A case of successful treatment of cutaneous Acanthamoeba infection in a lung transplant recipient.

Acanthamoeba species are known to cause 2 well-described entities: (1) granulomatous amoebic encephalitis (GAE), which usually affects immunocompromised hosts, and (2) keratitis, which typically follows trauma associated with contamination of water or contact lenses. Less common manifestations include pneumonitis and a subacute granulomatous dermatitis. We describe a case of granulomatous dermatitis secondary to Acanthamoeba infection in a lung transplant recipient and a successful outcome following treatment with lipid formulation of amphotericin B and voriconazole. We believe this is the second case report describing disseminated Acanthamoeba infection in a lung transplant recipient. We also describe successful outcome with a combination of lipid formulation of amphotericin B and voriconazole, drugs that have not been previously reported to treat Acanthamoeba.

Disseminated Acanthamoeba sp. infection in a dog.

Several species of free-living amoebae can cause encephalomyelitis in animals and humans. Disseminated acanthamoebiasis was diagnosed in pyogranulomatous lesions in brain, thyroid, pancreas, heart, lymph nodes, and kidney of a one-year-old dog. Acanthamoeba sp. was identified in canine tissues by conventional histology, by immunofluorescence, by cultivation of the parasite from the brain of the dog that had been stored at -70 degrees C for two months, and by PCR. The sequence obtained from the PCR product from the amoeba from the dog was compared to other sequences in the Acanthamoeba sp. ribosomal DNA database and was determined to be genotype T1, associated with other isolates of Acanthamoeba obtained from granulomatous amebic encephalitis infections in humans.

18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

Acanthamoeba strains isolated from organs of freshwater fishes.

Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.