[Molecular diagnosis of a fatal primary amoebic meningoencephalitis in Guadeloupe (French West Indies)]. / Diagnostic moléculaire d'une méningoencéphalite amibienne primitive à l'occasion d'un cas fatal en Guadeloupe.

We report the first case of primary amoebic meningoencephalitis in a 9-year-old boy in Guadeloupe. The outcome was rapidly fatal in 7 days. The patient presumably acquired the infection by swimming and diving in a basin supplied by natural thermal water 1 week before onset of the disease. The possibility of a free-living amoeba infection was suspected both on the negativity of all bacterial and viral initial tests and on the observation of peculiar cells in stained cerebrospinal fluid samples. Although the amoeba was not isolated, Naegleria fowleri could be identified by polymerase chain reaction with specific primers on DNA extracted from frozen cerebrospinal fluid samples. Furthermore, as the internal transcribed spacer (ITS1) region of DNA is variable in length between the different strains of N. fowleri, sequencing of the amplified ITS1 demonstrated that the responsible N. fowleri strain belongs to a common genotype present in the American and European continent.

Characterization of Naegleria species by restriction endonuclease digestion of whole-cell DNA.

Whole-cell DNA in Naegleria spp. and two related genera was examined by restriction endonuclease digestion and fractionation of the fragments by agarose gel electrophoresis. Visual inspection of ethidium bromide-stained gels shows differences in banding pattern between N. fowleri, N. lovaniensis, N. gruberi, N. jadini, N. australiensis, Didasculus thorntoni and Willaertia magna, and between the two subspecies of N. australiensis. Even between strains belonging to the same species differences could be observed. Significant differences were seen between strains of N. fowleri according to the continent of origin, and a hypothesis on the ancestry and the dispersal of N. fowleri was deduced from it. A N. fowleri strain isolated from one of the very few cured human infections showed the most distinct pattern within the species. The considerable variation detected with serological and biochemical techniques between strains of N. australiensis as well as between strains of N. gruberi, was confirmed in the analysis of their whole-cell DNA. With this technique the existence of N. jadini, D. thorntoni, W. magna and two Naegleria strains as separate systematic entities is substantiated.

Occurrence and transfection of a Giardia virus.

The presence of virus-derived RNA was investigated in 38 axenically growing Giardia isolates from different geographic areas. The RNA virus was demonstrated in Giardia strains from humans in the U.S.A., England and the majority of strains from Poland. Two strains isolated respectively from a cat and a cavia also contained it. Giardia strains from humans in Belgium and Israel did not contain this RNA virus. Transfection of the RNA virus was accomplished from English and Polish strains, as well as from the cat isolate to isolates lacking it. Differences were observed both in sensitivity of Giardia strains to transfection and in infectivity of the RNA virus from different Giardia strains. Transfection could be carried out with sonicated Giardia extract as well as with filter sterilized medium in which Giardia strains containing RNA virus had grown. The RNA virus did not replicate in Giardia-free medium. No correlation could be demonstrated between the presence of the RNA virus in Giardia isolates and their in vitro resistance to some antiprotozoal drugs, nor with the fact that the strain originated from symptomatic or asymptomatic carriers. The presence of the RNA virus in Giardia trophozoites did not influence the isoenzyme patterns or restriction endonuclease patterns of repetitive DNA. A correlation may exist with the length of time since the isolation in axenic culture of the strain.

Giardia isolates from primates and rodents display the same molecular polymorphism as human isolates.

Five Giardia isolates from primates and rodents were grown axenically and compared by different electrophoretic techniques. One isolate from a lemur (slow loris) contained a dsRNA virus also found in some of the Giardia of human origin. Using ethidium bromide stained gels and also Southern blots hybridized with a rDNA probe, two profiles of restriction fragment length polymorphism were found in the animal Giardia, which are identical to two profiles found previously in strains of human origin. Isoenzyme and total protein patterns obtained with agarose isoelectric focusing divided the strains in the same two groups. With pulsed field gradient gel electrophoresis, the isolates showed 6-8 chromosomal bands but none of the band patterns were identical. The size of the chromosomes varied from 0.8 to over 3.0 Mb. A ribosomal DNA probe hybridized with different bands.

Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns.

Ribosomal DNA (rDNA) plasmid restriction maps of 10 strains and rDNA hybridisation patterns of 61 additional strains have been used to assess inter- and intra-specific diversity and phylogenetic relationships in the genus Naegleria. The results obtained by this method largely confirm those of previous studies based on a variety of other criteria. They indicate that very little variation exists within the pathogenic species Naegleria fowleri despite its worldwide distribution and that it is closely related to the nonpathogenic Naegleria lovaniensis. Naegleria gruberi is most likely a polyphyletic grouping and care should be taken when using one strain as a reference point for this species. In addition, the two subspecies of the pathogenic Naegleria australiensis arose separately from within the range of variability encompassed by N. gruberi, as did the species Adelphamoeba galeacystis which should probably be assigned to the genus Naegleria. The species Naegleria jadini and Naegleria andersoni are not closely related to any other in the genus based on their rDNA patterns.

Sequence Variation in the Ribosomal Internal Transcribed Spacers, Including the 5.8S rDNA, of Naegleria spp.

The ribosomal internal transcribed spacer (ITS), including the 5.8S rDNA, from the majority of the 11 described species of the amoeboflagellate Naegleria and from Willaertia magna have a size between 300 and 450 bp. In N. jadini and N. minor these products are approximately 750 bp long. The products from strains of the pathogenic N. fowleri vary between 323 and 423 bp. These length variations in N. fowleri are due to insertions of short repeats in the ITS1, causing the elongation of one stem-loop in the putative secondary structure. In all other species the sizes were identical from strains of the same species. In N. jadini and N. minor there are long inserts in the ITS2. Naegleria italica, N. clarki and N. galeacystis have shorter inserts in the ITS2. These inserts cause the elongation of one stem-loop in the putative secondary structure proposed for the ITS2. Because of the small differences in sequence between N. fowleri and N. lovaniensis the ITS does not provide target sequences for specifically identifying the pathogenic N. fowleri. However, differences in ITS1 do allow to distinguish different N. fowleri isolates. The ITS and 5.8S rDNA sequences will be of additional help in describing new Naegleria spp., which becomes more based on molecular data because morphological differences are scarce in these organisms.

The amoeba-to-flagellate transformation test is not reliable for the diagnosis of the genus Naegleria. Description of three new Naegleria spp.

Trophozoites of several isolates from one location in Australia have failed consistently to transform into flagellates, although they display all other characteristics of the genus Naegleria. When changing the standard transformation test, flagellates were produced. In phylogenetic trees derived from partial small subunit ribosomal DNA (SSUrDNA) sequences, one of these strains branches close to a cluster comprising N. clarki, N. australiensis, N. italica and N. jadini. It is proposed that these Australian isolates represent a new species, named N. fultoni (strain NG885). Failing to form flagellates since their isolation, even when different transformation procedures are used, are two Naegleria strains from Chile and Indonesia. In SSUrDNA-based phylogenetic trees the Chilean strain clusters with N. pussardi and the Indonesian strain clusters with N. galeacystis, but the degree of sequence difference from these described species (3.5% and 2.2%, respectively) is sufficient to propose that both of the strains represent new species, named N. chilensis (strain NG946) and N. indonesiensis (strain NG945), respectively. The close relationships between each of the new species and the Naegleria species with which they cluster in SSUrDNA-based trees were confirmed by ribosomal internal transcribed spacer region (ITS) sequence comparisons. In France, several non-flagellating N. fowleri strains were isolated from one location. ITS rDNA sequence comparisons indicated that they correspond to a 'type' of N. fowleri found in both Europe and the USA. A redefinition of the genus Naegleria is proposed as a consequence of these and previous findings.

Primary amoebic meningoencephalitis in a young male from northwestern Mexico.

Primary meningoencephalitis caused by Naegleria fowleri was documented in a 16-year-old male from Mexicali in the state of Baja California in Mexico. In August 1978, seven days after sustaining moderate head trauma while swimming in a shallow, stagnant irrigation ditch on a hot summer day, the patient presented an acute illness with severe headache, fever and convulsions rapidly progressing into a comatose state. Actively moving trophozoites were observed in the spinal fluid on admission. The patient died shortly after admission to hospital on the third day of symptoms. Post-mortem examination revealed a meningoencephalitis with extensive destruction, haemorrhage and numerous parasites involving structures of the posterior fossa. Immunoperoxidase strains of trophozoites in meningeal and cerebellar tissue were positive for N. fowleri KUL and negative for N. gruberi, N. australiensis and Acanthamoeba rhysodes. This appears to be the first documented case of the disease in Mexico.

Naegleria australiensis: experimental meningoencephalitis in mice.

Naegleria australiensis was recently described as a new species of free-living amoeba pathogenic for mice. Infections of human brain by the free-living amoebae N. fowleri and Acanthamoeba spp. are well known. We here describe the clinicopathological features of experimental infection of the central nervous system of mice by N. australiensis. Weanling mice were inoculated intranasally and intracerebrally. The involvement of the nasal mucosa, olfactory neuroepithelium and lobes, cerebrum and cerebellum was detected in haematoxylin-eosin stained paraffin-embedded sections. Amoebic trophozoites sparsely located throughout the central nervous system were shown better by the immunoperoxidase method. Cysts were not detected. The histopathological changes differ from those produced by N. fowleri, especially in the degree of severity. They may be confused with those caused by Acanthamoeba spp. which usually produced subacute and chronic encephalitis with a prolonged clinical course.

Heterogeneity in the sensitivity of stocks and clones of Giardia to metronidazole and ornidazole.

The sensitivity in vitro to metronidazole and ornidazole of 7 stocks and of the cloned lines of 5 stocks of Giardia isolated from humans, rodents and monkeys was studied by the growth inhibition test. All 7 stocks of Giardia, irrespective of the host, differed in their sensitivity to these drugs, commonly used in therapy of human giardiasis. The differences were greater with ornidazole than with metronidazole. The 5 Giardia stocks from which clones were prepared were found to consist of populations with significantly (P less than 0.05) differing sensitivities to ornidazole and metronidazole. There was a positive correlation between high resistance in vitro to both drugs of all clones of one parent stock and treatment failures of giardiasis in the patient from which the parasite stock had been isolated. The spectra of sensitivity of Giardia to anti-giardial drugs may have implications concerning the suspected zoonotic character of human giardiasis.

Naegleria andersoni n.sp., a cosmopolitan amoebo-flagellate, with two subspecies.

A new Naegleria species, N. andersoni, is described that occurs world-wide. The new species consists of two subspecies and can be distinguished from previously described species on the basis of antigenic, isoenzyme and restriction enzyme DNA fragment differences and in maximum growth temperature tolerated. N. andersoni is not pathogenic in experimental animals and shows no cytopathic effect in cell culture. The electrophoretic karyotype differs between strains of the species, and most of all between the two subspecies, N. a. andersoni and N. a. jamiesoni.

Cloning of a 1.8 kb repeated sequence for the identification and comparison of Giardia intestinalis isolates.

Restriction enzyme digestion of bulk DNA from Giardia intestinalis reveals the presence of repeated sequences. A prominent 1.8 kb band in the Alu I profile was cloned into the pUC8 plasmid (pGI7) and used for comparing strains. When blots of DNA of 34 isolates from different geographic areas are probed with pGI7, hybridization with identical intensities can be detected. However, some strains give different hybridization patterns with several restriction enzymes. No hybridization of pGI7 can be detected with DNA from Trypanosoma brucei, Naegleria fowleri, Entamoeba histolytica and Trichomonas vaginalis. Therefore probe pGI7 may be useful in comparing different isolates as well as in screening for G. intestinalis infection.

Geographic origin and spread of pathogenic Naegleria fowleri deduced from restriction enzyme patterns of repeated DNA.

The restriction enzyme patterns of repeated DNA from 20 Naegleria fowleri and 13 N. gruberi strains were compared. On this basis strains of N. fowleri could be easily separated from N. gruberi. Although the restriction enzyme profiles of N. fowleri strains are quite homogenous, strains originating from Europe and from Australia had slightly different patterns. Both European and Australian profiles were found in the USA. In Australia a genetic variant was detected that is the same as the N. fowleri type present in New Zealand. Profiles of strains from India were identical with those from Europe. These results give additional information on the probable origin and dispersal of N. fowleri in the world. Strains of N. gruberi exhibit much more heterogenous banding patterns. Four European N. gruberi strains were, however, nearly identical while two strains from New Zealand had only a single band difference with one restriction enzyme. One Australian strain of N. gruberi was identical to an American strain that has been in culture for almost 30 years. The presence of a virus-like particle in a sister strain of this American isolate did not affect its banding patterns. Other strains of N. gruberi from the USA had diverse restriction enzyme patterns. Adelphamoeba galeacystis has restriction enzyme profiles distinct from those of the Naegleria strains investigated. Isoenzyme analysis in agarose isoelectric focusing confirmed the existence of intraspecific differences in N. fowleri.

A temporary flagellate (mastigote) stage in the vahlkampfiid amoeba Willaertia magna and its possible evolutionary significance.

A temporary flagellate (mastigote) stage has been observed in several isolates of the vahlkampfiid amoeba Willaertia magna. In an Australian isolate studied in detail, flagellates appeared synchronously, although later than in Naegleria fowleri or N. lovaniensis under similar conditions (half-maximal time, t50 = 168 min at 37 degrees C). The flagellates initially have four flagella and lack a cytostome, but undergo several successive divisions, the first of them synchronous, resulting in progressive reduction in cell volume. New flagella appear during and after division, and the number of flagella in daughter cells of later divisions is rather variable. Comparison of these observations with descriptions of other amoeboflagellates confirms that Willaertia is a valid genus. A likely sequence of morphological changes in the evolution of Willaertia and Naegleria from a hypothetical ancestral vahlkampfiid is proposed.

Ribosomal DNA sequences of Glugea anomala, G. stephani, G. americanus and Spraguea lophii (Microsporidia): phylogenetic reconstruction.

The microsporidian species Glugea anomala, G. stephani, G. americanus and Spraguea lophii were compared by using sequence data derived from their small subunit rDNA genes which were amplified by polymerase chain reaction and directly sequenced. These sequence data and published data of G. atherinae were analyzed and were used to infer a phylogenetic tree. The 5 microsporidian fish parasites appeared to be closely related. The higher sequence similarities demonstrated among G. anomala, G. stephani and G. atherinae suggest that these 3 parasites are in fact only 1 species of Glugea. Moreover, the higher sequence similarities between S. lophii and G. americanus support the transfer of the latter Glugea species into the genus Spraguea.

Epidemiological typing of Acanthamoeba strains isolated from keratitis cases in Belgium.

From the corneas of nine keratitis patients and from their contact lenses, contact lens boxes and saline solutions, 15 strains of Acanthamoeba have been isolated. An Acanthamoeba strain was isolated from the swimming pool where one of the patients swam, while in the tapwater of the houses of three patients investigated, no Acanthamoeba could be detected. All the Acanthamoeba isolates from the cornea belong to genotype T4, but are different subtypes of T4. The Acanthamoeba detected on the contact lenses (and/or associated paraphernalia) of a patient are of the same subtype as that isolated from the cornea. The only Acanthamoeba strain isolated from a contact lens which was not related to an Acanthamoeba keratitis infection proved to be another genotype. A strain of Hartmannella from a cornea and two vahlkampfiids isolated from contact lenses had no connection with keratitis. This study confirms that, as found elsewhere, only Acanthamoeba genotype T4 of the 12 known Acanthamoeba genotypes is responsible for keratitis in Belgium. Most cases of Acanthamoeba keratitis cases are due to poor hygiene in the treatment (cleaning and storage) of contact lenses.