Elemental minerals and microbial compositions as well as knowledge and perceptions regarding kaolin (clay) consumption by pregnant women in the Ho municipality of Ghana

Introduction Kaolin is a type of clay consumed mostly by women especially pregnant women of which the act of clay eating is termed geophagy. Different people use this type of clay for diverse purposes. Notwithstanding, most Ghanaians consume this clay out of cravings, taste and smell. There have been some attendant problems with the consumption of clay especially by pregnant women. This research sought to assess the mineral and microbial contents of kaolin and address the perceptions of pregnant women on geophagy. Methods This study employed a cross-sectional convenient sampling method to sample 217 pregnant women and sellers of clay (Ayilo). A face-to-face interview was conducted to administer a structured questionnaire to respondents. Mineral and microbial analyses were also conducted on the ore of the kaolin mined from Anfoega in the Volta Region of Ghana using standardized procedures. Results Results from this study suggests geophagy prevalence of 48.4% (n=217) among pregnant women in the Ho municipality of Ghana. Results obtained also suggest smell and taste of the clay, influenced the consumption by these pregnant women to a large extent. Strikingly, majority of the respondents had no knowledge on the adverse health implications clay consumption had on the human body. The physiological state of pregnancy also cause many to use it to treat nausea, other accompanying discomfort and in some instances to quench their hunger. Traces of Lead, Nickel and Arsenic were found in the clay. Common microorganisms identified were Bacillus, Pseudomonas, Mucor and Aspergillus spp. Conclusion Geophagy is a common practice among pregnant women living in the Ho municipality in the Volta region, Ghana. Most of them consumed it for varied reasons. Although there are beneficial minerals, accumulated effects of these heavy metals can lead to various complications in pregnancy. The clay also contained pathogenic microorganisms. These pathogens have a whole range of deleterious effects on the human body ranging from gastrointestinal infections to cancer and so may not be safe to consume clay products from Anfoega, Ghana.


Introduction
Pica is a general term which refers to the repeated ingestion of nonfood items normally insatiable and leads to its compulsive consumption [1]. Pica in humans has many different subgroups and each of these subgroups is defined by the substance ingested. Based on the items consumed [2], identified thirty-six (36) types of pica which consist of both non-food and food items. Several forms of pica exist and some classifications include amylophagia (starch), coprophagia (faeces), geophagia (dirt, soil and clay), hyalophagia (glass), lithophagia (stones), pagophagia (ice), etc. Furthermore, pica according to other researchers [2][3][4] include paint, hair, plaster, live wasps, chalk, vinegar, grass, and many others. Geophagy is a form of pica characterized by craving and eating of soil [5]. It is characterized as a form of pica (a term that comes from the Latin word magpie which has indiscriminate eating habits) [6]. The act of eating clay is common in developing countries because most pregnant women eat clay as an appetite suppressant and it is also observed in anorexia nervosa [6]. In Ghana, processed kaolin (white clay) ready for consumption is known as Ayilo (Ga), Agatawe (Ewes), Hyire (Akan) and several other names in other dialects. Kaolin can be industrially mined; it is usually dug out from the deep parts of the earth crust. It is mostly mined in parts of the Volta Region, especially Anfoega, where the freshly mined clayey soil is molded into lumps, oven-baked and distributed to markets across Ghana. It contains chemical elements such as Aluminium, Arsenic, Boron and Nickel which can be potentially harmful to humans [7]. Several scientists have warned against the consumption of clay neither by pregnant women nor ordinary persons, since there are diverse health implications associated with its consumption [8].
The craving for clay, is especially common in pregnant women and it is consumed by different people for different reasons. Most people consume it for its peculiar taste and smell and become addicted to it later on thus preventing them from avoiding it if need be. Kaolin is also perceived to possess some health benefits, it is believed to contain certain mineral elements which could be beneficial to human health, and some spiritual connotations are linked to its intake. They eat the clay because of some traditional beliefs. Some of these beliefs are; an antidote to diarrhoea, stomach discomfort, nausea and other discomforts associated with pregnancy [9]. Geophagy has some economic implications on some families since pregnant women save some monies for its purchase which may be disadvantageous to people with low income. An average of 230 grams of baked clay cost GHS 40 ($9.00) and the price vary with location [10] in Ghana. The objective of this paper therefore was to assess the knowledge, perceptions and mineral contents as well as microbiological quality of clay consumed by women especially pregnant women in Ghana.

Methods
Study area: the study area where this research was concluded was in Ho, the capital city of Volta Region (Figure 1). Volta Region is one of Ghana's ten administrative regions. The Kaolin samples were purchased from Anfoega in the Volta Region. Anfoega is situated in Dayi, Volta, Ghana. Its geographical coordinates are 6° 53' 0" North, 0° 18' 0" East and its original name (with diacritics) is Anfoega Akukome [11]. Other samples were taken from the Ho Asigame market.
Study design: this study was a cross sectional and experimental study in which both qualitative and quantitative data collection techniques were employed. Questionnaires for data collection were pre-tested by face-to-face interviews and the mineral and microbial contents analyses were done in the laboratory using standard methods. The perception of pregnant women on geophagy was also documented.
Study population and site: the study population included individuals who attended antenatal clinic at the Volta Regional and the Ho Municipal Hospitals. The sample size consisted of only pregnant women who had fair knowledge about geophagy. People between age 16 to 45 years were qualified to take part in this study source [12].
Sample size and sampling procedure: participants were chosen using convenient sampling and their willingness to take part in the studies. Consent forms were given, it was well explained and those who showed interest in the study were included. Estimated sample size: Where, n = estimated sample size Z = 1.96 (critical value) D = margin of error P = percentage picking a choice (50%) n = (1.96 ²*0.5(1-0.5))/0.05² = (3.8416 *0.5 (0.5))/0.05² = 384. 16   Sample preparation for mineral analysis: the clay ore samples were ground into powder with mortar and pestle and sieved using a 0.1 mm mesh. About 100 grams of the samples were weighed and added to 100 ml of distilled water. The samples were then placed on a shaker at 125 rpm for 12 hrs and then allowed to settle. The samples were filtered using a whatman 40 filter paper. The supernatant was then used to run the analysis.
Determination of mineral elements: the dry ashing method was used for atomic absorption spectrophotometer (AAS) analysis [13]. All glasswares were washed with 1% nitric acid followed by demineralised water. Three millilitres (3 ml) each of the clay supernatants were weighed into platinum crucibles. The crucible and the test portion were placed in a Muffle furnace at a temperature of 550°C for 8 hrs.
The crucible with ash was put in a desiccator to cool. Five millilitres (5 ml) of nitric acid of mass fraction not less than 65%, having a

Sample preparation for microbiological analysis
Enumeration of bacteria: the bacterial analysis was modified from [14]. One gram of each sample was serially diluted in 9 ml sterile distilled water and the resulting dilutions were plated on Blood Agar (BA; CM0271), Sabouraud Dextrose Agar (SDA; Oxoid CM 032) and Potato Dextrose Agar (PDA; Oxoid CM 0325) at 37°C for 48 hrs. The media plates were inspected daily over the 48-hour period. Colonies of resident microorganisms were calculated as CFU/g for each sample.
The mean counts of colonies on duplicate plates were determined and colonies were confirmed using appropriate confirmatory tests for the microorganisms present.
Enumeration of fungi: this was carried out according to the procedure outlined by [15] with media and process modifications. One (1) gram of each test sample was added separately to 99ml of 0.1% peptone in a 250 ml Erlenmeyer flask and allowed to settle for 5 minutes. Each flask was then shaken at 120 rpm for 10 minutes on an Orbital Shaker (Gallenkamp, England). The samples were serially diluted up to 1:10 4 and then plated on PDA and SDA followed by incubation at 3°C for 5 days. Moulds and yeast that appeared were identified by their culture and morphological characteristics using standard identification manuals [16].

Enumeration of parasites: method described by [17] with
modifications. This was done by pouring formol-saline in 15 ml test tube. Approximately 1 g of granulated kaolin was added to the formolsaline and mixed gently until dissolution. The suspension was left standing for about 10 minutes. The suspension was strained through gauze (350-450 nm). Three (3) ml of ether was added to the filtrate and vigorously shaken for 1 minute to obtain a homogeneous mixture.
The mixture was then centrifuged for 2 minutes at 2000 rpm. The supernatant was aspirated and discarded. A wet mount was prepared using the sediment and mounted for microscopy.

Results
Socio-demographic characteristics of respondents: a total of 217 pregnant women between the ages of 16-45 years were interviewed in this study. From the social demographics of the study (Table 1) Table 3 shows the associations between socio-demographic characteristics and awareness of side effects of pregnant women in the study. The age group of participants was significantly associated with awareness of side effects (χ 2 = 24.027; p = 0.008). Ethnicity of the pregnant women was significantly associated with awareness of side effects (χ 2 = 52.536; p < 0.001). Religion of participants was also significantly associated (χ 2 = 18.138; p = 0.001). The occupation of pregnant women was equally associated (χ 2 = 64.416; p < 0.001).

Association between demographics and geophagy practices:
The number of children pregnant women had was significantly associated (χ 2 = 11.523; p = 0.021). Total monthly income of pregnant women was significantly associated (χ 2 = 36.897; p < 0.001). Educational level had a significant association (χ 2 = 31.049; p < 0.001) and the locality of these pregnant women also had significant association (χ 2 = 19.754; p < 0.001). However, there was no significant association between household size and awareness of side effects (χ 2 = 0.624; p = 0.732).  Table 4.

Metal levels in clay samples
Microbiological analyses: predominantly, the species of bacteria which were common in all the samples were Baccillus and Pseudomonas spp. and their counts recorded ranged between 1.0x10 -1 -9.8x10 2 and 4.0x10 1 -9.8x10 2 CFU/g respectively. Likewise, Mucor spp. were predominant in samples not baked, collected from Anfoega and sold in retail shops (Anf/RET/NB) which recorded 2.0x10 1 CFU/g. Aspergillus spp. were isolated from samples from Ho market which had been baked (Ho/mkt/B) (Table 5). Generally, there was an observed decrease in microbial counts as the ore was pretreated before sale for consumption.
Parasitological analysis: no distinctive helminths, protozoans, cysts nor ova-like structures were identified. However, ova-like structures were seen but not confirmatory for routine parasites.

Discussion
Geophagy practices among pregnant women: most noted and commonly reported key symptoms of pregnancy include nausea, vomiting, headache, dizziness and spitting [18]. Studies by [19] established that depression and its allied factors are very severe in the first trimester of pregnancy. The findings of [18] also pointed in this direction that pregnancy symptoms are most severe in the first trimester. It is however critical to note that the second and third trimesters are not free of pregnancy symptoms. In view of this, pica is practiced by pregnant women to curb these associated discomforts.
Geophagy which is a common practice among pregnant women in Ghana and West Africa at large, is usually persistent. Data taken shows that most of these women heard of geophagy practices from either their friends or family or both. Most had consumed the clay once or several times in their lifetime. About half of them ate clay during childhood which indicates that geophagy is a practice that is built from an early age (childhood) before the woman reaches adulthood. This is in line with a research by [20] which suggests that the first geophagy experience is often during childhood due to influence of family members and friends or due to curiosity. Habitually, the consumption of clay ceases in adolescence, but is restarted during gestation. More than half of total participants who were interviewed had cravings to eat clay during pregnancy which support the claims made by [21] that women in their child bearing age have the desire to eat non-food items.
Results from our study also suggests a prevalence of 48.4% (n = 217) in the Ho municipality of Ghana which corroborates published findings of other researchers who reported prevalence of the same range in other parts of the country. In Ghana although information on pica is scanty, the few studies such as that conducted by [22] in Accra found 57% of her respondents practiced pica. Mensah et al. [23] reported a prevalence of 47.0% in Kumasi. Likewise, [18] also reported 52.12% among pregnant women in La-Nkwantanang municipality, Accra. However, relatively low prevalence of 28.49 % and 30.25% were reported by [24] (Accra) and [25]  Madugu [27] reported a rate of 50% among pregnant women studied in Zaria. In Kenya, a light yellow soft stone (odowa) dug out from excavation sites at Kajulu hills in Kisimu District, is reported to be eaten by pregnant women [28]. On an interesting note, various types of clay are consumed by pregnant women in Malawi and further states that the practice is so common among the locals to the extent that almost every pregnant Malawian woman is expected to practice pica as a sign of pregnancy [29].
Knowledge and perception about geophagy: the results obtained also suggest smell and taste of clay, influenced the consumption by these pregnant women to a large extent and has been observed earlier by [30]. Changes in smell and/or taste for pica substances from the onset of pregnancy till delivery has been observed by pregnant women in this category. Thus, physiological changes may play a key role in pica development as suggested by [31]. A related study [32], also observed that people with heightened olfactory sensitivity during pregnancy had substantially more cravings than women with no olfactory changes. Damp earth was an important trigger for geophagy; some women reported cravings triggered by the smell of wet earth after it rained [33]. The physiological theory about clay or non-food item consumption is that eating clay or dirt helps relieve nausea, control diarrhoea, increase salivation, remove toxins and alter odor or taste during pregnancy [34]. Most pregnant women in order to relieve the physiological changes within their body during pregnancy take nonfood items. Food Additives [36]. Results obtained in this study was slightly higher than findings of [37] who reported 0.0 (nil) mg/Kg of Arsenic in clay samples in Accra (Ghana). However, [38] reported a range of 218-271 ppm from clay samples in different parts of Kumasi Metropolis.
Furthermore [39], also reported Arsenic levels of range 2.7-22.74 µg/g in nine (9) samples of clay samples collected from both Greater Accra and Kumasi of Ghana. Ingestion of large amounts of Arsenic can result in gastrointestinal symptoms such as vomiting, obstruction in the circulatory and nervous systems and ultimately death [40]. Lead concentrations were also below the mean exposure levels. This was in contrast with findings reported by [38] with a range of 549-622.92 µg/Kg. In line with our results [37], reported 2.36 ± 0.08 mg/100g. Lead consumption can result in adverse health effects such as the dysfunction of some vital organs such as kidneys, liver and heart. Mahurpawa [37] reiterated that multiple organs in the body are targeted by Lead due to systemic toxicity. Manganese and Nickel levels were also below the set limits. In Tanzania, Nyanza et al. [41] reported ranges of 2.3-128 and 2.9-1400 mg/Kg for Nickel and Manganese respectively. Nonetheless, [39] did not detect any Manganese in the clay samples from Ghana. Manganese is an essential nutrient involved in bone formation and carbohydrate metabolism.
Pseudomonas spp. which were among the organisms identified also have pathogenic effects on humans. It can cause death among people with cystic fibrosis and immunocompromised people [46]. In Ghana, numerous pathogens and fecal coliforms were isolated from clay samples collected from different locations. Some of these fecal coliforms were Staphylococcus spp, Klebsiella, Escherichia, and Shigella and Enterobacter spp. [8]. Microbiological quality is affected since both sellers and buyers may affect the microbiological quality.
Mining sites of clay can be contaminated with fecal matter. Tano-Debrah & Bruce-Baiden, [14] isolated coliform bacteria, Staphylococcus species, and yeasts from the external surfaces of white clay in Ghana. The predominant bacterial species that were found in sampled clay in Nigeria were Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella [47]. There were two helminths that were found in the cultured clay which were lumbricoides and hookworm [47]. There is the likelihood of contamination of processed clays by Enterobacteriaceae, Staphyloccocus spp. and other sporeforming pathogenic species. A range of 5000 to 7000 species of bacteria per gram of natural soil was reported by [48] and [49].
Fungi have less effects on human but they can produce toxins which may be harmful to the host. Pathogenic Aspergillus spp. produce mycotoxins. Aflatoxin, the most well-known and well-investigated mycotoxin, is known to carry the most potent carcinogenic activity as a natural product. It also carries acute toxicity to various human cells [50]. Mucor spp. that was identified, is a common fungus which

What is known about this topic
• Pica practice is prevalent in Africa; • Geophagy is practiced in Greater Accra and Ashanti Regions Kumasi; • Parasites are linked to clay consumption by pregnant women in Ghana.

What this study adds
• Geophagy prevalence among pregnant women in Ho municipality of Ghana is 48.4%; • Results of this research points out that clay consumed by pregnant women in Ho Municipality is free from parasites; • Macro and micro elements investigated in the clay samples, were all within permissible levels recommended by World Health Organization (WHO).

Authors' contributions
Isaac Agyei Annor conducted the study, performed the analysis and  Table 1: socio-demographic characteristics of respondents