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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

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.

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.

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 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].

Purpose: To assess the efficacy of chlorhexidine monotherapy for Acanthamoeba keratitis, and to determine the therapeutic outcomes of concomitant topical corticosteroids. Methods: In this prospective interventional case series, 31 eyes of 31 patients with Acanthamoeba keratitis (AK) were treated with chlorhexidine 0.02% as monotherapy, from April 2010 to April 2011. The diagnosis of AK was made based on clinical manifestations and positive confocal microscopic (confoscan 3.4, Nidek Co. Ltd., Gamagori, Japan) results. We report the percentage of a favorable clinical response within two weeks of initiating treatment, worsening of the infection while receiving chlorhexidine, recovery of visual acuity (VA), duration of treatment with chlorhexidine and corticosteroids, necessity for addition of other anti-Acanthamoeba agents, presence of corneal scar at the end of the treatment, and need for penetrating keratoplasty (PK). Results: Two weeks after initiation of chlorhexidine, improvement in signs and symptoms was observed in 26 (83.9%) patients but 3 eyes required the addition of propamidine. After initial improvement in one patient, the infection worsened, necessitating the addition of Polyhexamethylene Biguanide (PHMB) and propamidine. A total of 26 (83.9%) patients received topical corticosteroids with mean duration of 65.8 ± 45.1 days. In 22 (71%) eyes, final visual acuity was ≥0.80. Improved VA occurred in 29 eyes (93.5%). Optical PK was considered in 3 (9.7%) eyes and a corneal scar developed in 8 (25.8%) eyes. Conclusion: Chlorhexidine is effective for monotherapy in AK and could be a good choice for initiating treatment. After the initial response to anti-Acanthamoeba agents, corticosteroids can be used as adjunctive therapy depending on the clinical condition.

The activities of therapeutic agents and myristamidopropyl dimethylamine (MAPD) against Acanthamoeba strains recalcitrant to medical therapy were studied. MAPD minimum cysticidal concentrations were 6.25 to 25 μg/ml; 10 to 30 μg/ml gave at least a 3-log cyst kill after 6 h, and 50 and 100 μg/ml gave at least a 3-log cyst kill within 2 and 1 h, respectively.

Background: The diagnosis of acanthamoeba keratitis has increased since 1985 due to the massive use of contact lenses and a better knowledge of the disease by ophthalmologists. The use of biassociated therapy has resulted in a better prognosis and lower complication rate. Aim: To report patients with acanthamoeba keratitis treated with the association of propamidine (Brolene(r)) and polyhexamethylbiguanide (PHMB) 0.02%. Patients and methods: Retrospective analysis of 27 patients (31 eyes) with acanthamoeba keratitis (bilateral in four cases), diagnosed by culture, biopsy or characteristic clinical features. Results: Ninety six percent of patients used rigid contact lenses. Acanthamoeba cultures were positive in 71% of cases. The delay in the diagnosis was between 1 and 5 months. Early treatment was possible in 29% of patients. Infection was erradicated in all cases with the biassociated therapy. A tectonic keratoplasty to treat a trophic perforation was done in eight eyes. No patient required therapeutic keratoplasty to resolve the infection. Visual acuity at the end of follow up was better than 20/40 in nine patients and in eight it was in the range of count fingers or less. Conclusions: In patients with the clinical picture of acanthamoeba keratitis, early or late antiamoebic treatment is warranted even in the absence of positive cultures. The visual results of the treatment are highly dependent on the precocity of treatment. Prevention is imperative and is based on a strict contact lens hygiene (Rev Méd Chile 2002; 130: 396-401)

Background: The diagnosis of acanthamoeba keratitis has increased since 1985 due to the massive use of contact lenses and a better knowledge of the disease by ophthalmologists. The use of biassociated therapy has resulted in a better prognosis and lower complication rate. Aim: To report patients with acanthamoeba keratitis treated with the association of propamidine (Brolene(r)) and polyhexamethylbiguanide (PHMB) 0.02%. Patients and methods: Retrospective analysis of 27 patients (31 eyes) with acanthamoeba keratitis (bilateral in four cases), diagnosed by culture, biopsy or characteristic clinical features. Results: Ninety six percent of patients used rigid contact lenses. Acanthamoeba cultures were positive in 71% of cases. The delay in the diagnosis was between 1 and 5 months. Early treatment was possible in 29% of patients. Infection was erradicated in all cases with the biassociated therapy. A tectonic keratoplasty to treat a trophic perforation was done in eight eyes. No patient required therapeutic keratoplasty to resolve the infection. Visual acuity at the end of follow up was better than 20/40 in nine patients and in eight it was in the range of count fingers or less. Conclusions: In patients with the clinical picture of acanthamoeba keratitis, early or late antiamoebic treatment is warranted even in the absence of positive cultures. The visual results of the treatment are highly dependent on the precocity of treatment. Prevention is imperative and is based on a strict contact lens hygiene (Rev Med Chile 2002; 130: 396-401)

Background: The diagnosis of acanthamoeba keratitis has increased since 1985 due to the massive use of contact lenses and a better knowledge of the disease by ophthalmologists. The use of biassociated therapy has resulted in a better prognosis and lower complication rate. Aim: To report patients with acanthamoeba keratitis treated with the association of propamidine (Brolene(r)) and polyhexamethylbiguanide (PHMB) 0.02%. Patients and methods: Retrospective analysis of 27 patients (31 eyes) with acanthamoeba keratitis (bilateral in four cases), diagnosed by culture, biopsy or characteristic clinical features. Results: Ninety six percent of patients used rigid contact lenses. Acanthamoeba cultures were positive in 71% of cases. The delay in the diagnosis was between 1 and 5 months. Early treatment was possible in 29% of patients. Infection was erradicated in all cases with the biassociated therapy. A tectonic keratoplasty to treat a trophic perforation was done in eight eyes. No patient required therapeutic keratoplasty to resolve the infection. Visual acuity at the end of follow up was better than 20/40 in nine patients and in eight it was in the range of count fingers or less. Conclusions: In patients with the clinical picture of acanthamoeba keratitis, early or late antiamoebic treatment is warranted even in the absence of positive cultures. The visual results of the treatment are highly dependent on the precocity of treatment. Prevention is imperative and is based on a strict contact lens hygiene (Rev Méd Chile 2002; 130: 396-401)

Liposomes can be used for the intracellular delivery of drugs into macrophages. Previously, we developed a liposome-mediated macrophage ‘suicide’ technique based on the intraphagocytic accumulation of the liposomally delivered bisphosphonate clodronate. Later we found that the diamidine propamidine is even more effective in this approach. In the present study it is shown that liposome-encapsulated 4′,6-diamidino-2-phenylindole (L-DAPI), another well known DNA-binding diamidine, is the most effective drug in killing liver macrophages (Kupffer cells), when intravenously administered in rat. Compared to liposome-encapsulated propamidine (L-propamidine) it showed about 10-fold more activity on a molar basis. Furthermore, L-DAPI was found to induce cell death by inducing apoptosis. The structurally strongly related phenanthridinium salts ethidium bromide (EB) and propidium iodide (PI) exert marked differences in their efficacy. Whereas liposome-encapsulated PI (L-PI) was about 5 times more active in killing macrophages than L-propamidine, liposome-encapsulated EB (L-EB) showed a strongly reduced activity (10 times less than L-PI). As is shown here, PI remains mainly encapsulated in liposomes, while substantial amounts of EB leak out of liposomes. This may very well explain the differences in in vivo activity between L-EB and L-PI.

The DNA sequences targeted by a complete homologous series of aromatic diamidines have been determined at single-nucleotide resolution via protection from cutting by the endonucleases DNase I, DNase II and micrococcal nuclease. Propamidine, pentamidine and to a lesser extent hexamidine bind selectively to nucleotide sequences composed of at least four consecutive A-T base pairs. In contrast, the binding to DNA of butamidine, heptamidine, octamidine and nonamidine is poorly sequence-selective. Sequences composed of only three consecutive A-T base pairs do not afford a potential binding site for propamidine or the longer homologues, and none of the drugs tolerate the presence of a G-C base pair within the binding site. Experiments with DNA molecules containing inosine in place of guanosine and 2,6-diaminopurine in place of adenine reveal that the lack of binding of propamidine to GC-containing sites is attributable to an obstructive effect of the exocyclic 2-amino group of guanosine. The present data support the view that the local conformation of the double helix (in particular the width of the minor groove) plays a dominant role in the binding reaction and that the capacity of diamidines to recognize AT-rich sequences selectively varies considerably depending on the length of the alkyl chain. The evidence indicates that binding to AT-tracts in DNA must play a role in the biological activity of these diamidines, but there is no simple correlation between binding and pharmacological efficacy.

Macrophages such as Kupffer cells in the liver are multifunctional cells. They are involved in host defense mechanisms and have a regulatory role in many biomedical processes. Their selective depletion, using liposome-encapsulated drugs, forms a widely accepted approach to studying their functional aspects in vivo. We have compared the Kupffer cell-depleting activities of liposome-encapsulated clodronate, propamidine, and ethylenediaminetetraacetic acid (EDTA) for this purpose. These molecules represent the drug families of bisphosphonates, diamidines (or aromatic polyamidines), and polyaminopolycarboxylic acid-chelating agents, respectively. The Kupffer cell-depleting activity of the liposome-encapsulated antimicrobial drug propamidine exceeded that of clodronate by about a factor of 10. EDTA appeared to be inefficacious for depletion of Kupffer cells in the rat.

Introduction. Following laboratory studies on new potential chemotherapy for Acanthamoeba keratitis, when chlorhexidine and propamidine provided an additive in vitro effect, a series of 12 patients with culture-proven Acanthamoeba keratitis from three UK centres was monitored during and after therapy. Methods. In all cases the clinical diagnosis was confirmed by amoebal culture. In some instances identification of the protozoa by direct microscopy of corneal tissue was possible. The medication was provided topically in drop form until the keratitis had resolved. In vitro sensitivity to chlorhexidine and propamidine was performed on all isolates and compared with sensitivity to a range of other drugs used for treatment of the infection. Results. In vitro drug testing confirmed that trophozoites and cysts of all 12 Acanthamoeba isolates were fully sensitive to chlorhexidine and propamidine. Therapy was satisfactory for controlling and eradicating the acanthamoebal infection in all patients. Three patients developed discrete stromal infiltration at the site of infection that resolved 1 week after commencing therapy, with or without use of steroids. Two patients developed a late inflammatory effect in the stromal scar at 6 months, which resolved with steroids. No clinical evidence of chlorhexidine toxicity was found in any patient. Conclusions. The combination of topical chlorhexidine and propamidine was very effective for treating Acanthamoeba keratitis provided the drugs were continued for a sufficient period. No drug toxicity or resistance of Acanthamoeba isolates was observed in the 12 treated patients.

Combinations of polymyxin B and dibromopropamidine isethionate exhibited synergistic inhibitory and bactericidal activity against Pseudomonas aeruginosa, Enterobacter cloacae, Proteus mirabilis, Escherichia coli and Staphylococcus aureus. Similar results were obtained with polymyxin B plus propamidine combinations except that propamidine was not as active as dibromopropamidine and the combination of polymyxin B plus propamidine against S. aureus only had additive activity. The antibacterial agents were tested in solutions and in a cream formulation. The findings indicate a potential for the use of selected combinations of these antibacterial agents in the treatment of wound and superficial eye infections.

Trophozoites and cysts of 20 isolates of Acanthamoeba from the cornea and five from related samples were tested in vitro for sensitivity to ten drugs (three aromatic dia-midines, two aminoglycosides, two macrolides, a polyene macrolide antibiotic, an organoarsenical and an antimetabolite) and two cationic antiseptics (chlorhexidine and polyhexamethylene biguanide, PHMB). Only chlorhexidine and PHMB showed uniform amoebacidal activity. Aromatic diamidines (pentamidine isethionate, propamidine isethionate and diminazene aceturate) generally proved effective against both forms of the amoeba; only pentamidine gave synergy with the biguanide while propamidine gave an additive effect. Other drugs tested proved erratic or ineffective against different isolates. Chlorhexidine alone, or together with propamidine, was subsequently used in two patients with proven Acanthamoeba keratitis; the causative isolates were sensitive to the individual compounds and to the combination in vitro. The treatment provided resolution of the clinical disease; amoebae were shown to be non-viable by histology and culture. The combination of chlorhexidine and propamidine is recommended for treatment of proven Acanthamoeba keratitis.

DNA sequences of three 18S rRNA gene alleles present in trophozoites obtained before and after therapy for Acanthamoeba keratitis substantiate a previous report that the infection was due to a single Acanthamoeba strain. Thus, the possibility that propamidine resistance which developed during therapy was due to a mixed infection was ruled out.

of their studies on the trypanocidal activity of aromatic diamidine compounds and was shown to possess well-marked activity against various protozoal organisms.2 Subsequently, Thrower and Valentine3 demonstrated that this compound had considerable antibacterial action against gram-positive cocci, both in vitro and clinically in the treatment of infected wounds and burns. These findings were verified in reports from several other clinical investigators.4*5,6 Kohn' and Bernheims,9 have recently shown that propamidine is capable of inhibiting the oxidative metabolism of susceptible organisms. Comprehensive studies of the pharmacological properties of this agent and other aromatic diamidine compounds have been reported by Wien'o and by Allen, Burgess and Cameron."1 Despite these extensive investigations, there have been no reports in the literature of studies concerned with either the general antibacterial spectrum of this agent or with its fungistatic activity against the common pathogenic fungi. In the course of investigations carried out in this laboratory, these

With the demonstration of their trypanocidal action by Lourie and Yorke in 1939 (1), a group of aromatic diamidines, notably stilbamidine (4,4'-stilbenedi-carboxamidine), propamidine (p, p'-trimethylenedibenzamidine) and pentamidine (pp'-pentamethylenedibenzamidine) has found a wide use in the treatment of trypanosomiasis. Subsequent studies revealed that these compounds possess as well activity against other protozoans as babesias and leishmanias, bacteria and fungi. Their development was recently reviewed by Schoenbach and Greenspan (2).

1. Green and blue-green algae which contaminate greenhouse cultures of Marchantia are named. 2. Copper acetate, tartrate, and sulphate are not sufficiently differential in their action, killing Marchantia as well as the algae they are to control. 3. Potassium permanganate in a saturated solution is useful in killing algae in stock cultures. Since all older portions of the Marchantia thallus are also killed, this treatment is not recommended for the control of algae in experiments involving mineral nutrition. 4. Propamidine, pentamidine, phenamidine, and stilbamidine stunt the growth of Marchantia and suppress algal growth at 100 p.p.m. At 10 p.p.m. most clones of Marchantia produce as much dry weight as the control plants. Algal growth is slight on treatments with propamidine and pentamidine; extensive on treatments with the other two diamidines. Treatments with 1 p.p.m. result in the production of much more dry weight in Marchantia, while many algae appear in small amounts in the cultures. 5. Propamidine...

Summary: A medium containing propamidine is described which has high selective activity for the species Bacillus anthracis. The spore form of the organism is essential as inoculum for the medium.

Demonstration of the fungistatic activity and therapeutic efficacy of stilbami-dine and 2-hydroxystilbamidine against Blastomyces dermatitidis in vitro and in vivo prompted an appraisal of the in vitro activity of other diamidine compounds. The objectives of this study were (1) to develop a reproducible in vitro technic for testing the drug sensitivity of fungi; (2) to investigate the mycelial and yeast phase sensitivities of the organisms simultaneously, under conditions as similar as possible; (3) to compare the fungistatic activity of the available diamidine compounds; and, (4) to compare the activity of all drugs employed against a number of strains of B. dermatitidis to determine the extent of strain variation in sensitivity.

A previously healthy 53-year-old man had keratitis of the right eye for six months, unresponsive to topical medical therapy. Acanthamoeba was grown from tissue obtained by corneal biopsy and from aqueous from an anterior chamber tap. The patient was treated with propamidine isethionate 0.1% drops and dibromopropamidine isethionate 0.15% ointment, and after two and a half months the ocular inflammation was continuing to resolve. This case supports a role for the diamidines in the treatment of acanthamoebic keratitis.

The synthesis of propamidinef (4-4' diamidino-diphenoxypropane) was reported in 1942 by Ewins and his co-workers in the investigation of trypanocidal drugs (1). In 1943, the therapeutic activity of propamidine as a topical antiseptic against Cram positive cocci in chronic wound sepsis was demonstrated by Thrower and Valentine (2) and by other clinical investigators (3, 4, 5). In 1945, Elson reported the in vitro sensitivity of Btastomyces dermetitidis to propamidine (6). B. dermafitidis was completely inhibited by 3.75 micrograms of propamidine per cc. of yeast extract agar. Because of the marked in vitro sensitivity of B. dermatitidis and the absence of toxic symptoms in cases of wound sepsis treated with topical application of propamidine, therapeutic trial in eases of cutaneous blastomycosis seemed warranted. It should be emphasized that, in spite of the lack of toxic symptoms reported, when propamidine is used in concentration of 0.1% as a local antiseptic, this drug and related aromatic diamidines produce general toxic effects when given systemically. After the intravenous injection of 2 mgm. of propamidine per kg. of body weight in the treatment of African sleeping sickness, Lourie (7) noted the production of immediate and severe collapse which was alarming but transitory. Late toxic effects have also have been reported with the use of the aromatic diamidines. Delayed kidney and liver damage and dissociated anesthesia of the trigeminal nerve have been particularly serious. An excellent review of these compounds has been published recently by Sehoenbaeh and Greenspaa (5).

The first medical cure of a corneal infection due to an Acanthamoeba species is reported. The 44-year-old patient developed a suppurative keratitis associated with an epithelial defect, hypopyon, and secondary glaucoma. Acanthamoeba was confirmed as the causative agent four months after presentation when positive cultures were obtained from the cornea and from the conjunctiva. Sensitivity studies of the isolated organism were performed, and the infection was successfully controlled by treatment with a combination of dibromopropamidine and propamidine isethionate ointment and drops and neomycin drops. Keratoplasty was performed 22 months after onset, and no viable acanthamoebae were present in the resected tissue, though possible cyst remnants were identified by immunofluorescent techniques.

Attempts at treatment of North American blastomycosis have been rather unsatisfactory in the past and the introduction of such potent drugs as propamidine and stilbamidine offered promise. In this connection, we have encountered no experimental work except Heilman's paper (3) on the effects of stilbamidine in blastomycosis in mice. Our own work was concluded prior to the publication of this paper.

M/80,000 propamidine inhibits the oxidation of the nitrogeneous constituents of the medium in which the bacteria grow. A certain latent period occurs before the inhibition reaches a maximum and the drug is more effective at pH 7.8 than at pH 6.7. As is shown in the accompanying article by Dr. Kohn, this concentration is in the minimal effective range for the inhibition of growth. A latent period and a similar pH effect also are present when growth is inhibited. It is thus possible to conclude that propamidine, unlike sulfanilamide and its derivatives, directly affects the oxidative metabolism of these bacteria.

CHLORDIMEFORM is a formamidine acaricide and insecticide used to control phytophagous mites, cattle ticks and certain lepidopterous insects. Because of its unique spectrum of biological activity with regard to selectivity, chlordimeform may be the forerunner of a new class of agricultural chemicals. Although its mechanism of action is unknown, Knowles and Roulston1, studying the action of chlordimeform and related compounds on the southern cattle tick, suggested that inhibition of monoamine oxidase (MAO) could be involved. This hypothesis was based on observations of ticks poisoned with formamidine compounds and on the known ability of amidine compounds, such as propamidine and pentamidine, to inhibit MAO from mammals2. We have observed that symptoms manifested by rats poisoned by chlordimeform and demethylchlordimeform are similar to those elicited by sympathomimetic agents including known MAO inhibitors (unpublished results of F. R. Johannsen and C. O. K.). Thus, it seemed appropriate to examine chlordimeform and related compounds as potential inhibitors of rat liver MAO in vitro.