Search

Objectives To report the negative fluorescein staining as an early sign of Acanthamoeba keratitis (AK). Methods Case report and brief review of related literature. Report of case A 30-year-old, contact lens wearer, woman presented with mild irritation and pain in the right eye. The best-corrected visual acuity (BCVA) was 20/20 in both eyes. Slit-lamp examination revealed a peripheral corneal infiltration. Empirical antimicrobial therapy was initiated. Within the third day, peripheral corneal opacity regressed but a Y-shaped linear epitheliopathy with a negative fluorescein staining, because of a ridge-like epithelial irregularity, was observed in the central cornea. Clinical findings progressed rapidly. Confocal microscopy revealed hyper-reflective cysts with the typical double-ring sign consistent with AK. Therefore, topical chlorhexidine and propamidine were initiated. Clinical findings regressed subsequently. The final BCVA was 20/20 in both eyes. Conclusion Acanthamoeba keratitis usually manifest as superficial epitheliopathy and progresses to the stroma. Findings may be obscure or atypical; comprehensive and careful examination may reveal mild findings in the early stages.

The aim of this study was to develop an in-vitro topical treatment for Acanthamoeba keratitis (AK) effective against cysts and trophozoites. Qualitative assays were performed with voriconazole, chlorhexidine, propamidine, cellulase, tobramycin, ciprofloxacin and paromomycin as monotherapy and various combinations. Riboflavin with ultraviolet-A (R + UV-A) as monotherapy or combined with voriconazole and moxifloxacin was also tested. Quantitative assays to assess cyst viability after treatment were performed for the chemicals that showed the highest activity in the qualitative assays. Paromomycin and propamidine did not show antiamoebic activity. Regardless of the total dose, no amoebicidal effect was observed for R + UV-A. Tobramycin, ciprofloxacin, voriconazole, chlorhexidine and cellulase were selected for quantitative assays because they appeared to cause greater damage to the structure of amoebae. Chlorhexidine and ciprofloxacin were the most active against Acanthamoeba spp. as monotherapy. Among the combinations evaluated, ciprofloxacin-voriconazole-chlorhexidine showed the greatest amoebicidal activity, with severe damage of the cellular membrane and an important decrease in cell concentration. In summary, ciprofloxacin as monotherapy and in combination with voriconazole and chlorhexidine has been classified as promising treatment. Additional in-vivo studies in animal models and clinical trials in patients with AK should be considered to confirm the efficacy of ciprofloxacin.

Purpose To compare the antimicrobial effect of topical anesthetics, antivirals, antibiotics, and biocides on the viability of Acanthamoeba cysts and trophozoites in vitro. Methods Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461). Test agents included topical ophthalmic preparations of common anesthetics, antivirals, antibiotics, and biocides. Organisms were exposed to serial two-fold dilutions of the test compounds in the wells of a microtiter plate to examine the effect on Acanthamoeba spp. In addition, the toxicity of each of the test compounds was determined against a mammalian cell line. Results Proxymetacaine, oxybuprocaine, and especially tetracaine were all toxic to the trophozoites and cysts of Acanthamoeba spp., but lidocaine was well tolerated. The presence of the benzalkonium chloride (BAC) preservative in levofloxacin caused a high level of toxicity to trophozoites and cysts. With the diamidines, the presence of BAC in the propamidine drops was responsible for the activity against Acanthamoeba spp. Hexamidine drops without BAC showed good activity against trophozoites, and the biguanides polyhexamethylene biguanide, chlorhexidine, alexidine, and octenidine all showed excellent activity against trophozoites and cysts of both species. Conclusions The antiamoebic effects of BAC, povidone iodine, and tetracaine are superior to the current diamidines and slightly inferior to the biguanides used in the treatment for Acanthamoeba keratitis. Translational Relevance Ophthalmologists should be aware that certain topical anesthetics and ophthalmic preparations containing BAC prior to specimen sampling may affect the viability of Acanthamoeba spp. in vivo, resulting in false-negative results in diagnostic tests.

Purpose: To report the first case of Acanthamoeba keratitis treated with oral miltefosine in the United States. Observations: A 17-year-old female with a history of orthokeratology contact lens wear presented after five months of left eye pain, redness, and photophobia. She was previously treated with antivirals and topical corticosteroids for presumed herpetic disease. She was found to have a large central ring infiltrate and corneal cultures were positive for Acanthamoeba. The infection progressed despite hourly PHMB 0.02% and chlorhexidine 0.02%, and oral vorizonazole. The patient was started on oral miltefosine 50 mg 3 times per day. Following one week of treatment, repeat cultures were positive for Acanthamoeba and therefore, the concentration of chlorhexidine was increased from 0.02% to 0.06% and PHMB was changed to propamidine isetionate (Brolene 0.1%). There was definite clinical improvement after five weeks of treatment with oral miltefosine, topical chlorhexidine 0.06% and propamidine isetionate 0.1%. Conclusions and importance: Acanthamoeba keratitis is a challenging entity to treat and often associated with a poor prognosis. Oral miltefosine may offer additional therapeutic benefit in cases of refractory Acanthamoeba keratitis. Keywords: Miltefosine, Acanthamoeba keratitis, Amoebic keratitis

In the current study, baseline sensitivity of Valsa mali to propamidine was determined using 80 strains collected from apple orchards in Shaanxi Province, China. The median effective concentration (EC 50 ) values for propamidine inhibiting mycelial growth ranged from 0.086 to 0.852 μg/mL, with a mean of 0.405 ± 0.137 μg/mL. After treated with propamidine, mycelia were contorted with an increased number of branches, loss of fruiting body production, and decreased cell membrane permeability. Moreover, the enzyme activities of the complexes I, II, IV and ATPase in the mitochondrial respiratory chain were increased significantly, while the enzyme activities of complexes III decreased. Importantly, both on detached leaves and branches of apple trees, propamidine applied at 100 μg/mL exhibited over 75% protective and curative efficacies, which were even better than the efficacies obtained by carbendazim at the same concentration. These results indicated that propamidine could be used as an alternative compound in controlling Valsa canker and mitochondrial respiratory chains might be correlated with the action mode of propamidine. This study encourages further investigation for the action mechanism of propamidine against plant pathogens and the information could be valuable for synthesis of new antifungal drugs with novel modes of action.

Acanthamoeba keratitis is a sight-threatening infectious disease. Resistance of the cystic form of the protozoan to biocides and the potential toxicity of chemical compounds to corneal cells are the main concerns related to long-term treatment with the clinically available ophthalmic drugs. Currently, a limited number of recognized antimicrobial agents are available to treat ocular amoebic infections. Topical application of biguanide and diamidine antiseptic solutions is the first-line therapy. We consider the current challenges when treating Acanthamoeba keratitis and review the chemical properties, toxicities, and mechanisms of action of the available biocides. Antimicrobial therapy using anti-inflammatory drugs is controversial, and aspects related to this topic are discussed. Finally, we offer our perspective on potential improvement of the effectiveness and safety of therapeutic profiles, with the focus on the quality of life and the advancement of individualized medicine.

WOS: 000299519800004, PubMed ID: 21748351, Acanthamoeba keratitis is a painful corneal infection and difficult to treat because no sufficiently efficient drug has yet been available. The aim of the study therefore was to assess the therapeutic potential of miltefosine on Acanthamoeba keratitis-infected hamster eyes. The cornea of hamsters were infected with Acanthamoeba hatchetti, a human corneal isolate. On the fifth day, all the cornea were microscopically examined in order to determine the degree of infections (G, from 0 to 3). Four groups were then prepared: miltefosine (160 mu M); 0.1% propamidine isetionate plus 0.02% polyhexnide; infected control (0.05% ethanol in PBS) and a non-infected control (0.05% ethanol in PBS) groups. The treatment was continued for 28 days. After the treatment, the cornea were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. Miltefosine treatment yielded much higher cure scores than propamidine isetionate plus polyhexanide. On the last day of treatment, 85% of the miltefosine-treated eyes were graded as G0; no changes were observed in the uninfected control group eyes; G3 eyes showed only a partial improvement. Furthermore, no Acanthamoeba cells could be recovered from the miltefosine-treated eye samples. Miltefosine appeared to hold necessary therapeutic properties for the treatment of Acanthamoeba keratitis., Orphanidis Pharma Research GmbH, Vienna, Austria, This work was supported by Orphanidis Pharma Research GmbH, Wilhelminenstr. 91/IIf, 1160 Vienna, Austria. The authors also thank Prof. Ali Fazil Yenidunya for his invaluable contribution in the preparation of the manuscript.

Propamidine, an aromatic diamidine compound, is widely used as an antimicrobial agent. To uncover its mechanism on pathogenetic fungi, Botrytis cinerea as an object was used to investigate effects of propamidine in this paper. The transmission electron microscope results showed that the mitochondrial membranes were collapsed after propamidine treatment, followed that mitochondria were disrupted. Inhibition of whole-cell and mitochondrial respiration by propamidine suggested that Propamidine is most likely an inhibitor of electron transport within Botrytis cinerea mitochondria. Furthermore, the mitochondrial complex III activity were inhibited by propamidine.

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC50 values of mycelial growth ranging from 0.182 to 1.460 μg ml−1, with a mean of 0.79 ± 0.27 μg ml−1. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10−9 and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC50 values of 5.0–15.0 μg ml−1, 15.1–75.0 μg ml−1 and more than 75.0 μg ml−1 respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.

Purpose To report a case of simultaneous Acanthamoeba and Fusarium keratitis associated with no-rub multipurpose contact lens solution and silicone hydrogel contact lens use. Method Observational case report. Results A 39-year-old woman was referred for worsening of a presumed bacterial corneal ulcer in the setting of silicone hydrogellens wear with occasional overnight wear, no-rub multipurpose contact lens solution use, and combined topical antibiotic/corticosteroid treatment. Initial corneal scrapings and culture confirmed Acanthamoeba and Fusarium solani, corroborated by in vivo confocal microscopy findings, yet despite topical chlorhexidine 0.02%, propamidine 1%,neomycin/polymyxin B ointment, and natamycin 5% along with oral itraconazole, the ulcer worsened. Four days after amoebic and fungal therapy initiation, it was discovered that the pharmacy accidentally dispensed neomycin/polymyxin B/dexamethasone, and despite immediate discontinuation, therapeutic penetrating keratoplasty from corneal melting was ultimately required. Corneal histopathology confirmed the presence of amoebic cysts and fungal elements. Conclusions Coexisting infection with Acanthamoeba and Fusarium species can occur in contact lens wear. Atypical infection must be considered in patients with corneal ulcers demonstrating poor therapeutic response in the setting of contact lens wear. Corticosteroids should be used with extreme caution in contact lens–related corneal infections, especially when the diagnosis remains unknown because they can lead to acceleration of active infection and keratolysis.

Background: We designed hybrid molecules between propamidine and benzimidazole in order to retain the antiprotozoal action, but decreasing the toxic effect of the molecule. Objective: Design and prepare 12 hybrids for testing their antiparasitic effect over three protozoa: Giardia intestinalis, Trichomonas vaginalis and Leishmania mexicana, as well as conduct several in silico simulations such as toxicological profile, molecular docking and molecular dynamics in order to understand their potential mode of action. Methods: Hybrids 1-3, 6-9 and 12 were obtained using a chemical pathway previously reported. Compounds 4, 5, 10 and 11 were prepared using a one-pot reduction–cyclization reaction. The in vitro antiparasitic and cytotoxic activities of these compounds were conducted. It was calculated several properties such as toxicity, PK behavior, as well as docking studies and molecular dynamics of the most active compound performed in a DNA sequence dodecamer in comparison with propamidine. Results: Compound 2 was 183, 127 and 202 times more active against G. intestinalis than metronidazole, pentamidine and propamidine. It was eleven times more active than pentamidine against L. mexicana. This compound showed low in vitro mammalian cytotoxicity. Molecular simulations showed a stable complex 2-DNA that occurred in the minor groove, analogous to propamidine-DNA complex. Conclusion: Compound 2, exhibited the higher bioactivity, especially towards G. intestinalis and L. mexicana. This study demonstrated that the replacement of benzimidazole scaffold instead of toxic amidine group in propamidine, results in an enhancement of antiprotozoal bioactivity. The preliminary molecular dynamics simulation suggests that the ligand–DNA complex is stable.

Acanthamoeba keratitis is a rare but serious complication of contact lens wear that may cause severe visual loss. The clinical picture is usually characterised by severe pain, sometimes disproportionate to the signs, with an early superficial keratitis that is often misdiagnosed as herpes simplex virus (HSV) keratitis. Advanced stages of the infection are usually characterised by central corneal epithelial loss and marked stromal opacification with subsequent loss of vision. In this paper, six cases of contact lens-related Acanthamoeba keratitis that occurred in Australia and New Zealand over a three-year period are described. Three of the patients were disposable soft lens wearers, two were hybrid lens wearers and one was a rigid gas permeable lens wearer. For all six cases, the risk factors for Acanthamoeba keratitis were contact lens wear with inappropriate or ineffective lens maintenance and exposure of the contact lenses to tap or other sources of water. All six patients responded well to medical therapy that involved topical use of appropriate therapeutic agents, most commonly polyhexamethylene biguanide and propamidine isethionate, although two of the patients also subsequently underwent deep lamellar keratoplasty due to residual corneal surface irregularity and stromal scarring. Despite the significant advances that have been made in the medical therapy of Acanthamoeba keratitis over the past 10 years, prevention remains the best treatment and patients who wear contact lenses must be thoroughly educated about the proper use and care of the lenses. In particular, exposure of the contact lenses to tap water or other sources of water should be avoided.

OBJECTIVES T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against pathogenic fungi, particularly Candida albicans. We previously reported that T-2307 uptake was mainly mediated by a saturable high-affinity carrier at the MIC for C. albicans. Since we hypothesized that the potent anticandidal activity arose from accumulation via the high-affinity carrier, we characterized the specificity and kinetic features of the carrier. METHODS The MICs of T-2307 for C. albicans strains were evaluated in the presence and absence of potential competitive substrates. The cells were exposed to [(14)C]T-2307, [(14)C]spermine or [(14)C]spermidine in the presence of unlabelled T-2307, pentamidine, propamidine, or competitive substrates if necessary, and the radioactivity in the cells was measured. C. albicans gene deletion was performed using a one-step PCR-based technique. RESULTS Coapplication with exogenous spermine or spermidine decreased the antifungal activity and uptake of T-2307 in C. albicans strains. T-2307 competitively inhibited spermine and spermidine uptake with inhibition constants similar to its Km for the high-affinity carrier. The comparison of MICs and kinetic values between T-2307 and other diamidine compounds suggested that the different antifungal properties could be partially attributable to the variations in their affinity with the carrier. Studies of gene deletion mutants revealed that T-2307 was transported into C. albicans by a high-affinity spermine and spermidine carrier regulated by Agp2. CONCLUSIONS Uptake of T-2307 via the high-affinity spermine and spermidine carrier regulated by Agp2 could contribute to its potent antifungal activity. Further investigation is required to identify the high-affinity carrier for potential targeting with novel therapies.

Background This study was done to evaluate the visual and anatomical outcomes of topical regenerating agents as a novel therapy for neutrophic corneal ulcer (NCU) secondary to acanthamoeba infection. Findings A 20-year-old woman with a history of contact lens wear was referred to our hospital for keratitis after responding poorly to conventional treatment. In vivo confocal microscopy images suggested acanthamoeba keratitis with double-walled cysts in the anterior corneal stroma. Acanthamoeba infection was confirmed by laboratory findings. She was started on 0.1 % propamidine and 0.02 % chlorhexidine drops every hour. The antibiotic and antifungal drops were stopped when bacterial and fungal cultures proved negative. A central neurotrophic corneal ulcers (NCU) appeared, and despite treatment with artificial tears, bandage contact lens, and autologous serum, the ulcer worsened and she was treated with topical CACICOL20 (1 drop every 2 days) for 8 weeks. The corneal defect was completely repaired in 3 weeks. The treatment was well tolerated, and no local or systemic side effects were noted. Visual acuity remained 20/400. Two months later, the defect was still closed and the patient continued with 0.1 % propamidine and 0.02 % chlorhexidine drops, bandage contact lens, artificial tears, and autologous serum. Conclusions Topical regenerating agents interact with components of the extracellular matrix, binding matrix proteins and protecting them from proteolysis, restoring the matrix environment, and improving tissue healing. In this case, CALCICOL20 was effective for vision stabilization, wound healing, and was well tolerated for NCU secondary to acanthamoeba infection.

Gezielte Anamneseerhebung und Spaltlampenmikroskopie erlauben in aller Regel die Diagnose einer Akanthamobenkeratitis bereits im Fruhstadium. In Zweifelsfallen konnen konfokale Mikroskopie, mikrobiologische und histopathologische Untersuchungen zur Sicherung der Diagnose beitragen. Mit konservativen Masnahmen (lokale Kombinationstherapie mit Aminoglykosiden, Polyhexamethylenbiguanid 0,02 %, Propamidinisoethionat 0,1 %) kann im Fruhstadium eine vollstandige Visusrehabilitierung erreicht werden. Im Spatstadium ist jedoch eine perforierende Keratoplastik oft unumganglich. An early diagnosis of an acanthamoeba keratitis can usually be made on the basis of the patient’s history and slit-lamp examination. Further helpful diagnostic steps are confocal microscopy, microbiological, and histopathological examination. In the early stages topical application of antimicrobials (combined application of aminoglycosides, polyhexamethylene biguanide 0.02 %, propamidine isoethionate 0.1 %) is effective. In the late stages a penetrating keratoplasty is often necessary.

The aromatic diamidines are a series of chemicals with high basicity. Among other properties they have, is that of intervening in the metabolism and transport of polyamines, since they inhibit s‐adenosyl‐L‐methionine decarboxylase (SAMDC) and diamine oxidase (DAO). They are of importance in pharmacology. Examples of this are: that pentamidine is used, for instance, in treating pneumonia in patients affected by the human immunodeficiency virus (HIV), that propamidine is utilized, among other things, to treat cornea infections, and that berenil is employed in veterinary medicine, though not in humans. Owing to their considerable interest, further chemicals belonging to this family are constantly being synthesized, with the aim of ensuring greater pharmacological capacities, better stability, and fewer secondary effects. It is essential to have available analytical methods for detecting these drugs in a range of media. In this study, attention was paid to nine aromatic diamidines that are heads of s...

Purpose To characterize the risk factors, clinical course, treatment outcome and the association between in vivo resistance and in vitro sensitivity for subjects with persistently culture-positive Acanthamoeba keratitis. Design Retrospective noncomparative case series. Participants Eleven subjects with repeatedly positive cultures for Acanthamoeba treated between January 1990 and December 2000, were reviewed. Only subjects with 2 or more positive cultures, availability of the clinical data, and availability of the last Acanthamoeba isolate were included in this study. Methods The medical records were analyzed, and the last isolate from each case was tested in vitro for the antiamoebic drugs used clinically: polyhexamethylene biguanide (PHMB), chlorhexidine, propamidine and hexamidine. Main outcome measures Risk factors, the clinical outcome and in vitro cysticidal drug sensitivity assay. Results Eleven subjects (11/180, 6.1%) had 2 or more positive cultures of whom 8 eyes of 8 subjects (8/180, 4.45%) were included in this study. Seven of eight (87%) subjects were diagnosed over 1 month from onset (late diagnosis). The most common presenting findings were diffuse stromal infiltrate (5/8, 62.5%), ring infiltrate (5/8, 62.5%), and corneal ulceration (3/8, 37.5%). The clinical course of the disease in all subjects consisted of recurrent episodes of corneal and scleral inflammation, with a mean duration of 13.4 ± 9 months. All subjects received PHMB, and 5/8 (62.5%) chlorhexidine too; hexamidine was used in combination in 6/8 (75%), and propamidine in 1/8 (12.5%). All subjects had topical steroids, and 5/8 (62.5%) systemic immunosuppression. The disease resolved with corneal scarring in 3/8 (37.5%) subjects, corneal (or impending) perforation treated with therapeutic keratoplasty in 4/8 (50%), and enucleation in 1/8 (12.5%). Final visual acuity was 0.43 ± 0.37. In vitro most isolates were resistant to propamidine, hexamidine was cysticidal in high concentrations, and PHMB and chlorhexidine had excellent sensitivity profiles. Conclusions In our large series of Acanthamoeba keratitis with a positive microbiologic diagnosis at presentation, nearly 5% developed recurrent episodes of corneal and scleral inflammation with viable Acanthamoeba in the cornea despite prolonged treatment with biguanides and/or diamidines. There was no correlation between in vitro drug sensitivities and the in vivo response for biguanides.

The treatment of Acanthamoeba keratitis has now been possible since the first successful therapy developed in the mid 1980s with a combination of propamidine 0.1% (Brolene) and neomycin 1%. However, only half the patients responded to this regimen as the cysts were often resistant to neomycin and relatively insensitive to propamidine. This led to research for better therapy, culminating in the mid 1990s with research in Glasgow demonstrating much increased effectiveness with use of the biguanide chlorhexidine 0.02% and in London and Bristol for similar effectiveness with the polymeric polyhexamethylene biguanide (PHMB) 0.02%. Both biguanides were combined with propamidine for enhanced effectiveness but were also shown to be effective as monotherapy. While this therapy inactivates the trophozoites and cysts in Acanthamoeba keratitis in the majority of patients (approximately 90%), there have been notable failures particularly when presentation is late with deep stromal infection. Additional highly acanthamoebicidal drugs are needed that can penetrate the stroma for synergistic action. This role may be taken up by certain antineoplastic drugs, such as alkylphosphocholine-1 (Miltefosine), that also have antiprotozoal activity.

The trypanocidal action of pentamidine is dependent on the rapid, selective accumulation of this drug by the parasite. We have investigated pentamidine transport by the bloodstream and procyclic life cycle stages of Trypanosoma brucei brucei. In bloodstream forms, 50 to 70% of [(3)H]pentamidine was transported by an adenosine-sensitive pentamidine transporter (ASPT1) that displayed a K(m) value of 0.26 +/- 0.03 microM and K(i) values of 0.45 +/- 0.04 and 2.5 +/- 0.8 microM for adenine and berenil, respectively. These values are very similar to those for inhibition of [(3)H]adenosine uptake by the P2 adenosine/adenine transporter, suggesting that ASPT1 and P2 may be identical. The remaining 30 to 50% of [(3)H]pentamidine transport was mediated by a low-capacity high-affinity pentamidine transporter (HAPT1) and a high-capacity low-affinity pentamidine transporter (LAPT1), with K(m) values of 36 +/- 6 nM and 56 +/- 8 microM, respectively. HAPT1 was inhibited by propamidine but displayed only low affinity to berenil and stilbamidine, whereas LAPT1 was not inhibited by any of these diamidines. Neither transporter was inhibited by melarsen oxide. In procyclics, an HAPT1-analog (procyclic pentamidine transporter; PPT1) was characterized, but no adenosine-sensitive pentamidine transport could be detected. Treatment with ionophores revealed that PPT1 may be a proton/pentamidine cotransporter.

A study was undertaken of the effects of varying those parameters that most affect analyses by capillary zone electrophoresis (CZE), electrolyte buffer, concentration of electrolyte, pH, voltage, temperature, and diameter of capillary, in respect to the following aromatic diamidines of therapeutic value: pentamidine, hydroxystilbamidine, propamidine, 6-amidino-2-(4-amidinophenyl) indole dilactate (DAPI), and stilbamidine. The data obtained permit the design of specific analytic methodology for each of these chemicals under differing conditions. As a demonstration, an analytical method is proposed in which all these chemicals are analyzed, simultaneously, in serum and urine under the following CZE conditions: citrate buffer 25 mM, pH = 3.75, T = 30°C, and V = 14 kV. The detection limits for the method for serum and urine are (μg/mL): pentamidine, 0.3; hydroxystilbamidine, 0.4; propamidine, 0.2; DAPI, 0.3; and stilbamidine, 0.3. Oasis HLB cartridges were used for prior treatment of both serum and urine samples.

Purpose: To determine the in vitro drug susceptibility of Australian corneal isolates of Acanthamoeba and to correlate the results with patient treatment and visual outcome. Methods: Acanthamoeba isolates were obtained from a reference laboratory. Cyst suspensions were prepared from 19 strains and exposed to 10 antimicrobial agents for 7 days. The minimum drug concentrations required to inhibit excystation were determined. Inhibited cells were then plated out to determine minimum cysticidal concentrations. Results: Overall, propamidine proved to be the most active anti-Acanthamoeba agent tested. The disinfectant polyhexamethylene biguanide, either pure or in Baquacil, was also effective. Pentamidine, hexamidine, chlorhexidine and chloroxylenol had intermediate activity, while neomycin, amphotericin B and povidone-iodine had poor activity. There was no clear relationship between in vitro susceptibility and visual outcome. Conclusions: Propamidine and polyhexamethylene biguanide drops are recommended as initial choices for the treatment of Acanthamoeba keratitis. The variability in the susceptibility to any one agent suggests that individual testing of isolates is necessary to identify the most appropriate treatment. A number of factors influence visual outcome in these cases; further studies are required to resolve the importance or otherwise of in vitro susceptibility.

Purpose To characterize patients with Acanthamoeba keratitis and to evaluate the safety and efficacy of propamidine isethionate 0.1% ophthalmic solution (Brolene) when administered concomitantly with neomycin-polymyxin B-gramicidin ophthalmic solution (Neotricin) in the treatment of Acanthamoeba keratitis. Design Prospective, noncomparative case series. Methods The authors report the clinical characteristics and outcomes of patients who entered this multicentered, open-label, clinical trial. Eighty-three patients with Acanthamoeba keratitis representing 87 infected eyes entered the trial. Results Sixty (69%) of the 87 eyes enrolled had data analyzed for treatment efficacy and safety. Of these 60 eyes, 50 (83%) experienced treatment success. Thirty (60%) patients successfully treated adhered to treatment protocol guidelines. Patients who broke protocol had disease exacerbation during the maintenance therapy phase. The only eyes lost/enucleated were 7 of 17 in which penetrating keratoplasty was performed before eradication of the infectious agent. Conclusion Propamidine isethionate and neomycin are an effective treatment for Acanthamoeba keratitis. Penetrating keratoplasty should be performed only for visual rehabilitation and not to "debulk" an active infection. The authors advocate treating patients with topical medications, mainly Brolene, until all organisms are eradicated. There should be no signs of infection for at least 3 months in the patients not receiving antiamebic medications before penetrating keratoplasty is performed.

Macrophages in the liver (Kupffer cells) can be depleted by a single intravenous injection with liposome encapsulated clodronate, an anionic bisphosphonate or by liposome encapsulated propamidine, a cationic diamidine. In the present study, it was investigated whether the efficacy of the liposome mediated depletion of Kupffer cells could be enhanced by modification of the phospholipid bilayers. The efficacy of liposome mediated depletion was evaluated by comparison of the percentages of ED2-positive Kupffer cells in the rat liver, depleted by the drugs encapsulated in the liposomes at different concentrations. It is demonstrated that anionic control liposomes, containing no drug at all and composed of phosphatidylcholine (PC), phosphatidylserine (PS) and cholesterol (C) in a molar ratio of 3:3:1, reduced the percentage of ED2-positive Kupffer cells in the rat liver to about 20% of their normal numbers. At certain drug concentrations, anionic liposomes are shown to be efficacious carriers for intraphagocytic delivery of clodronate, but not for propamidine. Optimal efficacy of the latter drug was achieved by encapsulation into neutral liposomes. Omission of cholesterol or mannosylation of the phospholipid bilayers did not improve the efficacy when compared to neutral liposomes. At high concentrations of encapsulated drugs, all liposome formulations induced a nearly complete depletion of Kupffer cells (>95%).

The hydration of the d(CGCAAATTTGCG)2 duplex and its complex with a propamidine reporter ligand has been examined in aqueous solution by two-dimensional NMR at two spectrometer frequencies and three temperatures. Quantitative analysis of ROESY and NOESY cross-peaks showed effective correlation times of approximately 0.5 ns at 283 K for DNA-water interactions in the major groove. In some cases the sign of the NOE inverts on changing either the temperature or spectrometer frequency. Larger effective correlation times of approximately 1 ns were observed for water interactions with A5(H2) and A6(H2) atoms located in the minor groove. Interproton NOEs and changes in chemical shifts showed that propamidine binds in the minor groove 5'-AATTT region of the host duplex, but does not displace waters adjacent to either A5(H2) or A6(H2). In the complex, the effective correlation times of these waters increase more than two-fold, possibly as a result of stabilisation due to H-bonded interaction with the amidine groups of the ligand. Hydration of the bound molecule was also found, suggesting that water may contribute to the DNA binding process for bis(amidine) drugs. Structure refinement by a NOE-restrained dynamic annealing procedure revealed that ligand binding is non-centrosymmetric with respect to the duplex, in accordance with the energetically favoured 5'-ATT (= 5'-AAT) sites predicted by analytical molecular modelling. In particular, the bound propamidine spans 3-4 base pairs in the A6-T7-T8 tract and makes close H-bonded contacts with A(N3/O4) acceptors positioned close to the minor groove floor. The refined NMR structure for the DNA-propamidine complex is compared with that determined recently using X-ray crystallographic methods.

A molecular modelling strategy has been developed to identify potential binding sites for bis(amidine) ligands in the minor groove of duplex DNA. Calculations of interaction energy for propamidine and pentamidine with d(CGCGAAT TCGCG)2 show that this duplex contains two symmetrically equivalent binding sites of identical affinity, each displaced by 0.3-0.4 bp from the centre of the AT segment. The ligands occupy groove sites spanning approximately 4 and 4-5 bp, respectively with asymmetric binding to the 5'-AATT sequence. The DNA-bis(amidine) interactions have been examined by high-resolution 1H-NMR. The patterns of induced changes in DNA proton chemical shift and the DNA-ligand NOEs confirm that both agents bind in the AT minor groove in a non-centrosymmetric fashion. Detailed structures were determined for each complex using a NOE-restrained simulated annealing procedure, showing that the B-type DNA conformation is not significantly altered upon complexation with either ligand. The free DNA duplex has previously been shown to be extensively hydrated in the minor groove [Kubinec, M.G. and Wemmer, D.E. (1992) J. Am, Chem. Soc. 114, 8739-8740 Liepinsh, E. Otting, G. and Wüthrich, K. (1992) Nucleic Acids Res. 20. 6549-6553]. We detect hydration water close to the A(H2) protons in the presence of propamidine, which may stabilise certain waters against exchange. This conclusion supports recent crystallographic analyses, suggesting that such ligands may use water molecules to bridge between amidinium protons and host DNA bases Details of the ligand interactions with AT-tract DNA duplexes can now be compared for the subsequences 5'-AAT, 5'-AATT and 5'-AAATTT.

Purpose To evaluate the effectiveness of topical agents for the treatment of Acanthamoeba keratitis (AK). Design Laboratory research. Participants Fifty-six Acanthamoeba isolates from 56 patients with clinically proven AK were studied. Methods The effectiveness of 7 agents against Acanthamoeba cysts was determined in vitro. The agents were 1.0% povidone-iodine, 0.05% benzalkonium chloride (BZC), 0.02% chlorhexidine gluconate (CHG), 0.1% propamidine isethionate, 0.02% polyhexamethylene biguanide (PHMB), 5.0% natamycin, and 1.0% voriconazole (VRCZ). These concentrations are those recommended for patients. In addition, 10-fold dilutions of each of the agents were tested. After exposing the cysts to each agent at 35°C for 1 hour or 24 hours, the agents were removed by centrifugal washing. The exposed cysts were observed by optical microscopy for 7 days. In addition, the fine structures of the exposed isolates were examined by transmission electron microscopy (TEM). The genotype of the isolates was determined by 18S rDNA fragment sequencing. Main Outcome Measures The in vitro susceptibility was determined by complete growth inhibition, and the morphologic appearance was determined by TEM. The genotypes of the 56 isolates were determined by 18S rDNA fragment sequencing. Results The Acanthamoeba cysts were most susceptible to natamycin, followed by povidone-iodine, BZC, PHMB, propamidine, and CHG. None of the strains was susceptible to VRCZ. The susceptibilities to PHMB and CHG may be time dependent and to propamidine may be concentration dependent. Transmission electron microscopy showed changes in the inner structure of the cysts exposed to natamycin and povidone-iodine. The Acanthamoeba genotype was T4 in 52 isolates, and cysts with the same genotype had different agent susceptibilities. Conclusions Natamycin and povidone-iodine had excellent cysti-static (or cystcidal) effects, and PHMB and propamidine did not. There was no correlation between agent effectiveness and Acanthamoeba genotype. Therefore, susceptibility tests of isolates are needed to choose the most appropriate agent, and our results can be a guideline for choosing the most appropriate agent for immediate empirical treatment of AK.

A method of reverse phase high performance liquid chromatography (RP-HPLC) was established to analyze the dissipation of propamidine residue in tomato. Residue of propamidine was extracted from tomato using methanol buffered and determined by RP-HPLC with UV detection at 262 nm. The results showed that the average recoveries of the samples fortified with propamidine at the concentration range of 25 to 300 mg kg-1 ranged from 87.972 to 106.341% with a relative standard deviation ranged between 0.169 to 3.503%. Initial deposit ranged from 2.45 to 5.70 mg kg-1. The dissipation of propamidine in tomato followed the first order kinetic equation. The dissipation rate constants in tomato treated with recommended and double recommended dose applied at 4 times and 2 times ranged from 0.110 to 0.151 days, and the corresponding half-lives from 4.589 to 6.300 days. At the day 14 after the last application the residue concentrations of propamidine in tomato ranged from 0.42 to 0.54 mg kg-1 from the two blocks for all treatments. These propamidine residues dissipated below the limit of detection of 0.07 mg kg-1 28 days after the last treatment. The results presented in this work and the low toxicity of propamidine for environment proved that propamidine will not pose any residual toxicity problem after 14 days of application and tomato fruits could be used safely for human consumption.

The first medical cure of Acanthamoeba keratitis was obtained by use of propamidine isethionate. Since then, it has been the basic drug recommended for use in treatment. Because some Acanthamoeba strains have been reported to be resistant to propamidine and propamidine was found to be only weakly cysticidal, superior homologs such as butamidine, pentamidine, hexamidine, heptamidine, octamidine, and nonamidine were tested for their amoebicidal effects on two Acanthamoeba strains isolated from patients with keratitis. Trophozoicidal and cysticidal efficiencies were found to be increased from propamidine to nonamidine; i.e., when the alkyl chain connecting the two benzene rings in their molecular structures was elongated, in comparison with propamidine, hexamidine and octamidine were the most amoebicidal molecules. As a result of these data, a kinetic study carried out on propamidine, hexamidine, and octamidine demonstrated that the amoebicidal effects resulted from two events: the diffusion of molecules through the plasma membrane or the double wall of trophozoites or cysts, respectively, and the lethal effects of molecules on amoebic protoplasm. The diffusion kinetics were increased when the alkyl chain was elongated, i.e., with an increase in the lipophilic properties of molecules. In contrast, the lethal effect kinetics were found to be unchanged by this elongation, indicating that they originated from the cationic surface-active properties induced by the protonated amidine groups attached to each benzene ring, which themselves remained unchanged from one molecule to the other. These results strongly advocate the immediate replacement of propamidine by hexamidine in the medical treatment of Acanthamoeba keratitis; in France, 0.1% hexamidine eyedrops are available (Desomedine). The results also advocate clinical investigations on the efficiency and toxicity of octamidine, which appears to be the most amoebicidal diamidine in vitro.

Purpose. To describe an immunocompetent male with bilateral microsporidial keratoconjunctivitis who responded to treatment with albendazole, propamidine, and fumagillin. Methods. Corneal and conjunctival epithelial scrapings from a man with bilateral keratoconjunctivitis previously treated with topical corticosteroids were evaluated by Gram stain and by fluorescence microscopy. Results. Gram stain and fluorescence microscopy of corneal epithelial scraping revealed organisms characteristic of microsporidia. Results of human immunodeficiency virus antibody testing were reported as nonreactive. Symptoms of ocular discomfort and clinical signs of keratoconjunctivitis resolved after five weeks of treatment that included systemic albendazole and topical propamidine isethionate 0.1% and fumagillin bicyclohexylammonium salt. A follow-up conjunctival scraping failed to detect any residual organisms 2 weeks after cessation of all treatment. Conclusion. Microsporidial ocular infection occurred in an immunocompetent non-contact lens wearer. Microsporidial keratoconjunctivitis should be considered in any individual with atypical multifocal diffuse epithelial keratitis, regardless of immune status or recent history of contact lens wear.

Purpose Acanthamoebae are ubiquitous free-living amoebae. As facultative pathogens, they are the causative agents of Acanthamoeba keratitis (AK), a sight-threatening ocular surface infection. AK can have a favorable prognosis when diagnosed and treated early in the disease course but available treatment options can remain ineffective even when started early. Methods Case presentation Results AK can have a favorable prognosis when diagnosed and treated early in the disease course but available treatment options can remain ineffective even when started early. We present a case of AK that was successfully treated with topical and systemic miltefosine after showing sight-threatening recurrences under recommended therapy including a combination of propamidine 0.1%, miconazole nitrate 1%, neomycin, diamide and cationic antiseptics over a 12 months period. Conclusion In previous studies, miltefosine (hexadecylphosphocholine), an alkylphosphocholine, approved for the oral and topical treatment of leishmaniasis, proved to be highly active against Acanthamoeba in vitro [Walochnik et al. 2002]. This has been confirmed by several other studies [e.g. Schuster et al. 2006, McBride et al. 2007, Walochnik et al. 2009, Polat et al.2012].

The effects of Brolene Tm on adherence of Candida albicans blastospores to buccal epithelial cells and on hyphal development were investigated in vitro. Brolene TM significantly reduced the number of adherent blastospores/ epithelial cell, increased the number of ‘clear’ epithelial cells, decreased percentage germination of blastospores and subsequent hyphal development.

Chromatographic separations of the components of polymyxin B sulphate are reported and it is shown that the separations are such that two of the three major components detected are specific for the intact drug in the presence of chemical decomposition products. The specificity of the separations with respect to other antibacterials, propamidine, dibromopropamidine and trimethoprim is also reported. Validation of the stability indicating assay based on this separation is described. Rate constants for the decomposition of polymyxin at various temperatures between 32 and 52°C and in solutions of different pH values from 2.0 to 10.3 are determined and the t 90 value at pH 7 and 4°C is estimated.

We developed an intensive treatment regimen of topical neomycin, propamidine, and polyhexamethylene biguanide that was tapered to a maintenance level over a 14- to 28-day period as toxicity developed. Since July 1991, we used this treatment on six eyes of five patients in whom Acanthamoeba keratitis was diagnosed clinically. All patients had positive cultures for microorganisms from their corneas or contact lens cases or had pathognomonic findings of pseudodendritic subepithelial infiltrates and radial keratoneuritis. After therapy, all patients improved within two to four weeks, with regression or resolution of neuritis and infiltrates, healing of epithelial defects, and lessening of pain. By three to four months, visual acuity had returned to 20/20 in all eyes. We believe the addition of polyhexamethylene biguanide to our treatment regimen in Acanthamoeba keratitis dramatically aided and hastened the clinical improvement in five consecutive patients and may, with early diagnosis, increase the number of medical cures.

A case of a 59-year-old Spanish patient who presented with severe ocular pain, blurred vision, eyelid swelling and foreign body sensation in the right eye is reported. She was a regular gas permeable contact lens [corrected] wearer who initially claimed to maintain standard lens care. After exploration, conjunctival injection, dendritiform corneal ulcers and stromal edema were observed. She was initially treated for a possible viral keratitis due to herpes simplex virus using 3% topical acyclovir and 0.1% dexamethasone eye drops 5 times a day. The patient did not respond to this treatment and six weeks later, corneal scrapings were positive for Acanthamoeba genotype T11. She was then treated with chlorhexidine 0.02%, propamidine 0.1% and 1% cycloplegic eye drops hourly which resulted in a significant improvement. After a month, ocular pain decreased and the clinical signs of keratitis ameliorated observed as a diminution of the size of the ulcer and also in the extension and opacity of the corneal infiltrates. The patient has been following this treatment for 3 months and it is possible that she will have to carry on with it for a whole year. To the best of our knowledge, this is the first case of severe keratitis due to Acanthamoeba genotype T11 in Spain .

The synthesis of 1,3-bis-[3,4,5,6-tetrahydroxyazepane-N-p-phenoxy] and 1,3-bis-[3,4,5,6-tetrahydroxyazepane-N-p-benzyloxy] propanes is reported. These compounds have been prepared to investigate the potential of incorporating iminosugars as useful recognition elements in DNA minor groove binding agents. The compounds were shown to have very moderate binding affinities for DNA in thermal denaturation and ethidium bromide displacement assays when compared with propamidine. They were also found to possess some in vitro anticancer activity that did not correlate with their DNA binding affinity.

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.