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Malarial anemia among pregnant women in the south-western coastal city of Mangaluru in India
Informatics in Medicine Unlocked 15 (2019) 100159
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Malarial anemia among pregnant women in the south-western coastal city of Mangaluru in India
T
Valleesha N. Chandrashekara,b, Kishore Punnatha,b, Kiran K. Dayananda,b, Rajeshwara N. Achura,∗, Srinivas B. Kakkilayac, Poornima Jayadevd, Suchetha N. Kumarib, D. Channe Gowdae a
Department of Biochemistry, Kuvempu University, Shankaraghatta, Shivamogga District, Karnataka, India Department of Biochemistry, K. S. Hegde Medical Academy, NITTE (Deemed to be University), Mangaluru, India c Light House Polyclinic, Light House Hill Road, Mangaluru, Karnataka, India d Sampoorna Clinic, Light House Hill Road, Mangaluru, Karnataka, India e Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pregnancy associated malaria Mangaluru P. vivax Anemia Placental malaria
Background: The worldwide burden of pregnancy associated malaria is largely underestimated. Objective: In this study, we assessed the incidence and severity of malarial anemia, and associated risk factors among pregnant women in Mangaluru, a city in India. Methods: A hospital-based cross-sectional study involving 105 pregnant women, including controls, was conducted at Government Lady Goschen Hospital, Mangaluru. The socio-demographic and hematological data were analysed to determine the prevalence of malarial anemia among pregnant women. Results: Of the 105 pregnant women, 71 were infected with malaria of which (48, 67.6%) had P. vivax, (13, 18.3%) P. falciparum and (10, 14.1%) mixed infections. Among those infected, 87.3% were anemic, in which 11.3% had severe anemia. Notably, malarial infection was higher among primigravidae (40.6%) women and they delivered low birth weight babies. Lack of knowledge about preventive measures and lack of awareness concerning antenatal care services, especially among rural women, were major determinants of pregnancy associated malaria. Conclusions: We conclude that there is an urgent need to strengthen the malaria surveillance measures and antenatal care services, and create awareness about pregnancy associated malaria. Further, increasing the number of rural health centers, establishing community outreach programs, distribution of bed nets, and early diagnosis and treatment, could reduce the pregnancy associated malaria burden in Mangaluru.
1. Introduction Malaria remains as a highly prevalent infectious disease, affecting billions of people worldwide, especially in the tropical regions. Among five human malarial infecting species, Plasmodium falciparum and P. vivax are the most prevalent species. In 2016, about 216 million cases and 445,000 deaths were reported from 91 countries across the world, in which India accounted for over 1 million cases and 331 malarial deaths [1]. In India, malaria is highly endemic and persists mostly in the Northern, Northeastern and Southwestern regions. In the Southwestern region of India, Karnataka state is a significant contributor of
malarial infections. Overall, among 30 districts in Karnataka state, the majority of malarial burden is reported from only two districts viz., Dakshina Kannada and Udupi [2]. Mangaluru, the administrative capital of Dakshina Kannada district, is situated on the Arabian seashore of Southern India. The city and its surrounding areas have a warm and humid climate, harboring high vector density leading to high rates of malarial transmission [3]. In 2016, Karnataka state reported a total of 15,816 malarial infections, of which Mangaluru alone accounted for 11,037 cases and the majority (82.4%) were due to P. vivax infections [4]. In malarial endemic regions, pregnant women and children less than
∗
Corresponding author. E-mail addresses: [email protected] (V.N. Chandrashekar), [email protected] (K. Punnath), [email protected] (K.K. Dayanand), [email protected], [email protected] (R.N. Achur), [email protected] (S.B. Kakkilaya), [email protected] (P. Jayadev), [email protected] (S.N. Kumari), [email protected] (D.C. Gowda). https://doi.org/10.1016/j.imu.2019.02.003 Received 26 December 2018; Received in revised form 13 February 2019; Accepted 14 February 2019 Available online 18 February 2019 2352-9148/ © 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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five years of age are at an increased risk of malaria associated severe complications [5]. It is well accepted that P. falciparum infections in pregnant women are known to result in maternal and fetal morbidity and mortality. However, there are emerging worldwide reports of P. vivax infections also resulting in maternal anemia and low birth weights [6–9]. The severity of malarial manifestations in pregnant women and the fetus depends on the acquired immunity level. It is well known that primigravidae (women in their first pregnancy) are more susceptible to malarial infections than second gravidae or multigravidae. The higher susceptibility of primigravidae women is due to lack of placental parasite-specific immunity in comparison to second gravidae or multigravidae women [10,11]. PAM is characterized by sequestration of Plasmodium falciparum-infected erythrocytes and infiltration of immune cells within intervillous spaces of placenta. Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP1) encoded by VAR2CSA, binds to CSA in placenta, thus avoiding the maternal immune response [12–14]. PAM results in placental basement membrane thickening, perivillous fibrinoid deposits, and syncytial knotting leading to impaired nutrient and gaseous exchange system between mother and fetus, causing intrauterine growth retardation (IUGR) and low birth weight (LBW) [15]. PAM can result in serious complications in the mother such as anemia, pulmonary edema, hypoglycemia, cerebral malaria and puerperal sepsis, which could lead to death. The fetal complications due to PAM could lead to abortion, still birth, IUGR, premature delivery, and LBW [15]. Vaccines for protection from PAM and its adverse birth outcomes, by using antibodies against VAR2CSA, which is known to be associated with PAM, are now in clinical trials (NCT02647489 and NCT02658253, https://www.clinicaltrials.gov). The burden of PAM in India is largely underestimated, as the National Vector Borne Disease Control Programme (NVBDCP) does not collect data on pregnant women with malarial infections and its associated abortions and still-births [16]. Few studies on PAM from endemic regions in India exist and have been confined only to regions such as Madhya Pradesh, Chandigarh, Surat, Gujarat and Jabalpur, whereas no systematic studies have been carried out in the southern part of India [17–20]. During PAM, anemia is very common, which is regarded as detrimental to fetal growth and poor pregnancy outcomes such as LBW and preterm delivery [21–23]. Keeping in view of the situation, this hospital-based study was undertaken to determine the clinical features, prevalence of malarial anemia, and its intensity, among pregnant women in the endemic settings of Mangaluru City.
2.2. Malarial diagnosis The malarial diagnosis was performed by examination of peripheral blood smears by conventional microscopy and rapid diagnostic test (RDT) kits (SD Bioline, India). Two blood slides (thick and thin) were prepared by finger prick, stained with 4% Giemsa stain (Sigma Aldrich, USA) and were observed under the microscope for the presence of Plasmodium parasite and the species type. The parasite densities were determined by counting the number of parasites and expressed as parasites/μL of blood (number of parasites counted/number of WBCs counted x total number of WBCs per μL of blood) or (number of parasites counted/number of RBCs counted x total number of RBCs per μL of blood). Percentage parasitemia was determined as (number of parasites per μL of blood/number of RBCs per μL of blood) x 100. The pregnant women were also screened by using a malaria Ag Pf/Pv RDT kit which detects histidine-rich protein II antigen of P. falciparum and lactate dehydrogenase of P. vivax in human blood. 2.3. Sample collection and hematological analysis About 1 ml of venous blood was collected into clot activator tubes for serum and sterile heparin coated vacutainers for plasma, centrifuged, and the supernatant stored at −70 °C until further use. The infected patients were treated by the attending physicians as per the NVBDCP recommendations. Within an hour of sample collection, the blood was used to determine the levels of hemoglobin (Hb), Red blood cells (RBCs), Hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC), by using an automated hematology analyzer (Mind Ray-Biomedical, Shenzhen, China). 2.4. Statistics The data was entered in Microsoft excel and the statistical analysis was performed using GraphPad Prism (GraphPad, Inc., San Diego California, USA). Quantitative variables were presented as number (percentages) and Mean ± Standard deviation. The comparison of non-parametric data between various groups was performed using the Kruskal-Wallis Test, and significance between any of the two groups was determined by the Mann-Whitney U Test with a 95% confidence interval with multiple testing. Spearman rank correlation was used to determine correlations between two continuous variables. A P-value less than 0.05 was considered to be significant.
2. Methods 3. Results 2.1. Study design 3.1. Characteristics of study participants During the period 2014 to 2017, a prospective hospital-based study was carried out at Government Lady Goschen Hospital in Mangaluru City, Karnataka, India. A total of 105 pregnant women aged between 18 and 40 years were randomly recruited into the study upon obtaining informed consent from the study participants or their relatives. All of the infected patients were febrile and received diagnosis and treatment at the hospital. The healthy pregnant women attending antenatal care (ANC) of Lady Goschen Hospital for general check-ups were recruited as healthy controls (HC). The study protocol was approved by the ethical committee of Kuvempu University, Shivamogga, Karnataka, the central ethics committee of NITTE University, Mangaluru, Karnataka, and the Institutional Review Board of Pennsylvania State University College of Medicine, Hershey, PA, USA. Pregnant woman aged > 18 years were included in the study, whereas the study participants with the use of any prophylaxis prior to diagnosis, and individuals testing positive for dengue, typhoid, HIV, hepatitis B and C infections, were excluded.
A total of 105 pregnant women were recruited into the study. Among the study participants, 71 were malaria infected, whereas 34 were HC. Among infected women, P. vivax (48, 67.6%) was the most prevalent infecting species. The mean age of the study participants was 25 years, with range between 17 and 40 years. Our observation correlated with the fact that women in their first pregnancy are susceptible to PAM, as most of the infected pregnant women were primigravidae (32, 45.1%), followed by second gravidae (25, 35.2%) and multigravidae (14, 19.7%). Among the 71 infected patients, (22, 31.0%) were in their first trimester, (32, 45.1%) were in second trimester and (17, 23.9%) were in third trimester. The mean birth weight of babies delivered by infected women (2.48 kg ± 0.26) was found to be significantly lower in comparison to birth weight of babies delivered by HC (2.64 kg ± 0.11, P = 0.0044). Among 71 infected women, 26 (36.6%) delivered babies with LBW (Table 1). In this study area, the majority of infected pregnant women were immigrants (55, 77.5%). Most of the infected pregnant women were engaged in household work (58, 82%) and a few (4, 5.6%) were also 2
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Table 1 Background characteristics of the study participants. Parameter
HC
P. vivax
P. falciparum
Mixed
Overall (Infected)
Number of study participants Mild malaria (MM) Severe malaria (MM) Mean age in years (range) 15-20 21-25 26-30 31-35 36-40 Gravidity Primi gravidae Second gravidae Multi gravidae Trimester First trimester Second trimester Third trimester Delivery outcome Mean birth weight Normal LBW
34 0 0
48 (67.6) 35 (72.9) 13 (56.5)
13 (18.3) 7 (14.6) 6 (26.1)
10 (14.1) 6 (12.5) 4 (17.4)
71 (100) 48 (67.6) 23 (32.4)
5 11 11 3 4
11 (78.6) 18 (66.7) 16 (76.2) 3 (37.5) 0 (0)
1 4 4 3 1
2 5 1 2 0
(14.3) (18.5) (4.8) (25) (0)
14 (19.7) 27 (38.0) 21 (29.6) 8 (11.3) 1 (1.4)
15 12 7
23 (71.9) 17 (68) 8 (57.1)
4 (12.5) 5 (20) 4 (28.6)
5 (15.6) 3 (12) 2 (14.3)
32 (45.1) 25 (35.2) 14 (19.7)
11 13 10
18 (81.8) 17 (53.1) 13 (76.5)
2 (9.1) 9 (28.1) 2 (11.8)
2 (9.1) 6 (18.8) 2 (11.8)
22 (31) 32 (45.1) 17 (23.9)
2.64 ± 0.11 34 0
2.5 ± 0.31 30 (66.7) 18 (69.2)
2.5 ± 0.20 9 (20) 4 (15.4)
2.4 ± 0.32 6 (13.3) 4 (15.4)
2.48 ± 0.26 45 (63.4) 26 (36.6)
(7.1) (14.8) (19) (37.5) (100)
Data expressed as number of study participants (percentages with respect to infected groups).
attendants. It was found that 57 (80.3%) infected pregnant women belonged to the below poverty level (BPL) group and were economically poor. We also observed that 50 (70.4%) infected women were not aware of preventive measures to be taken during pregnancy, and only 13 (18.3%) claimed the use of mosquito nets while sleeping (Table 2).
Table 2 Socio-demographic data across various infecting species. HC
P. vivax
P. falciparum
Residence, n (%) Native 25 11 (68.8) 3 (18.8) Immigrants 9 37 (67.3) 10 (18.2) Locality Urban 10 5 (45.5) 5 (45.5) Rural 24 43 (71.7) 8 (13.3) Education, n (%) No formal education 10 31 (81.6) 5 (13.2) Primary 9 9 (56.3) 3 (18.8) Secondary 9 2 (22.2) 3 (33.3) College level 6 6 (75) 2 (25) ANC visits Yes 24 4 (44.4) 4 (44.4) No 10 44 (71) 9 (14.5) Socioeconomic characteristics Poor 8 41 (71.9) 8 (14.0) Middle class 26 7 (50) 5 (35.7) Rich 0 0 (0) 0 (0) Knowledge of preventive measures to be taken Mosquito nets 15 6 (46.2) 3 (23.1) Removal of stagnant 12 2 (28.6) 2 (28.6) water window netting 28 4 (50) 2 (25.0) Use of mosquito 29 9 (50) 8 (44.4) repellents Chemoprophylaxis in 10 3 (50) 2 (33.3) pregnancy Does not know 5 39 (78) 5 (10)
Mixed
Overall (Infected)
2 (12.5) 8 (14.5)
16 (22.5) 55 (77.5)
1 (9.1) 9 (15)
11 (15.5) 60 (84.5)
2 4 4 0
(5.3) (25) (44.4) (0)
38 (53.5) 16 (22.5) 9 (12.7) 8 (11.3)
1 (11.1) 9 (14.5)
9 (12.7) 62 (87.3)
8 (14) 2 (14.3) 0 (0)
57 (80.3) 14 (19.7) 0 (0)
4 (30.8) 3 (42.9)
13 (18.3) 7 (9.9)
3.3. Effect of malarial infections on hematological parameters
2 (25) 1 (5.6)
8 (11.3) 18 (25.4)
Significant hematological changes in pregnant women during malarial infections are commonly observed. In this study, a significant
1 (16.7)
6 (8.5)
6 (12)
50 (70.4)
3.2. Clinical symptoms All of the infected patients (71,100%) were febrile. Apart from fever, 22 (31%) of patients had chills, 25 (35.2%) showed shivering, 32 (45.1%) had headache, 23 (32.4%) experienced weakness, 36 (50.7%) patients complained of myalgia, 20 (28.2%) had dizziness, 27 (38.0%) felt nausea, 18 (25.4%) had vomiting, 20 (28.2%) patients had inappetence, 11 (15.5%) patients had diarrhea, 18 (25.4%) patients were having cough and 10 (14.1%) patients experienced difficulty in breathing. Apart from various clinical symptoms, a majority of P. vivax patients experienced myalgia and nausea, whereas P. falciparum patients experienced chills, headache, weakness, dizziness, and inappetence (Table 3).
Table 3 Clinical symptoms among the malarial subjects.
Data expressed as number of study participants (percentages with respect to infected groups).
involved in agricultural activities. Interestingly, the bulk of the infected patients were from rural (60, 84.5%) areas. Most of the study participants (38, 53.5%) did not have any formal education, only 16 (22.5%) had primary level education, 9 (12.7%) had secondary level, and 8 (11.3%) had college level education. It was observed that the majority of infected pregnant women (62, 87.3%) did not visit hospitals for ANC. Only 7 (9.9%) pregnant women visited ANC during the first half of pregnancy, while 2 (2.8%) visited in the latter stages (i.e., gestational age of > 20 weeks). Among the 62 pregnant women not seeking care at ANC, 27 pregnant women (43.5%) sought help from traditional birth
Symptoms
P. vivax
P. falciparum
Mixed
Overall
Fever Chills Headache Weakness Shivering Myalgia Dizziness Nausea Vomiting Inappetence Diarrhea Cough Difficulty in breathing
48 (67.6) 12 (54.5) 17 (53.1) 12 (52.2) 14 (56.0) 30 (83.3) 12 (60.0) 22 (81.5) 10 (55.6) 8 (40.0) 8 (72.7) 12 (66.7) 8 (80.0)
13 (18.3) 8 (36.4) 11 (34.4) 8 (34.8) 6 (24.0) 3 (8.3) 5 (25.0) 3 (11.1) 4 (22.2) 10 (50.0) 2 (18.2) 3 (16.7) 1 (10.0)
10 (14.1) 2 (9.1) 4 (12.5) 3 (13.0) 5 (20.0) 3 (8.3) 3 (15.0) 2 (7.4) 4 (22.2) 2 (10.0) 1 (9.1) 3 (16.7) 1 (10.0)
71 22 32 23 25 36 20 27 18 20 11 18 10
Data expressed as number of study participants (percentages). 3
(100) (31.0) (45.1) (32.4) (35.2) (50.7) (28.2) (38.0) (25.4) (28.2) (15.5) (25.4) (14.1)
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Table 4 Influence of malarial infection on Hemoglobin, RBC levels and RBC indices among pregnant women with P. vivax, P. falciparum and mixed infections. Variable
Parasitemia (%) Hb (g/dL) RBC x103/μL HCT (%) MCV (fL) MCH(pg) MCHC (g/dL)
HC
P. vivax
0.0 ± 0.0 12.3 ± 0.5 4.7 ± 0.5 39.9 ± 4.0 84.6 ± 4.9 25.1 ± 2.1 30.1 ± 1.7
P. falciparum
Mixed
p value *(between anemic groups)
NA
A
NA
A
NA
A
Pv Vs Pf
Pv Vs Mixed
Pf Vs Mixed
0.1 ± 0.0 12.0 ± 0.6 3.7 ± 0.7 36.0 ± 8.6 86.3 ± 16.7 26.0 ± 4.5 30.1 ± 0.4
0.3 ± 0.2 7.8 ± 1.6 3.4 ± 0.7 28.9 ± 8.5 79.7 ± 9.5 23.5 ± 3.5 29.7 ± 1.2
0.2 ± 0.1 12.0 ± 0.7 3.1 ± 0.3 31.3 ± 1.8 81.3 ± 7.9 25.2 ± 3.1 30.2 ± 0.8
0.8 ± 0.4 6.8 ± 2.2 2.3 ± 0.3 27.1 ± 2.2 83.6 ± 9.2 24.8 ± 3.5 29.3 ± 1.1
0.2 ± 0.0 11.9 ± 0.8 3.4 ± 0.2 28.6 ± 4.1 77.1 ± 7.0 20.3 ± 1.5 28.9 ± 0.7
0.5 ± 0.3 6.7 ± 1.3 2.8 ± 0.5 25.0 ± 4.2 78.5 ± 7.8 21.1 ± 2.3 29.8 ± 1.5
< 0.0001 0.1263 < 0.0001 0.9358 0.3021 0.287 0.3279
0.009 0.044 0.021 0.22 0.748 0.062 0.911
0.126 0.946 0.019 0.303 0.184 0.02 0.371
Data expressed as Mean ± Standard deviation; Analysis between any two groups was compared by Mann-Whitney U Test; * p-value < 0.05 considered to be significant.
P. vivax (2, 40%), P. falciparum (1, 20%), and mixed (2, 40%) infections. Intrauterine growth retardation (IUGR) was observed in 21 (29.5%) infected women (P. vivax – 14, 66.6%; P. falciparum - 3, 14.4% and mixed - 4, 19%). There was no significant influence between the proportion of patients with IUGR and gravidity.
increase in parasitic burden was observed in anemic (A) group patients compared to the non-anemic (NA) group. Upon comparison between anemic groups of different infecting species, patients with P. falciparum infections had significantly increased parasitic levels (P < 0.0001) as compared to P. vivax and mixed infections. The levels of hemoglobin, in comparison with NA groups, were significantly decreased in the A group, irrespective of the infecting species. In comparison between A groups among various infecting species, the hemoglobin levels were significantly decreased during P. falciparum infections. It was observed that a significant negative correlation existed between increased parasitemia and Hb levels during P. vivax (r = −0.6844, P < 0.0001), P. falciparum (r = −0.8664, P = 0.0002) and mixed (r = −0.7052, P = 0.0251) infections. The levels of HCT, MCV, MCH and MCHC were also found to be significantly decreased in anemic patients in comparison with non anemic patients across various infecting species (Table 4). A significant positive correlation was observed between decreased Hb and RBC levels during P. falciparum (r = 0.8143, P = 0.0011) infections.
4. Discussion Several studies worldwide have shown that the majority malarial infections among pregnant women are due to P. falciparum, and the remaining are contributed by P. vivax infections [24]. However, the majority of studies have not focussed on the burden due to P. vivax malaria during pregnancy. In India, in spite of a high malarial burden in most part of the country, no systematic studies regarding the extent of malarial infections and clinical manifestations among pregnant women have been carried out. For the first time, the impact of malaria during pregnancy involving 71 infected pregnant women in Mangaluru City was carried out in this hospital-based study. In Mangaluru, although both P. falciparum and P. vivax exists, P. vivax is the major infecting species. The data obtained here indicates that P. vivax (67.6%) is the predominant infecting species, as compared to P. falciparum (18.3%) and mixed (14.1%) infections. The malarial infections present with a variety of clinical symptoms, which are similar to various other viral illnesses. In general, malarial infections in pregnant women in various regions across Asia have mostly been symptomatic. However, studies carried out in the Northeastern states of Orissa and West Bengal in India report the prevalence of asymptomatic infections among pregnant women [25]. In this study, although all of the infected patients were febrile and symptomatic, the fever was intermittent in nature, and its intensity ranged from a mild to severe level. Apart from fever, various other characteristic symptoms observed were myalgia and nausea during P. vivax, whereas P. falciparum patients presented with chills, headache, weakness, dizziness and inappetence. In this endemic setting, we found that the pregnant women residing in rural areas were highly susceptible to malarial infections as compared with those in urban localities, which is in agreement with earlier reports focusing on other regions of the country [24]. The infected women were mostly engaged in household activities and agriculture. Over 80% of pregnant women reported not to have regularly visited ANC during their pregnancy, and only 9.9% patients visited during their first trimester. Only 8% of pregnant women were aware that the ANC services are free of charge. Most of the women not seeking care at ANC did not report the use of any specific prophylaxis, multivitamins or iron supplements during pregnancy, and only 3% took self-medications such as paracetamol and multivitamins. Within the ANC users group, about 45% of women sought help from traditional birth attendants; however, there were incidences of non-compliance to medications prescribed by these local birth attendants as it sometimes resulted in vomiting, irritation, and tiredness. The major reason given for not
3.4. Malarial anemia intensity in pregnant women Anemia was defined as Hb levels of less than 11 g/dL. Based on the varying Hb levels, the study participants were grouped into (i) Nonanemic (NA) – study participants with hemoglobin ≥11g/dL. (ii) Mild anemia (MA) – hemoglobin range 8–10.9 g/dL. (iii) Moderate anemia (ModA) – hemoglobin range 5–7.9 g/dL and (iv) Severe anemia (SA) – hemoglobin < 5 g/dL. In this study, samples from 71 infected pregnant women were analzsed for the level of anemia. In the case of healthy controls, the majority were found be non-anemic (32, 94%), whereas only 2 (5.9%) had a mild level of anemia. Among the infected, only 9 (12.7%) were non-anemic and 62 (87.3%) were anemic. Upon classification based on varying hemoglobin levels, 29 (40.8%) were found to have mild anemia, 25 (35.2%) had moderate anemia and 8 (11.3%) had severe anemia (Table 5). Low birth weights were associated mostly with primigravidae (30, 40.6%), followed by second gravidae (9, 25.7%) and multigravidae (4, 28.5%). Among the 32 primigravidae women, an increased proportion of P. vivax (10, 31.2%) patients had given birth to LBW babies, in comparison to P. falciparum (1, 3.1%) and mixed (2, 6.3%) infections. Pre-term delivery was observed in 5 cases (7%), that were due to Table 5 Classification of study participants across varying degree of anemia. Anemia type
HC
P. vivax
P. falciparum
Mixed
No anemia (NA) Mild anemia (MA) Moderate anemia (Mod A) Severe anemia (SA)
32 (94) 2 (6) 0 (0) 0 (0)
3 (6.3) 23 (47.9) 18 (37.5) 4 (8.3)
3 4 3 3
3.0 2.0 4.0 1.0
(23.1) (30.8) (23.1) (23.1)
(30) (20) (40) (10)
Data expressed as number of study participants (percentages). 4
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localities. A review of national malaria drug policy is also recommended to include intermittent preventive therapy and nutrient supplementation during pregnancy, similar to African settings.
visiting an ANC was lack of awareness that ANC services in government clinics are free of charge, or financial problems, as most of these pregnant women relied on medications given by traditional birth attendants. In this study group, most of the women did not have any formal education, and hence they were not aware of the problem of malaria, its severity, and the necessary preventive measures to be taken. Altogether, the data suggests that the reason for higher prevalence of malarial infection in pregnant women among rural areas could be due to high transmission, lack of awareness about protective and preventive measures to be taken, decreased access to ANC clinics, poor socioeconomic status, decreased availability of preventive measures such as mosquito repellents, bed nets, ITN's, IRS, and limited access to antimalarials. The intensity and the timing of malarial infection in pregnant women are known to play an important role in IUGR and preterm delivery. Studies in Malawi suggest that women infected during their antenatal period are at increased risk of IUGR when compared to others, and preterm delivery was also associated with umbilical cord parasitemia [26]. In our study, overall, 29.5% pregnant women had IUGR and the majority was P. vivax (66.6%) patients, whereas about 36.6% had given birth to LBW babies. In general, primigravidae are highly susceptible to malarial infection, which is due to lower acquired immune responses and the sequestration of P. falciparum strains in placenta microvasculature [12–14]. The Plasmodium parasites have distinct antigenic and adhesive properties and are known to sequester in the intervillous spaces of placenta mediated by chondroitin 4-sulfate receptor [10,11]. With subsequent pregnancies, women are exposed to repeated infections and may gradually develop a certain level of immunity to prevent and control severe PAM complications [27,28]. There are reports of increased parasitic burden among primigravidae in comparison to multigravidae, due to lack of anti-parasitic antibodies during first pregnancy [28,29]. However, we found no correlation between gravidity and peripheral parasitemia levels. Malarial infections are known to cause significant changes in hematological parameters, and anemia in general is commonly associated with pregnancy. However, malarial anemia during pregnancy may result in severe life-threatening conditions to both mother and the fetus. The data obtained here has also shown that the hemoglobin levels are significantly decreased in pregnant women with malarial infections. Among the different infecting species, women with P. falciparum infections were found to have significantly decreased levels of Hb, RBC, and increased parasitemia. However, among 8 patients with severe anemia, P. vivax contribution was highest (50%) followed by P. falciparum (37.5%) and mixed (12.5%) infections. Our data support the studies that have shown P. vivax infections result in severe malarial anemia and may also lead to LBW [30].
Ethical statement The study protocol was approved by the ethical committee of Kuvempu University, Shivamogga, Karnataka; the central ethics committee of NITTE University, Mangaluru, Karnataka, and the Institutional Review Board of Pennsylvania State University College of Medicine, Hershey, PA, USA. All the study participants were recruited into the study only after obtaining informed consent. Funding This work was supported by the Grant D43TW008268 (2012–2018) from the Fogarty International Center of the National Institutes of Health, USA, under the Global Infectious Diseases Program. Conflicts of interest The authors declare that they have no competing interest. Acknowledgements The authors thank the study participants for their consent to participate in the study. We thank Dr.Shakuntala, superintendent of Lady Goschen Hospital for her support, Dr. Kishan Prasad, Department of Pathology, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Deralakatte, Mangaluru, India for his guidance, and Dr. Aravind Pallipady, Department of Pathology, A.J. Hospital, Kuntikan, Mangalore, India for his kind support. This work was supported by the Grant D43 TW008268 from the Fogarty International Center of the National Institutes of Health, USA, under the Global Infectious Diseases Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.imu.2019.02.003. References [1] World Health Organisation (WHO). World malaria re-port 2017. Geneva: WHO; 2017 Available from: http://www.who.int/malaria/publications/world-malariareport-2017/en/. [2] Directorate of Health and Family Welfare Services, Bengaluru. Malaria elimination plan in Karnataka 2016-2025: framework, strategies and policies. 2016 Available from: https://www.karnataka.gov.in/hfw/kannada/Documents/Malaria %20Elimination%20Plan%20in%20Karnataka%20(2016%20to%202025).pdf. [3] Dayanand KK, Punnath K, Chandrashekar V, et al. Malaria prevalence in Mangaluru city area in the southwestern coastal region of India. Malar J 2017;16(1):492https://doi.org/10.1186/s12936-017-2141-0. [4] Kakkilaya BS. Malaria in Mangaluru, in malaria site.com. 2017 [cited 2018 January 22]. Available from:. https://www.malariasite.com/malaria-mangaluru/. [5] Chaponda EB, Chandramohan D, Michelo C, Mharakurwa S, Chipeta J, Chico RM. High burden of malaria infection in pregnant women in a rural district of Zambia: a cross-sectional study. Malar J 2015;14(1):380https://doi.org/10.1186/s12936-0150866-1. [6] Brutus L, Santalla J, Schneider D, Avila JC, Deloron P. Plasmodium vivax Malaria during pregnancy, Bolivia. Emerg Infect Dis 2013;19(10):1605–11https://doi.org/ 10.3201/eid1910.130308. [7] Bardají A, Martínez-Espinosa FE, Arévalo-Herrera M, et al. Burden and impact of Plasmodium vivax in pregnancy: a multi-centre prospective observational study. PLoS Negl Trop Dis 2017;11(6):e0005606https://doi.org/10.1371/journal.pntd. 0005606. [8] Pincelli A, Neves PA, Lourenco BH, Corder RM, Malta MB, Sampaio SJ, et al. The hidden burden of Plasmodium vivax malaria in pregnancy in the amazon: an observational study in northwestern Brazil. Am J Trop Med Hyg 2018;99(1):73–83https://doi.org/10.4269/ajtmh.18-0135. [9] Stanisic DI, Moore KA, Baiwog F, Ura A, Clapham C, King CL, et al. Risk factors for malaria and adverse birth outcomes in a prospective cohort of pregnant women
5. Conclusion Malaria among pregnant women has emerged as a major hidden public health problem in Mangaluru City and its surrounding areas. Despite various comprehensive malarial control and elimination programs by the District Health Office, the National Vector Borne Disease Control Program Office, and local community leaders, the control measures specifically aimed at malaria during pregnancy are not properly addressed. The present study has shown that the lack of education among women, unavailability, and poor coverage of government ANC services, lack of knowledge about malaria and associated anemia, as well as non-compliance with preventive measures, are the major reasons for malaria prevalence among pregnant women in Mangaluru City and its surroundings. Thus, there is an urgent need to address this situation by scaling up malarial surveillance, providing training to health care workers, and spreading awareness through educational programs and ITN's distribution on a large scale. Further detailed studies are needed to understand the burden of malaria among pregnant women and fatal outcomes, particularly in malarial hotspot 5
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Malarial anemia among pregnant women in the south-western coastal city of Mangaluru in India
T
Valleesha N. Chandrashekara,b, Kishore Punnatha,b, Kiran K. Dayananda,b, Rajeshwara N. Achura,∗, Srinivas B. Kakkilayac, Poornima Jayadevd, Suchetha N. Kumarib, D. Channe Gowdae a
Department of Biochemistry, Kuvempu University, Shankaraghatta, Shivamogga District, Karnataka, India Department of Biochemistry, K. S. Hegde Medical Academy, NITTE (Deemed to be University), Mangaluru, India c Light House Polyclinic, Light House Hill Road, Mangaluru, Karnataka, India d Sampoorna Clinic, Light House Hill Road, Mangaluru, Karnataka, India e Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pregnancy associated malaria Mangaluru P. vivax Anemia Placental malaria
Background: The worldwide burden of pregnancy associated malaria is largely underestimated. Objective: In this study, we assessed the incidence and severity of malarial anemia, and associated risk factors among pregnant women in Mangaluru, a city in India. Methods: A hospital-based cross-sectional study involving 105 pregnant women, including controls, was conducted at Government Lady Goschen Hospital, Mangaluru. The socio-demographic and hematological data were analysed to determine the prevalence of malarial anemia among pregnant women. Results: Of the 105 pregnant women, 71 were infected with malaria of which (48, 67.6%) had P. vivax, (13, 18.3%) P. falciparum and (10, 14.1%) mixed infections. Among those infected, 87.3% were anemic, in which 11.3% had severe anemia. Notably, malarial infection was higher among primigravidae (40.6%) women and they delivered low birth weight babies. Lack of knowledge about preventive measures and lack of awareness concerning antenatal care services, especially among rural women, were major determinants of pregnancy associated malaria. Conclusions: We conclude that there is an urgent need to strengthen the malaria surveillance measures and antenatal care services, and create awareness about pregnancy associated malaria. Further, increasing the number of rural health centers, establishing community outreach programs, distribution of bed nets, and early diagnosis and treatment, could reduce the pregnancy associated malaria burden in Mangaluru.
1. Introduction Malaria remains as a highly prevalent infectious disease, affecting billions of people worldwide, especially in the tropical regions. Among five human malarial infecting species, Plasmodium falciparum and P. vivax are the most prevalent species. In 2016, about 216 million cases and 445,000 deaths were reported from 91 countries across the world, in which India accounted for over 1 million cases and 331 malarial deaths [1]. In India, malaria is highly endemic and persists mostly in the Northern, Northeastern and Southwestern regions. In the Southwestern region of India, Karnataka state is a significant contributor of
malarial infections. Overall, among 30 districts in Karnataka state, the majority of malarial burden is reported from only two districts viz., Dakshina Kannada and Udupi [2]. Mangaluru, the administrative capital of Dakshina Kannada district, is situated on the Arabian seashore of Southern India. The city and its surrounding areas have a warm and humid climate, harboring high vector density leading to high rates of malarial transmission [3]. In 2016, Karnataka state reported a total of 15,816 malarial infections, of which Mangaluru alone accounted for 11,037 cases and the majority (82.4%) were due to P. vivax infections [4]. In malarial endemic regions, pregnant women and children less than
∗
Corresponding author. E-mail addresses: [email protected] (V.N. Chandrashekar), [email protected] (K. Punnath), [email protected] (K.K. Dayanand), [email protected], [email protected] (R.N. Achur), [email protected] (S.B. Kakkilaya), [email protected] (P. Jayadev), [email protected] (S.N. Kumari), [email protected] (D.C. Gowda). https://doi.org/10.1016/j.imu.2019.02.003 Received 26 December 2018; Received in revised form 13 February 2019; Accepted 14 February 2019 Available online 18 February 2019 2352-9148/ © 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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five years of age are at an increased risk of malaria associated severe complications [5]. It is well accepted that P. falciparum infections in pregnant women are known to result in maternal and fetal morbidity and mortality. However, there are emerging worldwide reports of P. vivax infections also resulting in maternal anemia and low birth weights [6–9]. The severity of malarial manifestations in pregnant women and the fetus depends on the acquired immunity level. It is well known that primigravidae (women in their first pregnancy) are more susceptible to malarial infections than second gravidae or multigravidae. The higher susceptibility of primigravidae women is due to lack of placental parasite-specific immunity in comparison to second gravidae or multigravidae women [10,11]. PAM is characterized by sequestration of Plasmodium falciparum-infected erythrocytes and infiltration of immune cells within intervillous spaces of placenta. Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP1) encoded by VAR2CSA, binds to CSA in placenta, thus avoiding the maternal immune response [12–14]. PAM results in placental basement membrane thickening, perivillous fibrinoid deposits, and syncytial knotting leading to impaired nutrient and gaseous exchange system between mother and fetus, causing intrauterine growth retardation (IUGR) and low birth weight (LBW) [15]. PAM can result in serious complications in the mother such as anemia, pulmonary edema, hypoglycemia, cerebral malaria and puerperal sepsis, which could lead to death. The fetal complications due to PAM could lead to abortion, still birth, IUGR, premature delivery, and LBW [15]. Vaccines for protection from PAM and its adverse birth outcomes, by using antibodies against VAR2CSA, which is known to be associated with PAM, are now in clinical trials (NCT02647489 and NCT02658253, https://www.clinicaltrials.gov). The burden of PAM in India is largely underestimated, as the National Vector Borne Disease Control Programme (NVBDCP) does not collect data on pregnant women with malarial infections and its associated abortions and still-births [16]. Few studies on PAM from endemic regions in India exist and have been confined only to regions such as Madhya Pradesh, Chandigarh, Surat, Gujarat and Jabalpur, whereas no systematic studies have been carried out in the southern part of India [17–20]. During PAM, anemia is very common, which is regarded as detrimental to fetal growth and poor pregnancy outcomes such as LBW and preterm delivery [21–23]. Keeping in view of the situation, this hospital-based study was undertaken to determine the clinical features, prevalence of malarial anemia, and its intensity, among pregnant women in the endemic settings of Mangaluru City.
2.2. Malarial diagnosis The malarial diagnosis was performed by examination of peripheral blood smears by conventional microscopy and rapid diagnostic test (RDT) kits (SD Bioline, India). Two blood slides (thick and thin) were prepared by finger prick, stained with 4% Giemsa stain (Sigma Aldrich, USA) and were observed under the microscope for the presence of Plasmodium parasite and the species type. The parasite densities were determined by counting the number of parasites and expressed as parasites/μL of blood (number of parasites counted/number of WBCs counted x total number of WBCs per μL of blood) or (number of parasites counted/number of RBCs counted x total number of RBCs per μL of blood). Percentage parasitemia was determined as (number of parasites per μL of blood/number of RBCs per μL of blood) x 100. The pregnant women were also screened by using a malaria Ag Pf/Pv RDT kit which detects histidine-rich protein II antigen of P. falciparum and lactate dehydrogenase of P. vivax in human blood. 2.3. Sample collection and hematological analysis About 1 ml of venous blood was collected into clot activator tubes for serum and sterile heparin coated vacutainers for plasma, centrifuged, and the supernatant stored at −70 °C until further use. The infected patients were treated by the attending physicians as per the NVBDCP recommendations. Within an hour of sample collection, the blood was used to determine the levels of hemoglobin (Hb), Red blood cells (RBCs), Hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC), by using an automated hematology analyzer (Mind Ray-Biomedical, Shenzhen, China). 2.4. Statistics The data was entered in Microsoft excel and the statistical analysis was performed using GraphPad Prism (GraphPad, Inc., San Diego California, USA). Quantitative variables were presented as number (percentages) and Mean ± Standard deviation. The comparison of non-parametric data between various groups was performed using the Kruskal-Wallis Test, and significance between any of the two groups was determined by the Mann-Whitney U Test with a 95% confidence interval with multiple testing. Spearman rank correlation was used to determine correlations between two continuous variables. A P-value less than 0.05 was considered to be significant.
2. Methods 3. Results 2.1. Study design 3.1. Characteristics of study participants During the period 2014 to 2017, a prospective hospital-based study was carried out at Government Lady Goschen Hospital in Mangaluru City, Karnataka, India. A total of 105 pregnant women aged between 18 and 40 years were randomly recruited into the study upon obtaining informed consent from the study participants or their relatives. All of the infected patients were febrile and received diagnosis and treatment at the hospital. The healthy pregnant women attending antenatal care (ANC) of Lady Goschen Hospital for general check-ups were recruited as healthy controls (HC). The study protocol was approved by the ethical committee of Kuvempu University, Shivamogga, Karnataka, the central ethics committee of NITTE University, Mangaluru, Karnataka, and the Institutional Review Board of Pennsylvania State University College of Medicine, Hershey, PA, USA. Pregnant woman aged > 18 years were included in the study, whereas the study participants with the use of any prophylaxis prior to diagnosis, and individuals testing positive for dengue, typhoid, HIV, hepatitis B and C infections, were excluded.
A total of 105 pregnant women were recruited into the study. Among the study participants, 71 were malaria infected, whereas 34 were HC. Among infected women, P. vivax (48, 67.6%) was the most prevalent infecting species. The mean age of the study participants was 25 years, with range between 17 and 40 years. Our observation correlated with the fact that women in their first pregnancy are susceptible to PAM, as most of the infected pregnant women were primigravidae (32, 45.1%), followed by second gravidae (25, 35.2%) and multigravidae (14, 19.7%). Among the 71 infected patients, (22, 31.0%) were in their first trimester, (32, 45.1%) were in second trimester and (17, 23.9%) were in third trimester. The mean birth weight of babies delivered by infected women (2.48 kg ± 0.26) was found to be significantly lower in comparison to birth weight of babies delivered by HC (2.64 kg ± 0.11, P = 0.0044). Among 71 infected women, 26 (36.6%) delivered babies with LBW (Table 1). In this study area, the majority of infected pregnant women were immigrants (55, 77.5%). Most of the infected pregnant women were engaged in household work (58, 82%) and a few (4, 5.6%) were also 2
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Table 1 Background characteristics of the study participants. Parameter
HC
P. vivax
P. falciparum
Mixed
Overall (Infected)
Number of study participants Mild malaria (MM) Severe malaria (MM) Mean age in years (range) 15-20 21-25 26-30 31-35 36-40 Gravidity Primi gravidae Second gravidae Multi gravidae Trimester First trimester Second trimester Third trimester Delivery outcome Mean birth weight Normal LBW
34 0 0
48 (67.6) 35 (72.9) 13 (56.5)
13 (18.3) 7 (14.6) 6 (26.1)
10 (14.1) 6 (12.5) 4 (17.4)
71 (100) 48 (67.6) 23 (32.4)
5 11 11 3 4
11 (78.6) 18 (66.7) 16 (76.2) 3 (37.5) 0 (0)
1 4 4 3 1
2 5 1 2 0
(14.3) (18.5) (4.8) (25) (0)
14 (19.7) 27 (38.0) 21 (29.6) 8 (11.3) 1 (1.4)
15 12 7
23 (71.9) 17 (68) 8 (57.1)
4 (12.5) 5 (20) 4 (28.6)
5 (15.6) 3 (12) 2 (14.3)
32 (45.1) 25 (35.2) 14 (19.7)
11 13 10
18 (81.8) 17 (53.1) 13 (76.5)
2 (9.1) 9 (28.1) 2 (11.8)
2 (9.1) 6 (18.8) 2 (11.8)
22 (31) 32 (45.1) 17 (23.9)
2.64 ± 0.11 34 0
2.5 ± 0.31 30 (66.7) 18 (69.2)
2.5 ± 0.20 9 (20) 4 (15.4)
2.4 ± 0.32 6 (13.3) 4 (15.4)
2.48 ± 0.26 45 (63.4) 26 (36.6)
(7.1) (14.8) (19) (37.5) (100)
Data expressed as number of study participants (percentages with respect to infected groups).
attendants. It was found that 57 (80.3%) infected pregnant women belonged to the below poverty level (BPL) group and were economically poor. We also observed that 50 (70.4%) infected women were not aware of preventive measures to be taken during pregnancy, and only 13 (18.3%) claimed the use of mosquito nets while sleeping (Table 2).
Table 2 Socio-demographic data across various infecting species. HC
P. vivax
P. falciparum
Residence, n (%) Native 25 11 (68.8) 3 (18.8) Immigrants 9 37 (67.3) 10 (18.2) Locality Urban 10 5 (45.5) 5 (45.5) Rural 24 43 (71.7) 8 (13.3) Education, n (%) No formal education 10 31 (81.6) 5 (13.2) Primary 9 9 (56.3) 3 (18.8) Secondary 9 2 (22.2) 3 (33.3) College level 6 6 (75) 2 (25) ANC visits Yes 24 4 (44.4) 4 (44.4) No 10 44 (71) 9 (14.5) Socioeconomic characteristics Poor 8 41 (71.9) 8 (14.0) Middle class 26 7 (50) 5 (35.7) Rich 0 0 (0) 0 (0) Knowledge of preventive measures to be taken Mosquito nets 15 6 (46.2) 3 (23.1) Removal of stagnant 12 2 (28.6) 2 (28.6) water window netting 28 4 (50) 2 (25.0) Use of mosquito 29 9 (50) 8 (44.4) repellents Chemoprophylaxis in 10 3 (50) 2 (33.3) pregnancy Does not know 5 39 (78) 5 (10)
Mixed
Overall (Infected)
2 (12.5) 8 (14.5)
16 (22.5) 55 (77.5)
1 (9.1) 9 (15)
11 (15.5) 60 (84.5)
2 4 4 0
(5.3) (25) (44.4) (0)
38 (53.5) 16 (22.5) 9 (12.7) 8 (11.3)
1 (11.1) 9 (14.5)
9 (12.7) 62 (87.3)
8 (14) 2 (14.3) 0 (0)
57 (80.3) 14 (19.7) 0 (0)
4 (30.8) 3 (42.9)
13 (18.3) 7 (9.9)
3.3. Effect of malarial infections on hematological parameters
2 (25) 1 (5.6)
8 (11.3) 18 (25.4)
Significant hematological changes in pregnant women during malarial infections are commonly observed. In this study, a significant
1 (16.7)
6 (8.5)
6 (12)
50 (70.4)
3.2. Clinical symptoms All of the infected patients (71,100%) were febrile. Apart from fever, 22 (31%) of patients had chills, 25 (35.2%) showed shivering, 32 (45.1%) had headache, 23 (32.4%) experienced weakness, 36 (50.7%) patients complained of myalgia, 20 (28.2%) had dizziness, 27 (38.0%) felt nausea, 18 (25.4%) had vomiting, 20 (28.2%) patients had inappetence, 11 (15.5%) patients had diarrhea, 18 (25.4%) patients were having cough and 10 (14.1%) patients experienced difficulty in breathing. Apart from various clinical symptoms, a majority of P. vivax patients experienced myalgia and nausea, whereas P. falciparum patients experienced chills, headache, weakness, dizziness, and inappetence (Table 3).
Table 3 Clinical symptoms among the malarial subjects.
Data expressed as number of study participants (percentages with respect to infected groups).
involved in agricultural activities. Interestingly, the bulk of the infected patients were from rural (60, 84.5%) areas. Most of the study participants (38, 53.5%) did not have any formal education, only 16 (22.5%) had primary level education, 9 (12.7%) had secondary level, and 8 (11.3%) had college level education. It was observed that the majority of infected pregnant women (62, 87.3%) did not visit hospitals for ANC. Only 7 (9.9%) pregnant women visited ANC during the first half of pregnancy, while 2 (2.8%) visited in the latter stages (i.e., gestational age of > 20 weeks). Among the 62 pregnant women not seeking care at ANC, 27 pregnant women (43.5%) sought help from traditional birth
Symptoms
P. vivax
P. falciparum
Mixed
Overall
Fever Chills Headache Weakness Shivering Myalgia Dizziness Nausea Vomiting Inappetence Diarrhea Cough Difficulty in breathing
48 (67.6) 12 (54.5) 17 (53.1) 12 (52.2) 14 (56.0) 30 (83.3) 12 (60.0) 22 (81.5) 10 (55.6) 8 (40.0) 8 (72.7) 12 (66.7) 8 (80.0)
13 (18.3) 8 (36.4) 11 (34.4) 8 (34.8) 6 (24.0) 3 (8.3) 5 (25.0) 3 (11.1) 4 (22.2) 10 (50.0) 2 (18.2) 3 (16.7) 1 (10.0)
10 (14.1) 2 (9.1) 4 (12.5) 3 (13.0) 5 (20.0) 3 (8.3) 3 (15.0) 2 (7.4) 4 (22.2) 2 (10.0) 1 (9.1) 3 (16.7) 1 (10.0)
71 22 32 23 25 36 20 27 18 20 11 18 10
Data expressed as number of study participants (percentages). 3
(100) (31.0) (45.1) (32.4) (35.2) (50.7) (28.2) (38.0) (25.4) (28.2) (15.5) (25.4) (14.1)
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Table 4 Influence of malarial infection on Hemoglobin, RBC levels and RBC indices among pregnant women with P. vivax, P. falciparum and mixed infections. Variable
Parasitemia (%) Hb (g/dL) RBC x103/μL HCT (%) MCV (fL) MCH(pg) MCHC (g/dL)
HC
P. vivax
0.0 ± 0.0 12.3 ± 0.5 4.7 ± 0.5 39.9 ± 4.0 84.6 ± 4.9 25.1 ± 2.1 30.1 ± 1.7
P. falciparum
Mixed
p value *(between anemic groups)
NA
A
NA
A
NA
A
Pv Vs Pf
Pv Vs Mixed
Pf Vs Mixed
0.1 ± 0.0 12.0 ± 0.6 3.7 ± 0.7 36.0 ± 8.6 86.3 ± 16.7 26.0 ± 4.5 30.1 ± 0.4
0.3 ± 0.2 7.8 ± 1.6 3.4 ± 0.7 28.9 ± 8.5 79.7 ± 9.5 23.5 ± 3.5 29.7 ± 1.2
0.2 ± 0.1 12.0 ± 0.7 3.1 ± 0.3 31.3 ± 1.8 81.3 ± 7.9 25.2 ± 3.1 30.2 ± 0.8
0.8 ± 0.4 6.8 ± 2.2 2.3 ± 0.3 27.1 ± 2.2 83.6 ± 9.2 24.8 ± 3.5 29.3 ± 1.1
0.2 ± 0.0 11.9 ± 0.8 3.4 ± 0.2 28.6 ± 4.1 77.1 ± 7.0 20.3 ± 1.5 28.9 ± 0.7
0.5 ± 0.3 6.7 ± 1.3 2.8 ± 0.5 25.0 ± 4.2 78.5 ± 7.8 21.1 ± 2.3 29.8 ± 1.5
< 0.0001 0.1263 < 0.0001 0.9358 0.3021 0.287 0.3279
0.009 0.044 0.021 0.22 0.748 0.062 0.911
0.126 0.946 0.019 0.303 0.184 0.02 0.371
Data expressed as Mean ± Standard deviation; Analysis between any two groups was compared by Mann-Whitney U Test; * p-value < 0.05 considered to be significant.
P. vivax (2, 40%), P. falciparum (1, 20%), and mixed (2, 40%) infections. Intrauterine growth retardation (IUGR) was observed in 21 (29.5%) infected women (P. vivax – 14, 66.6%; P. falciparum - 3, 14.4% and mixed - 4, 19%). There was no significant influence between the proportion of patients with IUGR and gravidity.
increase in parasitic burden was observed in anemic (A) group patients compared to the non-anemic (NA) group. Upon comparison between anemic groups of different infecting species, patients with P. falciparum infections had significantly increased parasitic levels (P < 0.0001) as compared to P. vivax and mixed infections. The levels of hemoglobin, in comparison with NA groups, were significantly decreased in the A group, irrespective of the infecting species. In comparison between A groups among various infecting species, the hemoglobin levels were significantly decreased during P. falciparum infections. It was observed that a significant negative correlation existed between increased parasitemia and Hb levels during P. vivax (r = −0.6844, P < 0.0001), P. falciparum (r = −0.8664, P = 0.0002) and mixed (r = −0.7052, P = 0.0251) infections. The levels of HCT, MCV, MCH and MCHC were also found to be significantly decreased in anemic patients in comparison with non anemic patients across various infecting species (Table 4). A significant positive correlation was observed between decreased Hb and RBC levels during P. falciparum (r = 0.8143, P = 0.0011) infections.
4. Discussion Several studies worldwide have shown that the majority malarial infections among pregnant women are due to P. falciparum, and the remaining are contributed by P. vivax infections [24]. However, the majority of studies have not focussed on the burden due to P. vivax malaria during pregnancy. In India, in spite of a high malarial burden in most part of the country, no systematic studies regarding the extent of malarial infections and clinical manifestations among pregnant women have been carried out. For the first time, the impact of malaria during pregnancy involving 71 infected pregnant women in Mangaluru City was carried out in this hospital-based study. In Mangaluru, although both P. falciparum and P. vivax exists, P. vivax is the major infecting species. The data obtained here indicates that P. vivax (67.6%) is the predominant infecting species, as compared to P. falciparum (18.3%) and mixed (14.1%) infections. The malarial infections present with a variety of clinical symptoms, which are similar to various other viral illnesses. In general, malarial infections in pregnant women in various regions across Asia have mostly been symptomatic. However, studies carried out in the Northeastern states of Orissa and West Bengal in India report the prevalence of asymptomatic infections among pregnant women [25]. In this study, although all of the infected patients were febrile and symptomatic, the fever was intermittent in nature, and its intensity ranged from a mild to severe level. Apart from fever, various other characteristic symptoms observed were myalgia and nausea during P. vivax, whereas P. falciparum patients presented with chills, headache, weakness, dizziness and inappetence. In this endemic setting, we found that the pregnant women residing in rural areas were highly susceptible to malarial infections as compared with those in urban localities, which is in agreement with earlier reports focusing on other regions of the country [24]. The infected women were mostly engaged in household activities and agriculture. Over 80% of pregnant women reported not to have regularly visited ANC during their pregnancy, and only 9.9% patients visited during their first trimester. Only 8% of pregnant women were aware that the ANC services are free of charge. Most of the women not seeking care at ANC did not report the use of any specific prophylaxis, multivitamins or iron supplements during pregnancy, and only 3% took self-medications such as paracetamol and multivitamins. Within the ANC users group, about 45% of women sought help from traditional birth attendants; however, there were incidences of non-compliance to medications prescribed by these local birth attendants as it sometimes resulted in vomiting, irritation, and tiredness. The major reason given for not
3.4. Malarial anemia intensity in pregnant women Anemia was defined as Hb levels of less than 11 g/dL. Based on the varying Hb levels, the study participants were grouped into (i) Nonanemic (NA) – study participants with hemoglobin ≥11g/dL. (ii) Mild anemia (MA) – hemoglobin range 8–10.9 g/dL. (iii) Moderate anemia (ModA) – hemoglobin range 5–7.9 g/dL and (iv) Severe anemia (SA) – hemoglobin < 5 g/dL. In this study, samples from 71 infected pregnant women were analzsed for the level of anemia. In the case of healthy controls, the majority were found be non-anemic (32, 94%), whereas only 2 (5.9%) had a mild level of anemia. Among the infected, only 9 (12.7%) were non-anemic and 62 (87.3%) were anemic. Upon classification based on varying hemoglobin levels, 29 (40.8%) were found to have mild anemia, 25 (35.2%) had moderate anemia and 8 (11.3%) had severe anemia (Table 5). Low birth weights were associated mostly with primigravidae (30, 40.6%), followed by second gravidae (9, 25.7%) and multigravidae (4, 28.5%). Among the 32 primigravidae women, an increased proportion of P. vivax (10, 31.2%) patients had given birth to LBW babies, in comparison to P. falciparum (1, 3.1%) and mixed (2, 6.3%) infections. Pre-term delivery was observed in 5 cases (7%), that were due to Table 5 Classification of study participants across varying degree of anemia. Anemia type
HC
P. vivax
P. falciparum
Mixed
No anemia (NA) Mild anemia (MA) Moderate anemia (Mod A) Severe anemia (SA)
32 (94) 2 (6) 0 (0) 0 (0)
3 (6.3) 23 (47.9) 18 (37.5) 4 (8.3)
3 4 3 3
3.0 2.0 4.0 1.0
(23.1) (30.8) (23.1) (23.1)
(30) (20) (40) (10)
Data expressed as number of study participants (percentages). 4
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localities. A review of national malaria drug policy is also recommended to include intermittent preventive therapy and nutrient supplementation during pregnancy, similar to African settings.
visiting an ANC was lack of awareness that ANC services in government clinics are free of charge, or financial problems, as most of these pregnant women relied on medications given by traditional birth attendants. In this study group, most of the women did not have any formal education, and hence they were not aware of the problem of malaria, its severity, and the necessary preventive measures to be taken. Altogether, the data suggests that the reason for higher prevalence of malarial infection in pregnant women among rural areas could be due to high transmission, lack of awareness about protective and preventive measures to be taken, decreased access to ANC clinics, poor socioeconomic status, decreased availability of preventive measures such as mosquito repellents, bed nets, ITN's, IRS, and limited access to antimalarials. The intensity and the timing of malarial infection in pregnant women are known to play an important role in IUGR and preterm delivery. Studies in Malawi suggest that women infected during their antenatal period are at increased risk of IUGR when compared to others, and preterm delivery was also associated with umbilical cord parasitemia [26]. In our study, overall, 29.5% pregnant women had IUGR and the majority was P. vivax (66.6%) patients, whereas about 36.6% had given birth to LBW babies. In general, primigravidae are highly susceptible to malarial infection, which is due to lower acquired immune responses and the sequestration of P. falciparum strains in placenta microvasculature [12–14]. The Plasmodium parasites have distinct antigenic and adhesive properties and are known to sequester in the intervillous spaces of placenta mediated by chondroitin 4-sulfate receptor [10,11]. With subsequent pregnancies, women are exposed to repeated infections and may gradually develop a certain level of immunity to prevent and control severe PAM complications [27,28]. There are reports of increased parasitic burden among primigravidae in comparison to multigravidae, due to lack of anti-parasitic antibodies during first pregnancy [28,29]. However, we found no correlation between gravidity and peripheral parasitemia levels. Malarial infections are known to cause significant changes in hematological parameters, and anemia in general is commonly associated with pregnancy. However, malarial anemia during pregnancy may result in severe life-threatening conditions to both mother and the fetus. The data obtained here has also shown that the hemoglobin levels are significantly decreased in pregnant women with malarial infections. Among the different infecting species, women with P. falciparum infections were found to have significantly decreased levels of Hb, RBC, and increased parasitemia. However, among 8 patients with severe anemia, P. vivax contribution was highest (50%) followed by P. falciparum (37.5%) and mixed (12.5%) infections. Our data support the studies that have shown P. vivax infections result in severe malarial anemia and may also lead to LBW [30].
Ethical statement The study protocol was approved by the ethical committee of Kuvempu University, Shivamogga, Karnataka; the central ethics committee of NITTE University, Mangaluru, Karnataka, and the Institutional Review Board of Pennsylvania State University College of Medicine, Hershey, PA, USA. All the study participants were recruited into the study only after obtaining informed consent. Funding This work was supported by the Grant D43TW008268 (2012–2018) from the Fogarty International Center of the National Institutes of Health, USA, under the Global Infectious Diseases Program. Conflicts of interest The authors declare that they have no competing interest. Acknowledgements The authors thank the study participants for their consent to participate in the study. We thank Dr.Shakuntala, superintendent of Lady Goschen Hospital for her support, Dr. Kishan Prasad, Department of Pathology, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Deralakatte, Mangaluru, India for his guidance, and Dr. Aravind Pallipady, Department of Pathology, A.J. Hospital, Kuntikan, Mangalore, India for his kind support. This work was supported by the Grant D43 TW008268 from the Fogarty International Center of the National Institutes of Health, USA, under the Global Infectious Diseases Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.imu.2019.02.003. References [1] World Health Organisation (WHO). World malaria re-port 2017. Geneva: WHO; 2017 Available from: http://www.who.int/malaria/publications/world-malariareport-2017/en/. [2] Directorate of Health and Family Welfare Services, Bengaluru. Malaria elimination plan in Karnataka 2016-2025: framework, strategies and policies. 2016 Available from: https://www.karnataka.gov.in/hfw/kannada/Documents/Malaria %20Elimination%20Plan%20in%20Karnataka%20(2016%20to%202025).pdf. [3] Dayanand KK, Punnath K, Chandrashekar V, et al. Malaria prevalence in Mangaluru city area in the southwestern coastal region of India. Malar J 2017;16(1):492https://doi.org/10.1186/s12936-017-2141-0. [4] Kakkilaya BS. Malaria in Mangaluru, in malaria site.com. 2017 [cited 2018 January 22]. Available from:. https://www.malariasite.com/malaria-mangaluru/. [5] Chaponda EB, Chandramohan D, Michelo C, Mharakurwa S, Chipeta J, Chico RM. High burden of malaria infection in pregnant women in a rural district of Zambia: a cross-sectional study. Malar J 2015;14(1):380https://doi.org/10.1186/s12936-0150866-1. [6] Brutus L, Santalla J, Schneider D, Avila JC, Deloron P. Plasmodium vivax Malaria during pregnancy, Bolivia. Emerg Infect Dis 2013;19(10):1605–11https://doi.org/ 10.3201/eid1910.130308. [7] Bardají A, Martínez-Espinosa FE, Arévalo-Herrera M, et al. Burden and impact of Plasmodium vivax in pregnancy: a multi-centre prospective observational study. PLoS Negl Trop Dis 2017;11(6):e0005606https://doi.org/10.1371/journal.pntd. 0005606. [8] Pincelli A, Neves PA, Lourenco BH, Corder RM, Malta MB, Sampaio SJ, et al. The hidden burden of Plasmodium vivax malaria in pregnancy in the amazon: an observational study in northwestern Brazil. Am J Trop Med Hyg 2018;99(1):73–83https://doi.org/10.4269/ajtmh.18-0135. [9] Stanisic DI, Moore KA, Baiwog F, Ura A, Clapham C, King CL, et al. Risk factors for malaria and adverse birth outcomes in a prospective cohort of pregnant women
5. Conclusion Malaria among pregnant women has emerged as a major hidden public health problem in Mangaluru City and its surrounding areas. Despite various comprehensive malarial control and elimination programs by the District Health Office, the National Vector Borne Disease Control Program Office, and local community leaders, the control measures specifically aimed at malaria during pregnancy are not properly addressed. The present study has shown that the lack of education among women, unavailability, and poor coverage of government ANC services, lack of knowledge about malaria and associated anemia, as well as non-compliance with preventive measures, are the major reasons for malaria prevalence among pregnant women in Mangaluru City and its surroundings. Thus, there is an urgent need to address this situation by scaling up malarial surveillance, providing training to health care workers, and spreading awareness through educational programs and ITN's distribution on a large scale. Further detailed studies are needed to understand the burden of malaria among pregnant women and fatal outcomes, particularly in malarial hotspot 5
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