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.