Evaluation of a Diagnostic to Identify G6PD Deficiency in Brazil
Phase: NA
Conditions: G6PD Deficiency
Sponsor: PATH

 
 
 
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Evaluation of a diagnostic to identify G6PD 
deficiency in Brazil 
 
Version 5 
May 2019 
 
Investigators: 
  
 
 
 
  
  
 
  
 
 
Location of Research:  
Manaus and Porto Velho, Brazil  
 
Proposed Project Dates: 
May-December 2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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Study Title 
Validation of diagnostics to identify G6PD deficiency in Brazil  
Précis 
Cross-sectional diagnostic accuracy study with 2,000 patient participants and 15 
health worker participants. The participant population will be recruited at clinics and 
through a household survey using an enriched sample of a population with known 
G6PD status, established through previous epidemiological studies. The health 
worker participants will include trained intended users of the G6PD tests. Health 
workers will take capillary blood samples and conduct two point of care tests: 1) 
hemoglobin test, and 2) investigational point-of-care (POC) G6PD test. A thick 
blood slide will also be prepared for malaria microscopy. Venous blood will be 
collected and transferred to a laboratory where reference assays will be performed on 
venous samples using the Pointe Scientific G6PD Analyzer and hemoglobin tests. 
Trained health workers will also be surveyed to assess product usability through a 
questionnaire to assess label and packing comprehension as well as results 
interpretation.  
Objectives 
Primary Objective: To assess the accuracy and reliability of G6PD tests in detecting 
G6PD activity and classifying results when used by trained health care workers in 
Brazil.  
Component Objectives: 
• To determine the performance of G6PD tests in detecting G6PD activity and 
hemoglobin (Hb) compared to a reference assay 
• To assess the comprehension of the G6PD test packaging and labelling among 
intended users 
• To assess the usability of G6PD test result outputs among intended users 
Endpoints 
• Sensitivity and specificity of G6PD tests compared to the Pointe Scientific G6PD 
Analyzer  
o accuracy between POC G6PD test measure of G6PD activity and a 
reference assay  
o accuracy between POC G6PD test measure of hemoglobin and a 
reference assay  
• Comparison of POC G6PD test results using capillary and venous samples  
• Percent of trained health workers who can accurately comprehend key messaging 
included in the test packaging and labels 
• Percent of trained health workers who can accurately interpret the result output 
and classify results as either normal, invalid, deficient or intermediate 
Population 
Based on estimated G6PD prevalence, we expect to recruit approximately: 
• 1,700 people with normal G6PD activity levels 
• 200 people with intermediate G6PD activity levels 
• 100 people with deficient G6PD activity levels 
For the usability portion, we will recruit 15 trained health workers.  
Study sites  
 This study will be conducted in Manaus and Porto Velho.   
Study 
Duration 
9 months (estimated)  

 
 
 
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Table of Contents 
1.0 
Background and rationale for the study 
4 
2.0 
Study objectives 
6 
3.0 
Study design 
6 
4.0 
Research participants 
10 
5.0 
Study procedures 
11 
6.0 
Consent process 
21 
7.0      Study products 
22 
8.0 
Benefit and risk considerations 
25 
9.0 
Study and safety monitoring 
26 
10.0    Ethical considerations 
27 
11.0    Confidentiality and data management 
28 
12.0    Study limitations 
30 
13.0    Investigator responsibilities 
30 
References: 
31 
 
 
 
 

 
 
 
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1.0 Background and rationale for the study 
 
Glucose-6-phosphate dehydrogenase (G6PD) is a critical housekeeping enzyme in red 
blood cells (RBC) that supports protective systems against oxidative challenge by 
producing the reduced form of nicotinamide adenine dinucleotide phosphate [1, 2]. The 
most common human enzyme defect is G6PD deficiency, which affects more than 400 
million people worldwide [3,4]. Red blood cells are especially vulnerable to the effects of 
these mutations because they cannot replenish their supplies of the enzyme once they 
mature and enter the bloodstream. As a result, these cells are susceptible to hemolysis 
when subjected to oxidative stress, which can occur after therapy with antimalarial 8-
aminoquinolines such as primaquine, a few antibiotics, and some anti-inflammatories.  
Hemolysis can also be activated by other exogenous agents, including foods (e.g., fava 
beans), henna, and some infections (e.g., hepatitis A or B, pneumonia, and typhoid fever). 
In newborns G6PD deficiency often is first manifested in newborns as jaundice resulting 
from hyperbilirubinemia, which, if unchecked, can lead to kernicterus, a form of brain 
damage. In 1989, the World Health Organization (WHO) working group on G6PD 
deficiency recommended that “whenever possible, neonatal screening should be 
performed…in populations where G6PD deficiency is common (i.e., where it affects 
more than three to five percent of males).”[5]. While knowing the G6PD status of a 
patient is useful clinical information, access to testing for G6PD deficiency is very 
limited due to the price and complexity of the diagnostic products available for this 
condition, especially in malaria endemic populations and low resource settings.  
 
Uncomplicated malaria is typically treated by eliminating the asexual stage parasites that 
circulate in the blood and are the cause of symptoms. These are typically treated with 
artemisinin-based combination therapy (ACT) for all species of malaria or, in some 
countries, for Plasmodium (P.) falciparum malaria and chloroquine for P.vivax malaria 
depending on the country policy. While patients with P. falciparum infections are cured 
with ACT, in the case of P.vivax infection, patients are cured of their asexual parasites 
but some parasites remain sequestered in the liver which later release into the blood and 
cause relapse. 8-aminoquinoline-based malaria drugs such as primaquine and potentially 
in the future, tafenoquine, are the only ones with the capacity to prevent relapse and 
eliminate the liver stage parasites in P. vivax infections. Tafenoquine requires only a 
single dose regimen in comparison to a multi-day (7-14 days) regimen for primaquine. 
Tafenoquine is not currently available on the market.  
 
Because of the risks associated to G6PD deficiency for primaquine, the WHO 
recommends as good practice in the current malaria treatment guidelines: 
“the G6PD status of patients should be used to guide administration of primaquine for 
preventing relapse.” In recognition of (1) the diversity in prevalence of G6PD deficiency 
in malaria exposed populations and (2) the operational challenges in testing for G6PD 
deficiency in the context of malaria case management, the WHO also recommends: 
“when G6PD status is unknown and G6PD testing is not available, a decision to prescribe 
primaquine must be based on an assessment of the risks and benefits of adding 
primaquine.” 
 

 
 
 
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Considering these recommendations, some country malaria guidelines only treat P.vivax 
blood stage infections and do not provide primaquine. Other countries provide 
primaquine without testing for G6PD deficiency and others provide primaquine only if 
testing for G6PD deficiency is available.  
  
Currently, the G6PD status of a patient is most often defined by the patient’s G6PD 
phenotype, characterized by analysis of total activity in blood lysate [6,7]. This method, 
considered the gold standard, is a costly and complex quantitative laboratory based 
spectrophotometric test. While quantitative laboratory test can clearly identify subjects 
with all ranges of G6PD activities (including those with intermediate levels who may also 
be at risk of severe hemolysis), these methods require an equipped laboratory and skilled 
personnel.  
 
In field settings, the most commonly performed tests use qualitative devices such as the 
fluorescent spot test (FST) or the CareStart test, which can only discriminate gross 
deficiencies from all the other phenotypes [8-10]. These qualitative tests are adequate for 
males who are either deficient or normal in G6PD status. Because females carry two 
alleles of the G6PD gene, they can present as deficient, intermediate, or normal for G6PD 
activity. Qualitative tests cannot discriminate intermediate from normal G6PD activity. 
Quantitative tests are required for better case management for women, especially in 
anticipation of the availability of tafenoquine which is indicated for use only for women 
and men with normal G6PD activity.  
 
Given the target populations where antimalarial drugs are required, often in remote rural 
settings, point-of-care (POC) G6PD tests are required to support broader availability of 
primaquine and, in the future, tafenoquine. For treatment of women whose enzymatic 
activity is estimated to be normal by qualitative testing but is still too low for treatment of 
high-dose primaquine or tafenoquine, a quantitative portable device will be needed to 
obtain more accurate levels of G6PD activity to ensure appropriate and safe treatment.  
 
To date, no routine screening of G6PD deficiency is performed in Brazil before 
primaquine use for radical cure of P. vivax malaria. However, recent data suggest that 
G6PD screening could be cost-effective and that implementation of this additional step in 
malaria diagnosis and treatment is being considered by the Ministry of Health.  
 
In order to enable the use of radical cure treatment for P. vivax malaria, PATH is working 
to accelerate the development of G6PD tests that meet the following criteria: 
• Quantitative 
• Measures G6PD activity normalized by hemoglobin (IU/g Hb) 
• Measures hemoglobin (g/dL) 
• Corrects for enzyme temperature dependence 
• Finger stick (<10 ul) 
• Point-of-care 
• Easy-to-use 
• Affordable 
• Reviewed by WHO Prequalification Programme (WHO PQ) 

 
 
 
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PATH and the product developers have assessed POC test performance through 
laboratory evaluations using frozen specimens. Next steps in the validation of the POC 
tests include an assessment of diagnostic accuracy using clinical samples in target 
geographies. In addition to diagnostic accuracy, there is a need to assess the POC test to 
ensure its suitability for use among target end users. Data on both diagnostic performance 
and test usability will be required to support the registration of POC G6PD tests with key 
regulatory authorities in target countries.  
 
In Brazil, there is no routine screening to identify individuals with G6PD deficiency in 
the public health system and treatment with primaquine is recommended for all persons 
infected with P.vivax, with the exception of pregnant women and children less than six 
months of age [11-12]. In a recent cost study, Peixoto et al. estimate that the use of 
primaquine among the population with G6PD deficiency represents a great burden for the 
public health system on Brazil [13].  
 
 
2.0 Study objectives 
 
The hypothesis of this study is that the rapid test for G6PD and Hemoglobin, SD 
Biosensor, has equal accuracy by means of a comparison with the gold standard and 
high level of usability and acceptability among healthcare professionals who will execute 
it. The goal of this study is to contribute to a body of evidence that will support the 
submission of G6PD tests to the WHO PQ process and for product registration in target 
countries.  
 
The primary objective is to assess the accuracy and reliability of G6PD tests in detecting 
G6PD activity and classifying results when used by trained health care workers. 
Component objectives include: 
• To determine the performance of G6PD tests in detect G6PD activity compared to a 
reference assay 
• To assess the comprehension of the G6PD test packaging and labelling among 
intended users 
• To assess the usability of G6PD test result outputs among intended users 
 
3.0 Study design 
 
This is a cross-sectional diagnostic accuracy study that includes both participants and 
health worker participants. Health workers will include trained intended users of the 
G6PD tests. Health workers will take capillary blood samples and conduct POC G6PD 
testing on participant samples. Results will be compared to a reference assay. Trained 
health workers will also be surveyed to assess product usability through a questionnaire 
to assess label and packing comprehension as well as results interpretation for simulated 
G6PD tests. 
 
Study endpoints include: 

 
 
 
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• Diagnostic performance (sensitivity and specificity) of POC G6PD test compared to 
reference assay  
o Accuracy  between POC G6PD test measure of G6PD activity and a reference 
assay  
o Accuracy  between POC G6PD test measure of hemoglobin and a reference assay  
• Comparison of POC G6PD test results using capillary and venous samples  
• Percent of trained health workers who can accurately comprehend key messaging 
included in the test packaging and labels 
• Percent of trained health workers who can accurately interpret the result output and 
classify results as either normal, invalid, deficient or intermediate 
 
3.1 Statistical analyses 
Data will be entered into a Microsoft Excel database with built-in validation rules to 
minimize data entry errors. Descriptive statistical analysis, including calculating point 
estimates, distribution, and frequencies of responses, will be used to summarize and 
characterize the study population. 
 
3.1.1 Diagnostic accuracy and performance: 
For the purposes of this study, an individual will be considered G6PD deficient (case) if 
they test positive by the spectrophotometric gold standard. The primary success criterion 
will be focused on the ability to identify G6PD deficient participants correctly, such that 
both the POC G6PD test and the spectrophotometric gold standard test should both 
accurately identify all G6PD deficient specimens (with < 20% normal) as deficient. 
 
The performance of the POC G6PD against the spectrophotometric gold standard will be 
determined by calculating the sensitivity and specificity. Sensitivity and specificity of the 
SD Biosensor (SDB) G6PD assay were calculated as per Domingo et al. [14]. In 
summary, an adjusted male median will be calculated for both the POC G6PD and 
spectrophotometric gold standard test from where the 30%, 40%, 70% and 80% cutoff levels 
for the two tests will be used to categorically define G6PD deficient cases.  
 
Sensitivity calculations will be determined by the following method: TTP=true and test 
positive (positive by reference assays according to case definition and positive by the 
POC G6PD test), FNTP=false negative true positive (positive by reference assays 
according to case definition and negative by the POC G6PD test). Sensitivity 
=TTP/(TTP+FNTP).  
 
Specificity will be determined by the following method: TPTN=test positive true 
negative (negative by reference assays according to case definition and positive by POC 
G6PD test), TTN=Test and true negative (negative by reference assays according to case 
definition and negative by POC G6PD test). Specificity= TTN/(TTN+TPTN). 
 
Sensitivity and specificity results will be reported using 95% confidence intervals. 
 
3.1.2 Accuracy between G6PD activity and Hemoglobin methods: 

 
 
 
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Quantitative agreement for both G6PD activity and Hb values between the POC G6PD 
test and spectrophotometric gold standard will be graphically analyzed. Correlation 
graphs between the POC G6PD test and gold standard test will be plotted and an R-
squared value will be determined. An R-squared value of greater than 0.9 for both G6PD 
activity and Hb will be considered acceptable.  
 
Bland Altman plots, where differences between the G6PD POC tests and gold standard 
test are plotted against the gold standard value, together with the 95% limits of 
agreement. Acceptable limits of agreements for Hb should be within +/-1.0 g/dL (based 
on a 6% estimate for allowable method bias) and for G6PD activity should be within +/-
2.0 U G6PD/g Hb (based on a 15% estimate for allowable method bias). All statistical 
analyses will be performed using Stata 13.0.  
 
The data comparison for the analyses is outlined below: 
 
Table 1. Comparison methods  
Index test by 
sample type  
Reference method 
G6PD normalized for Hb 
Hb  
Venous  
Point Scientific G6PD from venous specimen 
normalized for Hb from venous specimen  
Hb from venous 
specimen  
Capillary  
Hemocue Hb from 
finger stick  
 
3.1.3 Diagnostic usability:  
Response choices to usability assessment questionnaires will include both multiple-
choice and open-ended responses. The usability questionnaire will include a brief 
assessment of health literacy using a validated health literacy instrument. Participants will 
be encouraged to comment on any aspects of the label or results they find confusing or 
inadequate. Success criteria are defined as 85% correct participant response to questions 
that assess key messages and results interpretation. Any participant that obtains 85% or 
above correct responses to the usability questionnaire will be considered to accurately 
comprehend the product IFU and labelling. Analyses will include descriptive statistics 
and a tabular presentation of findings.    
 
3.2 Sample size  
The sample size for this study is based on the expected prevalence of G6PD deficiency at 
the study site and by data requirements set by WHO PQ [15]. This requires obtaining 
samples from participants with a range of G6PD activity levels. The WHO PQ process 
defines these levels as such: 
 
Table 2. G6PD activity thresholds  
Females 
 
Deficient 
G6PD activity  < 30% of the adjusted male median 
Intermediate  G6PD activity 30%-80% of the adjusted male median 
Normal 
G6PD activity > 80% of the adjusted male median 
Males  
 

 
 
 
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Deficient 
G6PD activity < 30% of the adjusted male median 
Normal 
G6PD activity > 30% of the adjusted male median 
 
According to the Target Product Profile (TPP), the novel POC G6PD will need to be at 
least 95% sensitive for detecting G6PD activity levels at 30% to 80% of normal enzyme 
activity. Assuming a sensitivity of 95%, with a confidence interval of 95%, and a 1% to 
2% maximum marginal error, a minimum of 162 participants with deficient and 
intermediate G6PD activity will be needed. To account for any possible device/diagnostic 
failures or compromised blood samples due to insufficient blood, signs of blood 
degradation, or contamination, to the sample size will need to increase by 20%. In 
addition, to ensure there are sufficient deficient and intermediate participants in the case 
of a lower-than-expected enrollment rate of these two groups occurs, at least 200 
participants of each group should be included in the study. This additional enriched 
sample will also serve to meet the requirements of the WHO PQ. Eligible adults and 
children aged 2 years old and above will be recruited for potential participation though 
diagnostic performance analysis will not be segmented based on age.   
 
The estimated prevalence of G6PD activity at less than 30% of normal enzyme activity is 
5% in Brazil [16]. In Brazil, testing a minimum of 2,000 individuals is expected to 
generate approximately 100 deficient and 200 intermediate individuals. These data will 
be combined with data from other clinical evaluation sites in Ethiopia and India. The 
estimated prevalence of G6PD activity at less than 30% of normal enzyme activity is 
2.5% in Ethiopia and 5% in India [16]. In Ethiopia, testing a minimum of 1,500 
individuals is expected to generate approximately 38 deficient and 75 intermediate 
individuals and in India testing a minimum of 1,000 individuals is expected to generate 
approximately 50 deficient and 100 intermediate individuals. By combining the data from 
multiple clinical sites, these studies will reach the target number of samples required by 
WHO PQ.  
 
Per guidance from the WHO PQ and US FDA guidelines for usability testing, 15 
purposively selected intended users of POC G6PD tests will be sampled for the usability 
assessment across both study sites [15] [17]. As in the diagnostic performance 
assessment, the data from this usability assessment will be combined with data from a 
similar sample of health workers in other evaluations (a minimum of 15 in each Brazil 
and India) in order to reach the target number of users required by WHO PQ. 
 
Participant group  
Recruitment sample size 
Target enrollment sample 
size 
Participants  
2,200 
2,000 
Health workers trained in 
the use of the G6PD test  
18 
15 
 
3.3 Study sites  
This study will be conducted in Manaus and Porto Velho, reference centers for malaria 
diagnosis and treatment which already perform gold standard diagnosis of G6PD because 
of concomitant clinical trials in which this information is key (e.g., tafenoquine trials). 

 
 
 
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These centers are also responsible for technical supervision and training of other teams 
working in the primary care in the municipalities. Therefore, these sites have access to 
lower levels of health care service delivery. 
 
4.0 Research participants 
 
4.1 Characteristics of research participants 
Participants:  
Participants for this study will include both febrile patients seeking care at the Manaus 
and Porto Velho clinics and participants recruited via and enriched sample with known 
G6PD status established through previous epidemiological studies. Approximately half of 
the total participants will be recruited from each patient population. Participants with 
known G6PD recruited from the previous epidemiological studies have agreed to have 
their G6PD status retained and to be contacted again for participation in future studies as 
part of the informed consent process.  
 
Health workers:  
The health workers recruited for participation in this study will be representative of 
intended end users of point of care G6PD tests, as per requirements of the WHO PQ. This 
will include both health care providers such as community health workers, and laboratory 
technicians who perform malaria rapid diagnostic tests and other malaria testing near the 
point of care.   
 
4.2 Inclusion and exclusion criteria 
4.2.1 Participant with unknown G6PD status  
Inclusion criteria: 
• Seeking care at the Manaus or Porto Velho clinics 
• 2 years age or older  
• Willing to provide informed consent  
Exclusion criteria  
• Younger than 2 years of age 
• Participants who received a blood transfusion in the last 3 months, self-report  
• Unwilling to provide informed consent  
 
4.2.2 Participant with known G6PD status  
Inclusion criteria: 
• Included in previous G6PD surveys and provided consent to be contacted again  
• 2 years of age or older 
• Willing to provide informed consent or assent 
 
Exclusion criteria:  
• Younger than 2 years of age 
• Participants who received a blood transfusion in the last 3 months, self report 
• Unwilling to provide informed consent or assent or unavailable during study visit 
 

 
 
 
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to receive additional training. Records of the user proficiency assessment and 
certification will be kept as study documents and not shared with the laboratory managers 
or supervisors. Trained health workers will then be given a questionnaire to assess test 
label comprehension and results interpretation. At that time, the study staff will 
emphasize the participants that the questionnaire is being used to assess the effectiveness 
of the training content and test instructions, and not the skills or performance of the user.  
 
5.2 Diagnostic performance assessment 
5.2.1 Recruitment  
Screening of participants with unknown G6PD status will occur at the clinics in Manaus 
and Porto Velho and collaborating health facilities in the clinical catchment area. At 
clinics, the study team will use the recruitment script to recruit participants in the public 
areas, such as the waiting areas, of the clinics. Recruitment will be conducted in such a 
way as to not interfere with the potential participant’s care.  
 
Recruitment of participants with known G6PD status will take place at through home 
visits conducted by the study staff. Participants who were previously involved in 
epidemiological studies will be contacted in person by the study team and screened 
against inclusion criteria. Participants with known G6PD recruited from the previous 
epidemiological studies have agreed to have their G6PD status retained and to be 
contacted again for participation in future studies as part of the informed consent process.  
 
5.2.2 Screening and consent-clinics   
Study staff will screen participants for eligibility. The study team will explain the study 
and invite the person to participate. If the person expresses interest in participating in the 
study, she or he will be directed to a private space for consenting. Written consent or 
assent will be obtained from all participants. See detailed consent procedures and 
explanation of ethical considerations in sections 6.0 and 10.0.   
 
5.2.3 Screening and consent-household visits  
For participants with known G6PD status, the study team will use the recruitment script 
to recruit participants in their homes. Only members of the household who have a known 
G6PD status from past epidemiological studies will be eligible for participation. Other 
household members will not be asked to participate in the study. He or she will screened 
for inclusion in the study. The study team will explain the study and invite the person to 
participate. If the person expresses interest in participating in the study, she or he will be 
directed to a private space for consenting. Written consent or assent will be obtained from 
all participants. See detailed consent procedures and explanation of ethical considerations 
in sections 6.0 and 10.0. 
 
5.2.4 Testing procedures at the point of care  
All study procedures will be done in concordance with the health system; study 
procedures done at the clinics will be done in collaboration with clinic staff and the 
household surveys will be in collaboration by the health system agents and malaria 
control program. 
 

 
 
 
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Following completion of informed consent process, the study staff, who are trained in 
phlebotomy, will obtain a finger stick sample of no more than 30µl from the participant 
to perform the G6PD POC test, the POC hemoglobin test, and a thick blood film will be 
prepared to test for malaria as per the standard of care. Results of the POC tests will be 
recorded on data collection forms. If an invalid test result is obtained, a second finger 
stick sample will be obtained and a second test will be run. If an invalid test result is 
obtained a second time, the results will be recorded as invalid. The study staff will also 
draw a 4 mL tube of venous blood from the participant. The venous blood specimen will 
be taken using a standard venipuncture kit, and blood will be collected in the EDTA 
treated tube.  
 
Slides will be viewed at the nearest health facility by a microscopist. Results will be 
delivered to patients by the health agents as per standard routine.  
 
If the participants test positive for malaria or is found to be anemic based on hemoglobin 
thresholds from the WHO, the participant will be referred to a local health worker or the 
clinic for follow-up and case management (see Appendices C2 and C3).  
 
 
5.2.5 Testing procedures in the laboratory  
At the laboratory, aliquots of the venous blood sample will be used to run an additional 
replicate of the SBD POC test and reference tests in duplicate. Depending on availability, 
additional POC G6PD tests that are both commercially available and currently under 
development may be run as well. Hemoglobin [Hb value (g/dl)] will be determined 
within 24 hours using a hematology analyzer for each sample to estimate the G6PD 
activity. Aliquots of the venous blood will be used for running the spectrophotometric 
assay (reference standard) and the florescent spot test (FST). The reference assay, 
hemoglobin, and replicate of the G6PD quantitative test will be conducted at the same 
time. A thermometer and a hygrometer will be placed at the study site near the assays. 
Temperature and humidity will be observed at the time the tests are run and recorded in 
the study database. 
 
Test operators in the laboratory will be blinded to the results of the POC tests obtained 
and recorded in the field and operators of the reference assay will be blinded to the results 
of the replicate POC test run on venous sample. If there is a discrepancy between the 
results of the reference assay replicates, greater than allowed by the TPP, testing will be 
repeated. Performance characteristics and the results of additional testing of samples with 
discrepant results will be reported as per WHO technical specifications [15].  
 
Leftover sample will be stored for possible confirmatory or additional testing. Depending 
on availability of additional novel G6PD tests at the time of study start, additional assays 
may be run on remaining samples. If additional POC G6PD tests are added, in particular 
tests that are under investigation and not registered for use in Brazil, the study team will 
amend the protocol and advise the ethical review boards and appropriate regulatory 
authorities. For all novel assays to be included in the study, appropriate regulatory 
procedures will be followed in order to include the assays for research purposes only. 

 
 
 
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immediately after testing, as the test is registered for use in Brazil. Study staff will relay 
results and counsel participants who test positive for malaria or anemia according to the 
results return script (Appendix C3). Any participants identified as malaria positive or 
anemic will be counselled and referred for follow-up. Participants in the clinic will 
receive standard care at the clinic and participants recruited through household surveys 
will be referred for follow-up to the local health agent as per routine practice.  
 
In Brazil, no routine screening to identify individuals with G6PD deficiency is 
implemented in the public health system and treatment with primaquine is recommended 
for all people infected with P.vivax with the exception of pregnant women and children 
under six months of age. However, information regarding the G6PD status of a vivax 
patient is critical for informing safe and effective treatment recommendations. As per 
WHO guidelines for the treatment of malaria, standard treatment of primaquine should 
not be given to patients who are G6PD deficient in order to avoid primaquine-induced 
hemolysis and patients who are G6PD intermediate should be monitored closely for signs 
of hemolysis [18].  
 
Initial evaluations by PATH indicate that the POC G6PD test is able to identify G6PD 
deficiency (see section 7.1). As such, participants who test positive for P. vivax malaria 
and are found to be G6PD deficient or intermediate will be notified of their POC test 
result in order to make that information available to their health provider when 
determining their course of treatment for vivax malaria.  
 
Participants found to be vivax positive and G6PD deficient  
For vivax positive participants found to be G6PD deficient with the POC test (G6PD test 
result of below 30% of normal enzymatic activity), the study team will provide referral 
with the recommendation that primaquine treatment be withheld until confirmatory 
testing can be completed. Confirmatory testing will take no more than one week to 
complete. At that time, through referral to the health agent, P. vivax patients who are 
identified as G6PD deficient can receive treatment for the blood stage disease (treatment 
with chloroquine) immediately.  
 
If the results of the reference assay confirm the results of the POC G6PD test (enzymatic 
activity less than 30%) they will be contacted and informed of the result and their G6PD 
status by validated reference assay. If the results of the reference assay indicate that the 
participant is G6PD normal, they will be contacted, informed of the result, and referred to 
a health agent to receive with standard treatment for liver stage disease, as per national 
guidelines.   
 
Participants found to be vivax positive and G6PD intermediate  
For vivax positive participants found to be G6PD intermediate with the POC test (G6PD 
test result between 30 and 80% of normal enzymatic activity), the study team will 
provide referral with the recommendation that primaquine treatment be closely monitored 
for signs of hemolysis given the increased risk of hemolysis associated with moderate 
G6PD deficiency.  
 

 
 
 
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If the results of the reference assay confirm the results of the POC G6PD test (enzymatic 
activity between 30-80%) they will be contacted and informed of the result and their 
G6PD status by validated reference assay. If the results of the reference assay indicate 
that the participant is G6PD normal, they will be contacted and informed of the result and 
referred to a health agent to receive with standard treatment for liver stage disease, as per 
national guidelines.  
 
Among any vivax positive participants, those known to be G6PD intermediate or 
deficient based on previous studies may receive a false negative result on the POC 
investigational test in this study and found to be G6PD normal. In the event that this 
occurs, the study team will proceed in the same manner as for participants found to be 
vivax positive and POC G6PD deficient or intermediate. See Figure 3.  
 
Participants will be notified at recruitment that the study team will tell them the results of 
the G6PD test only if they are found to be G6PD intermediate or deficient. For 
participants who do not have P. vivax malaria, confirmatory testing will be done prior to 
informing the participant of this result. Participants who are found to be G6PD normal by 
the reference assay will not be notified of their result unless they were notified of a G6PD 
deficient and/or intermediate result by the POC test that needs to be corrected. If they 
were reported a false positive result from the POC test and found to be G6PD normal by 
the reference assay, the study team will follow up and provide the results of the reference 
assay and necessary counselling. Any participants found to be G6PD intermediate or 
deficient will be contacted by the study team to provide counselling and given a card with 
further information for patients and providers (Appendix C2).  
 
The study team will inform the participant that G6PD is a genetic condition and 
recommend that they may want to encourage their family to be tested as well. The study 
team will inform participants where and how their immediate family members can be 
tested free of cost. The card describes G6PD deficiency as it relates to contraindicated 
medications and other risk factors such as foods. The research team involved in the return 
of G6PD test results have significant experience working in G6PD research and 
counselling study participants as to the results. All staff involved in returning these 
results will receive dedicated refresher training aimed at providing the necessary 
information and delivering it in a way that facilitates comprehension. 
 

 
 
 
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5.5 Summary of study procedures  
Table 6. Summary of study procedures  
Participant group 
List of study 
procedures 
Estimated time  
Data 
collection 
forms used  
Health workers  
• Training on the use 
of POC G6PD tests 
• Consent and 
enrolment  
• Usability 
assessment 
questionnaire  
• Up to 5 hours  
 
• 20 minutes 
 
• 20 minutes  
• Appendix 
A2 
Children aged 2-18 
• Assent, parental 
permission, and 
enrollment  
• Conduct POC tests 
and blood draw 
• 20 minutes  
 
 
• Up to 30 min  
• Appendix 
A1 
Adults 
• Consent and 
enrolment  
• Conduct POC tests 
and blood draw  
• 20 minutes  
 
• Up to 30 min 
• Appendix 
A1 
 
 
 
 
 
 

 
 
 
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Figure 3. Participant flow and follow-up procedures 
 
 
 
*Vivax positive patients, recruited from the enriched sample of participants known to be G6PD deficient or intermediate, may receive a false negative G6PD 
normal result from the investigational POC test, In the event this occurs, this subpopulation will be considered G6PD intermediate/deficient and provided 
counseling and a counselling card as described in section 5.4.  
 

 
 
 
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5.6 Standard care and study procedures  
In Brazil, national guidelines for P. vivax malaria call for treatment of the blood stage of 
the parasite. For the prevention of relapse, the guidelines recommend treatment that 
includes 3 days of chloroquine (CQ) and then 7 days of primaquine (PQ). As part of 
standard malaria case management, suspected malaria cases are confirmed with 
microscopy. No G6PD testing is recommended or routinely available prior to treatment 
with primaquine.  
 
In Brazil, anemia screening is not routinely done at the point of care as part of case 
management for suspected malaria. The thresholds for anemia are as follows: 
 
• Mild anemia:  
o Hb 10.0 – 11.9 g/dl in women 
o Hb 10.0 – 10.9 in pregnant women and children 
o Hb 10.0 – 12.9 g/dl in men 
• Moderate anemia: Hb 7.0 – 9.9 g/dl  
• Severe anemia: Hb <7.0 g/dl 
 
There are no national guidelines regarding the treatment of anemia in Brazil. Treatments 
provided at the discretion of the medical care provider and generally involves iron 
supplementation, using oral iron tablets or parenteral injections, depending on severity. In 
this study, all diagnoses and treatment of anemia will be done through referral to a health 
agent or clinic.  
 
For the research study, in addition to conducting microscopy to screen for malaria, the 
study will test participants using an investigational POC G6PD test, an approved 
hemoglobinometer, and laboratory reference assays intended to assess the performance of 
the POC tests. Participants who test positive for malaria will be referred to a local health 
agent for further testing and case management. All treatment decisions will be made 
through referral to a local health worker or clinic.   
If any participants are confirmed P. vivax malaria positive and G6PD deficient at the 
point of care, the study team will relay this the information as part of the counselling. The 
study team will refer the participant to a health for treatment, equipped with counselling 
information that the participant may be G6PD deficient or intermediate and if given 
primaquine treatment is at an increased risk of severe hemolysis. This will enable the 
participant to receive immediate treatment for the blood stage infection and provide better 
information to the health worker responsible for case management, including any 
treatment for the liver stage infection. The participant’s G6PD status will then be 
confirmed by a reference assay to inform further case management as necessary. The 
table below outlines the study procedure and subsequent follow up action.  
  
Table 5. Case management  

 
 
 
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Study test 
Result 
Action 
Hemoglobin 
Normal hemoglobin levels  (by 
national guidelines) 
No action 
Mild moderate or severe anemia  
(by national guidelines) 
Counselling re: the results of the hemoglobin test 
and referral to the health system for follow-up 
case management. This may involve the 
provision of iron tablets or other treatment 
depending on severity.  
G6PD 
deficiency 
Negative test result on 
investigational device  (G6PD 
normal) 
No immediate action. If reference assay is 
discrepant and identifies G6PD intermediate or 
deficient status, the study team will conduct 
follow-up counselling (Appendix C2). 
Positive test result on 
investigational device  (G6PD 
deficient or intermediate) 
Result to be confirmed at the laboratory with the 
reference assay. If confirmed G6PD deficient or 
intermediate with the reference assay, the study 
team will conduct follow-up counselling 
(Appendix C2). 
Malaria  
 
Negative test result (no malaria)  
 G6PD and hemoglobin managed as above, 
otherwise no action 
Positive test 
result 
(confirmed 
malaria) 
POC G6PD 
normal  
Patient referred to health system for free 
treatment: 3 days CQ and 7 days PQ. 
If confirmed deficient or intermediate with 
reference assay, provide follow up counselling as 
described above.  
POC G6PD 
intermediate 
Patient referred to health system for free 
treatment: 3 days CQ and 7 days PQ. Provide 
counseling re: G6PD intermediate status and 
recommend additional monitoring and attention 
to possible signs/symptoms of hemolysis 
If confirmed deficient or intermediate with 
reference assay, provide follow up counselling as 
described above.  
POC G6PD 
deficient  
Patient referred to health system for free 3 days 
CQ with recommendation for health care provider 
to withhold PQ until confirmatory G6PD testing. 
Based on the reference assay, provide appropriate 
counselling for G6PD status as well as for any 
future treatment with PQ. 
 
6.0 Consent process 
 
6.1 Usability study  
Consent will take place, after health workers have be recruited, in a private setting in the 
health facility. Members of the study team trained in the protection of human subjects 
will conduct the consent procedure. During the consent process, the study team will 
explain the purpose of the study and what involvement will entail. It will be emphasized 
that participation is voluntary and that their decision will not affect their employment in 
any way. The potential participant will have the opportunity ask questions. Consent for 

 
 
 
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participation will be documented on a written informed consent form. The participant’s 
understanding of the study and their potential involvement will be assessed with brief 
questions on the consent forms. One copy of the signed informed consent form will be 
provided to the participant and one copy will be kept for study records. 
 
6.2 Diagnostic performance assessment  
Consent will occur, after participant recruitment, in a private setting in the health facility 
or home. Members of the study team trained in the protection of human subjects will 
conduct the consent procedure. Study staff will review the study details with the 
volunteer. The potential participant will be given an opportunity to review the informed 
consent form and ask questions. Written consent or assent will be obtained from all 
participants. Parent permission will be obtained for all participants under the age of 18. 
Child assent will be obtained for all participants between the ages of 5 and 11 with one 
form (Appendix B4.1) and between the ages of 12 and 17 using another form (Appendix 
B4.2).  Where appropriate, a conversational style oral presentation of consent information 
will be made in the local language to parents and children in order to account for any 
difficulties understanding written consent forms due to low literacy. Parents will confirm 
their permission for a child to participate in the study by signing or thumb printing on the 
consent forms. Children 5-17 will express their assent in writing in the presence of their 
parents as a witness, to ensure the assent process is without any coercion. The consent 
process will be conducted in Portuguese and consent forms were written in Portuguese 
and translated into English.  
 
During the consent process, the study team will explain the purpose of the study and what 
involvement will entail. It will be emphasized that participation is voluntary and that their 
decision will not affect the care they receive in any way. The potential participant will 
have the opportunity ask questions. Consent for participation will be documented on a 
written informed consent form. The participant’s understanding of the study and their 
potential involvement will be assessed with brief questions after the consent is reviewed. 
One copy of the signed informed consent form will be provided to the participant and one 
copy will be kept for study records. 
 
If a potential participant is illiterate, an independent literate witness will be asked to join 
the consent process. This witness will be a health care worker or other family member 
uninvolved in the study. The study staff will read the consent form aloud to the potential 
participant and the witness will verify that the information read aloud matches the 
information written on the consent form. The witness will affirm that the study 
participant chose to be in the research study, that he or she was present the whole time the 
study was being explained, and that the participant had a chance to ask questions. The 
participant will get a copy of this form to keep. The witness will also sign the consent 
form. 
 
7.0 Study products 
 

 
 
 
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7.1 SDB G6PD Analyzer  
The STANDARD G6PD Analyzer is designed to measure the quantitative determination 
of total-hemoglobin concentration and G6PD enzymatic activity in fresh human whole 
blood specimen based on reflectometry assays. The test is intended to aid in the 
identification of people with G6PD deficiency. The test is currently registered in Brazil 
but not used by the public health system or considered part of routine use in malaria case 
management. PATH has conducted some initial laboratory evaluations which suggest that 
the test aligns to performance specifications outlined in the target product profile [19]. 
The test has CE mark and is registered for use in other malaria endemic countries.  
 
Performance STANDARD G6PD test when stress tested under multiple temperature and 
humidity conditions. The STANDARD G6PD values were compared to the Pointe 
Scientific generated G6PD values for all 314 data points generated across the following 
ambient conditions: 22 °C, 32 °C 50% humidity, 37 °C 50% humidity, and 37 °C 75% 
humidity. ROC analysis was performed to generate the optimal thresholds for the 
STANDARD G6PD test.  CI refers to the confidence interval, NA, not applicable. 
 
Table 7. Performance of SD Biosensor STANDARD G6PD test 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Threshold 
(Pointe Scientific value U/gHb) 
Optimal STANDARD G6PD 
(U/gHb) 
Sensitivity 
(95% CI) 
Specificity  
(95% CI) 
30% (2.8) 
4.2 
100 
(95.1 – 100.0) 
96 
(93.0 – 98.3) 
70% (6.5) 
6.0 
98 
(92.4 – 99.7) 
99 
(96.8 – 99.9) 
80%  (7.4) 
6.2 
86 
(80.7 – 93.9) 
99 
(95.9 – 99.7) 
100%  (9.3) 
NA 
NA 
NA 

 
 
 
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Figure 4. SD Biosensor STANDARD G6PD Test 
 
 
 
 
 
7.2 Pointe Scientific  
The Pointe Scientific will serve as the reference assay to assess G6PD activity. Its 
intended use is for the quantitative, kinetic determination of G6PD in blood at 340 nm. 
For in vitro diagnostic use only. 
• A spectrophotometer capable of measuring at 340 nm with temperature 
controlled cuvette compartment is required to perform the assay 
• To determine G6PD activity, which is reported in terms of grams of Hb or the 
number of RBCs, the Hb or RBC count must be determined separately from 
performing the G6PD assay. Calculations are then performed to obtain the 
G6PD activity. For purposes of this study, the G6PD activity from the Pointe 
Scientific kit will be calculated in terms of grams of Hb. 
• US FDA cleared: k024006, Regulatory Class II, Product Code JBF. 
• Approved as the predicate device for POC G6PD test evaluation by the US 
FDA.  
 
7.3 Fluorescent Spot Test    
The fluorescent spot test (FST) or TrinityBiotech G-6-PDH Screening test is widely used 
for in vitro diagnosis of G6PD deficiency using whole blood or dried blood spots. The G-
6-PDH Screening test is a qualitative test performed by incubating a small amount of 
blood with glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate 
(NADP). Drops of the mixture are removed at 5-minute intervals, spotted on filter paper 
and then viewed under long-wave ultraviolet light. Fluorescence is clearly evident in 
mixtures prepared from normal blood, whereas deficient samples yield little or no 
fluorescence. The test is affordable and produces qualitative, visual results in minutes. 
This test will represent a qualitative standard care test that is currently available and 
cleared by the FDA. The test will be performed according to PATH’s guide to fluorescent 
spot testing for G6PD deficiency [20]. 

 
 
 
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7.4 HemoCue 
 
 
The HemoCue Hb 301 system is designed for quantitative POC whole blood Hb 
determination in primary care using a specially designed analyzer, the HemoCue Hb 301 
Analyzer, and specially designed microcuvettes, the HemoCue Hb 301 Microcuvettes. 
The HemoCue Hb 301 System consists of a small and portable analyzer (photometer) and 
plastic microcuvettes. The microcuvette serves both as a pipette and as a measuring 
cuvette. A blood sample is drawn into the cavity by the capillary action. The filled 
microcuvette is inserted into the HemoCue Hb 301 Analyzer. The measurement takes 
place in the analyzer, which measures the absorbance of whole blood at a Hb/HbO2 
isobestic point. The system is factory calibrated and needs no further calibration.  
 
7.5 Sysmex Hematology Analyzer  
The Sysmex hematology automated analyzer is used to perform full blood counts and 
reticulocyte counts. This analyzer is registered and available for use in Brazil. It is used 
for research purposes routinely at the sites in Manaus and Porto Velho.  
 
8.0 Benefit and risk considerations  
 
8.1 Benefit to study participants  
Participants in this study will have convenient access to a malaria test and a hemoglobin 
test. Malaria testing is available for free through the public health system. Hemoglobin 
testing and G6PD testing are not routinely offered as part of standard care.  Participants 
who test positive for malaria or are found to be anemic will be counselled and referred to 
a local health worker or clinic for follow up and case management. Participants who have 
malaria will benefit from having their treatment informed by G6PD status, measured at 
the point of care, thus lowering the risk of severe hemolysis associated with standard 
vivax malaria treatment for G6PD deficient individuals, see Figure 3.  
 
Participants in this study will have the opportunity to have their G6PD status tested by a 
reference assay. G6PD deficiency is a genetic condition that provides valuable clinical 
information for multiple clinical conditions beyond malaria treatment. If clinically 
relevant results are determined by the reference assay- that is G6PD activity is 
determined to be deficient or intermediate- participants will receive counselling regarding 
this information. G6PD testing will be offered to immediate family members of 
participants who are found to be G6PD deficient or intermediate free of cost.  
The study team will test all family members with the FST, which will be more readily 
accessible both at the study sites and allow for faster results return. For females who test 
normal on the FST, the study team will follow-up with the gold standard test to determine 
if they are intermediate or normal, and provide appropriate counselling. This information 
may inform the care they receive for malaria in the future.  
 
This research will also be advantageous for academic study and in the future for other 
people who will benefit from better G6PD tests and malaria treatment. Health workers 
will have the opportunity to receive training in the use of POC G6PD tests.  

 
 
 
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8.2 Risk and risk mitigation considerations 
Study procedures do not represent significant risks to the participants beyond those that 
are associated with normal blood draw, such as pain, discomfort, feeling light-headed, 
fainting, and infection at the site of finger stick or venipuncture. The risks associated with 
blood draws will be mitigated through adherence to standard clinic procedures for 
infection control and through the use of research staff who have been trained in best 
practices for blood collection. The volume of blood drawn as part of the study procedures 
is within the safely limits recommended by the WHO and other organizations for both 
adults and children [21]. In the unlikely event of a research related injury, cost of 
treatment will be covered by the study. All decisions regarding clinical care or malaria 
case management will be made through referral to the local health care facilities. 
 
There are no known reports of stigma associated with G6PD deficiency.  
 
The study staff are at risk for exposure to blood-borne pathogens in the course of their 
work. All study team members will adhere to standard procedures for infection control. 
Study staff exposed to blood-borne pathogens during the course of their study roles will 
follow their institutional guidelines for post-exposure prophylaxis. 
 
There is a minimal risk that health workers recruited for the usability study may feel 
compelled to participate in the study if their supervisor has recruited him or her. They 
may feel as through the usability study is intended to assess their performance rather than 
the usability of the test. We will mitigate these risks through the following measures: 
• Study staff will ask supervisors to explain that participation in the study is voluntary 
and will not affect employment in any way.  
• Consent procedures will be conducted in private to ensure confidentiality. During the 
consent procedure, participants will be informed that the aim of the study is to 
understand the user experience and the data will be used for purposes of product 
development only. The data will not be used to assess their competency or linked in 
any way to their job performance. 
 
9.0 Study and safety monitoring 
 
We anticipate that this evaluation poses minimal risk to participants, as it does not 
involve any medical intervention and blood draw volumes are within acceptable ranges. 
No data safety monitoring board will be used. PATH and the FMT/HVD and Fiocruz  
will conduct necessary staff training on study procedures prior to initiating the 
evaluation. Only trained users who have been certified as proficient in the use of the test 
will be involved in blood collection. The information participants will provide in the 
context of this evaluation is not considered sensitive and will not pose any significant risk 
to them personally or professionally. 
 
The study team will be supervised by the local study lead. Study data will be aggregated 
into a database and a monitoring report will be generated every week, summarizing key 
indicators for study compliance. PATH and FMT/HVD and Fiocruz will hold weekly or 

 
 
 
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bi-weekly study review calls to discuss data collection and data quality to date. These 
indicators include but are not limited to the number of participants consented, the number 
of samples acquired, any deviations from study procedures, and corrective actions taken. 
This also includes any adverse effects related to blood sampling. Compliance indicators 
related to counselling and follow up discussed on this call will include: 
• number of participants found to be G6PD deficient or intermediate by laboratory 
reference assay 
• number of counselling cards delivered or picked up by participants found to be 
G6PD deficient or intermediate by laboratory reference assay 
• proportion of participants found to be G6PD deficient or intermediate by 
laboratory reference assay that received appropriate counselling and counselling 
cards.  
 
 
 
PATH will designate trained and qualified personnel to monitor the progress of this 
clinical study in accordance with Monitoring SOPs and the study-specific Monitoring 
Plan. Prior to study start, a study initiation visit will be conducted to provide training to 
site staff about the protocol, the completion of study documentation and data collection 
forms, the monitoring schedule, and all regulatory requirements. During the study, 
routine monitoring visits will be conducted to assure the site continues to adhere to the 
protocol, the investigator agreement, and regulations regarding conduct of clinical 
studies. Assessments will be made regarding the subjects’ protection and safety, when 
relevant, as well as the quality, completeness, and integrity of the data. 
 
10.0 Ethical considerations  
 
10.1 Study conduct  
The investigators will ensure that this study is conducted in full conformity with the 
current revision of the Declaration of Helsinki, or with the International Conference for 
Harmonization Good Clinical Practice (ICH-GCP) regulations and guidelines, whichever 
affords the greater protection to the subject. Additionally, the investigator assures that all 
activities of this protocol will be guided by the ethical principles of The Belmont Report, 
45 CFR 46 and all of its subparts (A, B, C and D). Investigators and study staff are 
trained in the protection of human subjects. Training in the principles of informed 
consent and in the study procedures for obtaining informed consent will be conducted 
before study initiation.  
 
10.2 Informed consent  
Study team members trained in the principles of informed consent and human subjects 
protection will obtain written informed consent from all participants of both the usability 
and diagnostic performance assessment.  
 
10.3 Ethical review committees  
The protocol, informed consent form, and recruitment materials will be submitted to 
PATH Research Ethics Committee and the local ethics review boards in Manaus and 
Porto Velho, and the national IRB of the Ministry of Health in Brazil (CONEP)  

 
 
 
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10.4 Amendments 
All amendments and modifications will be submitted to the above institutional IRBs for 
review and approval. No changes in protocol conduct will be implemented until 
approvals by all IRBs are obtained. 
 
10.5 Continuing Review Reports  
The Primary Investigator will be responsible for submitting the required continuing 
review report and associated documents to the relevant IRBs, allowing sufficient time for 
review and continuation determination prior to the established continuing review date. A 
closeout report will be submitted at the end of five years, or upon completion of the 
study, whichever comes first. 
 
10.6 Deviations 
Any deviation from the protocol that may have an impact on the safety or rights of the 
subject or the integrity of the study will be reported to the appropriate IRBs within 72 
hours of when the deviation is identified.  All other deviations will be reported in the 
annual continuing review report.   
 
10.7 Unanticipated Events  
Any adverse events that are unanticipated, serious, and related or possibly related to 
participation in the research, any serious adverse events, or any incidents that suggest that 
the research places participants or others at risk, including breach of confidentiality will 
be promptly reported to the appropriate IRBs within 72 hours. A complete written report 
will follow the initial notification. Other incidents will be reported in the annual 
continuing review report.  
 
10.8 Compensation  
Compensation will be given in order to reimburse any costs related to the participation in 
the study, such as transportation costs for participants recruited through household 
survey. 
 
10.9 Genetic testing  
G6PD is a genetic condition. The diagnostics used at the point-of-care and in the 
laboratory diagnose G6PD deficiency through a measurement of G6PD enzyme activity 
in the blood, not full genome sequencing.  
 
11.0 Confidentiality  
 
11.1 Participant confidentiality 
The investigator will ensure that each participant confidentiality is maintained. Any 
clinically relevant information will be shared with the participant in a confidential setting. 
Participants will not be identified in any publicly released reports of this study. All 
samples and records labeled with a participant ID and will be kept confidential by the 
FMT/HVD and Fiocruz. PATH will not have access to records that identify the subjects. 

 
 
 
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All health workers will have the opportunity to participate in the POC G6PD test training 
regardless of their decision to participate in the research activities. Results of the usability 
assessment will be de-identified prior to sharing with anyone outside of the study team, 
including supervisors or other clinic staff.  
 
11.2 Data Entry 
Participant data are entered on paper forms at the time the sample is taken and included 
with the samples sent back to the lab. All paper forms will be tracked by study ID 
number. Paper forms will be stored for 1 year after the study ends in locked cabinets, 
after which time they will be destroyed following standard site procedures. 
 
All laboratory results will be entered into an electronic, password-protected database. 
Electronic study records will be de-identified upon completion of data collection. The 
electronic records will be maintained indefinitely in the databases and remain password-
protected. 
 
11.3 Data Monitoring 
The study team will enroll and conduct sample collection at each site and will be 
supervised by the local study lead. Study data will be aggregated into a database and an 
monitoring report will be generated every week, summarizing key indicators for study 
compliance. PATH and local investigators will hold weekly data review calls to discuss 
data collection and data quality to date. These indicators include but are not limited to the 
number of participants consented, the number of samples acquired, any deviations from 
study procedures, and corrective actions taken. In addition, a member of the PATH study 
team will conduct site-monitoring visits as needed to ensure compliance with the protocol 
and relevant SOPs. 
 
11.4 Access to data and data dissemination  
The data generated by this study will inform the commercialization of G6PD tests and 
may inform programmatic decisions around testing for G6PD deficiency. Data will be 
shared with study partners and the test developers. All data will be published in the open 
medical literature with the identity of the subjects protected.   
 
Groups that supervise the study may access the results. This includes members of the 
ethics committees of PATH, FMT / HVD, Ethics Committee Involving Human Beings 
from the Center for Tropical Medicine Research (CEP / CEPEM), and any other test 
auditor or regulator such as the US Food and Drugs Administration (FDA). Only de-
identified data will be shared with groups outside of the study team. 
 
In addition, a description of this study will be available on the website: 
http://www.ClinicalTrials.gov, as requested by the US government. On this site, there 
will be no personal information that identifies individual participation, only the 
description of the study and the results when available.  
 
De-identified data and samples will be stored in a secure location in FMT/HVD and 
Fiocruz  clinical research unit. If the data or specimen stored is planned to be used for 

 
 
 
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other purposes than this study, FMT/HVD and Fiocruz  and PATH will seek approval 
from the appropriate ethical committees.   
 
11.5 Quality control and quality assurance  
The study will be conducted in accordance with the current approved protocol, 
International Conference on Harmonisation (ICH) good clinical practices (GCP), relevant 
regulations and standard operating procedures. 
 
12.0 Study limitations 
 
 
There are some limitations to this diagnostic evaluation. With regard to any diagnostic 
accuracy evaluation, there are opportunities for bias. This study will rely on the 
quantitative spectrophotometer assay as the reference test rather than genetic sequencing 
and an imperfect reference test may lead to classification bias. Given the rates of G6PD 
prevalence in Brazil and the data requirements in the WHO verification guidelines, some 
purposive sampling will be required and we expect a significant number of the samples 
tested in Brazil to be G6PD normal. Finally, given the wide range of potential end users 
of POC G6PD tests globally and across different segments of the health system, the 
results of the usability assessment may not be generalizable outside of the study sites.  
 
13.0 Investigator responsibilities  
 
The two project partners involved in this evaluation are PATH (prime award recipient 
and evaluation lead), and FMT/HVD and Fiocruz (implementing research partner). 
Roles and responsibilities for each of the partners are listed below. L= lead; A= assist 
 
Task 
PATH 
FMT/HVD 
and Fiocruz   
Award oversight 
L 
A 
Study design and protocol development 
L 
A 
IRB submission--PATH 
L 
A 
IRB submission—Brazil  
A 
L 
Evaluation logistics arrangements 
A 
L 
Procurement of all study supplies  
L 
A 
Training on the use of study assays  
L 
A 
Conduct recruitment, consent, enrollment, and field 
data collection 
A 
L 
Conduct laboratory based data collection  
A 
L 
Data entry and cleaning 
A 
L 
Data analysis and reporting 
L 
A 
 

 
 
 
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3. Cappellini MD, Fiorelli G (2008) Glucose-6-phosphate dehydrogenase deficiency. Lancet 
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4. Howes RE, Piel FB, Patil AP, Nyangiri OA, Gething PW, et al. (2012) G6PD deficiency 
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5. Glucose-6-phosphate dehydrogenase deficiency. WHO Working Group. Bull World Health 
Organ. 1989; 67(6): 601–611.  
6. Beutler E, Blume KG, Kaplan JC, Lohr GW, Ramot B, et al. (1977) International Committee 
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7. Van Noorden CJ, Vogels IM (1985) A sensitive cytochemical staining method for glucose-6-
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Appendices:   
 
A. Data collection forms 
B. Consent forms and assent forms 
C. Recruitment and result return materials 
D. Investigator’s brochure