Assessing SARS-CoV-2 (COVID-19) Seroprevalence During Routine Antenatal Care Visits in Zambia
Conditions: SARS-CoV-2 Seroprevalence
Sponsor: Centre for Infectious Disease Research in Zambia

 
 
 
 
 
 
 
 
 
 
 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
 
Protocol Version 1.1 
 
13 June 2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
V4.1, 13 June 2021 
1 
 
 
 
 
Ministry of Health 
 
 
 
 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
 
Protocol Version 1.1 
 
13 June 2021 
 
 
 
 
 
 
 
Principle and Co-Investigators 
 
Affiliation 
Name 
Role 
Zambia National Public 
Health Institute 
Muzala Kapina, MBChB (PI) 
Protocol 
development, 
Nyambe Sinyange, MBChB, MSc 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
V4.1, 13 June 2021 
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John Simwanza, MBChB 
implementation, 
monitoring, data 
management and 
analysis, report 
writing 
Otridah Kapona, MSc 
Ministry of Health 
Lloyd Mulenga, MBChB, PhD, MMed (PI) 
Protocol 
development, 
implementation, 
monitoring, data 
management and 
analysis, report 
writing 
Sombo Fwoloshi, MBChB, MMed 
Aaron Shibemba, MBChB, MMed 
Kennedy Malama, MBChB, MPH 
Powell Choonga, MSc 
Angel Mwiche, MBChB, MMED 
Maureen Chisembele, MBChB, MMED 
Consity Mwale, MBChB, MMED 
Gideon Mwale, MBChB, MPH 
Centre for Infectious 
Disease Research in 
Zambia 
Izukanji Sikazwe, MBChB, MPH (PI) 
(CITI expiration: 9 Feb 2022) 
Protocol 
development, 
implementation, 
monitoring, data 
management and 
analysis, report 
writing 
Mwanza Wa Mwanza, MD   
Carolyn Bolton, MBChB, MSc 
Theodora Savory, MD 
Centers for Disease 
Control and Prevention 
Jonas Hines, MD 
(CITI expiration: 2 January 2022) 
Protocol 
development, 
implementation, 
monitoring, data 
management and 
analysis, report 
writing 
Elizabeth Heilmann, MPH 
(CITI expiration: 25 June 2023) 
Adam Wolkon, MPH 
(CITI expiration: 26 December 2021) 
Sam Yingst, DVM, PhD 
(CITI expiration: 31 October 2022) 
Warren Malambo, MPH 
(CITI expiration: 11 February 2022) 
Julie Gutman, MD MSc 
(CITI expiration: 25 June 2022) 
Laura Steinhardt, PhD, MPH 
(CITI expiration: 19 January 2022) 
 
 
 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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CDC’s role: CDC will not engage in data collection or have access to identifiable data; rather, 
CDC will provide technical leadership on protocol development, data collection procedures, 
data analysis, interpretation of results, and their dissemination. All data will be owned by 
the Zambia Ministry of Health. 
 
Funding source: Buffer component COVID-19 response Cooperative Agreement number: 
NU2GGH002251, Provincial Ownership to Uplift Delivery of HIV/TB Services in Zambia 
(PROUD-Z) through technical Assistance 
 
Conflict of interest: The primary investigators and co-investigators have no personal, 
financial, or other relationships that might pose a conflict of interest (or the appearance of a 
conflict) in their role in this activity. The contents in this protocol are those of the authors 
and do not necessarily represent the official position of the Centers for Disease Control and 
Prevention. 
 
Differences from CDC umbrella protocol 
This protocol was adapted from a generic protocol approved by CDC entitled, “Assessing 
COVID-19 seroprevalence from routine antenatal care visits,” approved on 12/17/2020 
(eClearance ID: 0900f3eb81c55548). No major methodological changes were made. Minor 
differences made in response to local feedback include:  
- 
Prevalence estimates at district level rather than health facility level 
- 
Twenty women each will be recruited from 40 health facilities among 4 districts each 
month compared to 30 women from 30 health facilities 
- 
First visit ANC routine lab tests (HIV, syphilis, Hepatitis B, and/or malaria) will be 
recorded on the questionnaire  
- 
Per guidance from the CDC-Atlanta team, several questions on potential exposure 
were removed from the questionnaire and COVID-19 vaccination history was added 
(compared to the generic protocol questionnaire) 
 

 
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Table of Contents 
Acronyms ................................................................................................................................... 6 
1.0 
Introduction ............................................................................................................ 7 
1.1 General information on research topic .................................................................................. 7 
1.2 Overview of research and research gaps ............................................................................... 8 
1.3 Overall purpose of the research ............................................................................................ 8 
2.0  
Statement of the Problem ....................................................................................... 9 
3.0 
Rationale/ Justification ........................................................................................... 9 
4.0 
Literature Review ................................................................................................... 9 
5.0 
Research questions ............................................................................................... 10 
6.0 
Research Aims and Objectives .............................................................................. 10 
6.1 Research Aim ...................................................................................................................... 10 
6.2 Primary Objectives .............................................................................................................. 10 
6.3 Secondary Objectives .......................................................................................................... 11 
7.0  
Methodology ........................................................................................................ 11 
7.1 Study Design ....................................................................................................................... 11 
7.2 Study Site and Population ................................................................................................... 11 
7.3 Selection of Participants, Sampling and Sample Size ............................................................ 13 
7.4 Implementation of Procedures ............................................................................................ 15 
7.5 Quality control and Assurance activities (supervisory, data and logistics monitoring) .......... 18 
7.6 Study staff roles and responsibilities ................................................................................... 19 
7.7 Data collection and management ........................................................................................ 20 
7.8 Data analysis ....................................................................................................................... 21 
7.9 Data Ownership .................................................................................................................. 21 
7.10 Sponsor monitoring ........................................................................................................... 21 
7.11 Dissemination of findings .................................................................................................. 22 
8.0  
Ethical Considerations .......................................................................................... 22 
8.1 
Ethical committee approvals ................................................................................. 22 
8.2 Risks & Benefits for Subjects ............................................................................................... 23 
8.3 Unexpected/Adverse Events ............................................................................................... 23 
8.4 Prevention of SARS-CoV-2 Infection in Investigation Personnel ........................................... 24 
9.0  
Timeline ................................................................................................................ 25 
10.0 Budget ..................................................................................................................... 26 
11.0 References  ............................................................................................................... 27 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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12.0 Appendices ...................................................................... Error! Bookmark not defined. 
Appendix I: Participant Information Sheet - English ......................... Error! Bookmark not defined. 
Appendix II: Participant Information Sheet - Bemba ........................ Error! Bookmark not defined. 
Appendix III: Participant Information Sheet – Chewa/Nyanja ........... Error! Bookmark not defined. 
Appendix IV: SARS-CoV-2 ANC Surveillance Questionnaire ............... Error! Bookmark not defined. 
Appendix V: Other, e.g. permission letters ....................................... Error! Bookmark not defined. 
Appendix VI: C.Vs ............................................................................ Error! Bookmark not defined. 
 
 
 
 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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Acronyms 
Acronym 
Explanation 
ANC 
Antenatal Care 
CIDRZ 
Centre for Infectious Disease Research in Zambia 
CDC 
U.S. Centers for Disease Control and Prevention 
CHWs 
Community Health Workers 
COVID-19 
Coronavirus Disease 
DHO 
District Health Office  
DBS 
Dry Blood Spot 
HIV 
Human Immunodeficiency Virus  
IRB 
Institutional Review Board 
MNCH 
Maternal, Newborn and Child Health 
MOH 
Ministry of Health 
MTA 
Materials Transfer Agreement 
NAAT 
Nucleic Acid Amplification Test 
NHRA 
National Health Research Authority 
ODK 
Open Data Kit 
PEPFAR 
U.S. Presidential Emergency Plan for AIDS Relief 
PHO 
Provincial Health Office  
PMTCT_STAT 
Prevention of Mother to Child Transmission of HIV 
PPE 
Personal Protective Equipment  
QA/QC 
Quality Assurance/ Quality Control 
SARS-CoV-2 
Severe Acute Respiratory Syndrome-Related Coronavirus-2 
SOPs 
Standard Operating Procedures  
UNZA BREC 
University of Zambia Biomedical Research Ethics Committee 
VTM 
Viral Transport Media  
ZNPHI 
Zambia National Public Health Institute 
 
 
 

 
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1.0 
Introduction 
1.1  
General information on research topic 
The novel coronavirus SARS-CoV-2, the cause of COVID-19, was first detected in Wuhan city, 
China in December 2019 and the first cases were confirmed in Zambia in March 2020. 
Current data suggest that as many as 50% of cases are asymptomatic [1]. Initial surveillance 
has focused primarily on patients with symptoms or severe disease, and, as such, the full 
spectrum of the disease, including the extent and fraction of mild or asymptomatic 
infections that do not require medical attention are not clear. Estimates of the case fatality 
ratio, and other epidemiological parameters, will likely be lower than current estimates 
once the full spectrum of disease is able to be included in the denominator. With any novel 
virus, such as SARS-CoV-2, initial population seroprevalence is assumed to be negligible. 
Thus, antibody seropositivity in a population can allow inferences to be made about the 
extent of infection, the cumulative incidence of infection in the population, and trends in 
infection over time.  
 
This protocol proposes to investigate the extent of SARS-CoV-2 infection, as determined by 
seropositivity, among pregnant women attending 1st antenatal care (ANC) visits, as a 
surrogate for the prevalence in the general population. The utility of ANC-based surveillance 
has been established for malaria [2-5], and there is no evidence to date suggesting that 
pregnant women are at higher risk of contracting SARS-CoV-2 than non-pregnant adults, 
suggesting that ANC-based surveillance could provide useful information for monitoring the 
spread of SARS-CoV-2 over time [6]. This will be a non-randomized assessment of the 
potential to leverage pregnant women attending 1st ANC as a pragmatic sentinel population 
to monitor prevalence of SARS-CoV-2 and to detect changes in transmission in selected 
areas. 
 
As the Zambian Ministry of Health (MOH) continues to focus on controlling and mitigating 
further spread of the SARS-CoV-2 through active surveillance for case detection, contact 
tracing, physical distancing and isolation while strengthening case-based management, it is 
important to build a national SARS-CoV-2 surveillance system that is crucial for containing 
transmission of the virus and preparing for future waves of the infection. 

 
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1.2 
Overview of research and research gaps 
This surveillance platform may be able to provide key information on the spread of the 
SARS-CoV-2 virus through the general population. By using serologic testing to understand 
trends in the proportion of pregnant women over time who were exposed to SARS-CoV-2, 
we will be able to understand not only the ultimate proportion of pregnant women who 
were ever infected, but also be able to track and potentially detect outbreaks (e.g. in areas 
with increasing positivity rates), unlike data that would be available from a single cross-
sectional survey, which could give the overall prevalence at a single point in time. In most 
places to date, testing has focused on symptomatic people, their contacts or others 
considered at high risk for infection, but there is limited understanding of the true 
proportion of asymptomatic cases.  
 
There are gaps in information about the prevalence and clinical characteristics of SARS-CoV-
2 in Zambia. We propose to conduct SARS-CoV-2 sentinel surveillance among first-time ANC 
attendees in selected Maternal, Newborn and Child Health (MNCH) departments in four 
districts (Lusaka, Chongwe, Chadiza and Chipata) of Lusaka and Eastern provinces to assess 
SARS-CoV-2 seroprevalence. Pregnant women will serve as a proxy for the healthy adult 
population of these districts, much as they have in sentinel surveillance for HIV and malaria. 
Because we will use serologic testing from blood and not nasal swabs, this collection will not 
substantially alter the risk for either the health worker nor the woman and will not deplete 
the supplies of SARS-CoV-2 Polymerase chain reaction (PCR) reagents used for diagnosis. 
 
1.3  
Overall purpose of the research  
The purpose of this project is to provide information on the seroprevalence of SARS-CoV-2 
antibodies in the community with pregnant women as the sentinel population. CIDRZ will 
work with facility in-charges in the selected sentinel sites to provide sensitization and SARS-
CoV-2 antibody testing to pregnant women presenting for their first ANC visit.  
 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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2.0  
Statement of the Problem  
Zambia is experiencing a SARS-CoV-2 epidemic. From March 18, 2020 to February 19, 2021, 
at total of 73,203 confirmed cases have been reported in Zambia. However, the true number 
is likely much larger, because surveillance systems for SARS-CoV-2 in Zambia are incomplete 
[7]. ANC surveillance of SARS-CoV-2 antibodies could provide needed insights into 
transmission trends in the general population. This surveillance will provide a means to 
systematically detect SARS-CoV-2 antibodies in pregnant women and notify relevant 
authorities about the prevalence.  
 
3.0 
Rationale/ Justification 
Since 2020, the coronavirus disease has become a major public health concern and 
sustainable and efficient surveillance platforms are urgently needed; assessing women 
attending 1st ANC should provide insights into transmission trends in the general population. 
This activity will determine what proportion of pregnant women demonstrate evidence of 
SARS-CoV-2 infection over time. The findings of this research may be used to change the way 
health system monitors the epidemic and ensures health services are made available with 
minimal disruptions.  
 
4.0 
Literature Review 
ANC surveillance has not been routinely utilized for SARS-CoV-2 in any country. A cross-
sectional study from Cape Town, South Africa found a seroprevalence of 38% among 
residual serum from antenatal clinic attendees in July and August 2020 [8]. Six African 
countries including Zambia (i.e., Kenya, Malawi, Mozambique, Nigeria, Uganda, and Zambia) 
are piloting ANC surveillance based on experience for surveillance of other infectious 
diseases (HIV, syphilis, malaria) [9,10]. ANC surveillance has been a key strategy for HIV for 
many years, because women attending ANC clinics represent an accessible healthy 
population [11-13].  
Prevalence estimates of SARS-CoV-2 infections have varied widely throughout Africa. This is 
likely a result of varying time, study population, and study design [14-20]. Nevertheless, 
SARS-CoV-2 infections are under-reported because of the large asymptomatic proportion of 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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infections, testing limitations, and surveillance gaps [21,22]. Africa is no exception, where 
several prevalence surveys (including in Zambia) have shown under-ascertainment of SARS-
CoV-2 infections [7,23]. For instance, during a population-based study in six districts in 
Zambia in July 2020, the ratio of reported cases to estimated infections ranged from 1:1012 
(Livingstone District) to 1:21 (Lusaka District) [7]; overall, only 1 case was reported for every 
92 infections. In Cape Town, 5% of residual serum specimens from persons living with HIV 
and women attending ANC clinics testing positive for SARS-CoV-2 antibodies had positive 
PCR test results in the public health surveillance system [24]. Other studies from other 
African countries have revealed substantial under-ascertainment of SARS-CoV-2 infections 
[18,25-27]. Strategies for improvement in surveillance have been called for by the Africa 
CDC and others [28,29]. 
 
5.0 
Research questions 
This study will assess the seroprevalence of SARS-CoV-2 in pregnant women aged 15-49 
years who present to the health facility for their first antenatal care visit. This surveillance 
will answer the following research question: “What is the seroprevalence of antibodies to 
SARS-CoV-2 among pregnant women attending first ANC visit in Zambia?” 
 
6.0 
Research Aims and Objectives  
6.1 
Research Aim 
The primary aim of this activity is to determine SARS-CoV-2 seroprevalence from women 
attending antenatal clinics for their first ANC visit as an easy-access population for routine 
monitoring. 
 
6.2 
Primary Objectives 
6.2.1 To estimate on a monthly basis the seroprevalence of SARS-CoV-2 among pregnant 
women aged 15-49 years attending their first ANC visit at selected health facilities in 
Chadiza, Chipata, Chongwe, and Lusaka districts of Eastern and Lusaka Provinces. 
6.2.2 To monitor trends in SARS-CoV-2 seroprevalence in the selected districts. 
 

 
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6.3 
Secondary Objectives  
6.3.1 To determine the feasibility and acceptability of SARS-CoV-2 antibody tests among 
pregnant women 
6.3.2 To estimate the prevalence of HIV-COVID 19 co-infection among pregnant women 
aged 15-49 years  
6.3.3 To determine sociodemographic factors associated with SARS-CoV-2 seropositivity 
among pregnant women 
6.3.4 To compare trends in seroprevalence among pregnant women with routine 
surveillance data to determine if ANC sentinel surveillance is a viable surveillance 
strategy for SARS-CoV-2 
 
7.0  
Methodology  
7.1 
Study Design 
This will be a passive surveillance of pregnant women attending their first antenatal care 
visit at the selected study sites in the four selected districts. Pregnant women attending 1st 
ANC will be used as a pragmatic sentinel population to monitor the seroprevalence of SARS-
CoV-2 over time. Women will be informed of this activity during group health education 
sessions and will be consented during individual HIV testing and counselling. To minimize 
discomfort to the participant, spots of blood will be collected from all women consenting to 
participate (up to 20 women per study site per month) on filter paper for a SARS-CoV-2 
serologic test using a finger prick or a venous blood draw, depending on the method being 
used to obtain blood for routine HIV and syphilis testing during first ANC visits. In addition, 
women will be asked to respond to a short questionnaire. The woman’s name will not be 
included on the sample or on the form; rather, a unique study ID will be used to link the 
questionnaire to the sample. Seroprevalence data will be aggregated at the district level on 
a monthly basis. 
 
7.2 
Study Site and Population 
7.2.1 Study Sites  
Study sites will be selected in each of four purposively selected districts using a probability-
proportional-to-size selection of 10 eligible facilities within each district. Eligible facilities will 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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have averaged at least 19 first ANC visits per month over the course of the previous year. 
Surveillance activities will be implemented in two urban and rural districts as follows: 
 
Table 1: Study sites and sampling interval for SARS-CoV-2 seroprevalence surveillance in 
ANC clinics in Chadiza, Chipata, Chongwe, and Lusaka Districts - Zambia 
District 
 
Facility 
Average 
monthly ANC 
attendance 
(2020)* 
Sampling 
interval 
Chadiza  
1 
Chanida Rural Health Centre 
19 
1 
2 
Nsadzu Rural Health Centre 
21 
1 
3 
Madzaela Health Post 
21 
1 
4 
Bwanunkha Rural Health Centre 
23 
1 
5 
Chanjowe Health Post 
24 
1 
6 
John Rural Health Centre 
24 
1 
7 
Mkumbuzi Rural Health Centre 
28 
1 
8 
Zemba Rural Health Centre 
36 
1 
9 
Chadiza Rural Health Centre 
44 
2 
10 
Miti Rural Health Centre 
46 
2 
Chipata  
1 
Champhande Rural Health Centre 
21 
1 
2 
Chikando Rural Health Centre 
23 
1 
3 
Lunkwakwa Urban Health Centre 
25 
1 
4 
Mwami Hospital Affiliated Health 
Centre 
28 
1 
5 
Chipata Hospital Affiliated Health 
Centre 
34 
1 
6 
Jerusalem Rural Health Centre 
35 
1 
7 
Walela Health Post 
56 
2 
8 
Mchini Health Post 
69 
3 
9 
Namseche Rural Health Centre 
92 
4 
10 
Kapata Urban Health Centre 
162 
8 
Chongwe  
1 
Nchute Health Post 
19 
1 
2 
Chikumbi Health Post 
22 
1 
3 
Kasisi Rural Health Centre 
23 
1 
4 
Palabana Rural Health Centre 
24 
1 
5 
Kanakantapa Rural Health Centre 
27 
1 
6 
Water Falls Rural Health Centre 
28 
1 
7 
Katoba Rural Health Centre 
32 
1 
8 
Chainda Rural Health Centre 
35 
1 
9 
Ngwerere Main Rural Health Centre 
109 
5 
10 
Chongwe Referral Rural Health Centre 
137 
6 
Lusaka  
1 
Coptic Hospital 
21 
1 
2 
Kuku Health Post 
98 
4 
3 
Chainda Urban Health Centre 
99 
4 
4 
Matero Main Urban Health Centre 
228 
11 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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5 
Chaisa Urban Health Centre 
231 
11 
6 
Kanyama West Health Post 
246 
12 
7 
George Urban Health Centre 
371 
18 
8 
Matero First Level Hospital 
407 
20 
9 
Kanyama First Level Hospital 
572 
28 
10 
University Teaching Hospital - Adult 
606 
30 
* Based upon PMTCT_STAT indicator from PEPFAR MER 
 
 
7.2.2 Study Population 
A sample of 20 pregnant women attending their first ANC visit at the selected study sites 
will be enrolled into the study until the monthly sample size is reached.  
 
7.3 
Selection of Participants, Sampling and Sample Size  
7.3.1 Selection of Participants 
Women presenting to the selected study sites for their first ANC visit at selected health 
facilities in the four districts will be eligible to participate in the study. A facility-specific 
interval for systematic sampling will be defined based on each facility’s monthly mean 
number of women attending their first ANC visit based on the historic monthly ANC 
attendance during fiscal year 2020. until the monthly sample size is met at a given facility. 
Data will be collected over a 12-15 months period of implementation to ensure that the 
number of required samples are met in all implementing sites. 
7.3.1.1 Inclusion criteria 
All women presenting to the selected study sites will be considered for inclusion 
into the study if they are: 
- 
Confirmed pregnant and are registered for their first ANC visit 
- 
Aged 15 to 49 years 
- 
Able to provide consent to participate and have blood sample collected for the 
SARS-CoV-2 antibody test 
 
7.3.1.2 Exclusion criteria 
- 
Not first ANC visit for the pregnant woman  
- 
Women less than 15 years or above 49 years old 
- 
Women not able to provide consent for study participation 

 
Assessing SARS-CoV-2 Seroprevalence during Routine Antenatal Care Visits in Zambia 
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7.3.2 Sample Size 
All pregnant women who meet the eligibility criteria presenting at the study sites will be 
approached for participation into the study. Monthly ANC attendance varies by district, as 
shown in Table 2. Study districts will be expected to recruit 200 pregnant women per 
month. The estimated seroprevalence is expected to vary by study site, as prevalence has 
been demonstrated to be different between urban and rural locations in Zambia [7]. A 
sample size of 200 women per district per month will be sufficient to detect a range of 
estimated proportions of SARS-CoV-2 seroprevalence (Figure 1). The confidence interval (CI) 
reflects the bounds of the true seroprevalence of COVID-19 among pregnant women 
attending ANC (and not the overall pregnant women population or general population). 
Where prevalence is expected to be low (i.e. rural districts), a sample size of 200 women 
produces a 95% CI half-width of 2.4% for 3% seroprevalence (lowest estimate). Where 
prevalence is expected to be higher (i.e. urban districts), a sample size of 200 women 
produces a 95% CI half-width of 7.0% for 50% seroprevalence. Women will be recruited 
evenly throughout the month from study sites selected for each district. If the monthly 
recruitment target is met, study sites will be expected to pause sample collection and 
reconvene at the start of the next month.  
 
 
 
Table 2: Average monthly number of 
first ANC attendees using PMTCT_STAT 
denominator 
District 
Average monthly  
ANC attendance 
Chadiza 
424 
Chipata 
759 
Chongwe 
653 
Lusaka 
6,378 
0.00
0.02
0.04
0.06
0.08
0.00
0.10
0.20
0.30
0.40
0.50
95% CI half-width (+/-)
Estimated Proportion
Figure 1. 95% CI half-width by 
estimated prevalence for sample size 
= 200

 
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7.4 
Implementation of Procedures 
7.4.1 Implementation of pre-survey activities 
7.4.1.1 Engage and mobilize stakeholders 
At the beginning of the study, we will systematically identify and engage stakeholders 
(Provincial and District Health Offices (PHO/DHOs) and other implementing partners) to help 
build involvement and allow them to participate in the planning of activities for 
implementation and ensure that their expectations are met. 
 
7.4.1.2 Community engagement and sensitization  
Information about the study will be cascaded to various levels of the community through 
sensitizations meetings with community leaders such as headmen, teachers and political 
leaders. Health care providers at all the sentinel sites will be part of the sensitization 
meetings with the community. Additionally, information about the study will be 
incorporated in routine health talks which are given to pregnant women as they come to 
register for ANC.  
 
7.4.1.3 Train DHO staff from sentinel sites 
Health facility in-charges and healthcare workers from the MNCH departments and 
laboratory hubs will be oriented in the approved study protocol by MOH/CIDRZ research 
teams and supported throughout the study time. They will also be trained in 
phlebotomy/specimen collection, labeling and storage, biohazard waste disposal, transfer of 
specimens, and quality assurance and control. Study staff will also be trained in data 
collection, documentation, storage and safety, reporting and monitoring. All staff 
participating in implementation of study activities will be required to obtain good clinical 
practice and human research protection training. Laboratory staff will be reoriented on 
running of assays to be used in the study. Additionally, on-site orientation will be conducted 
to enhance hands-on experience of study procedures. Lay healthcare providers and Peer 
Educators who have been providing support at the study sites will be selected and trained 
to provide support for study specific tasks such as participant identification and provision of 
health education. All staff will be provided with SOPs which will provide them step-by-step 
instructions on how to perform study procedures. 

 
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7.4.1.4 Develop data collection and management tools 
The following standard operating procedures will be developed to provide written step-by-
step instructions to help study staff to perform routine tasks at the study sites: screening 
and eligibility, informed consent, study enrolment, sample collection, handling and testing, 
data collection and management (questionnaire completion, data entry, security, QA/QC), 
roles and responsibilities SOP, etc).  
 
In addition, a basic questionnaire will be used to collect information from consented ANC 
attendees. This questionnaire contains information about symptoms of COVID-19, possible 
exposures to SARS-CoV-2, and risk mitigation behaviors. Additionally, the questionnaire will 
collect information on sociodemographic characteristics and routine ANC lab results (HIV, 
syphilis, Hepatitis B, and malaria). 
 
7.4.1.5 Procure study supplies  
Study sites will be supplied with all required commodities (i.e., assays, lancets, 
handwarmers, microtainers, dry blood spot (DBS) cards, protective clothing, polyester fiber-
tipped applicator, paper towel, waste segregation polythene bag, depressors, cool box, ice 
packs, bleach, hand sanitizer, scissors, cryovial with virus transport media (VTM), a fine 
tipped permanent marker, barcodes, bar code scanner, specimen collection and 
transportation log forms, etc.) to enable them to collect DBS samples for the SARS-CoV-2  
serologic tests. The study will outsource tablets for data entry purposes. Laboratories will be 
equipped with all necessary materials to process and run DBS samples.  
 
7.4.2 Implementation of survey activities 
7.4.2.1 Recruitment of study participants 
All study processes will be undertaken as part of routine ANC so as not to disrupt patient 
flow. Women will be informed of this activity during group health education at the 
beginning of the first ANC visit. Health education on SARS-CoV-2 will cover symptoms of 
active infection, testing, and prevention methods, but will emphasize that this study looks 
for antibodies to SARS-CoV-2 in the blood which are NOT an indicator of current infection. 

 
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Women experiencing symptoms of COVID-19 will be managed according to national 
guidelines (such women will not be recruited in the study because they will not proceed 
with routine ANC given their acute complaints). Women will be informed that the quality of 
their antenatal care will not be impacted by their decision whether or not to participate in 
the study. If they do participate, they will not receive their test results as this study aims to 
measure population level seroprevalence and does not impact individual clinical care. 
Women will be approached for consent during individual counseling and testing sessions 
and blood will be collected on filter paper for a SARS-CoV-2 serologic test using blood from 
the same finger prick as that for HIV and syphilis testing. While waiting for HIV test results, 
consenting women will be asked to respond to a short questionnaire. Study participants will 
then continue with the remainder of the ANC visit as usual.  
 
Sites should have regular access to serological and other routine tests required for care of 
pregnant women. As a test of past infection, serology results will not be communicated to 
the facility or participants because they do not affect clinical care (i.e. vaccine eligibility). 
Any woman that has symptoms of COVID-19 will be referred to relevant testing facility and 
will follow the national SARS-CoV-2 testing and treatment guidelines. Because they will 
receive evaluation for their acute complaints per national guidelines, they will not undergo 
routine ANC services, and, thus, will not be eligible for participation in this study. 
 
7.4.2.2 Sample collection, labelling, storage and courier   
All staff (i.e., study and health facility staff) involved in the collection and transportation of 
specimen will be trained in safe handling practices and spill decontamination procedures. 
After obtaining consent from the participant and completing the study questionnaire and 
while maintaining rapport with the participant, the research nurse or phlebotomy staff will 
collect blood samples onto a dry-blood spot (DBS) card using finger stick procedures by 
trained staff. DBS samples collected on filter paper using a standard operating procedure 
will be used for SARS-CoV-2 antibody detection. Specimen and questionnaires will be 
labeled at time of collection with an identification sticker that has a pre-printed unique 
study ID and laboratory specimen ID, and current date. A paper log will be completed by 
study staff that contains the date, study staff ID, study ID, and a lab label to create a paper 

 
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trail in case of mislabeling. This paper log will be stored at the health facility and collected 
by the study team on a monthly basis. DBS cards will be placed in a bag prior to 
transportation to district hub laboratories for storage and testing using the existing viral 
load sample courier system supported by PEPFAR. 
 
DBS cards will be tested for anti-SARS-CoV-2 antibodies using a serologic assay. The specific 
assay has not been determined yet, as potential cross-reactivity is an emerging issue in 
Africa [30,31]. Ongoing studies at CDC in Atlanta will inform assay selection. Analyses will be 
done at a hub or central laboratory. Samples will be kept until the study procedures 
outlined in the protocol are completed or for up to three years, at which time any remaining 
samples will be destroyed. The samples will not be used for another purpose than what is 
stated in the protocol.  
 
Sample analysis will be batched as appropriate; total antibodies or IgG antibodies to SARS-
CoV-2 will be assessed using laboratory-based serological assays such as enzyme linked 
immunosorbent assay (ELISA), immunofluorescence (IFA), chemiluminescent assays (CLIA), 
multiplexed bead-based assays (the specific assay to be utilized in the study in pending 
guidance from CDC in Atlanta). All testing will be conducted under appropriate and 
recommended biosafety conditions per CDC guidance.  
 
7.5 
Quality control and Assurance activities (supervisory, data and logistics 
monitoring) 
Project supervisors will review the data collection forms and aggregate results on a monthly 
basis to assess for completeness, accuracy, and consistency. Periodic site visits will be 
conducted by the research team to assess for adherence to protocol procedures. Deviation 
from study protocols will be documented in an incident report and reported to the IRB 
within three working days. Additionally, up to 20% of samples may be run in duplicate at a 
central laboratory in Lusaka for the purposes of quality assurance. 
 

 
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7.6 
Study staff roles and responsibilities  
CIDRZ will support the Ministry of Health/Zambia National Public Health Institute to 
undertake this surveillance by providing technical assistance, training and mentoring of staff 
based in the health facilities and DHOs, project oversight, and supplies procurement. The 
project may be supplemented by PEPFAR funded activities as well as with additional 
technical support from CDC Zambia. The roles and responsibilities of CIDRZ supported are as 
outlined: 
 
7.6.1 Technical Lead 
The technical lead will be responsible for technical oversight throughout the study period. 
Before implementation, the technical lead will lead protocol development and submission 
for ethical approval. The technical lead will participate in developing SOPs and training study 
staff as well as monitoring data reporting and quality control activities. Upon completion of 
the study, the technical lead will coordinate preparation and dissemination of the final 
report. 
 
7.6.2 Study Coordinator 
The study coordinator will support study implementation by ensuring that all study activities 
are being implemented as laid out in the approved protocol. He/she will prepare the ethics 
application and ensure that study staff are working in compliance with, and be responsible 
for, tracking the project milestones. The study coordinator will manage the study budget, 
provide support for recruiting and training of staff. Additionally, he/she will be the point of 
contact between the principal investigator and staff at study sites. 
 
7.6.3 Research Nurse 
One research nurse will be employed for each of the study sites. Their role will be to ensure 
site compliance with the approved protocol by reviewing regulatory requirements. They will 
be responsible for communicating information about the study to potential participants and 
ensuring that staff involved with study implementation have the correct credentials and 
study related training. The research nurse will draw a blood sample onto filter paper via 
finger prick for SARS CoV-2 antibody testing and will adhere to sample storage and 

 
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transportation procedures. They will also ensure that correct source documents are used 
and that data is accurately captured in the questionnaires. They will act as the point of 
contact between the study coordinator and site staff.  
 
7.6.4 Community Health Workers 
Community health workers advocate for underserved communities to receive services and 
will be engaged from the already existing cadres known at the facility to supplement efforts 
of MOH HCWs and research nurses. For this study, they will conduct health education to 
pregnant women attending ANC visits. They will also be trained on how to collect 
information from the ANC attendees and administer the questionnaire.  
 
7.6.5 Data Coordinator  
The data coordinator will provide support for data management. He/she will ensure that 
ANC COVID folder on the central server is updated routinely and resolve any problems with 
the database. He will conduct quality improvement activities and liaise with the study 
coordinator about any identified quality problems with the data.  
 
7.6.6 Program Assistant 
The program assistant will provide administrative support for the study. He/she will submit 
request for supply purchases, make regular follow ups about delivery timelines and keep 
stock of supplies. In addition, he/she will submit cash requests for various activities and 
ensure that receipts are submitted to the Finance office as per stipulated guidelines. The 
program assistant will identify venues for trainings and communicate with the vendor about 
scheduled trainings.   
 
7.7 
Data collection and management 
The study questionnaire and database will be designed using Open Data Kit (ODK)–a free, 
open-source suite of tools that will allow collection of data using android mobile devices 
without Internet connectivity. CHWs will be trained to administer the electronic 
questionnaire that will be uploaded onto password protected android devices (see appendix 
IV) which will be piloted to ensure that data is correctly captured and stored when data 

 
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21 
collection begins. Individual respondents will be assigned a unique identifier to assist with 
data confidentiality. Respondents’ names and other personally identifying information will 
not be recorded in the questionnaire. Data will be uploaded to a server after each day of 
ANC data collection. Data collection devices (tablets, storage card) will be handled only by 
facility or study staff and kept in a locked office, in a lockable cabinet. Data will be exported 
to MS Excel for cleaning once every week. Any missing variables will be communicated to 
the research nurse for follow up and action with the CHW.  
 
7.8 
Data analysis 
Data will be analyzed on a monthly basis using a statistical program. Basic descriptive 
statistics, by district, including prevalence point estimates, confidence intervals, and relative 
standard errors will be tabulated. Variance and confidence intervals will be adjusted for 
health facility clustering. Prevalence estimates will be disaggregated by sociodemographic 
variables, routine ANC lab results, and risk factors. 
 
7.9 
Data Ownership 
All data collected through this protocol are the property of the Zambian Ministry of Health. 
All reviews and decisions regarding data sharing, for example at the request of a journal at 
the time of publication, will be made by the MoH as the owners of the primary data 
according to NHRA policies and procedures. 
 
7.10 Sponsor monitoring 
CDC is the primary sponsor for the activities described herein. As the sponsor, the CDC may 
conduct monitoring or auditing of activities to ensure the integrity of the data collected and 
to ensure the rights and protection of individuals. Monitoring and auditing activities may be 
conducted by:  
7.10.1 CDC staff ("internal") 
7.10.2 Authorized representatives of CDC (e.g., a contracted party considered to be 
“external") 
7.10.3 Both internal and external parties. 
 

 
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Monitoring or auditing may be performed by means of on-site visits to government facilities 
or through other communications such as telephone calls or written correspondence. The 
visits will be scheduled at mutually agreeable times, and the frequency of visits will be at the 
discretion of CDC. During the visit, any related materials may be reviewed and the CoAg 
along with the relevant staff should be available for discussion of findings. To ensure 
compliance with national and international regulatory guidelines, the activities outlined in 
this proposal may be subject to inspection or review by authorized Zambian or international 
regulatory authorities, including the UNZA BREC, NHRA, and US IRBs. 
 
7.11 Dissemination of findings 
Monthly summary data from the ANC visits at all participating facilities will be monitored to 
ensure that data are being collected correctly, however, no formal interim analyses will be 
conducted. Results will be presented by CIDRZ through tables and disseminated through 
reports to the Ministry of Health, ZNPHI, CDC, and other stakeholders. Results will also be 
disseminated through conference presentations and through publications in peer reviewed 
journals. 
 
8.0  
Ethical Considerations 
8.1 
Ethical committee approvals 
The study protocol will be reviewed by the local IRB (UNZA BREC) and CDC before data 
collection starts for appropriate input and approvals. An information sheet with details 
about the study will be offered to eligible participants in their preferred language (i.e., 
(Chewa/Nyanja, Bemba) while study staff provide detailed explanation about the study 
procedures Participants will be told the general purpose, the possible risks, and benefits in 
the local language (Nyanja/Chewa or Bemba). Individual written consent will be obtained 
before sample collection and administration of the questionnaire. Pregnant and recently 
pregnant women are considered emancipated minors for the purpose of providing their 
own informed consent. Women will be offered a copy of the consent form if they wish to 
take it. Participation is voluntary and refusal will not affect a participant’s health care. 
Additionally, participants may refuse to answer specific questions during the questionnaire.  
 

 
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8.2 
Risks & Benefits for Subjects 
This investigation poses minimal risk to participants, involving the collection of a small 
amount of blood. Every attempt will be made to utilize the finger prick being made for 
routine HIV and syphilis testing during the first ANC visit. During the blood collection, 
women may experience temporary discomfort, and there is a very small risk of infection. 
The area will be cleaned thoroughly prior to drawing blood to prevent infection. There are 
no economic risks associated with this study. Pregnant women will not be asked to make 
any additional visits to the health facility specifically for the purposes of the study. However, 
the participants may spend additional time (estimated 10 minutes) at the facility during the 
first ANC visit to complete the questionnaire. The primary benefit of the study is indirect in 
that data collected will help improve and guide efforts to understand the extent of SARS-
CoV-2 infection, which might help contribute to efforts to prevent further transmission of 
the virus in Zambia. There is no specific direct benefit to participants. 
 
Of note, this activity will NOT contribute to clinical care of the participants in real-time. 
Among other reasons, this is because serologic conversion in general does not occur until 
after acute illness; because serologic tests are not diagnostic for current SARS-CoV-2 
infection, they are inappropriate for clinical decision making; and because lab analysis would 
take place remotely and results would not be available until at best days or weeks after the 
clinic visit. Further, as we will not record any identifiers on the filter paper samples, it will 
not be possible to link the results of SARS-CoV-2 serologic testing back to the woman from 
whom the sample was taken. 
 
8.3 
Unexpected/Adverse Events 
This study does not entail substantial risk to any participants. However, any major 
deviations from the protocol will be treated as an unanticipated problem/event and will be 
reported to all IRBs of record within the timeframe specified by the IRB. This also includes 
any problem or event that: 1) poses harm or risk of harm to subjects or others; 2) is 
unanticipated (not described in research plan or consent form); and 3) related to the study 
procedures. 
 

 
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8.4 
Prevention of SARS-CoV-2 Infection in Investigation Personnel 
All personnel involved in the investigation will be trained in infection prevention and control 
procedures for SARS-CoV-2 as detailed by the Zambian Ministry of Health, including proper 
correct use of masks, physical distancing, limiting time in enclosed and/or crowded spaces, 
and hand hygiene. Personnel who develop symptoms will be isolated, tested, and referred 
appropriately. They will be allowed to resume involvement after resolution of symptoms 
and completion of recommended days in isolation. 

 
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9.0  
Timeline 
Activities 
Responsible Person 
Duration in Months 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Adaptation of protocol to UNZA BREC 
PI/Technical Lead 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Submission and approval 
PI/ Technical Lead 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Training for all DHO staff and facility in 
charges from sentinel sites 
Technical Lead/ Study 
Coordinator 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
On site orientation for all MCH staff in the 
sentinel sites 
Study Coordinator/ 
Study Nurses 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Enrolment of participants 
Facility Staff/  
Study Nurses 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Supervisory visits to sentinel sites 
All 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Monitoring availability of all logistics and 
surveillance activities 
Study Coordinator/ 
Study Nurses/  
Program Admin 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Monthly data reporting and monitoring  
Study Nurses 
 
 
X  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Quality control and assurance activities 
Technical Lead/ Study 
Coordinator 
 
 
X  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Data analysis and preparation of the final 
report 
Technical Lead/ SI 
team 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Presentation of final report by CIDRZ to MoH 
Senior staff, ZNPHI and CDC 
PI/ Technical Lead 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Presentation of the results by MoH/ZPHI to 
various stakeholders 
MoH/All 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Submission of the copy of report to Scientific 
and Ethical Committee  
Technical Lead 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Dissemination of findings (Journal publication, 
abstract and poster) 
Technical Lead 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Posting findings on Government and CIDRZ 
websites 
Technical Lead 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 

 
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10.0 Budget 
We are requesting a total of US$300,992.02 which will be allocated to personnel, travel, 
supplies and other costs. The budget has been broken down as follows:  
 
Category 
Cost (US$) 
% 
Personnel 
 
60,897.93 
20.23 
Equipment 
 
- 
- 
Supplies 
 
125,366.00  
41.66 
Travel 
 
42,120.00 
13.99 
Other  
 
 
24.12 
 
Trainings 
26,508.07 
 
 
Stakeholder meetings 
1,500.00 
 
 
Communication 
3,600.00 
 
 
Administrative costs 
9,000.00 
 
 
Stipends for ANC TS and transport 
refunds  
35,000.00 
 
CIDRZ IDC 
 
- 
 
Total 
 
300,992.01 
100 
 
 
 
 

 
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