4. DISCUSSION
FLA are cosmopolitan microorganisms ubiquitous in all matrices of natural and anthropogenic environments, including water resources. The presence of FLA in pools and recreational waters is worrying, since some of these microorganisms are human pathogens, as well as being widely implicated in persistence and / or pseudo-resistance of pathogenic bacteria, viruses and fungi in water, including water treated with disinfectants (Thomas et al., 2004; Staggemeier et al., 2016; Mavridou et al., 2018; Gomes et al., 2020; Hubert et al., 2021).
The studies included in present review are distributed by five continents, however, they have a heterogeneous spatial distribution within the territories of the continents, this can suggest differences in the level of FLA importance for health in the contexts of different countries, as well as differences in the frequency of cases diseases associated with the FLA. The frequency of cases of FLA related diseases can be influenced by the difference in the predominance of risk factors, the sensitivity of the health surveillance strategy of each country, as well as the heterogeneous distribution of trained professionals carrying out research in this area. In addition, the ease of confusing symptoms of diseases associated with the FLA with those caused by other microorganisms, combined with some cases of rapid deterioration of the patient’s health and death (Jahangéer et al., 2020) can contribute to the rarity of reports or even the lack of association of diseases with FLA, especially in contexts where post-mortem study policies are not robust.
Our findings show that the general prevalence of FLA in swimming pools and recreational waters is 44.785%, however, a higher (51.54%) and lower (37.95%) prevalence value was obtained when considering the data from studies published up to 2010 and studies published after 2010, respectively (Table 2). A similar result was reported in a study that aimed to determine the prevalence of Naegleria spp. in water resources (Saberi et al., 2020). This reduction in the prevalence reported in most recent studies was attributed to the most accurate diagnosis and reduction of false positive results (Jahangeeer et al., 2020; Saberi et al., 2020), as contrary to studies published up to 2010, the vast majority of studies published after 2010 used molecular methods for FLA identification. Curiously our results show that the general prevalence of FLA considering studies that they have simultaneously used morphological and molecular methods coincides with the average prevalence obtained considering data from studies that used only one of the methods (Table 2). This may suggest that the simultaneous use of these two methods reduces the extreme values obtained separately by each of the methods, and that these methods can be complementary, especially in studies that aim to assess the presence or absence of viable FLA in water samples. The authors agree that the morphological method (generally based on culture) is more laborious and less precise than molecular methods in the identification of FLA (Saberi et al., 2020; Hikal & Dkhil, 2018).
The subgroup analysis considering the distribution of the studies by the continents showed that FLA are more prevalent in the swimming pools and recreational water from America (59.52%), followed by Europe (53.88%). In relation to countries, the highest value of the prevalence of FLA was obtained in Malaysia (89.33%), followed by Italy (82.10%) and France (69.62%), and the lowest values were obtained in Taiwan (27.91), Iran (30.33) and Turkey (30.55). As for the sample source, the indoor hot swimming pools presented a higher value (52.27%) of FLA prevalence, followed by public swimming pools (45.31%) and hot springs (40.93%), recreational waters presented a relatively low value (34.75%). These results are in accordance with other authors whose studies reported high prevalence of FLA (Acanthamoeba spp. 48.5%, Naegleriaspp. 46.0%, Vermamoeba spp. 4.7% and Balamuthia spp. 0.7%) in hot springs (Fabros et al., 2021). Saberi et al. (2020) reported the following prevalence values for Naegleria spp. 44.80%, 32.88% and 21.27%, in swimming pools, hot springs and recreational waters, respectively. The subgroup analysis showed that prevalence values are statistically different (p <0.001) for all variables studied (Table 2). These findings are in accordance with other studies that reported a variable distribution in abundance and diversity of FLA species around the world (Jahangéer et al., 2020; Saberi et al., 2020; Fabros et al., 2021).
Our results also show that Vermamoeba spp., Hartmannellaspp. and Acanthamoeba spp. are more prevalent, presenting the following prevalence values, 16.06%, 16.40% and 15.38%, respectively (Table 3). The lowest prevalence value was for Naegleria spp. (10.01%). These results are in disagreement with the findings of other authors who reported higher prevalence values (Saberi et al., 2020; Fabros et al., 2021). The lower prevalence values found in this study can be explained by the fact that only data from studies that included molecular methods in the identification of amoeba were used to calculate the prevalence of different genera of FLA. As discussed in the previous paragraphs, studies based on molecular methods for identifying FLA report lower prevalence values.
The global prevalence of FLA reported in the present study (44.79%) is worrying, since direct contact between humans and these waters is often established. In addition, several studies have reported the isolation of several potentially pathogenic FLA (Caumo et al., 2009; Alves et al., 2012; Behniafar et al., 2015;) and others with proven pathogenicity inex-vivo and in-vivo trials (Brown and Cursons, 1977; Janitschke et al., 1980; Rivera et al., 1983; Rivera et al., 1993; Gianinazzi et al., 2009). Most of these FLA are identified as N. fowleri , Acanthamoeba spp. and Balamuthia mandrillaris . Most isolates of Acanthamoeba spp. reported as pathogens are distributed among the T5, T11, T15, T3 and T4 genotypes, and among them, the T4 genotype is more prevalent in hot springs (Mahmoudi et al., 2015; Fabros et al., 2021) and is associated with most cases ofAcanthamoeba keratitis (Diehl et al., 2021).
The presence and abundance of FLA in swimming pool water clearly indicates that in addition to these microorganisms being resistant to chlorine in the dosage used in the treatment of drinking water (Thomas et al., 2004; Gomes et al., 2020) they are also resistant to chlorine, and other disinfectants in the dosage used for swimming pools and artificial recreational waters (Rivera et al., 1983; Kiss et al., 2014).Acanthamoeba castellanii trophozoites and cysts have been reported to be resistant to exposure for more than 2 h to NaOCl and NaCl at concentrations up to 8 mg/L and 40 g/L, respectively. On the other hand, exposure to the combined effect of NaOCl or NaCl with ultraviolet C (UV-C) radiation resulted in rapid inactivation of trophozoites even when lower concentrations of NaOCl and NaCl were used (Chaúque & Rott, 2021). Cyst inactivation was achieved by twice as long exposure (300 min) to the combined effect of NaOCl or NaCl and UV-C, with redosing of NaOCl. Despite having demonstrated that both methods are effective, and that they have a strong potential to be used in the effective disinfection of swimming pool water, it was found that the use of NaCl is more cost-effective, as it is cheaper, has a residual effect, redosing is not necessary and is simple to apply (Chaúque & Rott, 2021).
The main aspects that constituted limitations for the present study are: the lack of studies carried out in most countries of the world; the heterogeneous distribution of the number of studies among the included countries; difference in FLA identification methods among many studies and discrepancy in the number of samples considered positive by the morphological and molecular method in the same study. The loss of isolates from positive samples in some studies, due to fungal contamination of non-nutrient agar plates prior to molecular identification of the amoebae, was also a limitation.
It is concluded that the prevalence of FLA in swimming pools and recreational waters is high and, therefore, of concern, since there is a risk of contracting infection by pathogenic amoebae or other pathogens (such as fungi, bacteria and viruses) that may be harbored and dispersed by FLA in water (Mavridou et al., 2018). Thus, it is necessary to implement disinfection techniques that are effective in eliminating microorganisms, including FLA, in swimming pools and artificial recreational waters. The use of the combined effect of NaCl and UV-C has great potential to be used to eliminate or minimize the risk of infection by FLA in swimming pools and other artificial recreational waters. The potential risk of infection by FLA in natural recreational waters needs to be routinely quantified by health surveillance. Warning signs need to be placed where there is minimal risk of infection by free-living amoebae, and people using these water bodies need to be educated about the potential risk and possible safety measures. These measures include not diving in recreational waters wearing contact lenses, preventing water from entering the airways and eyes, and avoiding jumping into the water. Health care workers (especially those working near recreational water use sites with risk of infection by FLA) need to be trained to be on the lookout for symptoms suggestive of infection by FLA, especially in summer.