Free-living amoebae isolated in the Central African Republic: epidemiological and molecular aspects

Among the many species of free-living amoebae infecting humans, only Naegleria fowleri, a few species of Acanthamoeba, Balamuthia mandrillaris recently Sappinia diploïdea and Paravahlkampfia Francina are responsible for human diseases especially deadly encephalitis outside of Acanthamoeba keratitis related. In the Central African Republic (CAR), no studies have previously been conducted about free amoebae and no suspicious cases of encephalitis or amoebic keratitis was reported even though the ecosystem supported the proliferation of these microorganisms. The objective of this study was to identify free-living amoebae present in CAR and to define the molecular characteristic. Bathing sites and cerebrospinal fluid from patients died of bacterial meningitis untagged were explored by culture and PCR and the amplicons were sequenced which allowed to characterize the species found. Only species of the genus Tetramitus, namely T. Entericus, T. waccamawensis and T.sp similar to those already described in the world and not pathogenic for humans were found in bathing sites, the cerebrospinal fluid meanwhile remained negative. Although no pathogen species such as Naegleria fowleri or species of Acanthamoeba have been isolated, this study worth pursuing because this investigation was very limited in space because of the insecurity in the country.


Introduction
Free-living amoebae, unlike parasitic amoebae, complete their entire cycle in nature and do not require a host [1]. Some free-living amphizoic amoebae can, however, accidentally infect humans and cause neurological, ocular and cutaneous infections [2,3]. The main organisms involved are Naegleria, Acanthamoeba, Balamuthia, several amoebae of the genus Sappinia (S. diploidea, S. pedata) and a species of the genus Paravahlkampfia, P. francina, which was recently incriminated in cases of encephalitis [4][5][6]. Some of the cases of encephalitis were opportunistic infections in immunodepressed individuals and consisted of granulomatous encephalitis due to Acanthamoeba and Balamuthia, which evolves chronically and is usually fatal. In contrast, primary amoebic meningoencephalitis due to Naegleria fowleri is an acute condition in healthy children and adults, manifesting several days after infection and rapidly evolving to severe disease in the absence of early treatment. In the Central African Republic (CAR), the presence of free-living amoebae has not been studied, and no suspected cases of primary amoebic meningoencephalitis have been reported, although cases have been reported in Nigeria, Zambia and South Africa [7,8]. The presence in the CAR of hot springs and numerous warm-water lakes with abundant organic matter would indicate that such organisms might exist there also. The objective of this study was to identify and characterize the free-living amoebae that are present and to determine whether they include N. Fowleri and try to assess the risk of the population.

Methods
Context geographic: The CAR is a landlocked country in the heart of the African continent in an intertropical zone. It covers an area of 623 000 km 2 and is bordered on the east by the Republic of Sudan

Results
The temperature of the water at the sampled sites did not exceed 40°C, and that at 47% of the sites was ≥ 30°C (Table 1,Table   2, Table 3). Samples from only eight of the 32 sites were positive on culture. Five of the cultures were from algae and three from filtered water, but the samples of algae and water were from different sites.
Only the cultures incubated at 37°C for 72 h were positive; all those incubated at 44°C were sterile, as were those of CSF. DNA of freeliving amoebae was identified by PCR in seven of the eight positive cultures with JITS primers. Naegleria DNA was found in two samples (ITS primers), but N. fowleri was not identified (Table 4).
Furthermore, no amoebic DNA was found in CSF, and sequencing showed no Naegleria species, only the species of Tetramitus namely T. waccamawensis, T. entericius and .T.
SP. were identified with the analysis of the sequences ( Table 4).
The physicochemical characteristics of the various sites did not offer any clues, as the species found were all of the same genus (Table   1, Table 2, Table 3). The phylogenetic study showed that the species found in the CAR were identical to those found in Australia and the USA and were very similar to other Tetramitus isolated elsewhere in the world (Figure 2).

Discussion
This preliminary search for free-living amoebae in the aquatic environment is the first of its kind in the CAR. It was limited spatially because of the lack of security in the country. The temperature of the water in most of the bodies studied was 25-35°C, and none had a temperature superior at 40°C. No Naegleria or Acantamoeba species was isolated, even though these species are ubiquitous and they are probably present in the CAR. The results show that the conditions at certain sites are favourable for the growth of N. fowleri, with abundant organic matter and a temperature superior at 30°C; the water at two sites was even superior at 35°C [5,8,9]. A case of infection with N.
fowleri described in Guadeloupe occurred after bathing in water at 27°C, and N. fowleri has been found in water at 26.9-34.9°C [10,11]. Almost all the sites studied were compatible with the presence of this amoeba, which would justify continuation of this study. It will therefore be extended to other sites and particularly hot springs, once the security situation improves. In the cases of primary amoebic meningoencephalitis seen in Madagascar and Guadeloupe, the amoebae were visible in fresh CSF, and PCR of frozen CSF showed the presence of N. fowleri [10,12].  [13]. The unidentified strain is also similar to these two species; a further study will be conducted to identify this strain.  Possible diagnosis at the Institute Pasteur of Bangui.

Competing interests
The authors declare no competing interests.

Authors' contributions
Alain Farra was the principal investigator, working from levies on sites, cultivation, the realization of the PCR, the sequence analysis as well as the drafting of this article. Alain Farra was accompanied