Cosmopolitan Nature of the Eggs of Ascaris lumbricoïdes Identified in Wells Water Destined for Domestic Usage in Sub-Urban Areas in the Center Region of Cameroon

: In order to evaluate the cosmopolitan character of the eggs of Ascaris lumbricoïdes identified in the wells of four (04) sub-urban areas (Mbalmayo, Mbankomo, Okola and Soa) of the Central region of Cameroon, a study was conducted from September 2018 to March 2019. Sampling was carried out on 16 wells following a monthly sampling frequency. Eggs of Ascaris lumbricoïdes were identified using the formalin-ether concentration and the Kato-Katz identification methods. The result of the physico-chemical analysis shows that the water has an average temperature of 25.31°C and an acidic pH (6.7). Parasitological analysis shows the presence and predominance of the species Ascaris lumbricoïdes at any place and at any time in these waters. The highest parasitological densities were obtained during the rainy season. Statistical analysis shows that orthophosphates are significantly and positively correlated with the densities of the eggs of Ascaris lumbricoïdes (p≤0.01). While nitrates, ammonia nitrogen, colour, turbidity and Dissolved Total Solids are significantly and positively correlated with the densities of the eggs of Ascaris lumbricoïdes (p≤0.05). Water from these wells is therefore a source of contamination by Ascariasis.


I. Introduction
Intestinal parasitosis is a serious public health problem. These intestinal parasitosis are usually caused by intestinal helminths and protozoa. Among the helminths, Ascaris lumbricoïdes is the largest of the nematodes and the largest of the helminthiasis that affect humans (Khuroo, 1996). While overall health status and levels of health and hygiene have improved over the past centuries, at least in industrialized countries, 350,000 people are severely affected by Ascaris-like worms and 60,000 deaths per year are believed to be directly associated with this parasite (CDC, 2015). This parasitosis is predominant in developing countries, where heat, humidity and poor hygiene all contribute to its transmission (Ndiaye et al., 2012). Ascariasis is endemic in sub-Saharan Africa, South-East Asia and Latin America. It affects about a quarter of the world's population Verhaeghe et al. (1989), with high prevalence in all the age groups in poor, sub-urban, and even tropical rural areas, reaching up to 80% of the population (Sun, 1980;Messou et al., 1997;Ravaoalimalala et al., 2002). In Bangladesh Aubry and Gaüzère (2018), for example, the prevalence is 80%. Fertilized females of Ascaris lumbricoïdes lay eggs that are disposed of in the external environment. These eggs may accidentally end up in aquatic environments. As a result, water can act as a vector for potentially dangerous parasites such as. The present study aims at assessing the cosmopolitan character of the eggs of Ascaris identified in well water in relation to abiotic factors in four sub-urban areas of the Central region Cameroon (Mbalmayo, Mbankomo, Okola and Soa). More specifically: to measure the main physicochemical variables of these waters, to identify the eggs of Ascaris lumbricoïdes in these waters and to evaluate the influence of abiotic variables on the distribution of the eggs of Ascaris lumbricoïdes in these wells water.  (BUCREP, 2005). Soa is a municipality of Cameroon located in the Central region and the Mefou and Afamba department, 14 km northeast of Yaoundé. It lies between 3°58' North latitude and 11°35' East longitude. These cities are characterized by a four-season Guinean climate comprising: a long dry season (LDS) from mid-November to mid-March, a short rainy season (SRS) from mid-March to the end of May, a short dry season (SDS) from June to August, and a long rainy season (LRS) from September to mid-November with an average annual rainfall of 1564.7 mm (Foahom, 1983;Abossolo, 2015). The soils are predominantly ferralitic and hydromorphic (Onguene Mala, 1993). Ferralitic soils are formed on moderately or strongly denatured acidic rocks. They have good physical properties. They are deep soils with good permeability. Hydromorphic soils are found in swampy areas and along riverbanks.

II.2. Selection of sampling points
Sixteen wells were retained (designated as MW1, MW2, MW3, MW4 in Mbalmayo locality; BW1, BW2, BW3, BW4 in Mbankomo locality; OW1, OW2, OW3, OW4 in Okola locality and SW1, SW2, SW3, SW4 in Soa locality. These water points have been chosen in different districts according to criteria such as: the great interest that the population shows for these water points, accessibility, level of urbanization. Nowadays, well water is increasingly used by the population to meet their domestic needs, dental hygiene, drinking, cooking and bathing, among others. For the identification of eggs of Ascaris lumbricoïdes, water samples were taken from wells at locations characterized by an accumulation of organic matter or the presence of the herbarium. The water samples collected were immediately placed in 1000 mL sterile polyethylene bottles and preserved by the addition of 2 mL of 10% formalin and returned to the laboratory in a cooler.

II.4. Parasitological analysis
The samples were left to stand at room temperature for 24 hours to settle. The supernatant was poured and the remaining pellet was measured. The concentration of helminths eggs was then determined using Kato-Katz and formalin-ether techniques, followed by observation under the Olympus CK2 inverted microscope at 40X magnification.

II.4.1. Kato-Katz technique
A pellet fragment resulting from centrifugation at 500 rpm for 5 minutes was removed and deposited in the center of a slide. The slide was covered with a rectangle of glycerol-soaked cellophane paper and the pellet was spread between the slide and the cellophane in a smear using a test tube. The preparation thus obtained was directly observed under an optical microscope for the identification of the eggs of Ascaris lumbricoïdes.

II.4.2. Formol-ether concentration technique
After homogenization of the pellet, 5 mL were collected with a graduated syringe and introduced into a 10 mL test tube. To this, 2 mL formalin and 3 mL ether were successively added. The resulting mixture was centrifuged at 500 rpm for 5 minutes

II.4.3. Identification and enumeration of helminth eggs and larvaes
The identification of these eggs was carried out using the plates of (OMS, 1994). Thus, the enumeration was carried out using the formula proposed by (Ajeagah et al., 2010). The number (x) of the eggs of Ascaris lumbricoïdes contained in 1L of sample is obtained by: being the volume of the pellet in 1 L of sample, = the volume of the pellet used for observation and the number of the eggs of Ascaris lumbricoïdes observed in .

II.4.4. Data analysis
The data obtained were grouped by season: LRS (Long Rainy Season) from September to November and LDS (Long Dry Season) from December to February. Spearman's ''r''correlation test was performed using SPSS 20.0 software and is used to evaluate the physico-chemical parameters that influence the distribution of parasites in water depending on the degree of binding between the different parameters.

III.1. Results of physico-chemical analysis
Water temperatures ranged from 24. 25° C in wells SW3 and SW4 during the main rainy season (LRS) to 27° C at the OW1 station during the main dry season (LDS). Suspended Solids (SS) and turbidity fluctuated from 3 to 39 mg/L and 3 to 75.5 mg/L respectively with the highest values obtained in LRS. The greatest value of colour (394.5 Pt.Co) were obtained at well OW4 during the long rainy season (LRS) while well SW3 had the lowest colour value (3.5 Pt.Co) during the long dry season (LDS). In general, the highest colour values are obtained during the Long Rainy Season. Dissolved Total Solids (DTS) ranged from 19 mg/L in well BW4 to 366.5 mg/L in well MW1 during the long rainy season ( Table 2). The pH values recorded during the study ranged from 4.92 CU at well OW1 to 6.80 CU at well SW1 during the long rainy season. These pH values vary from one sampling point to another during both seasons. Dissolved oxygen ranged from 35.15 mg/L (LDS) and 67.10 mg/L (LRS) in wells OW1 and OW3 respectively. Nitrate and orthophosphate levels ranged from 0.09 to 9.50 mg/L NO 3 and 0.02 to 7.50 mg/L PO 4 3respectively, with high levels obtained in LRS. Oxidability levels in the water ranged from 1.57 mg/L at OW4 well in LDS to 5.04 mg/L at MW3 well in LRS (Table 3).

III.2. Results of biological analysis
Results regarding the identification of the eggs of Ascaris lumbricoïdes in well water revealed the presence during the study period in all four localities. Water samples taken throughout the study period were found positive for eggs in both seasons with the exception of wells SW1, SW2 and SW3 in Soa. The highest densities were obtained during the long rainy season (Table 4).  Legend: Temp = Temperature, Oxy = Dissolved Oxygen, STD = Total Dissolved Solids, TSS = Suspended Solids, Colour = Colour, Turb = Turbidity, PO 4 3-= Orthophosphates, NO 3 -= Nitrates, NH 4 + = Ammonium; Ascaris = eggs of Ascaris lumbricoïdes. * = correlation is significant at threshold p ≤ 0.05 and **= correlation is significant at threshold p ≤ 0.01. The temperature of the wells is globally similar (around 25°C) during all seasons but is highly variable in LDS (between 24.25 and 27°C) in all study localities except the Soa locality. This variation in temperature could be explained by the fact that the wells are shallow and open, so that the light energy reaches the water table. Similar results were obtained by Tuekam Kayo (2013) in the groundwater of Mbalmayo. The well water of the 04 localities analysed have an overall acidic pH. This acidity would be linked to the nature of the soil in the central region. In this respect, Nola et al. (1998) point out that the pH of water depends on the nature of the land it crosses. TSS, colour and turbidity were high in the different study areas during both seasons with the exception of wells MW2, MW4 and SW4 for colour and MW1, BW4 and SW4 for turbidity. These high values would be explained by the absence or inadequacy of wells. This is because the development of the water points by the construction of curbs at least 0.5 m high, the presence of a cover and the concreting of the structure may prevent the risks of pollution. The low nitrate, orthophosphate and oxidability values obtained overall could be justified by the low use of agricultural inputs during the study period by the populations surrounding the various wells. The high values of dissolved oxygen obtained in all localities (LRS) except Okola locality and well BW1 in Mbankomo (LDS) would be related to the decrease in organic matter. Indeed, when the quantity of organic matter is low in the environment, the quantity of oxygen consumed by the microorganisms in the mineralization process is low.

IV.2. Parasitological analysis of the water from the wells studied
The results of the parasitological research of the sampled waters allowed to highlight the eggs of Ascaris lumbricoïdes. These eggs are believed to come mainly from human and animal faeces that accidentally end up in well water.
The wide dispersion of concentrations in the various samples would depend on several factors: the number of infested individuals and/or animals, the number of eggs laid daily by the helminth species and the number of eggs removed per gram of infested human faeces. In this regard, Desoubeaux and Duong (2011) point out that for agents whose transmission is linked to the fecal-oral hazard, the promiscuity found in certain communities is conducive to the spread of the parasite. Ascaris lumbricoïdes eggs have been identified in the waters of all the wells studied and in all the 4 localities and this would testify to the cosmopolitan nature of this organism. Indeed, Ascariasis is cosmopolitan and is endemic in sub-Saharan Africa, in poor, sub-urban or even tropical rural areas (Ndiaye et al., 2012). Only wells SW1, SW2 and SW3 in Soa do not harbour Ascaris lumbricoïdes eggs. This may be explained by the good state of protection of these water points. Certainly, these two wells comply with the well construction standards as recommended by the WHO. The absence of Ascaris eggs in these wells may also be justified by the fact that these wells are deep and not located in swampy areas; therefore, they cannot be receptacles for waste water carrying any substance. Eggs of Ascaris lumbricoïdes were present in the wells sampled during both seasons. High densities were obtained during the long rainy season (LRS) compared to the long dry season (LDS). This could be explained by the increased parasitic prevalence of helminthiasis in the rainy season, as during the rainy season it is the run-off water carrying all kinds of substances that pollute the well water. The distribution of environmental forms of helminths vary from one point to another according to the level of contamination It would also be crucial to mention that these muds could support contamination of the water table by the process of infiltration or draining water into unprotected wells via domestic effluents (Ajeagah et al., 2018). Eggs of Ascaris lumbricoïdes were abundant in all the wells and in all the study localities, which would justify its resistance in the environment. According to Dold and Holland (2011), embryonated eggs can remain in this state for 2 to 6 years while waiting to be ingested by the final host. The resistance of this organism can also result from the structure of the membranes and certain abiotic factors. In this respect, the wall of Ascaris is thick and hilly, if the environmental conditions are favorable, the eggs can remain infectious for up to six years. Most of the eggs of Ascaris lumbricoïdes identified have been modified by the environment. This modification has been marked by the loss of the hill on the external envelope, by the modification of the shape and the decrease in diameter among other things.

IV.3. Influence of physico-chemical variables on the density of eggs of Ascaris lumbricoïdes
Statistical analyses (p ≤ 0.05) have shown that the distribution of Ascaris lumbricoïdes eggs in the wells water is a function of the physico-chemical characteristics of the water. Thus, the high densities of eggs found in well MW1 may be favor to the high oxygenation of the water at this sampling point. This resulted in significant and positive correlations found between dissolved oxygen (r = 0.343; p = 0.007) and the eggs of Ascaris lumbricoïdes. The positive correlations obtained between ammoniacal nitrogen, orthophosphates, nitrates and densities of eggs of Ascaris lumbricoïdes would explain the parasite loads observed in the water points. Indeed, the high concentrations of mineral elements in the environment can increase the activation and development of forms of parasite resistance. The positive correlations obtained between SS, colour, turbidity and egg densities of Ascaris lumbricoïdes show that helminth eggs are most often adhered to organic matter suspended in water (Ajeagah et al., 2016).

V. Conclusion
At the end of this study on the evaluation of the cosmopolitan character of Ascaris lumbricoïdes eggs identified in well water in relation to abiotic factors in four sub-urban areas of Central region of Cameroon (Mbalmayo, Mbankomo, Okola and Soa), it turned out that the water has an average temperature of 25, 31° C, an acid pH and is oxygenated. A parasitological analysis shows the presence of the species Ascaris lumbricoïdes everywhere and along the period of study. In addition, these well waters are contaminated with eggs of the Ascaris lumbricoïdes through domestic effluent and through the use of cannon and flush latrines, usually located downstream and less than 15 m from the water points. This contamination is also due to the non-compliance of wells with construction standards. The distribution of helminth eggs in the waters studied is a function of certain physicochemical parameters such as dissolved oxygen, temperature, turbidity, colour, and ammoniacal nitrogen. The populations that use these waters on a daily basis may be therefore exposed to an increased health risk. To mitigate this, it is necessary to treat these waters before any use by usual methods.