Point of Care Diagnostic to Identify the Causative Agent of the Plague in Blood Samples
Conditions: Plague, Plague, Bubonic, Plague, Pneumonic, Yersinia Pestis Plague, Yersinia Pestis; Bubo, Yersinia Pestis; Pneumonia, Yersinia Sepsis, Yersinia Pestis Infection, Bubo; Yersinia Pestis, Bubonic; Plague, Skin, Pneumonic Plague
Sponsor: Brimrose Technology Corporation

1 
 
Document:  Protocol Document 
 
Brief Title:  Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in 
Madagascar 
 
Project Title:  Evaluation of Diagnostic Tools " Lateral Flow Immunoassay (LFI)  
  
for Human Plague in Madagascar” (Testing Multi-Echelon Diagnostics in a   
Resource Limited Endemic Setting: Human Plague in Madagascar 
 
NCT Number: NCT04562012 
 
Date Last Modified: 19AUGUST2020 
  
 

2     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
 
 
August 19, 2020 
 
 
Dear Clinical Trial Reviewer, 
 
We are registering this novel research proposal conducted in Madagascar as a clinical trial 
study. It is our goal to present a description of this study in a straightforward way so that you 
can effectively evaluate the logic of the scientific design and how compliance to regulatory rules 
are met.  
 
This is an investigational medical device study of a non-FDA cleared in vitro diagnostic (IVD)    
device/assay called Lateral Flow ImmunoAssay (LFI) for plague detection using a design concept 
similar to a home pregnancy test https://www.brimrosetechnology.com/detection-diagnostics. 
The study has been approved by an accredited Institute Review Board (Northern Arizona 
University FWA00000357) as a non-significant risk device study and minimal risk to human 
subjects (project ID 1582884). Since this study involves human subjects in Madagascar, this 
study was also approved by the local Malagasy ethics committee (CERBM - Comite D’Ethique de 
la Recherche Biomedicale FWA # 00000330). NAU is the IRB of record therefore is the overall 
lead institute for the human subject research component for this study. To conform to the 
requirements of the IRB regulations and gain the approval of two IRB boards, adequate 
measures are built in this study to ensure maximum protection of human subjects. This includes 
proper consenting, minimizing risk of participant harm (physical, psychological, social), 
preservation of confidentiality, data security, and a response plan.   
 
Submitted by, 
 
David P. Trudil 
Brimrose Technology Corporation 
Principal Lead and Project Central Contact 
 
 
Dawn J Birdsell, Ph.D. 
Study Principal Investigator 
Northern Arizona University 
 
David M Wagner, Ph.D. 
Study Director 
Northern Arizona University 
 
Minoarisoa Rajerison, Ph.D. 
Study Director 
Institut Pasteur de Madagascar 
 
Voahangy Andrianaivoarimanana, Ph.D. 
Study Principal Investigator 
Institut Pasteur de Madagascar   
 

3     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
STUDY PURPOSE 
The purpose of this study will be to generate the data required to thoroughly validate the 
ability of plague LFI assay to accurately diagnose human infections with Y. pestis. These 
validation data will eventually be presented to the FDA (along with data from other types of 
studies not from human subjects) to seek approval for commercial license.  
 
STUDY OBJECTIVE 
The objective will be to validate this assay on the capillary blood of humans suspected to have 
plague.  
 
SPECIFIC AIMS 
The specific aims of this study are to enroll up to 300 participants who present clinical signs of 
illness based on specific inclusion criteria. We will collect two types of blood samples from 
enrolled participants 1) capillary blood from a finger prick and 2) venous blood. The capillary 
blood will be used for direct testing on the LFI assay and the venous blood will be used to 
perform independent validations using FDA approved methods. This study is designed as a 
correlation study to understand 1) how LFI assay results compare with results from traditional 
diagnostic methods based on DNA detection methods and bacterial culture isolate and 2) 
effectiveness of capillary blood to serve as a diagnostic clinical sample as compared with 
traditional biological samples (venous blood, bubo and sputum). The study is designed to 
evaluate the outcome of LFI (SMART™-PRT, New Horizons Diagnostics Corp. 
https://www.nhdiag.com/index.htm) and how LFI results correlate with the standard plague 
diagnostics methods used in Madagascar and two other FDA-approved methods. We are not 
examining the relationship between the results of the LFI and health outcomes of the 
participants. Decision of participant’s medical treatment is solely based on the clinical 
judgment of the physician and guidelines set forth by Madagascar National Plague Control 
Program (PNLP). All participants who are tested by LFI will have received medical treatment 
prior to the start of the study and the continuation of their medical treatment is guided by 
PNLP and physician judgment only.  
 
BACKGROUND 
Yersinia pestis, which causes the disease plague, is ecologically established in rodent 
populations worldwide, including on all continents except Australia and Antarctica. Plague is a 
rapidly progressing highly feared infectious disease that historically was responsible for the 
deaths of millions world-wide due to natural outbreaks during three major pandemics. Within 
the last century, several nation states developed plague as a biological weapon and the US 
government currently lists Y. pestis as a potential bioweapon (1) that could be used against US 
war fighters. As a natural disease, plague continues to be a threat in modern times with the 
World Health Organization (WHO) classifying it as a re-emerging infectious disease . Human 
plague outbreaks occur naturally on an annual basis worldwide with the vast majority of cases 
(4,5) located in rural remote low-resource settings of Madagascar, an island nation off the east 
coast of Africa. Madagascar has more human plague cases than any other country in the world. 
In 2015 alone, WHO reported 85.93% of global cases came from Madagascar (2). Over a five-

4     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
year period, there have been more than 3,500 suspected human plague cases in Madagascar 
(Rajerison et al., unpubl).  
 
In nature, humans acquire plague by being bitten by fleas carrying Y. pestis. The bacterium 
travels to the nearest lymph node (LN) from the wound site and causes LN swelling, known as a 
bubo (hence, bubonic plague) within 3-6 days post infection with a 50%-70% mortality rate if 
untreated. If bubonic plague is not medically treated, it can further progress to infect the lungs 
(pneumonic plague) for which person to person transmission can ensue. Pneumonic plague, is a 
rapidly progressing disease (1-3 days) with 100% mortality rate; for this reason, aerosolized Y. 
pestis is of concern as a potential biological weapon. Importantly, there are no licensed 
vaccines currently available for plague, so antibiotics are the only means to medically defeat 
this disease. Key to preventing mortality and further transmission in rural settings is the ability 
to rapidly diagnose at the very early acute phase of the disease. Such a diagnostic assay would 
need to be highly simple and usable in a low-resource setting to effectively service the human 
populations most affected (local rural inhabitants and possibly warfighters).   
 
The existing rapid diagnostic assay generally relies on detection of the F1 antigen of Y. pestis or 
serodiagnosis of anti-F1 antibody (6).  A rapid assay, F1RDT (3) is not licensed for commercial 
use but is used in low-resource settings worldwide. This assay uses non-blood clinical samples 
(bubo aspirates and sputum) to diagnose. Procuring bubo aspirate for diagnosis requires an 
invasive collection procedure that is significantly more painful than blood sample collection. 
The research here aims to evaluate the diagnostic effectiveness of a blood-based rapid 
diagnostic assay. This is a US-developed assay based on a Lateral Flow ImmunoAssay (LFI) 
design concept similar to a home pregnancy test. The biological sample target is capillary blood 
from a finger prick. The simplicity of design enables this assay to be used in low-resource 
settings. We will evaluate the expression levels two Y. pestis proteins in the capillary blood of 
humans suspected to have plague and determine how these levels correlate with results from 
traditional diagnostic assays. 
 
Lay Summary: Plague is a deadly but highly treatable disease caused by the bacterium Y. pestis. 
Due to the historical development of Y. pestis as a bioweapon by several nation states, it is 
listed by the US as a potential bioweapon that could be used against US warfighters. Although 
this bacterium is ecologically established worldwide, it mostly affects impoverished people who 
live in rural low-resource areas of Madagascar. Plague is acquired directly from bites of infected 
fleas but, if left untreated, it can progress to the highly lethal pneumonic form that can result in 
human to human transmission. With the dangers of pneumonic plague in the context of both 
natural outbreak and as a bioweapon used against warfighter, the goal of this study is to 
investigate a diagnostic test that is able to rapidly and locally diagnose this disease in low-
resource settings. This study aims to evaluate a US-developed new LFI assay intended for 
capillary blood to diagnose humans infected with Y. pestis. We will rigorously validate with 
assay on human populations from active plague sites and correlate the results with the results 
of paired clinical samples used in standard medical workup using existing diagnostics tests.  
 
RESPONSIBLE INSTITUTES  

5     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
Brimrose is the sponsor for this clinical trial study and will be the entity to manufacture the LFI 
device.  David Trudil, Director Biotechology Division at Brimrose, will be responsible for 
overseeing the device component of this entire study. He will take the data generated from this 
study to the FDA for clearance request. Northern Arizona University (NAU) is the lead institute 
for overseeing the field work conducted in Madagascar which involve enrolling human subjects 
suspect to be plague positive and test the LFI device on their clinical samples. NAU has 
partnered with Institut Pasteur de Madagascar (IPM) to perform this field work. Madagascar is 
the region with the most active plague site in the world documented by the WHO and, for this, 
IPM is the world’s expert on managing human plague outbreaks, and they have the field work 
expertise necessary to successfully validate this LFI assay. The technical expertise for the LFI 
assay will come from the Naval Health Research Center (NHRC). They will provide quality 
training and will serve as a technical liaison. As the lead institute, NAU (https://in.nau.edu/pmi/) 
will oversee the work performed by our International Partner (IPM) and will coordinate the 
training of IPM personnel. Dr. Wagner will be responsible for the global scientific direction for 
the field work and Dr. Birdsell will be responsible for the coordination of the collaboration for 
the human subject research that occur in Madagascar.  
 
International Partner: IPM is the lead plague research organization in Madagascar and has 
collaborated with NAU on several previous plague studies in Madagascar. Diagnosing and 
reporting of human suspect plague cases is mandatory by Madagascar national law under 
Plague National Control Program (PNLP). The Madagascar Ministry of Public Health (MMPH), as 
part of PNLP, requires declaration of all suspected human plague cases and mandates the 
collection of biological samples (sputum and bubo aspirates, but not blood) from these cases as 
part of the confirmation procedure. Human plague outbreaks primarily occur in rural low-
resource settings. These cases are treated at local rural health centers located throughout 
Madagascar. Local physicians collect clinical specimens (bubo aspirates and/or sputum), which 
are then transported to IPM, located in Antananarivo, where sample workup is performed. IPM 
facility serves as the national plague reference laboratory, and has stored 90% of human 
samples and bacterial isolates from recent cases across five years. Drs. Minoarisoa Rajerison 
(head of the plague unit – Science officer) and Voahangy Andrianaivoarimanana (senior 
scientist - PI) are key IPM personnel. Personnel conducting the field study include: Angeltine 
Lalao Ralafiarisoa and Soloandry Rahajandraibe. All the IPM field investigators are medically 
and/or paramedically trained and have > 8 years of field experience. IPM personnel, Jerry 
Rakotoniaina, will serve as study coordinator and will handle highly sensitive medical 
information. Because IPM is charged by the Madagascar government to oversee all operations 
involved with managing active plague outbreaks, IPM personnel are well connected with rural 
local public health officials. They communicate regularly with rural health officials to provide 
consultations (via email and cell phone) and support (medical supplies and personnel).  
 
MANAGEMENT PLAN 
The Directors and PIs at each of the participating institutions are well experienced and will be 
responsible for their respective entity’s participation in the project. David Trudil from Brimrose 
will serve as the sponsor for this clinical trial study. Dr. Wagner will serve as the overall PI (and 
co-PI for the human subjects research) and will be responsible for the global scientific direction 

6     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
and coordination of the collaboration for the field work in Madagascar. Dawn Birdsell will serve 
as PI for the human subjects research component for this project. The PIs of the respective 
institutions will meet bi monthly by videoconference and also regularly communicate via email. 
In addition, NAU, and IPM will meet together in person annually or biannually at agreed 
location site once the COVID pandemic subsides. 
 
TRAINING ON LFI ASSAY IMPLEMENTATION 
A formal training on LFI device will be given. The NAU group will meet with the Naval Health 
Research Center (NHRC) to develop LFI assay protocol and train on the LFI assay. NHRC will 
provide training for the assay and will serve as a technical liaison for both NAU and IPM, with 
NAU overseeing training of IPM personnel. NHRC will ensure that overall protocols developed 
by NAU are being implemented correctly. This training includes proper use of the LFI assay, 
accuracy in reading of LFI results, and proper method of data collection. NHRC will provide NAU 
and IPM with onsite training at both facilities (NAU and IPM) at a sufficient timing to meet the 
requirements of the statement of work.  
 
FIELD INVESTIGATION PROTOCOL 
IPM has developed a field sampling protocol which closely mirror those from past field missions 
conducted for retrospective plague investigations. IPM and NAU will work together to finalize 
the field sampling protocol that will involve enrolling up to 300 participants across two plague 
seasons (Table 1). Five IPM personnel with field mission experience were trained according to 
IRB requirements. 
 
Timeline of study activity: Human plague in Madagascar has a distinct seasonal component and 
is associated with specific environmental conditions. The human plague season typically starts 
to take off in September, peaks from October to January, and tapers off in March.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

7     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
 
 
Table 1: Timeline for the study divided across three performance periods   
(Base period = 18 months; Option Period 1 (OP1) = 12 months; Option Period 2 (OP2) = 
6 months). Months are aligned with plague seasons, performance periods and study 
activities. 
 
 
 
Location: The study will be carried out mostly in endemic plague outbreaks regions in 
Madagascar (i.e., the Central Highlands region) but can extend to any region in Madagascar 
where local clinical health officials report human plague.  
 

8     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
Study population: Active plague sites tend to be located in low resource rural settings. The 
study population will be comprised of local people presenting signs of illness consistent with 
plague. They will be recruited at rural health centers located throughout Madagascar. The 
Madagascar Ministry of Public Health requires declaration of all suspected human plague cases 
and mandates the collection of biological samples (sputum and/ or bubo aspirates) from these 
cases as part of the medical workup for official confirmation. Our proposal will work within this 
existing mandatory confirmation and reporting system to obtain the requisite enrollment 
number. Since blood is a novel biological sample for plague diagnostics, we will include a 
traditional clinical sample type as a positive control for plague detection. We will test the LFI on 
residual de-identified clinical samples (bubo and/or sputum) from participants who are enrolled 
in our study. This would thoroughly document the diagnostic capabilities of the LFI assay as 
compared to paired capillary blood. 
 
We plan to enroll up to 300 participants across the entire study which encompasses two plague 
seasons (Aug 2020-March 2021; Aug 2021-March 2022). We aim to enroll at least 150 
participants per plague season but if the opportunity presents to enroll more within the first 
plague season, then we will proceed to enroll up to 300 participants.  
 
• The inclusion criteria for this study  
o Adults 18 to 75 years old (male and female) 
▪ Be able to receive and give verbal communication 
o Children 5 to 17 years old (vulnerable population) 
▪ Parents or legal guardian be available to give permission. 
▪ Parents or legal guardian to consent for children (5-6 years)  
o Identified as a suspect human plague case by the local medical professional 
 
o Will include at least one of the following symptoms:  
▪ For bubonic plague: High fever, chills, and/or the presence of a painful 
bubo 
▪ For pneumonic plague: High fever, chills, cough for less than 5 days, 
bloody sputum, and/or chest pains. 
• Patients may be recruited from both plague surveillance program 
and non-plague surveillance programs. 
o For this study, patients from the plague surveillance 
program will have higher priority for enrollment than 
patients from the non-plague surveillance programs. 
 
• The exclusion criteria for this study 
o Children under the age of 5 years old 
o Children between the age of 5 years to 17 years without a parent or legal 
guardian 
o Not compliant with the study procedure (blood sampling) 
 

9     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
Adults 18 to 75 years old (male and female), Children 5 to 17 years old with parent or legal 
guardian, Malagasy nationals across all ethnic groups can participate. This study does not 
exclude pregnant women but they are not considered vulnerable population because their 
pregnancy status is not a selection criterion. For this reason, Appendix B is not included in this 
application.  
 
Participants will initially learn about the study from their physician during their medical 
consultation. They will then receive an information sheet by the physician who will then be 
referred to an IPM personnel to receive more information about the study. 
 
When will recruitment occur? Recruitment will most likely occur during the regular plague 
season (Aug 2020-April 2021 and Aug 2021-April 2022) when plague activity is high but it can 
happen any time of the year as sporadic cases can arise outside of the normal season. Local 
physicians will alert IPM of a suspect plague case at their health center. IPM personnel will be 
dispatched to this local health center and begin the informing, consenting, and enrolling 
process.  
 
Where will recruitment take place? The recruitment will take place at the site of regional and 
local health centers throughout Madagascar where local people seek medical assistance. It is 
local practice for IPM research personnel to visit these healthcare facilities to provide additional 
medical assistance (during outbreaks) at the invitation of the local physicians and perform 
plague investigations to fulfill Malagasy government mandates. 
 
Identifying partnering healthcare centers. During the low plague season (May – Aug), IPM will 
contact the local physicians from regional health center to inform them of this study. Physicians 
will learn about the study’s purpose, design, and the referral role for which they would serve. 
From these communications, IPM will know in advance which health centers plan to 
participate. 
 
Identifying suspect plague case and providing immediate medical treatment. When the local 
physician has identified a suspect plague case based on clinical presentation, he/she will 
perform the national protocol for declaring suspect plague case. This includes collecting 
medical workup samples (sputum and/or bubo aspirates) and completing a TLO declaration 
form. The sample and TLO form are to be given to IPM for medical workup for official 
confirmation, irrespective if the patient enrolls in the study. The physician will then provide 
standard medical care according to PNLP. This includes administration of antibiotics. Blood 
collection is not part of the standard medical workup. 
 
Role of physicians as providing referrals. The physician is not part of the research but will 
provide prospective subjects with information about the research (which, in this case, includes 
the information section of the informed consent document but will not obtain subjects’ consent 
for the research or act as representatives of the investigators. This practice is in alignment with 
guidance on engagement described here:  https://www.hhs.gov/ohrp/regulations-and-
policy/guidance/guidance-on-engagement-of-institutions/index.html. After the start of medical 

10     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
treatment, the local physician will alert the Plague-IPM Unit of suspect plague case(s) at their 
health center. IPM will immediately dispatch field investigators who will arrive as soon as 
possible. The physician will give a leaflet to the patient that will provide information about the 
study conducted by IPM. If the patient is illiterate, then the physician will read the leaflet aloud. 
The local physician will then will refer them to IPM personnel for more information. This is a 
process approved by the local ethics committee. The physician will not participate in the 
consenting and enrollment process but rather refer patients to the study. Medical treatment 
already started before introduction of the study and will continue regardless of study 
enrollment status.  
 
Consenting participants. The informing, consenting, enrolling, and sample collection process 
will be performed in a room at the local health center and will require two to three hours total. 
If the participant meets the inclusion criteria, the potential participant will undergo the 
consenting process. He/she will meet face to face with an IPM personnel who will verbally take 
them through the study information (the objectives of the study, proposal to participate in the 
study, and the type of sample to be taken – as laid out in the flyer leaflet. He/she will be 
provided a hard copy of the informed consent form that contains the signature and give them 
time for review. For those who are illiterate, the IPM personnel will verbally communicate the 
consent form information and the signature page at a language level that is understood by the 
participant. After confirming that each participant understands the nature and details of the 
study, they will be given the opportunity to ask questions and to receive answers. Part of this 
confirmation is to ensure they understand that entities outside of Madagascar may review their 
data records and health information (age, gender, location, clinical form of the disease, medical 
treatment). No identifiable information will be provided to outside entities. FDA and DoD are 
not specifically mentioned in the consent form due to a lack of familiarity of this organization 
for the typical participant in this study. After this comprehension check, each person will be 
asked if they would like to enroll in the study. To be enrolled, each participant must sign the 
informed consent form. For those who are illiterate, they will sign by marking an X or provide a 
finger print on the signature line. For minor children ages 5-17, the parents or legal guardian 
will sign the consent form to authorize permission. The minors ages 7-17 will provide assent by 
signing the assent form. For children ages 5-6, a parent or legal guardian signs the assent letter 
in their place. Two duplicate consent form (and assent form if applicable) will be signed, with 
one copy be given to the patient or parent / legal guardian and a second copy retained by IPM) 
for long-term storage for five years. 
 
Post Enrollment Procedure.  Once a participant is enrolled, the IPM personnel will assign a 
numeric code to represent, a unique identifier code (TLO #) to track data collected to preserve 
the participant confidentiality. Then the IPM personnel will measure the body temperature 
using an axillary thermometer and proceed to perform a one-time collection of two types of 
blood (venous and capillary) that will total less than 5 ml. This is equivalent to approximately 
one teaspoon of blood. Each blood sample type will be labeled with the participant’s TLO #. 
Approximately 4 ml of venous blood will be drawn out of the vein of an arm into a storage tube 
designed for blood preservation and ~ 0.1 ml of capillary blood will be captured from a finger 
prick into a capillary tube. These blood collection procedures are done using standard medical 

11     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
practices performed by medically or paramedically trained IPM personnel. Specimen sampling 
protocols are designed in such a way as to not endanger the health of patients or IPM 
personnel. These practices are safe, minimally invasive, and the blood volume falls below the 
minimal risk category. The participants may experience some level of discomfort but this level 
does not exceed discomfort experienced with these low risk medical procedures. When 
collecting blood from the participants, IPM personnel will wear proper protective gear (gloves 
and masks) and, when appropriate, prophylactically take antibiotics when needed. These are 
standard practices of IPM research personnel when conducting field investigations. 
 
Once the blood collection is complete, the enrolled participant will be given their compensation 
and congratulated for completing the study. Immediately afterwards, in a separate room or at 
the IPM facility, the capillary blood will be tested on the LFI assay. Venous blood along with the 
residual medical workup samples (bubo or sputum) will be transported to IPM facility for further 
testing.  
 
Prior to leaving the health care center, IPM personnel will collect the medical workup specimen 
(bubo aspirate or sputum) from the physician along with the completed TLO declaration form. 
The medical workup specimens are normally shipped to IPM facility but this step will be 
bypassed for enrolled patients. Instead IPM personnel will transport the medical workup 
specimen and TLO declaration form during their return to IPM facility. IPM is responsible for 
conducting the medical workup for all suspect plague cases to provide official confirmation. The 
TLO declaration form, containing highly sensitive information (including patient’s full name), is 
stored at IPM in a secured location according to Madagascar protocol. Every medical workup 
sample is de-identified by assignment of a unique TLO # which is recorded on the TLO 
declaration form to link samples and resulting data to the patient. This is the normal procedure 
of formal confirmation mandated by Madagascar Law. TLO# will be used as ID for collected 
blood samples and data of LFI results from capillary blood. In addition, each enrolled patient 
will also be assigned a unique Study ID#. From the TLO form, a limited metadata will be 
collected to populate a data collection form (Table 2). No identifiable metadata will be 
included. To further safeguard the confidentiality and privacy of enrolled participants, data will 
be further de-identified using the unique study ID# when shared with entities outside of 
Madagascar (including NAU). The TLO# will be removed.  
 
Table 2: Data collection form. 
 
 
 
 

12     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
Diagnostic testing of biological samples. For the venous blood, these tests include two primary 
confirmation method (Table 3): 1) Real-time qPCR and 2) Culture for bacterial isolation. Venous 
blood samples will be processed for secondary confirmatory methods: 1) DNA testing using 
FilmArray NGDS Warrior Panel (FDA-approved), 2) testing for presence of plague antibodies, and 
3) DNA analysis for genetic studies and genome discovery. FilmArray warrior panel will serve as 
one of several independent diagnostic confirmation methods to compare LFI results. The medical 
workup samples will be largely consumed for confirmation tests mandated by Madagascar law 
(Table 3 – gray highlights). Remaining residual amounts will be utilized to test on LFI and extract 
DNA for genetic studies.  
  
Table 3: Four human specimen types collected in the field and their respective analytical fates. 
Each field sample will be tested directly or after further processing "Analyzed Sample". 
Each analytical fate is classified as the new device diagnostic test (LFI = Lateral Flow 
ImmunoAssay), primary confirmation (qPCR = DNA tests; Culture = bacterial isolate), 
and secondary confirmation (RDT = Rapid diagnostic test; ELISA = antibody detection in 
blood; FilmArray Warrior Panel PCR, canSNP = Genetic typing; WGS = Whole genome 
sequencing to discover novel regions). Region in gray indicate standard medical workup 
confirmatory testing mandated by Madagascar law. Region in black are tests not 
subject to IRB regulations due to the use of de-identified residual medical workup 
specimens. 
 
 
 
 
Description of assay 
• New investigative in vitro (outside of human) diagnostic assay: Lateral Flow 
ImmunoAssay (LFI) (Fig 1) 
o Not FDA approved 

13     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
▪ This Madagascar study will inform decision on FDA clearance path for the 
LFI. 
▪ Until FDA approval – the device risk determination was determined as 
non-significant by the sponsor (Brimrose) and the IRB of record (NAU).  
o Biological Sample Type and Volume: 
▪ Human biological samples (capillary blood, sputum, bubo aspirate) 
• Volume (30 ul - 50 ul) is equivalent to 3-5 eye drops of liquid 
o Device characteristics: Design concept is very similar to a home pregnancy test 
▪ Result will be read by eye after 15 minutes 
▪ This assay was developed by New Horizons Diagnostics Corp.  and targets 
two plague proteins, F1 and LcrV. (https://www.nhdiag.com) 
• The F1 protein is well characterized but the characteristics of LcrV 
protein in terms of expression and stability in the context of 
human infection is not known. 
o Results from this study can provide knowledge in this area. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data Analysis: 
These human specimen samples will then be used to test the LFI assay directly at the remote 
field locations and IPM will read the results by eye after 15 minutes.   
 
Statistical analysis of the data 
• For evaluation of the LFI compared to the reference test (culture on the same sample), a 
2 * 2 contingency table will be constructed from the laboratory results.  
Figure 1.  New Horizons Diagnostics Corp has developed a rapid point-of-care 
lateral flow immunoassay (LFI) to diagnose plague infections. This assay 
(SMART™-PRT) is a multiplex LFI capable of detecting two Y. pestis antigens; F1 
and LcrV. The picture illustrates results with laboratory-purified antigens (top) 
and control (bottom). The test requires a single drop of blood from a finger stick 
and is read in 15 minutes.  

14     PROTOCOL DOCUMENT Point-of-Care rapid LFI (Lateral Flow Immunoassay) for Human Plague in Madagascar 
 
The sensitivity of the test LFI sera evaluated against the reference test (bacteriology). It 
expresses the proportion of confirmed plague cases correctly identified by the reference 
test. 
 
• Results data from other standard diagnostic tests (Table 3) will be compared with LFI 
results to determine the diagnostic effectiveness of the LFI device.  
 
• A professional statistician will be employed for data analysis as a subcontractor from the 
University of Maryland-Baltimore, Department of Mathematics & Statistics. 
 
 
References: 
1. Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, 
Friedlander AM, Hauer J, Koerner JF, Layton M, McDade J, Osterholm MT, O'Toole T, 
Parker G, Perl TM, Russell PK, Schoch-Spana M, Tonat K. Plague as a biological weapon: 
medical and public health management. Working Group on Civilian Biodefense. JAMA. 
2000 May 3;283(17):2281-90. Review.   PubMed ID: 10807389 
 
2. WHO International meeting on preventing and controlling plague: the old calamity still 
has a future. Wkly Epidemiol Rec. 2006 Jul 14;81(28):278-84. English, French.   PubMed 
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