PWS_Cusco_Rev.docx

PERFORMANCE WORK STATEMENT

Introduction Infectious diarrhea remains a major threat to deployed military forces worldwide, in addition to their impact on travelers and populations (predominantly children) living in the developing world. Despite advances in our understanding of the incidence, prevalence, and biology of infectious diarrhea, obstacles remain to the development of effective countermeasures. Due to the mission-aborting potential of diarrhea disease, the U.S. military has placed a high priority on the development of an effective vaccine against the most common enteropathogens associated with travelers’ diarrhea like ETEC, Campylobacter and Shigella species. However, prior to any vaccine clinical trial, a better understanding of the true incidence of disease and better characterization of the true clinical spectrum of disease in respect to etiology should occur.

Background Infectious diarrhea remains a major threat to deployed military forces worldwide, in addition to their impact on travelers and populations (predominantly children) living in the developing world. Despite advances in our understanding of the incidence, prevalence, and biology of infectious diarrhea, obstacles remain to the development of effective countermeasures. Due to the mission-aborting potential of diarrhea disease, the U.S. military has placed a high priority on the development of an effective vaccine against the most common enteropathogens associated with travelers’ diarrhea like ETEC, Campylobacter and Shigella species. However, prior to any vaccine clinical trial, a better understanding of the true incidence of disease and better characterization of the true clinical spectrum of disease in respect to etiology should occur.

To accomplish this goal, over the past 3 years we have executed a longitudinal prospective study using foreign language schools in Cusco which enroll students from developed nations as they arrive in Peru. The Amauta Spanish School with sites both in Peru and Argentina is the largest and most prominent language school in the Cusco region. It receives as a minimum 20 students each week to participate in 5 to 8 week courses of varying levels of Spanish. Students are between 20 and 50 years of age and are comprised almost entirely of students from the United States and Europe. Over one year of time, about 1400 relatively young students attend this school which would be able to participate. The other characteristic of this school is that most students are hosted within two housing facilities, each across the street from each other, which facilitates the capture of diarrheal episodes. Given this information, we propose to continue with the longitudinal, prospective study enrolling subjects on arrival, and establishing a surveillance system to identify and study all diarrheal episodes in which they will develop during their stay in Cusco to expand upon our existing dataset with the intent to publish the findings and develop the site as a venue for future clinical trials. The study will be conducted using the same methodology of a vaccine trial done in this setting, identifying the incidence of diarrhea associated with each enteropathogen and the prevalence of asymptomatic carriage of these enteropathogens in the same study group. Questionnaires’ would also be employed to further identify risk factors as well as severity of disease and overall impact of their infection. Given the collection of basic epidemiologic and demographic data over time, epidemics of diarrheal disease will be able to be fully characterized as they spread through the school and determine mitigating factors which might be capitalized on to prevent transmission.

This project is to be conducted with the services of Peruvian researchers in Cusco . We will also be working in collaboration with local laboratories within Cuzco and increasing their overall capacity. Information for the trial should also benefit the evaluation and treatment of traveler’s to Cuzco at the local travel clinics.

Methods Spanish School cohort

Volunteers will be recruited upon arrival at the Amauta Spanish School in Cusco, Peru. The study will include students that are enrolled in an on-site/in-residence Spanish language training program in Cusco. Amauta Spanish School with sites both in Perú and Argentina is the largest and most prominent language school in the Cusco region. It receives as a minimum 20 students each week to participate in 5 to 8 week courses of varying levels of Spanish. The demographic of the student population is between 20 and 50 years of age and is comprised almost entirely of students from the United States and Europe. Over one year of time, approximately 1400 students would attend this school and could potentially be recruited into the study.

Inclusion Criteria:

1. Male and female travelers 18 years of age and older traveling to Cusco as part of an in-residence Spanish language training program.

2. Travelers’ proficiency in the English language, able to communicate fluidly in English and understand written test.

3. Consent form signed by volunteer.

Exclusion Criteria:

1. Those individuals who do not wish to participate or have given consent.

2. Those who do not understand English to a sufficient degree to understand the consent or fill out the projects forms.

a. Field work:

Enrollment

The contents of the consent form will be explained by the study doctors to each group of students as soon as arriving into Cusco, where the study doctor may answer any questions they may have (see Appendix A). The purpose, procedures, and expected risks and benefits of the study will be explained in detail and each student will then be individually invited to participate in the study. If they agree, a written informed consent will be obtained and demographic and medical history data will be collected using a standardized questionnaire. The consent will be in English given that it is the most prevalent language spoken and a copy of the signed informed consent will be provided to each participant for future reference.

b. Data collection:

Procedures after Enrollment

After enrollment, the study doctors will check for inclusion and exclusion criteria. For those who will meet all inclusion and none of the exclusion criteria, a questionnaire will be applied which will address the following demographic data and questions. Enrollment questionnaire varies slightly for participants enrolling in travelers’ diarrhea verses respiratory illness study or both. (see Appendix B):

Travelers’ Diarrhea

· Age, Sex, Race, Nationality, Country of Residence.

· Recent travel history. Date of arrival to Cusco.

· Prior vaccination for Hepatitis A, Cholera and Typhoid.

· The exact place where they are staying in Cusco (house, room number or alternative address).

· List of medical problems, allergies, current medications.

· Advice received to prevent traveler’s diarrhea prior to travel.

Respiratory Illness

· Age, Sex, Race, Nationality, Country of Residence.

· Recent travel history. Date of arrival to Cusco.

· Prior vaccination for influenza, Hib Flu, pneumococcal and pertussis.

· The exact place where they are staying in Cusco (house, room number or alternative address).

· List of medical problems, allergies, current medications.

· Advice received to illness prior to travel.

In addition to applying this questionnaire, travelers’ diarrhea study subjects will receive a detail explanation of the mechanics of stool collection and the definition of a diarrheal episode as defined by this study, so they can collect a stool sample as soon as diarrhea develops, before taking any type of medication, particularly antibiotics. Materials for stool collection to include a “napoleon hat” will be given to each enrollee while being trained in its use. In addition, extra stool hats and collection material will be placed in the bathrooms of each dormitory space. All participants will also be informed about the services to be provided by the study physicians at the on-site medical clinic and given contact numbers in which they can call with questions if needed. To assist in providing this information a pamphlet and wallet side card with pertinent information and their enrollee number will be given to the each student enrolled in the study. After the enrollment process, each participant will be asked to provide a baseline stool sample with their next bowl movement.

Those that choose to enroll in the respiratory part of the study will be asked to provide a respiratory sample as part of the enrollment process for baseline analysis consisting of both a nasal and throat swab. For the nasal swab, a health worker will introduce a Dacron swab into the nostril parallel to the pallet; it will then be slowly withdrawn, after leaving it in place for 10-15 seconds, using a rotating motion. For the throat swab, the field worker will place a clean Dacron swab in the posterior pharynx, rotate it vigorously and then withdraw the swab. One nasal and one throat swab will be placed into the same commercially available universal virus transport medium (VTM) and the applicator sticks will be broken off near the top of the tube, an approach that should maximize influenza virus detection. Samples will be kept in portable coolers with Ice packs to maintain samples cool until they reach the laboratory. All participants will be encouraged to present to the study physicians with any symptoms of an upper respiratory infection or if they just feel sick. To assist, a thermometer will be given to each participant so that they may take and record their temperature in the absence of the physician.

Subject Follow-up

Travelers’ Diarrhea Study An episode of diarrhea will be defined as one or more liquid or semiliquid stool (which is defined as those stools that will take the shape of the container). By using a definition requiring only one episode of diarrhea, we hope to make our study as sensitive as possible to determine the true clinical scope of disease. A separate episode of diarrhea will be defined as another episode of unformed or loose stool after a period of 2 full days of only formed or no stools (not having diarrhea). Stools are to be collected by the patient using a napoleon stool hat and brought to the physician as soon as possible. If the physician is not available it should be placed in a designated refrigerator for processing as soon as the physician or technologist returns to the school to process the sample. A stool sample will be obtained at the beginning of each diarrheal episode, as well as weekly if the episode continues. Study subjects will also be interviewed daily to document their symptoms and signs on each day of the diarrheal episode. The laboratory technician and/or physician will also visit all school classes to investigate if any enrolled subjects have had any diarrheal episode in which they have not reported to our study clinic.

For the determination of asymptomatic carriage, incidence, and pathogenicity of a given organism, enrolled participants will also be asked to provide one stool each week on a specified day irrespective of its consistency, throughout their time at the school, using the same method used to obtain a sample during a diarrheal episode. A brief questionnaire, to determine if they were ill that week or had any travel along with current eating behaviors will be asked (see Appendix C). Those who are determined to have asymptomatic carriage of bacteria known to cause diarrhea will not be treated unless they develop disease. This is because there is limited to no evidence to show the risk of treating for the carriage of certain bacteria with antibiotics in someone who is well out ways the risk of becoming ill with that bacteria. The only exception will be if the bacteria isolated are of a public health risk, principally Salmonella typhi which causes typhoid fever.

If a participant is having diarrhea, they will be asked to complete a questionnaire in addition to providing a stool sample for analysis.

This questionnaire will address specific questions including (see Appendix D):

· The start of their illness and type of symptoms (nausea, vomiting, bloody stools, cramping, fever, etc.).

· The number and consistency of their stools for each 24 h interval since inception.

· The use of any medications or fluids for the treatment of their diarrhea.

· Whether medical attention was sought and to what level of care.

· Document any recent travel history.

· Note any interaction with other sick contacts.

Our study physician will also take vital signs and perform an exam on those with diarrhea to document other signs or symptoms of their illness and note the severity of their diarrhea. The physician should also note in the subject’s medical record the level of care and treatment provided. During the course of their illness they will continue to have interaction and follow up with the physician and will repeat the stool culture for diarrhea which does not resolve in 7 days.

We also plan to follow each participant after they have left the school out to 9 months to screen for possible sequelae which might be associated to their travelers’ diarrhea. Participants will be asked to complete a brief online survey the day that they are to return home, at 1 and 6 months after their stay in Cusco to see if they have developed any chronic symptoms or symptoms consistent with reactive arthritis or chronic fatigue syndrome.

The questionnaire will address the following (see Appendix E):

· Whether gastrointestinal problems have continued.

· Changes in sleeping patterns, energy and mental state since travel.

· The presence of any chronic physical symptoms (muscle and join pain, headaches, and swollen glands).

· Presence of influenza like symptoms.

· Any developed medical problems, other than travelers’ diarrhea while in Cusco.

To screen for functional bowel problems or irritable bowel syndrome (IBS), a second survey called the “Rome III Diagnostic Questionnaire for the Adult Functional GI Disorders (including Alarm Questions) and Scoring Algorithm” will be given in its entirety at 3 and 9 months after participants have left Cusco.

The questionnaire will address the following (Appendix F):

· Presence and severity of any chronic physical symptoms.

· Recent illnesses or changes in health.

· Brief relevant family health history

Respiratory Illness Study All participants will be asked to take a brief weekly questionnaire to determine if they were ill that week, recent travel and sick contacts (Appendix C). Students presenting with symptoms at this time will be referred for further respiratory illness follow up; however, participants are encouraged to present anytime they feel sick with fever or a respiratory illness to the study physicians for evaluation. To assist, all participants will be given a thermometer to be able to check and record their temperature if they feel they have a fever.

An episode of respiratory illness will be defined as one or more of the following symptoms: fever, sore throat, congestion, runny nose or cough. Using a broad definition for respiratory illness will increase our sensitivity to capture all disease in order assess the full clinical spectrum related to each pathogen. Physicians will collect nasal and pharyngeal swabs as outlined above from all cases in addition to filling out a separate respiratory illness episode questionnaire. Sputum may also be taken if pneumonia is suspected.

The respiratory episode questionnaire will address the following questions (Appendix G):

· The start of their illness and type and severity of symptoms (headache, cough, expectoration, chest congestion, rhinorrhea, etc.).

· The use of any medications or fluids for the treatment of their respiratory illness.

· Whether medical attention was sought and to what level of care.

· Note any interaction with other sick contacts.

Our study physician will also take vital signs and perform an exam on participants with any respiratory illness to document other signs or symptoms of their illness. The physician should also note in the subject’s medical record the level of care and treatment provided. During the course of their illness they will be followed daily by the study site physicians with their signs and symptoms documented on a respiratory episode questionnaire and flowchart until the participant has fully recovered.

BOTH STUDY COMPONENTS

For convenience of the students and reliability of sample collection, physicians will be hired to help manage the program on site and provide free clinical treatment and medications to those suffering from diarrhea and respiratory illnesses. Study physicians will also provide general medical advice for other illnesses that the study subject may experience free of charge but prescriptions for medications that are not related to the treatment of diarrhea or diagnosed respiratory illness will be at the patient’s expense. Study physicians will also be available by phone or within the clinic on a 24 hour on call basis for emergencies with more severe cases being referred to higher levels of care. Laboratory results gained will be given to the study physician, including antibiotic sensitivity of isolated strains, so as to inform study subjects and to ensure appropriate treatment. To provide clinic space, we will rent one of the dormitory rooms within the main part of the school and convert it into a clinic so that the study doctor can examine subjects, administer questionnaires, fill out and store medical records, collect the stool and respiratory samples, process the stool and respiratory samples for transport within transport media, and store collected samples under refrigeration to await twice per day pickup and delivery to the local laboratory for processing. Medical records will also remain confidential and secured behind lock and key when authorized personnel are not present or the records are not in use.

Treatments for diarrhea and upper respiratory illness that are offered during the study will be routine and the standard of care and only administered by trained and licensed physicians who have the proper credentials to practice medicine at the clinic. This clinic will also only be able to handle non-urgent medical problems with more severe or more urgent medical problems being referred to a higher level of care for treatment. The clinic will also undergo proper certification by local and state health departments and certifying bodies to meet all regulations required by Perú to practice medicine.

At the end of the study or if participants decide to voluntarily leave the study, we will administer a brief questionnaire or “close out” form in which they can choose to express criticisms or strength in the study or describe how their eating or sick habits have changed during their participation within the study(Appendix H).

Laboratory diagnosis: Travelers’ Diarrhea Study

Collected fecal samples in most cases will be brought to the lab immediately (<3 hours) for processing or stored under refrigeration after an aliquot has been placed in Cary-Blair medium. Once in the lab aliquots will be plated on selective agar and placed in 2.5% potassium dichromate, and sodium acetate/acetic acid/formalin transport medium (SAF). Fresh stool will also be sampled for microscopy and aliquots placed in cryovials and frozen at -80 for future molecular testing. Stool samples will undergo routine stool culture as outlined below and antimicrobial sensitivity testing. To assist in the orderly collection of data, a lab order form will be used to track the sample and results (see Appendix I). The remainder of the stool sample may be discarded or stored for future use if consent for this purpose is obtained from the subject. More complex molecular testing, genotyping, or serotyping will be performed on isolates shipped back to NAMRU-6 on dry ice.

Stool samples will be observed macroscopically and characterized by consistence and appearance. Hemoccult cards and gross examination will be used to check for gross and occult blood. Stool will also be examined microscopically by direct and stained methods for parasite detection and the presence of fecal leukocytes. Cultured enteropathogens will be biochemically identified, and antibiograms will be performed. Stool samples will be cultured for bacterial enteropathogens by conventional microbiologic techniques [Gray, 1995; Nachamkin, 1995]. Routinely, five lactose-fermenting colonies morphologically typical of E. coli will be selected from MacConkey agar (MAC) and tested for the presence of ETEC heat-labile (LT) and heat-stable (ST) enterotoxin gene sequences by polymerase chain reaction [Stacy-Phipps, 1995].

Laboratory processing of stool will include streaking onto Hekton Enteric Agar (HEA), thiosulfate citrate bile salt sucrose agar (TCBS), MacConkey agar (MAC), Salmonella-Shigella agar (SS), and Campylobacter blood – free agar base (CBA). Inoculation into selenite enrichment broth and in pepton water will be performed. Sub-cultures from the selenite broth and pepton water will be performed 18 - 20 hours after inoculation and incubation at 37°C. Inoculated Campylobacter blood-free agar base plates will be incubated at 42°C for 48 hours in a microaerophilic atmosphere. If only a small amount of stool is collected, microbiological examination will be first, followed by viral detection and parasitological detection last. Bacterial cultures will be examined for the following agents: Salmonella, Shigella, Campylobacter, Aeromonas, Plesiomonas, pathogenic E. coli and Vibrio. Serotyping will be performed for Shigella spp. and V. cholerae. Colonies displaying an appearance consistent with Shigella by standard biochemical testing will be confirmed by agglutination with type-specific antisera. Shigella flexneri isolates will then be further typed by agglutination with type-specific antisera.

Five aliquots of stool will be frozen at –80°C until ELISA or molecular tests for bacteria, viruses and parasites at the NAMRU-6 laboratories in Lima. Campylobacter isolates will be capsular typed using a standardized multiplex PCR assay (Poly, 2011). Shigella isolates will be serotyped using commercially available typing sera. All non-typeable Campylobacter and Shigella isolates will have further characterization. Antibiotic sensitivity testing against 17 antibiotics (amoxicillin-clavulanate, ampicillin, azithromycin, ceftriaxone, chloramphenicol, cephalothin, cefepime, ciprofloxacin, rifaximin, amikacin, , gentamicin, imepenem, tetracycline, and trimethoprim-sulfamethoxazole, ticarcillin, ticarcillin/clav, furazolidone) will be performed by disk diffusion testing as described elsewhere [Woods, 1995]. Both disk diffusion methods and automated microbiological identification and sensitivities with minimum inhibitory concentrations (MIC) may occur using the Phoenix™ by Becton, Dickinson and Company (BD) which may include sensitivities against additional antibiotics as clinically appropriate. Culture and sensitivity results will be reported to the medical officer who will in turn notify volunteers of their results.

After microbiology testing is completed, all samples and strains will be sent to NAMRU-6 Lima for further characterization.

Storage of samples for future use

Consent for storage and future use of samples will be requested from all participants. Sample aliquots will be stored at -80 °C for up to 20 years. These samples will be used for studies on infectious agents not yet described, host-pathogen interactions or immune responses to the different pathogens causing travelers’ diarrhea, individual susceptibility to each pathogen, and diagnostic markers for specific pathogens. Approval from NAMRU-6 IRB and other pertinent IRBs will be sought whenever using these samples in future studies.

Data Entry

All data from surveys and case report forms will be collated and entered by the on-site contractor into an online RedCap database that will be reviewed by NAMRU-6 investigators. Hard copies of all data forms will be stored on-site until a to be determined date when they will be transferred to NAMRU-6 in Lima.

Period of Performance

This Contract is for a period from 30 September 2015 to 29 September 2016 with one option of extension from 30 September 2016 to 29 September 2017..

NAMRU-6 agrees to:

1. Provide laboratory technical assistance and help and assist in the development of standard operating procedures for techniques required by the protocol for the study. Will also provide database support and management for the collected information.

2. Provide molecular support for the study for more advanced testing.

3. Cover the cost of consumables used in the testing associated with the protocol.

Contractor agrees to:

1. Implement the study “Incidence, etiology, and severity of travelers’ diarrhea among students at a Spanish language school in Cuzco, Perú” by:

a. Employing the services of a seasoned researcher to serve as the project manager and co-investigator within Cusco to help run the protocol and to participate in any publications which may come from the data. Hire all physicians, nurses, laboratory personnel, and data entry persons needed to execute the study.

b. Monitoring of the study site and managing an onsite health clinic to treat diarrheal cases, administer questionnaires, and perform the initial processing and storage of samples.

c. Assisting in the enrollment of study participants into the study to include providing instructional material, training, and administering the consent to participate within the study.

d. Performing the routine microbiological work-up of stool samples to include routine culture and antimicrobial susceptibilities of pathological organisms and parasitological work-up. Current estimates, anticipate 50 samples per month.

Deliverables To continue carrying out study titled “Incidence, etiology, and severity of travelers’ diarrhea among students at a Spanish language school in Cuzco, Peru”, contractor shall be required to:

Monitoring of the study site and managing an onsite health clinic to treat diarrheal cases, administer questionnaires, and perform the initial processing and storage of samples.

Assisting in the enrollment of study participants into the study to include providing instructional material, training, and administering the consent to participate within the study.

Performing the routine microbiological work-up of stool samples to include routine culture and antimicrobial susceptibilities of pathological organisms and parasitological work-up.

Maintaining all records, databases, archived samples, and assist in the data analysis.