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PREVENTING MALARIA AND ANEMIA IN PREGNANCY_

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					PREVENTING MALARIA AND ANEMIA
        IN PREGNANCY:
   A CASE-CONTROL STUDY OF
 INTERMITTENT PREVENTATIVE
 TREATMENT ON BIOKO ISLAND,
     EQUATORIAL GUINEA

    TRAVELING SCHOLARS PROPOSAL

     JOSEPHINE CZECHOWICZ, SMSI
       DR. YVONNE MALDONADO
         DR. LUIS BENAVENTE

           APRIL 17, 2006
                 TABLE OF CONTENTS

MEDICAL SCHOLARS COVER PAGE           3



RELEVANT EXPERIENCE                   4



INVESTIGATORS                         5



FIELD SITE                            6



LEARNING OBJECTIVES                   7



PROPOSAL

 ABSTRACT                             8

 SPECIFIC AIMS                        9
 BACKGROUND AND SIGNIFICANCE          9
 EXPERIMENTAL DESIGN                 11

   INTERPRETATION OF RESULTS         14

   POTENTIAL SOURCES OF ERROR        14
 RELEVANCE TO MEDICINE                 15
 JUSTIFICATION FOR AWAY EXPERIENCE     15
 BIBLIOGRAPHY                          16



APPENDICES

 APPENDIX I: QUESTIONNAIRES

     ENGLISH                           18

     SPANISH TRANSLATIONS              20
 APPENDIX II: IRB APPLICATION RECORD   22
 APPENDIX III: CONSENT FORMS           23
Please see paper copy for signatures.
                         RELEVANT EXPERIENCE
Prior Institutions: Carleton College
Major, Concentration: Biology, Biochemistry
Minor: Spanish
Degree: B.A. June 2004

Research         Where and      Duration   Average     Scholarly          Publication
Topic            When           in         hours       Contributions
                                Months     per week
Synthesis of     -Carleton      Total of   Summer:     -Synthesized       Abstract and Poster at
lead             College        10         50h/week    substrate          American Chemical Society
compounds for    Department     months                 analogue           Conference, 2004
drugs against    of Chemistry              Academi     inhibitors
trypanosomes                               c year:
                 -Summer                   10h/week    -Tested
                 2002, 2003                            inhibitory
                                                       kinetics
                 -Throughout
                 2003
                 academic
                 year
Antibiotic       -Center for    12         40-50       -Tested 404        -Abstracts and Posters:
resistance of    Vaccine        weeks      hours per   isolates of S.     Northern California
Streptococcus    Development               week        pneumoniae for     International Health
pneumoniae in    Santiago,                             resistance to 16   Conference, Chilean
Santiago,        Chile                                 antibiotics        Infectious Disease
Chile                                                                     Conference
                 -Summer                               -Generated
                 2004                                  antibiotic         -Manuscript in preparation
                                                       resistance
                                                       profile for
                                                       Santiago, Chile
Molecular        -National      1 year     40-50       -Investigated      -Abstract and Poster: Young
mechanisms of    Cancer                    hours per   role of BAG-3      Investigators Meeting, 2005
ovarian cancer   Institute,                week        protein in
metastasis       NIH, lab of                           apoptosis          -Presentation: NIH Post-
                 Dr. Elise                                                baccalaureate seminar
                 Kohn                                                     series

                 -2004-2005                                               -Publications:
                                                                          Pharmacogenomics,
                                                                          Women’s Health, manuscript
                                                                          in preparation
                            INVESTIGATORS
Protocol Director: Josephine (JoAnn) Czechowicz

I am a first-year medical student. My main interests are women’s health,
infectious disease, international health and pediatrics. Before coming to Stanford,
I did research on drug design, molecular signaling and epidemiology. The project
that intrigued me most was a study of antibiotic resistance I conducted as part of
a Fogarty International Research Fellowship in Santiago, Chile. My work in Chile
ignited my interested in international health.

Here at Stanford, I am pursuing a concentration in Women’s Health. I have
already taken several classes in the concentration including Women and Health
Care (the McGann lecture series) as well as Current Topics and Controversies in
Women's Health and Sex-based Biology, Current Issues in Reproductive Health,
and Mommies and Babies in the winter. I so enjoyed the Mommies and Babies
class that I have applied for the TA position for next year. My interest in women’s
health inspired me to found a new student group called “Women and Medicine”
focused on educating my peers about major health issues pertinent to women in
the US and around the world.

Stanford Advisor: Yvonne Maldonado, M.D.

Dr. Maldonado is Associate Professor of Pediatric Infectious Disease at Stanford.
She has significant expertise in childhood infection and perinatal transmission of
disease, most specifically HIV, but also malaria. Additionally, Dr. Maldonado has
done a large amount of fieldwork, with field sites in Mexico and Zimbabwe. Dr.
Maldonado is the founder and director of the Stanford Infectious Diseases and
Immunology Center. She serves on federal, national and international advisory
committees relevant to pediatric infectious diseases, pediatric vaccines and
international health.

On-Site Advisor: Luis Benavente, M.D.

Dr. Luis Benavente has over 20 years of experience in public health, as well as
maternal and child health. Accredited as a Specialist in Epidemiology, Luis has
extensively published in epidemiology of nutritional disorders, nutrition and
infection, and micronutrient deficiencies. He served as both Professor and Head
of the Department of Public Health at the Universidad Peruana Cayetano
Hereida, and worked at the Peruvian Institutes of Health in their Research Center
for 13 years. Luis then went on to work with Project HOPE in Guatemala,
Nicaragua, Peru and in the US from 1996-2002. Luis currently is the Senior
Health Program Officer for Medical Care Development International. He holds a
Medical Degree and a Masters of Science Degree in Microbiology and
Immunology.



                                  FIELD SITE
Obstetrics and Family Planning Clinic, Malabo Regional Hospital

This facility is the central prenatal facility on Bioko Island, providing prenatal care
to over 70% of the island’s pregnant women annually. The clinic staffs three
obstetricians and typically sees 40-50 patients per day. The facility has agreed to
provide me with access to patients upon check in and a private space for me to
conduct malaria and anemia tests, as well interview my subjects.

Medical Care Development International (MCDI) and the Bioko Island Malaria
Control Project (BIMCP)

The government of Equatorial Guinea, Medical Care Development International
and Marathon Oil Corporation have developed and are actively engaged in a
comprehensive malaria control project for Bioko Island, Equatorial Guinea. This
joint effort is expected to contribute significantly to the overall health and quality
of life of the people of Equatorial Guinea.

The malaria control program will adopt a strategic plan designed around three
program goals:

      Reduce the transmission of the malaria parasite through mosquito control
       programs and improved preventive measures at the household and the
       community level
      Improve the health care delivery systems for malaria cases by establishing
       malaria treatment centers that will use standardized protocols, diagnosis
       and referrals
      Enhance surveillance systems and operational research to strengthen
       Equatorial Guinea’s capacity to conduct surveys and implement an
       effective reporting and monitoring system

My on-site collaborator in this project, Luis Benavente, is Senior Health Project
Officer of MCDI. Our academic inquiry has the potential to positively impact the
second and third of the goals of the BIMCP.
                       LEARNING OBJECTIVES
To understand the process of community-based research from conception to
publication.

This objective will be attained through the process of completing the project. At
present I have conceived of my project, set up collaboration with a community-
based partner, written a research proposal, planned appropriate statistical
analysis and filed for IRB approval. Once I arrive in Bioko, I will learn about the
obstacles to research in a resource-poor setting and how to overcome them to
complete my investigation. Finally, upon my return, I will work with my statistical
partners, Dr. Meira Falcovitz-Halpern here at Stanford and Dr. Luis Benavente in
Africa, to analyze my data. Finally Drs. Maldonado and Benavente will guide me
through the preparation and submission of a manuscript.

To gain an inside perspective on the reality of public-private collaboration in
humanitarian aid work.

Health economists have touted the Bioko Island Malaria Control Project as a
model of public-private collaboration in humanitarian development. My research
partner in this study, Medical Care Development International, is a major player
in this project. Through my work with MCDI researchers, I will see first hand how
development projects are planned and implemented in a public-private
partnership.

To observe what comprises the standard of obstetric care in a resource-poor
setting.

My data collection will be based at the obstetrics clinic of Malabo Regional
Hospital. In addition to collecting data and interviewing patients, I will also have
the opportunity to shadow Dr. Gloria Nsteng in clinic and on the labor and
delivery ward of the hospital. Dr. Ntseng is the chief obstetrician and a Bioko
native. My interactions and conversations with her will give me insight into the
quality and delivery of obstetric care in the developing world.
                             PROPOSAL
       PREVENTING MALARIA AND ANEMIA IN
                 PREGNANCY:
           A CASE-CONTROL STUDY OF
INTERMITTENT PREVENTATIVE TREATMENT ON
    BIOKO ISLAND, EQUATORIAL GUINEA

                                  ABSTRACT
Malaria during pregnancy poses great risks to both mother and child, the most
common of which is anemia. Anemia increases the mother’s risk for placental
abruption, preterm labor and maternal death and puts the developing baby at risk
for low birth weight and miscarriage. One dose of intermittent preventative
treatment (IPT) with the drug combination sulfadoxine-pyrimethamine during the
first 26 weeks of pregnancy has been shown to decrease a woman’s risk of
malaria by 85% and anemia by 50%. The life cycle of P. falciparum as well as
socio-behavioral factors differ throughout sub-Saharan Africa. In order to
optimize IPT delivery, the operational efficacy of IPT in preventing malaria and
anemia must be assessed on a local level.

This case-control study examines a population of pregnant women in their third
trimester on Bioko Island, Equatorial Guinea. We aim to determine whether
treatment with at least one dose of IPT during the first two trimesters of
pregnancy correlates with reduced proportions of malaria and anemia.

70 participants will be recruited for each of two groups: a study group that
previously received at least one dose of IPT in the first two trimesters and a
control group that received none. We will determine malaria infection status using
an immunochromatographic test and measure hemoglobin levels with a
HemoCue detector. Additionally, to better understand contributors to anemia
other than malaria, we will gather information with a quantitative survey. The
knowledge gained in this study will allow for more effective implementation of
malaria prevention measures for pregnant women and ultimately better outcomes
for women and children.
                             SPECIFIC AIMS
      Specific Aim 1: To determine whether pregnant women in Malabo,
       Equatorial Guinea who have taken Intermittent Preventative Treatment
       (IPT) with sulfadoxine-pyrimethamine have lower proportions of malaria
       than those who have not taken IPT. Malaria status will be determined
       using a rapid Immunochromatographic Test (ICT).

      Specific Aim 2: To determine whether pregnant women in Malabo,
       Equatorial Guinea who have taken IPT have higher hemoglobin levels
       than those who have not taken IPT. Hemoglobin levels will be measured
       with a rapid HemoCue test.

      Specific Aim 3: To investigate factors other than malaria that contribute
       to maternal anemia among pregnant women on Bioko Island using a
       quantitative survey.

   Hypothesis: Women who receive IPT with sulfadoxine-pyrimethamine
   during pregnancy will have lower proportions of malaria and higher
   average hemoglobin levels than those who do not receive IPT.



              BACKGROUND AND SIGNIFICANCE
Each year in sub-Saharan Africa, approximately 30 million pregnant women are
at risk for contracting malaria and suffering its adverse consequences (World
Health Organization/UNICEF, 2003). Pregnant women are particularly vulnerable
to malaria and are more likely to be infected with the pathological P. falciparum
than non-pregnant women [1]. The most common complication of malaria during
pregnancy is maternal anemia. P. falciparum infection increases a woman’s risk
of anemia by 450% [2]. Anemia secondary to malaria puts pregnant women at
greater risk of other morbidities including placental abruption, placenta previa,
premature labor and maternal death. Placental malaria, a condition in which
parasites infect the placenta, poses further risks, particularly for the developing
fetus [3, 4]. Malaria in pregnancy can result in poor fetal outcomes, specifically
miscarriage, low birth weight, and neonatal mortality [5]. Pregnancy presents an
important opportunity for health-related interventions that can simultaneously
improve outcomes for both mother and child.

Numerous strategies have been employed to prevent maternal malaria including
the use of insecticide-treated bed nets to prevent mosquito bites and indoor
residual spraying. Both interventions have been shown to dramatically decrease
the risk of malaria [6, 7]. Past pharmaceutical interventions have been
controversial. Weekly prophylaxis with chloroquine was effective throughout the
1980s. However, the emergence of chloroquine resistant malaria has decreased
the effectiveness of this option. Further, the logistic constraint of weekly drug
administration has made chloroquine an unrealistic choice even in areas with
little resistance [8].

Intermittent preventative treatment (IPT) with sulfadoxine-pyrimethamine (SP)
has been proven to be a safe and effective treatment in many malaria-endemic
areas. Three IPT doses of SP in Malawi were shown to decrease a woman’s risk
of active parasitemia by 72% [9]. A more recent study conducted in Kenya
showed that as little as one dose of SD-IPT in the first two trimesters of
pregnancy decreased risk for malaria by 85%. The same study showed a
beneficial effect of IPT usage on hemoglobin level, decreasing risk for anema by
50% [10]. In 2000, the WHO recommended that IPT be implemented in malaria
endemic regions as the standard of care for prophylaxis during pregnancy (WHO,
2000). However, since the announcement of this policy, there has been little
assesment of its effectiveness or implementation [11]. The life cycle of P.
falciparum and numerous sociobehvioral parameters differ between different
regions and climates, therefore local assessment of the operational efficacy of
IPT is essential to determine optimal IPT implementation in a particular
community.

Equatorial Guinea (EG) is a Central African country located on the Gulf of Guinea
(Figure 1) consisting of a continental region and an island region. Bioko Island,
30km from the coast of Cameroon, is home to the EG capital, Malabo. Malaria on
Bioko is holo- to hyper-endemic with an infection rate of approximately 50%
among children [12]. The infection rate among pregnant women has not been
directly measured but is estimated at 25% [13]. P. faliciparum infection is nine
times more common on Bioko than infection with P. vivax. The life cycles of P.
falciparum and P. vivax differ: P. falciparum infects and then is eliminated while
vivax may become dormant and recur within a host [14].

A recent population-based study estimated the maternal anemia prevalence on
Bioko to be approximately 75% [13]. Although the anemia rate among pregnant
women is high, little research has been done to determine the contribution of
malaria to anemia. Examining a possible correlation between IPT use and
increased hemoglobin levels may provide insight into the contribution of malaria
to the high anemia rate. Surveying the subjects on other possible contributors to
anemia, may provide insight into other methods of combating maternal anemia
(e.g. iron supplementation) if little correlation between ITP use and hemoglobin
level is observed.
Figure 1. Relative location of Equatorial Guinea

Since 2003, a public-private partnership to eradicate malaria has existed
between the EG Ministry of Health and Marathon Oil: the Bioko Island Malaria
Control Project (BIMCP). The $8.3 million project has been implemented and
assessed by Medical Care Development International (MCDI), a Washington DC-
based non-profit organization. The main strategies of the program have been
indoor residual spraying and improved prophylaxis distribution among children.
The program was implemented in 2003 and has had tremendous success. In just
one year, the prevalence of malaria among children fell by 33% [13]. There is a
good deal of political will in support of malaria eradication on Bioko. Research in
support of IPT would likely result in its implementation by BIMCP. Further,
identifying major barriers to IPT access among women would allow for
optimization of IPT delivery.



                      EXPERIMENTAL DESIGN
Overview:
This project is a case-control study to determine whether IPT use correlates with
lower proportions of active P. falciparum malaria and reduced hemoglobin levels
in pregnant women in the city of Malabo, on Bioko Island, Equatorial Guinea. We
will survey participants to investigate contributors to anemia during pregnancy
other than malaria, as well as to identify barriers to IPT use.
Timeline:
 1/1/06– 4/17/06     6/26/06 – 8/11/06   8/14/06-8/31/06        9/1/06-12/1/06
 Study design,       Data collection;     Data entry,      Dissemination of results
proposal writing,      50 hours per      and analysis;      to BIMCP; Manuscript
 IRB approval,          week. 150        40 hours per           preparation for
medical scholars     subjects in MRH        week              submission to peer-
application and       Obstetrics and                         reviewed journals; 10
study setup; 10      Family Planning                            hours per week
hours per week            Clinic


Population:
Participants will be recruited from the obstetrics and family planning clinic based
at Malabo Regional Hospital. The clinic is the main prenatal care facility in
Malabo, providing prenatal care to 70% of the city’s pregnant women. The clinics
typically see between 30 and 50 obstetrics patients per day. Data will be
collected over a period of seven weeks. Patients that fit the criteria of the study
will be asked to participate upon check-in for pre-natal visits. Interviews and
sample collection will be performed on site at the clinic. Over 95% of Malabo
residents speak Spanish, so Czechowicz, who is fluent, will conduct interviews in
Spanish.

Inclusion Criteria
     Pregnant women gestational age 26 weeks or greater (third trimester)
     Age 18 or older
     Study Group: Received at least one dose of sulfadoxine-pyrimethamine
       IPT during the first two trimesters of pregnancy
     Control Group: Received no SP-IPT during first two trimesters
Exclusion Criteria
     Use of malaria prophylaxis other than SP-IPT during first two trimesters
     Active malaria at time of conception
     Severe pregnancy complication

Subjects will be sorted into “Study” and “Control” groups based on whether or not
they previously received any IPT, as summarized in this table:

                        Study                   Control



Size                    70                      70



IPT utilization prior Yes                       No
to visit?

All patients will be consented according to the Stanford IRB standards for Human
Subjects Research (for consent form, see Appendix III) and identifiers will be
coded on site. Approval from the Stanford IRB is pending (record of IRB
application, Appendix II).

Data Collection:
Malaria Diagnostic Testing:
The subjects’ malaria status will be determined by Immunochromatographic Test
(ICT) for P. falciparum (a simple assay with a sensitivity of 100% and specificity
of 99.7% relative to the standard lab technique of thick smear microscopy) [15].
ICT requires a finger stick and subsequent collection of less than 100L of blood.
A drop of blood is placed on the chromatographic strip, and lines on the strip are
visualized in response to P. falciparum antibodies. Based on the results of this
test, patients will be classified as either positive or negative for active malaria.
Those subjects with active malaria will be informed and encouraged to follow up
with their physician.

Hemoglobin Concentration Testing:
Hemoglobin concentration will be determined using a HemoCue portable
hemoglobin meter. Approximately 10m of blood from the original finger stick
capillary tube will be tested. The numerical value of the subjects’ hemoglobin
concentration will be recorded. Those subjects with hemoglobin concentrations
below 10mg/dL (anemic) will be informed of their status and encouraged to follow
up with their physician.

Identifying anemia risk factors other than malaria:
An investigation of factors other than malaria that contribute to low hemoglobin
levels will be assessed with a quantitative survey (Appendix I, Part B, “Anemia
Questionnaire”). Subjects in both Study and Control groups will be interviewed.
Factors to be considered include dietary iron intake and iron supplementation.
Subjects will be classified as Low, Moderate or High risk for dietary iron
deficiency.

Identifying barriers to IPT:
Barriers to IPT access in Malabo will be investigated using a qualitative survey
(see Appendix I, Part C, “Barriers to IPT Questionnaire”). The survey will be
administered to patients in both the Control and Study groups. The survey
contains both open and closed ended questions.

Power Value:
To determine if IPT use correlates with lower proportions of malaria we look to
see a difference between study and control groups (i.e. an effect size) of 20% or
greater. The prevalence of malaria among pregnant women on Bioko is
estimated to be 25% [12, 16, 17]. To achieve a power value of 80% and an alpha
of p<0.05, the sample size must be at least 69 subjects in each group of the
study.

To be convinced that IPT use correlates with higher Hemoglobin levels, we look
at see an effect size of 0.75 g/dL or greater. The standard deviation of
Hemoglobin concentration on Bioko is 1.7 g/dL [18, 19]. To achieve a power
value of 80% and an alpha of p<0.05, the sample size must be at least 64
subjects in each group.


Data Analysis:
Data will be analyzed in collaboration with Dr. Maldonado’s colleague, Dr. Meira
Falkovitz-Halpern, Ph.D. A Two-tailed Fischer’s Exact Test will be used to
determine whether the proportion of women who have malaria among IPT users
is different than that among those who did not use IPT. The mean hemoglobin
concentrations between IPT users and non-users will be compared using a T-
test. Proportions of women at Low, Moderate and High risk for dietary anemia
will be calculated for both Study and Control groups.

Interpretation of Results:
If IPT use correlates with lower incidence of malaria and anemia, this will
demonstrate that the operational efficacy of the treatment is comparable to its
efficacy in randomized clinical trials. If IPT use does not correlate with lower
proportions of malaria or anemia, this will suggest that there is an obstacle to
operational efficacy specific to Bioko.

Maternal anemia is attributable to numerous factors, but malaria is typically its
regarded as its primary cause in Bioko. Differences in active malaria proportions
between the study and control groups can be attributed to IPT. If we observe
lower proportions of malaria and anemia in the IPT group it follows that the
reduction in anemia can be proportionately attributed to the reduction in malaria.
Our survey of the dietary and behavioral contributors to anemia will help to
explain why women who are uninfected with malaria may be anemic.

Potential Sources of Error:
The following are potential sources of error in this investigation:

      Clinic-based study. The study is clinic-based and therefore could
       potentially not represent a cross-section of the population. The subjects
       could potentially be in better health on average than the general
       population. However, according to a 2004 BIMCP population-based
       survey, the vast majority (approximately 90%) of women receive prenatal
       care at a hospital or clinic, so hospital goers are not an elite subset. Also,
       since the study does not measure rates of IPT utilization across the
       population, but rather correlations between IPT and certain health
       outcomes, this issue is less problematic.
      HIV and malaria. It is well established that HIV infection increases one’s
       risk of contracting malaria, and vice versa. Furthermore, HIV positive
       women who are also infected with malaria are more likely to be anemic
       than HIV negative women [20]. However, the HIV infection rate among
       women in Malabo is 3.4%, relatively low compared to other countries in
       sub-Saharan Africa (CIA world fact sheet, 2001). For this reason, we
       expect that HIV will be only a minor confounding factor, if a factor at all.

      Inaccuracies from self-reported data. Because of Recall Bias, subjects are
       probably more likely to overestimate than to underestimate their use of
       IPT. Therefore, if a significant correlation between IPT and lower malaria
       and anemia rates is observed, it is not likely to be undermined by the self-
       reported nature of the study.

                    RELEVANCE TO MEDICINE
Malaria and anemia take a great toll on women and children in sub-Saharan
Africa. Pregnancy presents a unique opportunity in which one intervention can
improve two lives. The results of this study could have important implications for
implementation of IPT in Equatorial Guinea. The Ministry of Health, Marathon Oil,
and MCDI began the Bioko Island Malaria Control Project (BIMCP) in 2004. The
project’s primary tool has been indoor residual spraying (IRS), which has been
implemented with great success. IRS combined with vigorous prophylaxis
distribution among children has resulted in a 33% decrease in malarial infection
among children (MCDI, unpublished data). The political momentum to eradicate
malaria on Bioko is strong. Solid research supporting the effectiveness of IPT in
preventing malaria and anemia among pregnant women would likely result in
enthusiastic implementation of such measures by BIMCP.

Malaria-related anemia during pregnancy contributes to poor outcomes for both
the mother and the fetus. The mother has an increased risk of morbidity and
mortality, and the child has increased risk of miscarriage, low birth weight, and
stillbirth. Understanding the optimal methods of malaria prevention in pregnant
women will allow for better planning and outcomes on Bioko and throughout sub-
Saharan Africa.

         JUSTIFICATION FOR AWAY EXPERIENCE
This study requires travel to Bioko Island, EG. My project is designed to examine
an epidemiological problem unique to pregnant women on Bioko. My
collaboration with the BIMCP will result the immediate application of my findings
to improve malaria prophylaxis delivery to pregnant women there. Further, my
partner in the study, MCDI, is based in Malabo, EG.
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