Prevalence of malaria and associated factors among symptomatic pregnant women attending antenatal care at three health centers in north-west Ethiopia.

BACKGROUND: Malaria is the disease caused by Plasmodium species and primarily transmitted by the bite of female Anopheles mosquitoes. During pregnancy, malaria causes life threatening outcomes to the mother, the fetus and the new born. Even though, malaria symptomatic pregnant women highly attract mosquitoes and have higher potential of transmitting the disease in communities, most of the previous studies focused on pregnant women with asymptomatic Plasmodium infections. Therefore, the aim of this study was to assess the prevalence of malaria and associated factors among symptomatic pregnant women attending antenatal care at three health centers in northwest Ethiopia.
METHODS: A health facility based cross-sectional study was conducted from February to April, 2021. A total of 312 malaria symptomatic pregnant women were involved from three health centers and enrolled by convenient sampling technique. A questionnaire was used to collect socio demographic and clinical data through face to face interview. Capillary blood samples were collected and used to prepare thin and thick blood smears, which were then stained using 10% Giemsa and examined under light microscope. Logistic regression was used to assess factors associated with malaria. Adjusted odds ratio with 95% confidence interval was calculated and P-value < 0.05 was considered statistically significant.
RESULTS: The prevalence of malaria among symptomatic pregnant women was 20.8% (65/312) of which 12.2% (38/312), 4.8% (15/312) and 3.8% (12/312) were P. falciparum, P. vivax and mixed infections, respectively. Being illiterate (p< 0.001), first trimester (p = 0.036), primigravidae (p<0.001), living far from health center (p<0.001), not sleeping under long lasting insecticide treated nets (p<0.001) and living near irrigation areas (p = 0.006) were significantly associated with prevalence of malaria in malaria symptomatic pregnant women.
CONCLUSIONS: Even though prevalence of malaria is decreasing in the country because of scale-up of intervention and prevention measures, this study showed that, malaria is still the major public health problem among pregnant women. Being illiterate, first trimester, primigravidae, living far from health centers, not sleeping under long lasting insecticide treated nets and living near irrigation areas were factors that increased the prevalence of malaria in malaria symptomatic pregnant women. Therefore, special attention should be given to pregnant women prone to these factors.

Background

Malaria is the disease caused by intracellular parasites of five species of the genus Plasmodium namely; Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium ovale (P. ovale), Plasmodium malariae (P. malariae) and Plasmodium Knowlesi (P. knowlesi) [1]. According to the World Health Organization (WHO) 2020 report, malaria is endemic in 87 countries in the world with 229 million cases and 409,000 deaths in 2019 [2]. Of this global burden, 215 million cases and 384,000 deaths occurred in Africa [2]. Malaria is transmitted to humans by the bite of female Anopheles mosquitoes. Anopheles mosquitoes feed on blood for survival and production of eggs. During feeding, infected female Anopheles mosquito inoculates the infective sporozoites from its salivary gland into human circulation. After inoculation, parasites circulate with blood and those reaching to the liver undergo one cycle development. Then, parasites infect and multiply inside red blood cells to bring the characteristic signs and symptoms [3]. The clinical manifestations include fever, joint pain, chills, headache and vomiting which usually appear between 10 and 15 days after the Anopheles mosquito bite. In addition, malaria may also be transmitted through blood transfusion and congenitally [4].

In Ethiopia, P. falciparum and P. vivax are the two dominant species causing malaria and accounted for 60% and 40%, respectively. Anopheles arabiensis is the main malaria vector; An. pharoensis, An. funestus and An. nili play a role as secondary vectors [5].

Malaria diagnosis is done using microscopic examination, rapid diagnostic test and molecular techniques. The detection of Plasmodium parasites by light microscopy from capillary or venous blood is still the primary method in most health facilities across the world [6]. Early diagnosis and treatment play an important role in malaria prevention and control. Artemisinin based combination therapy and chloroquine are first-line anti-malaria treatments for P. falciparum and P. vivax, respectively. Oral quinine is used as the first-line treatment for pregnant women during the first trimester [5]. Vector control using long lasting insecticide treated nets, indoor residual spraying and larval source management are used for prevention of malaria in Ethiopia [5].

Malaria affects both sexes and all age groups. However, infection during pregnancy poses severe and life threatening outcomes to the mother, the fetus, and the new born. Because, during pregnancy, there is decline in immunity and sequestration of infected erythrocytes to the placenta [7, 8]. Maternal complications of malaria in pregnancy include; anemia, cerebral malaria and death [4]. For instance, malaria related anemia is estimated to cause 10,000 maternal deaths each year in Africa [9]. Major complications among fetuses and newborns include; low birth weight, intrauterine growth retardation, abortion, premature delivery and fetal death [10]. In sub-Saharan Africa, among the 11 million pregnant women exposed to malaria in 2018, about 872,000 newborns were born with low birth weight [11]. Although, pregnant women have higher potential of transmitting malaria than their counter parts [12], community chemotherapy campaigns often exclude them. So, they may contribute to persistent malaria transmission in the area.

The prevalence of malaria among asymptomatic pregnant women has been well documented in Ethiopia [13-16]. However, prevalence among symptomatic pregnant women remains poorly considered. So, there is scarcity of data on prevalence of malaria and associated factors among symptomatic pregnant women. In addition, Ethiopia has set goals for malaria elimination and conducting confirmatory testing for 100% of suspected malaria cases and treat as per the guide line is one of the main strategic objectives [5]. Therefore, this study aimed to assess the prevalence of malaria and associated factors among symptomatic pregnant women attending antenatal care at three health centers in northwest Ethiopia.

Materials and methods

Study design, period and area

A health facility based cross-sectional study was conducted at three health centers (Tis Abay, Zenzelma and Hamusit) in northwest Ethiopia from February to April, 2021. The three health centers were selected purposively due to logistic reasons. Zenzelma and Hamusit are located at 15km and 40 km, respectively North of Bahir Dar city. Tis Abay is located at 35km east of Bahir Dar city. The climate of all the three areas is Woyna dega (1500m-2500m). The Altitude of Hamusit, Zenzelma and Tis Abay areas is 2077 meters, 1800 meters and 1653 meters, respectively above sea level. The three study areas have similar climatic condition with main rainy season occurring from June to August. Similarly, the study areas have an intensive irrigation system used for cultivation of rice, onion, sugarcane etc. Malaria transmission occurs throughout the year in the areas but the peak incidence and epidemics occur seasonally at the end of September and beginning of May during planting and harvesting seasons. About 43,128, 11,650 and 55,426 number of catchment populations get health service from Tis Abay, Zenzelma and Hamusit health centers, respectively. There were 336, 90 and 776 pregnant women diagnosed for malaria from Tis Abay, Zenzelma and Hamusit health centers, respectively in 2019/2020.

Sample size determination and sampling technique

Using single population proportion formula with 50% prevalence, 95% confidence level, 5% margin of error and 10% non-response rate the total sample size was 422. However, the total number of symptomatic pregnant women in the study areas was 1202 based on 2019/2020 data from all health centers which is finite population or less than 10.000. So, adjustment (correction) formula was used and 312 malaria symptomatic pregnant women have participated in the study. Finally; using proportionate allocation, 87, 24 and 201 study participants were involved from Tis Abay, Zenzelma and Hamusit health centers, respectively and enrolled by convenient sampling technique.

Dependent and independent variables

The dependent variable was malaria infection status. The independent variables were age, residence, educational status, occupation, gestational age, gravidity, LLINs use, distance from health centers and Irrigation.

Operational definitions

Symptomatic: Women who exhibited at least one of the signs and/or symptoms of malaria like fever (axillary temperature ≥37.5°C), joint pain, malaise, vomiting, chills etc.

Pregnant: Women of confirmed urine Human Chorionic Gonadotropin (HCG) hormone positive in laboratory.

Distance was estimated as:

Far = Distance from health centers to pregnant women’s home covering 2 hours and more on foot

Medium = Distance from health centers to pregnant women’s home covering 1–2 hrs on foot

Near = Distance from health centers to pregnant women, s home cover less than 1 hour on foot

Data collection and processing

Clinical and socio-demographic data collection

Women confirmed of being pregnant by using Human Chorionic Gonadotropin (HCG) hormone test from urine sample in laboratory attend their follow up. So, pregnant women exhibiting signs and/or symptoms of malaria during their visit were asked for willingness to participate in the study. From volunteered pregnant women, clinical and socio- demographic data were collected through face to face interview by the midwives using a structured questionnaire.

Blood sample collection

For detection and identification of Plasmodium species, capillary blood was collected by trained and experienced medical laboratory technicians from each health center and the principal investigator. Pregnant women’s finger was cleaned with 70% ethyl alcohol and the side of fingertip was pricked with a sterile lancet. The first drop of blood which contains tissue fluids was wiped away. One μl and 2 μl of blood were used for preparation of thin and thick blood films, respectively. The prepared blood films were air dried and thin films were fixed with absolute methanol. The smears were then stained by 10% giemsa stain and examined under light microscope following standard operating procedures. A negative result was reported after checking at least 100 oil immersion fields. Thick blood films were used for parasite detection and thin blood films were used for species identification.

Data quality control

Before starting data collection, training was given to data collectors (Midwives and Medical Laboratory Technicians) by principal investigator on how to collect socio-demographic and clinical data and process laboratory data. To ensure the quality of Giemsa stain, a quality giemsa stock solution prepared in Amhara public health institute (APHI) was used for preparation of 10% Giemsa (working solution) and prepared every 8 hours. Buffered water with PH- value of 7.2 was used for preparation of Giemsa stain working solution and filtered before use. At the end of data collection, all the slides were re-examined by experienced malaria microscopists in APHI who have not been engaged during data collection. This review (re-examination) was taken as final.

Data analysis

Questionnaire containing socio-demographic characteristics, clinical data and associated factors was checked for completeness. The data were coded, entered, cleaned and analyzed using Statistical Package for Social Sciences version 20 (SPSS 20).

Descriptive statistics (frequencies, mean and percentage) was used to explain the study participants in relation to the variables. Logistic regression was used to determine factors associated with malaria. Adjusted Odds ratios (AORs) with 95% confidence interval were calculated and P- value < 0.05 was considered statistically significant.

Ethical considerations

Ethical approval was obtained from College of Medicine and Health Sciences, Bahir Dar University, Institutional Review Board (protocol number 157/2021). Support letter was also obtained from APHI, South Gondar and Bahir Dar city administration Zonal health departments, Dera district health office and the three Health centers. Written informed consent was obtained from study participants after explaining the purpose of the study by data collectors. Study participants with positive results were treated according to the national malaria treatment guideline.

Results

Sociodemographic characteristics of study participants

Overall, 312 malaria symptomatic pregnant women comprising of 87.8% (274/312) rural and 12.2% (38/312) urban dwellers participated in the study. Of these, 58% (181/312) were illiterate, 70.2% (219/312) were in the age group of 21–30 years old and 81.1% (253/312) were farmers (Table 1).

Table 1

Sociodemographic characteristics of study participants attending ANC at three health centers in northwest Ethiopia, February to April 2021.

Variables	Category	Frequency (%)
Age group	≤20	43 (13.8)
	21–30	219 (70.2)
	31–40	50 (16.0)
Residence	Rural	274 (87.8)
	Urban	38 (12.2)
Educational status	Illiterate	181 (58.0)
	Primary	68 (21.8)
	Secondary and above	63 (20.2)
Occupation	Farmer	253 (81.1)
	Private business	43 (13.8)
	Employed	16 (5.1)

Prevalence of malaria among symptomatic pregnant women

The overall prevalence of malaria was 20.8% (65/312) (95% CI: 16.7%-25.6%). The highest relative proportion of malaria was found among 1st trimester of pregnancy, 44.6% (29/65). In relation to gravidity, the highest relative proportion of malaria was detected among primigravidae, 67.7% (44/65) (Table 2).

Table 2

Prevalence of malaria among symptomatic pregnant women attending ANC at three health centers in northwest Ethiopia from February to April 2021 (n = 312).

Result		Proportion %
Total positive		20.8% (65/312) (95% CI: 16.7–25.6)
P. falciparum		12.2% (38/312) (95% CI: 9–16.2)
P. vivax		4.8% (15/312) (95% CI: 2.9–7.8)
Mixed (P. falciparum & P. vivax)		3.8% (12/312) (95% CI: 2.2–6.6)
Gestational age	First trimester	44.6% (29/65)
	Second trimester	32.3% (21/65)
	Third trimester	23.1% (15/65)
Gravidity	Primigravidae	67.7% (44/65)
	Secundigravidae	10.8% (7/65)
	Multigravidae	21.5% (14/65)

Factors associated with malaria among symptomatic pregnant women

Illiterate Pregnant women were more affected than those who had attended secondary school and above (AOR = 7.9; 95%CI: 2.55,-24.50, p<0.001). Pregnant women in 1st trimester and primigravidae had 2.6 and 8.3 times increased susceptibility to malaria infection than pregnant women of 3rd trimester and multigravidae, respectively. However, age and residence of pregnant women were not statistically associated with malaria infection (Table 3).

Table 3

Factors associated with malaria among symptomatic pregnant women attending ANC at three health centers in northwest Ethiopia, February to April 2021.

Variables	Category	Malaria		COR (95%CI)	P-value	AOR (95%CI)	P-value
		Positive	Negative
Age (Yrs.)	≤20	13	30	3.9 (1.26, 12.08)	0.018*	1.5 (0.33,7.05)	0.589
	21–30	47	172	2.5 (0.92, 6.54)	0.072	1.7 (0.51,5.57)	0.398
	31–40	5	45	1
Residence	Rural	62	212	3.4 (1.02, 11.47)	0.047*	0.6 (0.13,2.48)	0.442
	Urban	3	35	1
Educational status	Illiterate	51	130	3.7 (1.51, 9.18)	0.004*	7.9 (2.55,24.50)	< 0.001*
	Primary	8	60	1.3 (0.41, 3.88)	0.679	0.9 (0.25,3.04)	0.818
	secondary and above	6	57	1
Gestational age	1st trimester	29	67	2.6 (1.3, 5.17)	0.008*	2.6 (1.07, 6.17)	0.036*
	2nd trimester	21	91	1.4 (0.66,2.83)	0.395	1.4 (0.55, 3.43)	0.497
	3rd trimester	15	89	1
Gravidity	Primigravidae	44	90	4.0 (2.04, 7.65)	< 0.001*	8.3 (3.11, 22.25)	< 0.001*
	Secondi gravidae	7	44	1.3 (0.49, 3.39)	0.614	1.9 (0.57, 6.02)	0.307
	Multigravidae	14	113	1
Distance from HCs to home	Far	20	20	5.1 (2.43, 10.55)	< 0.001*	2.7 (1.00, 7.26)	< 0.001*
	Medium	15	75	1.0 (0.51, 2.00)	0.969	0.9 (0.41, 2.14)	0.883
	Near	30	152	1
Sleeping in LLINs	No	56	140	4.8 (2.25,10.04)	< 0.001*	5.8 (2.25,15.05)	< 0.001*
	Yes	9	107	1
Spend outside at dusk	Yes	60	184	4.1 (1.6, 10.69)	0.004*	2.0 (0.66, 6.23)	0.216
	No	5	63	1
Irrigation within 500m from home	Yes	24	38	3.2 (1.7, 5.93)	< 0.001*	3.0 (1.38, 6.67)	0.006*
	No	41	209	1

HCs = Health centers.

Discussion

Malaria remains a leading cause of morbidity and mortality especially among pregnant women and children in the developing world [17]. In the present study, the prevalence of malaria was 20.8% which is in line with the prevalence in Ghana (22%) [18]. However, the prevalence of malaria in this study is lower than the prevalence in Burkina Faso (49%) [19] and Mali (28.1%) [20]. This difference might be due to the difference in overall prevalence and burden of malaria in countries. Compared to other endemic countries in Sub-Saharan Africa, malaria prevalence in Ethiopia is relatively low; but, Burkina Faso and Mali are among the top ten countries with the highest number of malaria cases and deaths. Burkina Faso takes up (3% of the global cases and 4% of global deaths) and Mali covers (3% of the global cases and deaths, and 6% of cases in West Africa) [2]. In addition, the difference may be due to the difference in study period. For example, the current study was done from February to April (local dry season) and low malaria transmission season; whereas, the study in Burkina Faso was done during both low and high malaria transmission seasons and the study in Mali was done during high malaria transmission season (September and May).

On the other hand, the prevalence of malaria in the present study is higher than the prevalence in Pawe Hospital, northwestern Ethiopia (16.3%) [21] and North Gonder, northwestern Ethiopia (11.5%) [22]. This variation might be due to low coverage and utilization of LLINs and existence of favorable vector breeding sites like irrigation in the current study area. Since, 63% of pregnant women were not sleeping under LLINs mainly due to unavailability and this might have increased the prevalence in the current study.

Plasmodium falciparum mono-infection was the most predominant species with overall prevalence of 12.2%, which is in line with the study done in Pawe Hospital, northwestern Ethiopia (9.67%) [21]. But, this result is lower than the study conducted in Burkina Faso (26.9%) [19]. This variation might be due to the difference in inclusion criteria, study period and study site (type of health facility). The present study was conducted in health centers and pregnant women with severe malaria were excluded. However, Tahita and his colleagues’ study was conducted in hospital and included pregnant women with severe malaria which mostly occurs due to P. falciparum. In addition, the overall malaria burden is higher in Burkina Faso compared to Ethiopia as explained above.

The prevalence of P. vivax in the current study was 4.8% which is in line with the study in North Gonder, northwestern Ethiopia (3%) [22]. However, this result is higher than the study done in Pawe hospital (1.6%) [21]. This variation might be due to variation in vector competence, rain fall, temperature and study site. The prevalence of mixed infections in the current study was 3.8%. This is in line with studies in North Gonder, northwestern Ethiopia (2.3%) [22] and Pawe hospital, northwestern Ethiopia (3.03%) [21].

Analysis of factors associated with malaria among symptomatic pregnant women showed that, educational status, gestational age, gravidity, distance, LLINs and irrigation were significantly associated with malaria. In the current study, educational status of pregnant women has significant association with malaria. Illiterate pregnant women had increased malaria susceptibility than women who attended secondary school and higher education. A similar finding was reported from studies done in Nigeria [23] and Benishangul Gumuz, northwest Ethiopia [24].

Pregnant women in the first trimester of pregnancy were at increased odds of having malaria than women in third trimester. This finding is in line with the study in Mali [20], Jawi district, northwestern Ethiopia [13] and Benishangul Gumuz, northwest Ethiopia [24]. In relation to gravidity, primigravidae were at higher odds of developing symptomatic malaria compared to multi-gravidae. Similar association was found in studies done in Mozambique [25], Ghana [18], Burkina Faso [19], Jawi district, northwestern Ethiopia [13] and Benishangul Gumuz, northwestern Ethiopia [24].

Distance, where pregnant women live relative to the health centers was statistically associated with symptomatic malaria infection. However, distance in relation to malaria was not assessed in any studies. According to the present study, pregnant women who live in far distances were 2.7 times more likely to have increased malaria susceptibility compared to pregnant women living in nearby health centers. This might be due to the assumption that, women of far areas may not attend health centers regularly for their ANC follow up because of transportation problem. In addition, the health centers are situated in the towns and as we go far, there might be increased vector breeding sites that in turn might have made the association.

In the present study, pregnant women who do not sleep under LLINs have significantly increased susceptibility to malaria infection. This finding is in agreement with the study done in Benishangul Gumuz, northwest Ethiopia [24]. However, according to the studies done in Burkina Faso [19] and Jawi district, northwestern Ethiopia [13], the association between bed net utilization and malaria was not significant. This variation might be due to higher coverage and utilization of LLINs in the above studies compared to the present study (72% versus 37%, respectively).

According to the present study, the presence of irrigational activity in nearby houses was significantly associated with prevalence of malaria. Pregnant women who live in areas where irrigational activities are practiced within 500 meters in nearby houses were 3 times more likely to have malaria than those living in irrigation free areas. This might be due to the fact that, irrigation might serve as important factors for Anopheles mosquito breeding and might make the association significant. However, other studies have not investigated the association between irrigation and malaria.

Conclusions

Even though prevalence of malaria is decreasing in the country because of scale-up of intervention and prevention measures, this study showed that, malaria is still the major public health problem among pregnant women. P. falciparum was the predominant species causing malaria infection in the area. Illiteracy, first trimester, primigravidae, living far from health centers, not sleeping under LLINs and living in areas within 500m near irrigation were factors that increased the prevalence of malaria infection in malaria symptomatic pregnant women. Therefore, special attention should be given to pregnant women prone to these factors.

(ZIP)

Click here for additional data file.

14 Dec 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please describe how verbal consent was documented and witnessed, and why written consent was not obtained. If your study included minors, state whether you obtained consent from parents or guardians.

3. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed the survey or questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. If the questionnaire is published, please provide a citation to the (1) questionnaire and/or (2) original publication associated with the questionnaire.

4. Thank you for stating the following financial disclosure:

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

6. We note that you have referenced (ie. Bewick et al. [5]) which has currently not yet been accepted for publication. Please remove this from your References and amend this to state in the body of your manuscript: (ie “Bewick et al. [Unpublished]”) as detailed online in our guide for authors

http://journals.plos.org/plosone/s/submission-guidelines#loc-reference-style

7. We note that Figure in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Major comments

1. The information flow in the background of the main body is not adequate. Try to arrange in such a way that paragraphs connect and also within the paragraphs there is a connection between sentences. The authors may start with malaria epidemiology, then biology of the parasite, clinical presentation, etc.

2. Grammatical errors should be corrected.

Minor Comment

Line 15: Rephrase the sentence to increase clarity. I suggest the sentence read, "Malaria is a disease caused by Plasmodium species…….

Line 17: Symptomatic pregnant women……however, it is not clear these women are symptomatic for what infection/condition. Clarify.

Line 18: Symptomatic pregnant women are highly potential transmitters…..but symptomatic individuals normally seek medical attention, thus it is not clear how can they become an important cause of epidemics. It would be clear if they were asymptomatic.

Line 27: Questionnaire data was collected…..this sentence is not clear, re-write it to increase clarity. For instance, “A questionnaire was used to collect information/data through a face-to-face interview.

Line 28: Thin and thick films from…..re-write to read…”Capillary blood samples were collected and used to prepare thick and thin blood smears, which were then stained using ……”

Line 30: Simply write….”Logistic regression was used to assess……..”

Line 36: Pregnant women who couldn’t read………just write…”Illiterate pregnant women…..”

Line 38-39: Change “lived far from”…to…. “living far from”, “didn’t sleep under”…..to…”not sleeping under”, “lived near”…to…”living near”.

Line 40: I presume these were factors associated with confirmed malaria infection, therefore, should not be written as odds of symptomatic malaria but rather of malaria infection in malaria symptomatic pregnant women.

Lines 42-45: The conclusion does not include the contribution of the socioeconomic factors on the presence of confirmed malaria infection in these malaria symptomatic pregnant women.

Line 50: In which year were the 229 million cases and 409,000 deaths were reported, and where is the reference?

Line 64: RDTs should be written in a long form since it appears for the first time.

Line 53-57: During feeding……Then parasites infect and multiply…..This section is redundant, should be removed.

Methods

Study design

Line 101: The climate is Woyna Dega………can you explain what does this suppose to mean in climatic terms?

Line 104: Rephrase the sentence to read, “Mean annual rainfall and temperature of……………is 1800 mm and 20.10C, respectively.”

Line 128-29: Using proportionate allocation 87, 24, and 201 study participants were involved……where were these proportions involved from each of the three study sites, respectively? If so please state that, and explain what was the criteria for having such proportions per site.

Data collection

Line 141: Questionnaire…..can you explain whether the questions were open or close-ended? Please also give a summary of the information the questionnaire was trying to gather, I mean the type of demographic and socioeconomic or clinical data.

Line 143: Blood sample collection….Of the blood slides (thin/thick), which one was used for the detection and counting of the parasite density, and which one was used for species identification?

Did you count the parasite density? If yes, how was it done?

Results

Line 198: The prevalence of malaria………..the percentages should be out of the brackets, and the whole numbers in brackets and given as proportions with the denominator being the total number of malaria infections e.g. 44.6% (29/65), etc.

Line 198-201: This information should be given in a form of a Table, and preferably be added to Table 2. Table 2 needs to be restructured by removing the parasite stages prevalence i.e. Gametocytes and schizonts as they add no value to the data presented. Likewise, remove the number of malaria negative results and instead provide the proportions of malaria positives and their percentages e.g. P. falciparum 12.2% (38/312), P. vivax 4.8% (15/312), and mixed infection…..

Line 204-205: Provide the odds ratio, confidence interval, and p-value.

Discussion

Line 257-60: Can you explain why primigravidae had higher odds of having malaria infection than other multigravidas?

Conclusion

Should be summarized and made more clear.

Reviewer #2: Dr Almaw and colleagues have examined symptomatic pregnant women for malaria and describe the associated characteristics of the women found to have malaria. The study is quite interesting, and the results clearly identify certain groups of symptomatic pregnant women as being at particular risk of malaria. There are some missing pieces of information that will strengthen the paper.

Major comments

1. The authors need to provide a clear definition of “symptomatic”. How did they define women of interest? Also, how did they identify “pregnant”? Were pregnancy tests done in women of childbearing age, for example? This is important for interpreting relative proportions of infection by trimester. (It would also be helpful to define cutoffs used for different trimesters).

2. The manuscript contains unnecessary detail in places. For example, lines 52-60 provide a “textbook” description of the parasite life cycle, which can all be removed, and similarly lines 64-73 contain much generic information on malaria diagnosis and management in general and in pregnancy. The detailed description of the study sites can be condensed substantially (lines 97-119)- but please include the size of populations in the catchments here.

3. Sample size, Line 123. These determinations are often hard to follow. It seems the researchers assumed a parasite prevalence of 50% which was high, and was not observed, and the sample size was not obtained. How does this affect study interpretation?

4. Line 198: these data are relative proportions by trimester, not prevalence.

5. Line 216-7: This statement is manifestly incorrect and should be removed. Even if it is the first study of a very specific question such assertions are best avoided.

6. In the discussion it is generally unclear whether studies from elsewhere used similar recruitment criteria or not. This is critically important for comparing the studies. For example reference 7 appears to be a simple cross sectional study, while reference 21 (Tagbor) compared symptoms in parasitemic and aparasitemic women. References by Tahita (22) and Bardaji seem most directly comparable in design.

7. Line 219-220: differences in season relative to transmission of malaria is a possible explanation (and worth mentioning) but it would make sense to start with differences in overall transmission between countries. Isn’t disease burden much higher in the mentioned countries?

8. Line 224-5: what was the design of the other studies in Ethiopia, if cross sectional or observational rather than focussed on symptomatic women this needs to be taken into account.

9. Line 238-242: this paragraph is confusing in its intentions, as to whether the findings are similar to the two previous studies, or different than them. Keeping the comparison simple and not trying to explain “differences” is easier to follow, especially given the size of one of the earlier studies.

Minor comments

1. There are many minor English language errors throughout the manuscript. An edit by an expert English speaker or professional editor is needed. I have confined my suggestions to the scientific content.

2. Abstract and conclusion: the term “considerably high” is poor, please change it.

3. Check use of abbreviations and only use them if repeated 3 or more times. IRS, LSM, and probably LLINs are examples of unnecessary abbreviations.

4. Line 81: the figure of 11 million women exposed to malaria refers to infected pregnant women in Africa, please revise.

5. Line 133 how is “irrigation” defined?

6. Line 158-160: how was the QC implemented? What happened in case of disagreements?

7. Table 1: the lines for “total” are really superfluous and could be removed.

8. Table 3 “Spent outside at dusk” needs better description and definition.

9. Line 288-290: are these symptoms authors used to define malaria?

10. The following references need additional detail or correction: 1, 4 (journal name spelled out); 2 and 12: 2 different editions of World Malaria Report- both needed?; 9: second author surname missing; 18, 25: no source given- journal or other publication??; 21: incomplete citation- published?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Richard Mwaiswelo

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

14 Jan 2022

Point by point response to editor and reviewers comments

1. Point by point response to editor comments

First, we authors would like to thank editor and both reviewers for comments to make this manuscript more plausible. As much as possible, we authors would like to address your comments here after.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: well taken. Dear editor, we have now added requirements that were previously missed.

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please describe how verbal consent was documented and witnessed, and why written consent was not obtained. If your study included minors, state whether you obtained consent from parents or guardians.

Response: well taken. Dear editor, our study subjects were pregnant women who came to health center for Ante Natal Care visit seeking diagnosis and treatment for malaria. They are sick and do not refuse to give data like history, signs and/or symptoms, blood sample etc. But, to participate in the study, we explained the purpose of the study and requested their willingness without signing on consent form. This was what we want to mean by Verbal consent.

3. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed the survey or questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. If the questionnaire is published, please provide a citation to the (1) questionnaire and/or (2) original publication associated with the questionnaire.

Response: well taken. We have included as supportive file.

4. Thank you for stating the following financial disclosure:

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: well taken. We declare that, the authors received no specific funding for this work.

5. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Response: Well taken. dear reviewer, all data are available without restrictions

6. We note that you have referenced (ie. Bewick et al. [5]) which has currently not yet been accepted for publication. Please remove this from your References and amend this to state in the body of your manuscript: (ie “Bewick et al. [Unpublished]”) as detailed online in our guide for authors

http://journals.plos.org/plosone/s/submission-guidelines#loc-reference-style

Response: well taken and removed

7. We note that Figure in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth)…………..

Response: dear editor, we have removed it.

Thank you

2. Point by point response to reviewer#1 comments

Authors’ Response to the reviewer#1`s comments

First, we authors would like to thank reviewer for comments to make this manuscript more plausible. As much as possible, we authors would like to address your comments here after.

Reviewer #1: Comments to the author

Reviewer #1: Major comments

1. The information flow in the background of the main body is not adequate. Try to arrange in such a way that paragraphs connect and also within the paragraphs there is a connection between sentences. The authors may start with malaria epidemiology, then biology of the parasite, clinical presentation, etc.

Response: well taken and corrected.

2. Grammatical errors should be corrected.

Response: well taken. We have tried to edit

Minor Comments

Line 15: Rephrase the sentence to increase clarity. I suggest the sentence read, "Malaria is a disease caused by Plasmodium species…….

Response: Well taken and we have corrected as you suggested

Line 17: Symptomatic pregnant women……however, it is not clear these women are symptomatic for what infection/condition. Clarify.

Response: well taken and it is to mean malaria symptomatic

Line 18: Symptomatic pregnant women are highly potential transmitters…..but symptomatic individuals normally seek medical attention, thus it is not clear how can they become an important cause of epidemics. It would be clear if they were asymptomatic.

Response: Well taken. Dear reviewer, Symptomatic pregnant women have higher potential of attracting mosquitoes because of different physiological changes like increased abdominal temperature and hence sweating, increased breath etc. so that they can be easily bitten and that vector again may take infection and transmit to the others.

Line 27: Questionnaire data was collected…..this sentence is not clear, re-write it to increase clarity. For instance, “A questionnaire was used to collect information/data through a face-to-face interview.

Response: Well taken and we have corrected as you suggested

Line 30: Simply write….”Logistic regression was used to assess……..”

Response: well taken and corrected

Line 36: Pregnant women who couldn’t read………just write…”Illiterate pregnant women…..”

Response: well taken and corrected

Line 38-39: Change “lived far from”…to…. “living far from”, “didn’t sleep under”…..to…”not sleeping under”, “lived near”…to…”living near”.

Response: well taken and changed

Line 40: I presume these were factors associated with confirmed malaria infection, therefore, should not be written as odds of symptomatic malaria but rather of malaria infection in malaria symptomatic pregnant women.

Response: well taken and corrected

Lines 42-45: The conclusion does not include the contribution of the socioeconomic factors on the presence of confirmed malaria infection in these malaria symptomatic pregnant women.

Response: well taken and corrected

Line 50: In which year were the 229 million cases and 409,000 deaths were reported, and where is the reference?

Response: well taken; it was in 2019 and the reference is WHO 2020

Line 64: RDTs should be written in a long form since it appears for the first time.

Response: well taken and corrected

Line 53-57: During feeding……Then parasites infect and multiply…..This section is redundant, should be removed.

Response: Well taken

Methods

Study design

Line 101: The climate is Woyna Dega………can you explain what does this suppose to mean in climatic terms?

Response: well taken. Dear reviewer, Woyna dega (Amharic term) is climatic zone covering areas of 1500m -2500m

Line 104: Rephrase the sentence to read, “Mean annual rainfall and temperature of……………is 1800 mm and 20.10C, respectively.”

Response: well taken and rephrased

Line 128-29: Using proportionate allocation 87, 24, and 201 study participants were involved……where were these proportions involved from each of the three study sites, respectively? If so please state that, and explain what was the criteria for having such proportions per site.

Response: well taken. Dear reviewer, it is to mean that; 87, 24, and 201 study participants from Tis Abay, Zenzelma and Hamusit health centers, respectively. This proportions per site were obtained by taking previously diagnosed malaria symptomatic pregnant women from each health center, i.e 336, 90 and 776 ( line 118 previous document) malaria symptomatic pregnant women from Tis Abay, Zenzelma and Hamusit health centers, respectively were diagnosed in precious year( 2019/2020). Total (336+90+776 = 1202). So, if there are 336 (for Tis Abay health center) from 1202, how many will be selected from 312(sample size)? It is equal to 76. Using similar way for getting number of malaria symptomatic pregnant women to be enrolled from each health center; i.e 24 and 201 for Zenzelma and Hamusit health centers, respectively.

Data collection

Line 141: Questionnaire…..can you explain whether the questions were open or close-ended? Please also give a summary of the information the questionnaire was trying to gather, I mean the type of demographic and socioeconomic or clinical data.

Response: dear reviewer, questionnaire was closed ended. The socio demographic data we gathered include; age, educational status, occupation, residence, etc. the clinical data were signs and/or symptoms of malaria. For more information, can you please see the questionnaire we have attached as supportive file? All the data we gathered are available there.

Line 143: Blood sample collection….Of the blood slides (thin/thick), which one was used for the detection and counting of the parasite density, and which one was used for species identification?

Did you count the parasite density? If yes, how was it done?

Response: Thick blood films were used for detection and counting of parasite density; whereas, thin blood films were used for species identification. Malaria parasite count was performed on Giemsa-stained thick blood film against 200 WBCs. Malaria parasite density was calculated based on the assumption of 8,000 WBC per micro-liter (µl) of blood.

Parasites/ µl = Parasite counted against 200 WBCs×8000 WBCs/ µl

200

Results

Line 198: The prevalence of malaria………..the percentages should be out of the brackets, and the whole numbers in brackets and given as proportions with the denominator being the total number of malaria infections e.g. 44.6% (29/65), etc.

Response: well taken and corrected

Line 198-201: This information should be given in a form of a Table, and preferably be added to Table 2. Table 2 needs to be restructured by removing the parasite stages prevalence i.e. Gametocytes and schizonts as they add no value to the data presented. Likewise, remove the number of malaria negative results and instead provide the proportions of malaria positives and their percentages e.g. P. falciparum 12.2% (38/312), P. vivax 4.8% (15/312), and mixed infection…..

Response: well taken; dear reviewer, we have corrected as you suggested. Please see pages 11 line 186-187

Line 204-205: Provide the odds ratio, confidence interval, and p-value.

Response: Dear reviewer, do you mean from the table? If so, there is COR, AOR and p-value.

Discussion

Line 257-60: Can you explain why primigravidae had higher odds of having malaria infection than other multigravidas?

Response: dear reviewer, this may be related to the development of pre-immunity and antibodies in multigravidae. So, the development of pre-immunity to malaria with increased gravidity and previous exposures may help multigravidae women to be in reduced risk of malaria. But, primigravidae lack this pre-immunity. It might be also linked to infection-specific immunological factors. Plasmodium-infected erythrocytes sequester in the maternal placenta by producing surface antigens mainly variant surface antigens (VSA) that bind to chondroitin Sulphate-A (CSA) receptors in the placenta. These antibodies are associated with protection against placental infection. Therefore, primigravidae mothers lack these anti-adhesion antibodies against CSA binding parasites, which develop only after successive pregnancies and this makes them more susceptible to infection (Fried et al., 1998).

Conclusion

Should be summarized and made more clear

Response: well taken: we have summarized

Thank you

3. Point by point response to reviewer#2 comments

Authors’ Response to the reviewer`s comments

First, we authors would like to thank reviewer#2` for comments to make this manuscript more plausible. As much as possible, we authors would like to address your comments here.

Reviewer #2: Dr Almaw and colleagues have examined symptomatic pregnant women for malaria and describe the associated characteristics of the women found to have malaria. The study is quite interesting, and the results clearly identify certain groups of symptomatic pregnant women as being at particular risk of malaria. There are some missing pieces of information that will strengthen the paper.

Major comments

1. The authors need to provide a clear definition of “symptomatic”. How did they define women of interest? Also, how did they identify “pregnant”? Were pregnancy tests done in women of childbearing age, for example? This is important for interpreting relative proportions of infection by trimester. (It would also be helpful to define cutoffs used for different trimesters)

Response: well taken. We defined “symptomatic” as Women who exhibited at least two of the following signs and /or symptoms of malaria;

Fever (temperature ≥37.5c0 as recorded by electronic thermometer), joint pain, headache, vomiting, malaise, chills…

Pregnant: women of confirmed urine HCG positive

2. The manuscript contains unnecessary detail in places. For example, lines 52-60 provide a “textbook” description of the parasite life cycle, which can all be removed, and similarly lines 64-73 contain much generic information on malaria diagnosis and management in general and in pregnancy. The detailed description of the study sites can be condensed substantially (lines 97-119) but please include the size of populations in the catchments here.

Response: well taken. Dear reviewer, we have edited study area and included number of catchment populations getting service from each health center. Please see page 6. But, we haven’t omitted life cycle, diagnosis and management section. Because, the second reviewer suggested that, this section is in adequate and should even be added. We also believe that, this is relevant in back ground section. We hope that, you will suggest as again and we authors will accept and re-write.

Sample size, Line 123. These determinations are often hard to follow. It seems the researchers assumed a parasite prevalence of 50% which was high, and was not observed, and the sample size was not obtained. How does this affect study interpretation?

Response: dear reviewer, we were unable to address the sample size obtained by taking 50% because of logistic reasons (like time and other resources). But, we have reduced it according to statistical reasons.

4. Line 198: these data are relative proportions by trimester, not prevalence.

Response: well taken. Dear author, we have corrected as you suggested.

5. Line 216-7: This statement is manifestly incorrect and should be removed. Even if it is the first study of a very specific question such assertions are best avoided.

Response: well taken and corrected

6. In the discussion it is generally unclear whether studies from elsewhere used similar recruitment criteria or not. This is critically important for comparing the studies. For example reference 7 appears to be a simple cross sectional study, while reference 21 (Tagbor) compared symptoms in parasitemic and aparasitemic women. References by Tahita (22) and Bardaji seem most directly comparable in design.

Response: well taken. Dear reviewer, it is true that some studies we used do not have similar study design as well as recruitment criteria and even the objectives are different. We simply took the overall and species specific prevalence and compared. Because; as our understanding, we couldn’t get studies which are closely related to our study by study design, recruitment criteria, objective… So, to discuss the prevalence, we have used those studies for comparison.

7. Line 219-220: differences in season relative to transmission of malaria is a possible explanation (and worth mentioning) but it would make sense to start with differences in overall transmission between countries. Isn’t disease burden much higher in the mentioned countries?

Response: Alright! Overall, Burkina Faso and Mali are among the ten countries with the highest number of malaria cases and deaths. Burkina Faso takes up (3% of the global cases and 4% of global deaths) and Mali covers (3% of the global cases and deaths, and 6% of cases in West Africa) (WHO 2020). Compared to other endemic countries in Sub-Saharan Africa, malaria prevalence in Ethiopia is relatively low.

Line 224-5: what was the design of the other studies in Ethiopia, if cross sectional or observational rather than focused on symptomatic women this needs to be taken into account.

Response: Well taken. It was similar study design (cross-sectional).

9. Line 238-242: this paragraph is confusing in its intentions, as to whether the findings are similar to the two previous studies, or different than them. Keeping the comparison simple and not trying to explain “differences” is easier to follow, especially given the size of one of the earlier studies.

Response: well taken. Dear reviewer, We have removed this part as we found it not important

Minor comments

1. There are many minor English language errors throughout the manuscript. An edit by an expert English speaker or professional editor is needed. I have confined my suggestions to the scientific content.

Response: well taken and we have tried to edit

2. Abstract and conclusion: the term “considerably high” is poor, please change it.

Response: well taken; dear reviewer, we have changed it. Please see in page 3

3. Check use of abbreviations and only use them if repeated 3 or more times. IRS, LSM, and probably LLINs are examples of unnecessary abbreviations.

Response: well taken and accepted

4. Line 81: the figure of 11 million women exposed to malaria refers to infected pregnant women in Africa, please revise.

Response: well taken and we have corrected

5. Line 133 how is “irrigation” defined?

Response: well taken; irrigation in our study terms is defined as “agricultural activity whereby farmers use controlled amount of water through canals for watering and cultivation of rice, onion and sugar cane.”

6. Line 158-160: how was the QC implemented? What happened in case of disagreements?

Response: well taken. Dear reviewer, the main goal of examining the slides by malaria microscopists in APHI was to confirm results and give final decision. So that, the disagreements were all confirmed and decided by those malaria microscopists.

7. Table 1: the lines for “total” are really superfluous and could be removed.

Response: well taken and removed

8. Table 3 “Spent outside at dusk” needs better description and definition.

Response: well taken. Dear reviewer, there are people who do not be indoor at dusk/evening/ especially in rural areas in Ethiopia. They spend most of the time in evening performing different activities outside. They may even sit together and play with family members and neighbors. Because, this is the only time they can have a rest after working whole days in farm. They may be bitten by mosquitoes during the time. This is what we want to mean by “spend outside at dusk”

9. Line 288-290: are these symptoms authors used to define malaria?

Response: well taken. Of course! But, it seems that this should not be put here and we removed.

10. The following references need additional detail or correction: 1, 4 (journal name spelled out); 2 and 12: 2 different editions of World Malaria Report- both needed?; 9: second author surname missing; 18, 25: no source given- journal or other publication??; 21: incomplete citation- published

Response: well taken. References 1&4 are corrected.

*We have used two different editions of WHO. Because, we have used different data from both editions

* Reference 9; well taken and corrected as Hyiid L,

Reference 18: well taken and we have removed as it is un-necessary

Reference 21 from previous manuscript (reference19 from revised manuscript after reviewers’ comments); dear reviewer, we are un clear with mistakes from this reference. Can you please suggest us? Reference 21(reference19 from revised manuscript); ??? Please see page20, line 333-335

Reference 25 (ref 23 from revised manuscript, page 21): has been corrected

Thank you

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

1 Feb 2022

8 Mar 2022

Point by point response to reviewer#2 comments

First of all, we authors would like to thank reviewer#2` again for comments to make this manuscript more plausible. As much as possible, we authors would like to address your comments here.

Minor comments

1. Line 48: the paper shows that malaria is A significant problem, not THE significant problem.

Response: well taken; dear reviewer, we have corrected as you suggested. “Malaria is the disease…”

2. I still contend that lines 63-71 are text book descriptions of malaria which do not belong in this article. Reviewer 1 suggests also that some of this text is redundant.

Response: well taken; dear reviewer, this section is about the transmission and life cycle in human host. Actually, it is in lines 49-55 from revised manuscript. We understood that the idea was… “Malaria is transmitted to humans by the bite of female Anopheles mosquitoes. Anopheles mosquitoes feed on blood for survival and production of eggs. During feeding, infected female Anopheles mosquito inoculates the infective sporozoites from its salivary gland into human skin. After inoculation, parasites circulate with blood and those reaching to the liver undergo one cycle development. Then, parasites infect and multiply inside red blood cells to bring the characteristic signs and symptoms. The clinical manifestations include fever, joint pain, chills, headache and vomiting which usually appear between 10 and 15 days after the Anopheles mosquito bite. In addition, malaria may also be transmitted through blood transfusion and congenitally”. Even we haven’t got redundancy here. We hope we have got your idea. Thank you!

3. Line 136 Sample size: I still do not find this clear, and the authors’ written response is also unclear (response to reviewers, page 11).

Response: well taken; dear reviewer,

The sample size was determined using single population proportion formula based on 50% prevalence, 95% confidence level and 5% margin of error with 10% non-response rate as follows.

n = (Zα/2)2 P (1-P)

d2

n= (1.96)2 (0.5) (1–0.5)2 = 384 plus 10% non-response rate =422

(0.05)2

Where;

n = number of sample size.

P= the proportion (p = 0.5) taken as 50% prevalence

d= marginal error between the sample and population (0.05).

Z= critical value at 95% certainty (1.96), considering 10 % non-responsive rate.

But, according to information obtained from health centers, the total number of symptomatic pregnant women diagnosed in the three study areas in 2019/2020 was 1202 which is finite population or less than 10.000. So, according to statistics, adjustment (correction) formula is used to calculate sample sizes for population of less than 10,000. This is because a given sample size provides proportionately more information for small population than for a large population and we have reduced the sample size based on the following formula.

nf = n/(1+(n/N)) where, nf= final sample size i.e. sample size from finite population

n= sample size from an infinite population i.e. 422

N= total number of target population from all three health centers who visited in previous year i.e. 1202; So, nf= 422/1+ (422/1202)) = 312 (total number of symptomatic pregnant women participated in the study).

4. Line 185-6: The wording is not clear here. If the expert microscopist’s review was taken as final, please say so. Or describe who confirmed discordant results and how.

Response: Well taken. Dear reviewer, the review (re-examination) by microscopists in APHI was taken as final. Because, it is the APHI that assesses malaria quality assurance and gives final decision about species type in every health institutions around.

Thank you!

Submitted filename: Response to reviewers.docx

Click here for additional data file.

18 Mar 2022

21 Mar 2022

Point by point response to reviewers, comments

Authors’ Response to the reviewers, comments

First, we authors would like to thank reviewers` for their comments to make this manuscript more plausible. As much as possible, we authors would like to address your comments here.

Reviewer #1: Abstract

Background

Line 18......There is no need of having this sentence since the previous sentence in line 15-17 has already explained the problem that prompted this study to be conducted.

Response: Well taken. Dear reviewer, we have removed it

Results

There should be no space between the numbers and the percentage symbol, but rather there should be a space between the percentage symbol and the brackets. The correction should be done throughout the document.

Response: Well taken. We have corrected

Conclusion

Lines 38-40.should state that...."malaria is still a public health problem among pregnant women" ....instead of ..."in the study area

Response: Well taken and we have corrected as you suggested

Line 42....change "are" to "were", "increase" to "increased/increasing" the prevalence........

Response: Well taken and changed

Main body

Line 46.....the sentence is left hanging, here it would be good to mention the five Plasmodium species infecting human.

Response: Well taken. We have included five species.

52.....Does the Anopheles inoculate the infective sporozoites into the skin?

Response: Well taken. Dear reviewer, it is to mean human circulation and we have corrected it.

Line 69-70...This sentence is not supposed to to be in that paragraph as it brings confusion. Whereas the previous sentence is on control measures, this sentence is on the risk factors for malaria infection in pregnant women.......thus there is no continuation.

Response: Well taken. We have removed it.

Line 133.....What is HCG? Spell it out.

Response: Well taken. Dear reviewer, it is to mean Human Chorionic Gonadotropin hormone and we have included it.

Line 176.....Remove the word "have".

Response: Well taken and we have removed it.

Table 2.....Remove the frequency column as the figures in this column have already presented in the next column.

Response: Well taken and we have removed.

Line 189...change "have" to "had". Do the same for line 191.

Response: Well taken. We have changed.

Line 193....change "are" to "were"

Response: Well taken. We have changed.

Line 200....."this study results"...should be changed to..."the prevalence of malaria in this study".

Response: Well taken and changed.

Line 201...."than study results"....to...."than the prevalence in Burkina Faso".

Response: Well taken and changed.

Line 212...."than the study results".....to..."than the prevalence in".

Response: Well taken and changed.

Line 233....."shown"...to..."showed"

Response: Well taken and changed

Reviewer #2: The authors have responded to my comments. I don't think textbook description of the malaria life cycle is a good use of space but will let this pass.

Response: Dear reviewer, we have received. Thank you!

Submitted filename: Response to reviewers.docx

Click here for additional data file.

22 Mar 2022

Prevalence of malaria and associated factors among symptomatic pregnant women attending antenatal care at three health centers in North-west Ethiopia

PONE-D-21-30318R3

Dear Dr. Almaw ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

29 Mar 2022

PONE-D-21-30318R3

Prevalence of malaria and associated factors among symptomatic pregnant women attending antenatal care at three health centers in north-west Ethiopia

Dear Dr. Almaw:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Luzia Helena Carvalho

Academic Editor

PLOS ONE