ACCEPTED. Microbiology, Medical University of Vienna, Austria, II. Medical Dept., Pulmonological

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1 JCM Accepts, published online ahead of print on November 00 J. Clin. Microbiol. doi:0./jcm.0-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. GAE CAUSED BY ACANTHAMOEBA GENOTYPE T 0 Acanthamoeba genotype T as causative agent of GAE in an HIV negative patient Running title: GAE caused by Acanthamoeba genotype T Julia Walochnik, * Alexander Aichelburg, Ojan Assadian, Andrea Steuer, Govinda Visvesvara, Norbert Vetter and Horst Aspöck Dept. of Med. Parasitology, and Dept. of Hospital Hygiene, Clin. Inst. of Hygiene and Med. Microbiology, Medical University of Vienna, Austria, II. Medical Dept., Pulmonological Centre SMZ Baumgartner Höhe, Otto Wagner-Hospital, Vienna, Austria and Div. of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA *Corresponding author. Mailing address: Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Kinderspitalgasse, 0 Vienna, Austria. Phone: () Fax: () julia.walochnik@meduniwien.ac.at

2 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T Abstract. Acanthamoeba genotype T was identified as the causative agent of Acanthamoeba skin lesions and granulomatous amoebic encephalitis (GAE) in an HIV negative patient with an underlying tuberculosis. To our knowledge this is the first GAE case involving genotype T.

3 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T 0 0 Acanthamoebae are the causative agents of Acanthamoeba keratitis and of granulomatous amebic encephalitis (GAE), a condition predominantly occurring in immunocompromised individuals. In GAE most probably skin and olfactory neuroepithelium function as portals of entry, and occasionally inflammation can be observed at these primary foci. Approximately 0 cases of Acanthamoeba GAE have been published worldwide and less than 0 of these patients have survived (). Recently, the first GAE case in Austria was diagnosed (Aichelburg et al. in preparation). The patient was a -years old man from India with respiratory and neurological symptoms and widespread granulomatous dermal lesions. He had a CD+ count of cells/ µl, but was negative for HIV. The final diagnosis included miliary tuberculosis, tuberculous meningitis, granulomatous amebic encpehalitis (GAE) and Acanthamoeba skin lesions. The patient did not improve after tuberculostatic treatment and treatment with streptomycin, fluconazole, trimethoprim/ sulfamethoxazole, amphotericin B, flucytosine and sulfadiazine, as potentially anti-acanthamoeba effective drugs. However, he finally was treated successfully with oral and topical miltefosine and intrathecal and intravenous amikacin. Altogether six CSF samples, two dermal punches, one bronchoalveolar lavage (BAL), one lung and one liver biopsy and one biopsy from the main brain lesion were investigated for acanthamoebae by lactophenol cotton blue (LPCB) stain (), culture and PCR. For culture, specimens were inoculated onto the centre of non-nutrient agar plates (.%) precoated with Escherichia coli. The plates were sealed with parafilm, incubated at 0 C for d and examined every h. For PCR whole-cell DNA was isolated as described previously () and a fragment of the S rdna was amplified using the JPD and JPD primers (). Acanthamoeba strain ATCC PRA-0, genotype T was used as a positive control. Amplicons were sequenced using a 0 ABI PRISM automated sequencer (Applied Biosystems, Langen, Germany) and obtaining sequences from both strands.

4 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T 0 0 Multiple sequence alignment was performed with the CLUSTAL X application () excluding primer sites from the analysis. Genotypes were assessed with the model assumption of a <% sequence dissimilarity within one genotype (). Sequence data were deposited in GenBank and are available under the following accession number: EF. Lung, liver and brain biopsies were also examined by immunofluorescence staining using a polyclonal rabbit anti-a. castellanii serum at :0 and :00 dilutions. Sequential patient serum samples at dilutions between : and :000 were tested for anti-acanthamoeba IgG and IgM antibodies by immunoblotting as described previously (0), using antigens of all three Acanthamoeba groups (group I: strain Am, group II: ATCC PRA-0, group III: ATCC 00). Pooled serum from 0 healthy individuals was used as a negative control. Drug susceptibility assays were carried out at 0 C in microtiter plates with 0 trophozoites (ATCC PRA-0) per well. Antimicrobials were used at the following final concentrations (corresponding to the concentrations suggested for treatment): 0 mg/ ml amikacin, mg/ ml amphotericin B, mg/ ml caspofungin, mg/ ml fluconazole, 0 mg/ ml flucytosine, 0 mg/ ml rifampicin, 0 mg/ ml streptomycin, 00 mg/ ml sulfadiazine, and 0 mg/ ml voriconazole. Viability of amoebae was recorded after h by phase contrast microscopy and 00% eradication was confirmed by transferring the respective suspension to E. coli-coated plates and recording amoebal growth for d. Assays were performed in two independent experiments, each time in triplicate. LPCB stains, culture and immunofluorescence were consistently negative. However, the first four CSF samples, dermal specimens, BAL, and lung and brain biopsies were positive by Acanthamoeba-specific PCR. Sequencing identified the causative agent ( strain BS0) as Acanthamoeba genotype T. All obtained sequences were identical. In cluster analysis BS0 clusters with strain CCAP 0/c (GenBank AF00) (Fig. ). The liver biopsy and the two final CSF samples obtained five and nine months after admission to the

5 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T 0 0 hospital, respectively (both after therapy), were negative. Serological testing revealed high IgG (:000) and particularly IgM (:000) titers on a serum obtained three days after admission to the hospital, titers then gradually declining in consecutive sera to being comparable to the negative control (IgM titer of :) after one year. Interestingly, the patient showed highest immunoreactivity against Acanthamoeba morphological group III (corresponding to genotype T). Since the patient did not show any improvement even after treatment with drugs that had been shown to be useful in previously published cases of successful treatment, miltefosine () and amikacin were finally used to successfully treat our patient. In the present case, three aspects are of particular interest: (i) the co-infection with Mycobacterium tuberculosis, (ii) that acanthamoebae were only detectable by PCR, while culture remained negative, and (iii) that the acanthamoebae were genotype T not T, the genotype commonly associated with disease. Co-infection with M. tuberculosis is not only important as the reason for the severe immunosuppression of our patient (thus having contributed to the establishment of the Acanthamoeba infection), but also with respect to the fact that acanthamoebae can act as hosts for mycobacteria protecting them from antibiotics and disinfectants and even increasing their virulence (,, ). Other cases of Acanthamoeba-Mycobacterium co-infections have been reported, but these affected AIDS patients (). The fact that culture was consistently negative is not entirely unexpected, as acanthamoebae usually sit deep in the tissue, mainly around blood vessels and their isolation from the CSF is generally uncommon (, ). It is tempting to speculate that this is particularly true for non-aids patients, where amoebae cannot disseminate ad libitum. When the biopsy sample was obtained in the present case, the CD+ counts had already reached. As the location of the lesion obviated a total excision, the site of active infection was

6 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T 0 0 probably missed. This would also explain negative immunofluorescence testing and highlights the importance of molecular methods in the diagnosis of Acanthamoeba infections. In a recent case of disseminated Acanthamoeba infection in an HIV-negative patient even multiple biopsies with detailed histological analysis and culture did not enable a premortem diagnosis (). Acanthamoeba in the present case was identified as genotype T. It has been proposed to subdivide T in a highly pathogenic and a weakly pathogenic cluster (). The sequence of BS0 clusters with the weaker pathogenic subgroup what might also have contributed to the survival of our patient. It has been shown that of the Acanthamoeba genotypes T is the predominant genotype in GAE (as well as in AK), followed by T, T0 and T (). The two latter also correspond to morphological group III, as does T. A recent study revealed that while in the UK T is the most abundant genotype, T predominates in environmental samples in Iran (). Our patient, who originated from India, might have acquired his infection outside Europe and in this respect it is interesting to note that several GAE cases in HIV negative patients have been reported from India, while in Europe and the USA GAE is mostly linked with AIDS (0-, ). ACKNOWLEDGMENTS The authors wish to thank Susanne Glöckl and Iveta Häfeli (Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Vienna) and Rama Sriram (CDC, Atlanta) for excellent technical assistance. LITERATURE CITED. Adekambi, T., S. Ben Salah, M. Khlif, D. Raoult, and M. Drancourt. 00. Survival of Environmental Mycobacteria in Acanthamoeba polyphaga. Appl Environ Microbiol :-.

7 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T Bloch, K. C., and F. L. Schuster. 00. Inability to make a premortem diagnosis of Acanthamoeba species infection in a patient with fatal granulomatous amebic encephalitis. J Clin Microbiol :00-.. Booton, G. C., G. S. Visvesvara, T. J. Byers, D. J. Kelly, and P. A. Fuerst. 00. Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections. J Clin Microbiol :-.. Cirillo, J. D., S. Falkow, L. S. Tompkins, and L. E. Bermudez.. Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect Immun :-.. Gast, R. J., D. R. Ledee, P. A. Fuerst, and T. J. Byers.. Subgenus systematics of Acanthamoeba: four nuclear S rdna sequence types. J Eukaryot Microbiol :-0.. Maghsood, A. H., J. Sissons, M. Rezaian, D. Nolder, D. Warhurst, and N. A. Khan. 00. Acanthamoeba genotype T from the UK and Iran and isolation of the T genotype from clinical isolates. J Med Microbiol :-.. Mura, M., T. J. Bull, H. Evans, K. Sidi-Boumedine, L. McMinn, G. Rhodes, R. Pickup, and J. Hermon-Taylor. 00. Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga. Appl Environ Microbiol :-.. Schroeder, J. M., G. C. Booton, J. Hay, I. A. Niszl, D. V. Seal, M. B. Markus, P. A. Fuerst, and T. J. Byers. 00. Use of subgenic S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol :0-.. Schuster, F. L., and G. S. Visvesvara. 00. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. International Journal For Parasitology : Sharma, P. P., P. Gupta, M. V. Murali, and V. G. Ramachandran.. Primary amebic meningoencephalitis caused by Acanthamoeba: successfully treated with cotrimoxazole. Indian Pediatr 0:-.. Shirwadkar, C. G., R. Samant, M. Sankhe, R. Deshpande, S. Yagi, F. L. Schuster, R. Sriram, and G. S. Visvesvara. 00. Acanthamoeba encephalitis in patient with systemic lupus, India. Emerg Infect Dis :-.. Singhal, T., A. Bajpai, V. Kalra, S. K. Kabra, J. C. Samantaray, G. Satpathy, and A. K. Gupta. 00. Successful treatment of Acanthamoeba meningitis with combination oral antimicrobials. Pediatr Infect Dis J 0:-.. Sison, J. P., C. A. Kemper, M. Loveless, D. McShane, G. S. Visvesvara, and S. C. Deresinski.. Disseminated acanthamoeba infection in patients with AIDS: case reports and review. Clin Infect Dis 0:0-.. Steinberg, J. P., R. L. Galindo, E. S. Kraus, and K. G. Ghanem. 00. Disseminated acanthamebiasis in a renal transplant recipient with osteomyelitis and cutaneous lesions: case report and literature review. Clin Infect Dis :e-.. Thomas, P. A., and T. Kuriakose. 0. Rapid detection of Acanthamoeba cysts in corneal scrapings by lactophenol cotton blue staining. Arch Ophthalmol 0:.. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins.. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. :-.. Velho, V., G. K. Sharma, and D. A. Palande. 00. Cerebrospinal acanthamebic granulomas. Case report. J Neurosurg :-.. Walochnik, J., M. Duchene, K. Seifert, A. Obwaller, T. Hottkowitz, G. Wiedermann, A. Eibl, and H. Aspock. 00. Cytotoxic activities of

8 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T 0 alkylphosphocholines against clinical isolates of Acanthamoeba spp. Antimicrobial Agents And Chemotherapy :-0.. Walochnik, J., A. Hassl, K. Simon, G. Benyr, and H. Aspock.. Isolation and identification by partial sequencing of the S ribosomal gene of free-living amoebae from necrotic tissue of Basilliscus plumifrons (Sauria: Iguanidae). Parasitol Res : Walochnik, J., A. Obwaller, E. M. Haller-Schober, and H. Aspock. 00. Anti- Acanthamoeba IgG, IgM, and IgA immunoreactivities in correlation to strain pathogenicity. Parasitol Res :-.

9 GAE CAUSED BY ACANTHAMOEBA GENOTYPE T LEGENDS FIG.. Maximum likelihood cluster analysis of the obtained sequence and various Acanthamoeba strains with special reference to genotype T and using Acanthamoeba genotypes, and as an outgroup. BS0 is the sequence from the presented case. Bootstrap values are based on 00 replicates and are given at the nodes (ML/NJ/MP). The asterisk indicates values below 0%.

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