José E. Piñero

Acanthamoeba keratitis: an emerging disease gathering importance worldwide?

Acanthamoeba keratitis (AK) is increasingly being recognized as a severe sight-threatening ocular infection worldwide. Although contact lens wear is the leading risk factor for AK, Acanthamoeba parasites are also an important cause of keratitis in non-contact lens wearers. Diagnosis of AK is challenging, and the available treatments are lengthy and not fully effective against all strains. The pathogenesis of Acanthamoeba is still under study, and the identification of the key factors involved in this process should be useful for the development of fully effective therapies. This review focuses on recent developments on AK pathogenesis and diagnosis as well as novel strategies for the evaluation of anti-amoebic agents that could be applied in the near future against these pathogens.

Antileishmanial activities of dihydrochalcones from piper elongatum and synthetic related compounds. Structural requirements for activity

Two dihydrochalcones (1 and 2) were isolated from Piper elongatum Vahl by activity-guided fractionation against extracellular promastigotes of Leishmania braziliensis in vitro. Their structures were elucidated by spectral analysis, including homonuclear and heteronuclear correlation NMR experiments. Derivatives 3-7 and 20 synthetic related compounds (8-27) were also assayed to establish the structural requirements for antileishmanial activity. Compounds 1-11 that proved to be more active that ketoconazol, used as positive control, were further assayed against promastigotes of Leishmania tropica and Leishmania infantum. Compounds 7 and 11, with a C(6)-C(3)-C(6) system, proved to be the most promising compounds, with IC(50) values of 2.98 and 3.65 microg/mL, respectively, and exhibited no toxic effect on macrophages (around 90% viability). Correlation between the molecular structures and antileishmanial activity is discussed in detail.

PCR-ELISA for diagnosis of mucocutaneous leishmaniasis

In this work we demonstrate that the PCR-ELISA technique is sufficiently sensitive and specific for use as a diagnostic test in cases of mucocutaneous leishmaniasis. DNA was extracted from cultures of Leishmania braziliensis, Leishmania infantum, Leishmania tropica, Leishmania mexicana, Trypanosoma cruzi, and blood samples from individuals who presented a clinical diagnosis of leishmaniasis as well as from healthy individuals. The DNA was PCR amplified and the product obtained was hybridised with a biotin-labelled probe, the sequence of which was designed in our laboratory. The result of the hybridisation was visualised by means of an ELISA technique using antifluorescein antibody labelled with alkaline phosphatase and p-nitrophenylphosphate (pNFF) as chromogen. The optical density of the products of the pNFF hydrolysis was quantified in a spectrophotometer at a wavelength of 405 nm. Using this technique the percentage of detection was 83.3% in blood samples from patients clinically diagnosed as having mucocutaneous leishmaniasis. No false positive results were obtained.

Isolation and characterization of Acanthamoeba strains from soil samples in Gran Canaria, Canary Islands, Spain.

Free-living Amoebae of Acanthamoeba genus include non-pathogenic and pathogenic strains that are currently classified in 18 different genotypes, T1–T18. In this study, a survey was carried out to evaluate the presence of Acanthamoeba strains in soil samples collected between 2012 and 2013 in Gran Canaria Island, Canary Islands, Spain. Samples were inoculated onto non-nutrient agar (NNA) plates and were checked for the presence of Acanthamoeba. Identification of Acanthamoeba strains was based on the morphology of the cyst and trophozoite forms. Subsequently, positive samples were cloned for their molecular characterization at the genotype level by sequencing the DF3 region located in the 18S rDNA gene of Acanthamoeba as previously described. Sequencing results revealed the presence of T2, T5 and T4 genotypes within the studied samples. To the best of our knowledge, this is the first report demonstrating the presence of Acanthamoeba in Gran Canaria Island and the first study at the genotype level in the Canary Islands.

Evaluation of the in vitro activity of commercially available moxifloxacin and voriconazole eye-drops against clinical strains of Acanthamoeba

PurposeAcanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as “Acanthamoeba keratitis” (AK) and, more rarely, an infection of the central nervous system called “granulomatous amoebic encephalitis” (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. Moxifloxacin, a fourth generation fluoroquinolone, has been used with other drugs to treat GAE, but its efficacy as a treatment for AK is not known. Voriconazole has been used to treat AK; however, its cysticidal efficacy is not known. Both drugs are commercially available as eye-drops. The aim of this study was to evaluate the in-vitro activity of these eye-drops against Acanthamoeba compared to two reference drugs (chlorhexidine and amphotericin B) which are currently used to treat AK and GAE.MethodsThe sensitivity of two clinical and one type strain of Acanthamoeba to the commercial concentrations of the four drugs was evaluated with a colorimetric assay. Mature cysts were incubated with voriconazole to determine their sensitivity to this drug. The effects on cell proliferation and cell toxicity were determined using standard procedures with commercial kits.ResultsThe four compounds were active against the Acanthamoeba strains in this study. Although it prevented encystation, moxifloxacin’s amoebicidal activity was low. Voriconazole activity was greater than that of the other drugs, even at a concentration lower than in commercial eye drops. It was effective against cysts and decreased cell proliferation, with low cellular cytotoxicity.ConclusionVoriconazole could be used against AK as a first-line treatment or in combination. Moxifloxacin is an interesting adjuvant to consider as it is effectively prevents encystation of the amoeba which often complicates infection resolution. In addition, moxifloxacin is effective in preventing secondary bacterial infections.

In vitro activity of perifosine: a novel alkylphospholipid against the promastigote stage of Leishmania species

Perifosine is a novel alkylphospholipid. Perifosine has displayed significant antiproliferative activity in vitro and in vivo in several human tumor model systems and has recently entered phase II clinical trials. Other alkylphospholipids have been previously used as antileishmanial agents, and miltefosine (Impavido) is now established as the first oral drug for the treatment of visceral and cutaneous leishmaniasis. Perifosine showed the higher activity against all tested strains. This study demonstrates for, the first time, an in vitro activity of perifosine against different species of Leishmania in the promastigote stage.

Is Balamuthia mandrillaris a public health concern worldwide

Balamuthia mandrillaris is an opportunistic, free-living amoeba that can cause skin lesions and the typically fatal Balamuthia amoebic encephalitis (BAE) both in immunocompromised and immunocompetent individuals. Available data for BAE cases indicate that this disease is difficult to detect because knowledge of predisposing factors is lacking, causing a challenge for diagnosing BAE. The number of reported BAE cases is increasing worldwide, and this is a major concern because little is known about the pathogen, no standardized detection tools are available, and most of the treatments are almost empirical. The recently reported cases, novel diagnostics tools, and successful therapeutic approaches against BAE infections are reviewed here.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Successful Monitoring and Treatment of Intraocular Dissemination of Acanthamoeba

I ntraocular spread of Acanthamoeba after keratitis is rare, being previously documented and microbiologically confirmed in 1 case of chorioretinitis and 1 case of endophthalmitis. Both occurred several months following penetrating keratoplasty (PK), were initially treated as suspected sterile inflammations, did not receive specific oral or intraocular treatment for Acanthamoeba, and finally required evisceration. We report the first documented case, to our knowledge, of successful management of intraocular Acanthamoeba dissemination. Intraocular and systemic treatment was monitored through microbiological testing and polymerase chain reaction analysis of serial aqueous taps.

Acanthamoeba castellanii Neff: In vitro activity against the trophozoite stage of a natural sesquiterpene and a synthetic cobalt(II)-lapachol complex.

In this study, the in vitro activities of a natural sesquiterpene, alpha-cyperotundone, isolated from the root bark of Maytenus retusa and a cobalt(II)-complex of a natural occurring prenyl hydroxynaphthoquinone (lapachol) were evaluated against the trophozoite stage of Acanthamoeba castellanii Neff using a previously developed colorimetric 96-well microtiter plate assay, based on the oxido-reduction of Alamar Blue(R). The obtained activities showed that these two compounds were able to inhibit the in vitro growth of the amoebae at relatively low concentrations. Further identification of the molecular targets of these products and their effects on acanthamoebae should be determined to evaluate their possible therapeutic use.