex ® twice a day 
(total dose is 1.2 g/day)  for 4 weeks. 
 
STUDY PROCEDURES 
 


 
VERSION 1.0: 7/15/2013 
5
8) In this section you need to clearly and comprehensively describe exactly what will happen to participants 
once they are enrolled in your study. Depending on the study it might include how potential participants will be 
approached, when they will be randomized, the frequency and duration of visits or whether they are expected to 
self-complete a daily diary at home, the duration of the study or follow-up, and any measurements taken at each 
visit (e.g. questionnaires, physical measurements, biological samples).  
 
You should include precise details of the treatment(s)/intervention(s) intended for each group/participant. You 
should also provide details of any follow-up schedule (i.e. time between visits) and consider how you will 
monitor participants’ adherence with the treatment schedule. You might also describe under which 
circumstances participants may be withdrawn and how this will occur. A schematic diagram or flow chart may 
be useful for this section. 
 
RESPONSE: To test the hypothesis that  Mucinex ®administration will reduce the signs and symptoms of 
filamentary keratitis, patients with the diagnosis of filamentary keratitis will be enrolled. At the initial visit (Day 
#1), visual acuity, number of filaments on the cornea, schirmers test, ocular surface symptoms, these are 
standard, non-invasive common clinic procedures that will be recorded. The patient will then be instructed to 
continue their current medication regimen, with no alterations. The patient will then be given 600mg tablets of 
extended-release Mucinex®  to take home, and will be instructed to take them twice a day with abundant water 
until the next visit in 4 weeks. If patient misses a dose, they are not required to make up for missed dose and can 
continue to take the next dose. At the follow up visit the visual acuity, number of filaments on the cornea, and 
ocular surface symptoms will be assessed.  
 
The investigators will screen the patients for a history of nephrolithiasis, a contraindication of this therapy prior 
to their enrollment.  Patients will continue their usual eye treatment regimen while participating in this study.  
 
9) Randomization (if applicable) 
Include the method (including any software) used to generate the random allocation sequence. Describe type of 
randomization performed, ratio of assignment to groups, block size permutation and stratification if applicable. 
Explain the methods used to conceal group allocation until assignment. Also, include information on who will 
generate the allocation sequence and who will assign participants into their groups.  
 
This section should also discuss if participants, the investigator, and those assessing/analyzing the outcome(s) 
will be blind (or masked) to group assignment or if the study will be an open-label study (investigators and 
subjects know their assigned group). 
 
 
RESPONSE: N/A 
 
10)  Study Specific Procedures:  List all procedures (interventions, tests, surveys, etc.) to be performed ONLY 
for research purposes. 
 
RESPONSE: None 
 
11)   Standard of Care Procedures:  Clearly list all standard of care (standard therapy) procedures 
(interventions, tests, etc.) to be performed regardless of the subject’s enrollment in the study, but that will be 
included in the research assessment. 
 


 
VERSION 1.0: 7/15/2013 
6
RESPONSE: 1) Dry eye symptoms assessment (Ocular Surface Disease Index questionnaire). 
 
MEASUREMENT TOOLS 
 
12) It is essential to state how the data will be collected to assess the primary and secondary outcome(s) of the 
study (e.g. patient questionnaire, medical charts, routinely collected hospital/research database, biological 
specimens). Describe at what point(s) of the study data collection will occur. You should make statements that 
justify the validity of the study measure/instrument. If not, you will have to verify how you will ensure the 
validity and quality of data being collected. Also, mention here if you are going to have one or more assessors 
to collect data, their level of training/experience (or how they will be trained), and if you are planning to assess 
inter-rater reliability (if applicable).  
 
RESPONSE: Data will be collected during the two clinical visits (baseline and follow-up), in which ocular 
surface symptoms score and number of corneal filaments will be will be recorded in a data collection sheet.  
 
13) Sample size and statistical power - A sample size or power calculation should be 
performed. This calculation is used to estimate the number of subjects required to answer your primary study 
hypothesis with an accepted power. Conversely, it also allows you to estimate what power can be achieved with 
a limited number of participants. This number is calculated by specifying the magnitude of the effects that are 
expected (i.e. informed and clinically significant), variability of the measurements and the acceptable degree of 
type I and II errors. You need to specify the assumptions made for the calculation. It is recommended that you 
consult with a statistician for this section. Also keep in mind the estimated recruitment rate and whether you 
need to adjust for anticipated non-responders and losses to follow up.  
 
RESPONSE:  
  This will be a first-of-its-kind study evaluating an FDA approved and over-the-counter available drug 
(guaifenesin; Mucinex®) to treat cough as part of the symptoms of common cold. Our study will be the first to 
evaluate the effect of Mucinex ®  alleviating the symptoms of filamentary keratitis. No data are available to 
estimate the changes expected after initiation of Mucinex ®. This is a pilot study and represents minimal to no 
risk to participants, and involves no additional procedures other than the routinely performed clinical 
assessments. In this pilot study we aim to enroll 15 participants. 
 
14)  Statistical methods - The statistical methods used for the study 
objectives/hypotheses (e.g. t-test, chi-squared, multivariate modeling) must be sufficiently detailed. If 
conducting a randomized controlled study, you should state whether methods will include an “intention to treat” 
(ITT) analysis, per protocol analysis, or both. An ITT analysis is preferred as it compares all subjects in the 
groups to which they were originally randomly assigned (despite withdrawal, treatment failure or cross-over). 
Consultation with a statistician is strongly recommended. See Harvard Catalyst Statistical Consulting for more 
information. 
 
RESPONSE: We will use a paired T test or a Wilcoxon signed-rank test, depending on the distribution of the 
data, to assess whether there is a significant difference between pre- and post-therapy symptoms of filamentary 
keratitis.   
 
DATA SAFETY MONITORING PLAN (DSMP)  


 
VERSION 1.0: 7/15/2013 
7
 
Complete question 15 OR 16 depending on the risk of the study. If your study is submitted as minimal risk and 
determined by the HSC to be more than minimal risk you will be asked to complete question 16 during the 
review process.  
 
15) If the research is no more than minimal risk, please describe any provisions in place to ensure the safety 
of participants and the validity and integrity of data. If safety monitoring will occur a safety monitoring plan 
may include elements such as: parameters for safety review, the frequency in which safety review will occur, 
the person(s) responsible for safety review, and the plan (including the person(s) responsible) for reporting 
adverse events, protocol deviations, or noncompliance to the HSC and others (where applicable). Data 
monitoring may include the specific elements that will be reviewed (e.g., informed consent documentation, 
verification of the accuracy of data), the frequency of data monitoring, and the person(s) responsible.  
 
RESPONSE: The PI will assess for adverse events as per protocol. OCRO study staff will document any 
protocol deviation, patient’s non-compliance and report them to HSC per policy. 
16) If the research is more than minimal risk, a more detailed data and safety monitoring plan is required. 
Please detail the plan for this study below:  
 
QUESTION 
RESPONSE 
Please identify the types of monitoring (e.g., 
centralized1 , on-site2, a mix of both) that will be 
utilized for this study and the rationale for use 
(why these practices are appropriate given the 
nature (e.g., multisite) and risk of the study): 
If a multisite study and centralized 
monitoring practices will be employed, 
describe how processes and expectations 
for site record keeping, data entry, and 
reporting will be communicated (e.g., 
through a Manual of Operations and/or 
SOP’s): 
N/A 
Which individual(s) or group will be responsible 
for data and safety monitoring (e.g., PI, specific 
members of the study team, independent 
monitor(s), DSMB)? Please be specific and 
indicate who is responsible for what aspects of the 
study, including what responsibilities are shared: 
N/A 
Provide information related to the expertise and 
qualifications of the individual(s) and/or groups 
listed above relative to monitoring.  Provide a 
description of any specific training/qualifications 
required for personnel carrying out monitoring 
activities including personnel conducting internal 
N/A 
                                                 
1 Centralized monitoring means a remote evaluation carried out by study personnel or representatives at a location other than the site(s) at which the clinical 
investigation is being conducted. 
2 On-site monitoring means an in-person evaluation carried out by study personnel or representative(s) at the site at which the clinical investigation is being conducted. 


 
VERSION 1.0: 7/15/2013 
8
data monitoring, statistical monitoring, or other 
monitoring activities and the presence of scientific 
and/or clinical knowledge needed to adequately 
monitor the trial: 
For those who have monitoring responsibilities 
please describe the plan to provide mentoring, 
feedback, and additional training to investigators 
and study staff:  
N/A 
If this is a multisite study, explain the plans in 
place to facilitate timely communication of routine 
monitoring results and immediate reporting of 
significant monitoring issues to other sites and 
stakeholders (i.e., CRO, IRB(s)) as well as 
communication from sites to monitors: 
N/A 
Please explain how monitoring activities for this 
study will be documented (i.e., a monitoring log 
kept in the regulatory binder): 
N/A 
How are monitoring results communicated to 
stakeholders (investigator(s), IRB): 
Please note that the MEE HSC requires 
that monitoring reports be received within 
5 business days of receipt by the MEE 
PI/study team. 
N/A 
What processes are in place for addressing 
unresolved or significant issues (e.g., significant 
noncompliance with the protocol) identified by 
monitoring at MEE or across study sites (when 
applicable): 
N/A 
If applicable, describe the composition of the Data 
and Safety Monitoring Board (DSMB) including 
each member’s relevant expertise, experience in 
clinical research and in serving on other DSMB’s 
and the absence of serious conflicts of interest. 
Explain if any of the members have relationships 
with those sponsoring, organizing, conducting or 
regulating the trial and the nature of these 
relationships or if the members have no 
involvement in the design and conduct of the 
study: 
Note whether or not a DSMB is required 
(e.g., by the funding agency):  
 
Note whether or not a DSMB Charter 
exists for this study and include it as an 
appendices or indicate if it will be 
submitted separately to the HSC at a later 
date (if for example, the charter will be 
developed by the DSMB at the initial 
meeting): 
N/A 


 
VERSION 1.0: 7/15/2013 
9
Address the process for communication of Data 
and Safety Monitoring Board determinations to the 
investigator and to the HSC: 
N/A 
If applicable, please describe the electronic data 
capture systems you will be using and what 
training will be provided for those who will be 
using these systems: 
N/A 
Include specific data elements (e.g., review of 
informed consent documentation, confirmation of 
subject eligibility) that will be reviewed: 
N/A 
Specify the frequency of data monitoring (a 
specific number of times, at defined time points, 
after a certain number of subjects have been 
recruited or as needed): 
N/A 
Who will be responsible for data review (e.g., PI, 
study staff, DSMB)? If this is a shared 
responsibility indicate which specific elements 
each person or group is responsible for: 
N/A 
Specify the parameters (i.e., procedures, laboratory 
tests, or other measures) that will be used to 
evaluate the safety of the study treatment(s) or 
interventions; to include the methods and timing 
for assessing, recording, and analyzing these 
parameters: 
N/A 
Specify the frequency of safety observation (a 
number of times, at defined time points, after a 
certain number of subjects have been recruited or 
as needed): 
N/A 
Explain who will be responsible for safety review 
(e.g., PI, DSMB, monitor): 
N/A 
Address any “stopping rules” for individual 
research subjects including who will be 
responsible for making these determinations: 
N/A 
Address any “stopping rules” for parts of the 
study, or for the entire study including who will be 
responsible for making these determinations: 
N/A 
Address the frequency and process for eliciting 
adverse event information from research subjects:  
N/A 
Please acknowledge that adverse events and 
unanticipated problems will be reported to the 
HSC in accordance with the HSC policy on 
Reporting Adverse Events and Unanticipated 
Problems:  
N/A 
 

SAP_001.pdf: 
Cover Sheet 
 
Study Title: MUCINEX for Treatment of Filamentary Keratitis 
NCT No: NCT02859246 
Date: 7/21/2019 


 Statistical Analysis Performed  
 
 
 
 
 
 Statistical Test of Hypothesis  
 
We calculated the difference in the mean number of filaments and OSDI score between 
baseline and follow-up visits and evaluated their change over time. We used the paired-
sample Wilcoxon signed-rank test if data did not follow a normal distribution and the 
paired Student t-test in the case of normally distributed data. For all the analyses we 
considered the average value of both eyes for all variables with the exception of 
symptom scores (OSDI), which were considered per patient. Data are presented as the 
mean ± standard deviation (SD), a range for continuous variables, and percentages for 
categorical variables. A twotail P value of less than 0.05 was considered statistically 
significant in all statistical tests. The statistical analysis was performed using STATA 
13.0 (StataCorp, College Station, TX).