299 Rayad Barakat Microbiome Manipulation: Antibiotic Effects on Cutaneous Leishmaniasis [email protected] Tel: (301) 295-5895 Presentation and Healing Fax: (301) 301-0680 4301 Jones Bridge Rd Bethesda, MD 20814 R. Barakat1, C. Olsen1, N. Aronson1,2 1F. Edward Hebert School of Medicine, Uniformed Services University, Bethesda, MD 2Walter Reed National Military Medical Center, Bethesda, MD Discussion Results Abstract Clinical Presentation Rationale: Cutaneous ulcers of leishmanial infection are chronic wounds with an Table 1. Demographic Data Figure 1. Study Flow Diagram Somewhat more African-American men with cutaneous leishmaniasis received antibiotic treatment as anaerobe-predominant microbiome, further impacted by the immunomodulatory effects compared to those without antibiotic exposure. The most commonly prescribed antibiotic was amoxicillin- of Leishmania and superinfecting bacteria. Leveraging a policy to give presumptive Abx+ Abx- p-value Enrollment Assessed for eligibility (n=418) clavulanate (n=123 courses in 94 subjects), generally given in one- or two-week courses, and with several pyoderma treatment prior to evaluation for leishmaniasis, we assessed the effect of patients receiving multiple courses or multiple antibiotics. It was usually not possible to determine whether antibiotic therapy upon clinical presentation or treatment outcome among those with Total 195 165 Excluded (n=58) antibiotics were given per command policy to r/o pyoderma or for clinical illness such as superinfected cutaneous leishmaniasis. Methods: Data were obtained from a 418-patient open label Sex 0.13 Received Prior Treatment (n=53) ulcers. Doxycyline was given per command policy for malaria prophylaxis and received by 29 subjects, study of 10 or 20 days of sodium stibogluconate (SSG) therapy conducted at Walter Non-cutaneous leishmaniasis (n=3) Male 189 (97%) 164 (99%) Consent withdrawn (n=2) those individuals receiving it from the start of their time in theater until their return for CL treatment+28 Reed Army Medical Center. Subjects with parasitologically confirmed leishmaniasis days. Female 6 (3%) 1 (1%) were undergoing their first treatment course. We assessed clinical presentation by We observed no significant differences in duration of illness, baseline lesion size, number of CL lesions, Race 0.01 number and aggregate size of lesions, appearance, and lymphatic involvement. We Allocation regional lymphadenopathy, or presence of ulceration compared between those with or without antibiotic evaluated treatment outcome by patient report of healing at 2, 6, 12 months post SSG. Caucasian 133 (68%) 123 (75%) exposure. Patients were divided into those receiving antibiotics prior to treatment, those receiving Asian 3 (2%) 1 (<1%) antibiotics during treatment, and those who had not received any antibiotics. We Allocated to antibiotic group (n=195) Allocated to no-antibiotic group Treatment Outcome Hispanic 16 (8%) 18 (11%) (n=165) evaluated the two pre-treatment populations with Mann-Whitney Tests and Fisher’s Those receiving antibiotics during SSG treatment were assumed to have clinical bacterial infection, in Exact Test and the three posttreatment populations with χ2 and Kruskal-Wallis tests. contrast to those receiving antibiotics prior to SSG treatment (who may have received antibiotics per African-American 38 (19%) 20 (12%) Results/Conclusions: The cohort was 360 mainly young white males, with 4 median Antibiotic received pre-presentation command policy). Although at the 2-month follow-up timepoint non-antibiotic receiving patients appeared to lesions in both groups. The most commonly used antibiotics were doxycycline (n=29 Other 5 (3%) 3 (2%) (n=137) trend to lower cure rates than those with prior antibiotic treatment in both 10-day SSG and 20-day SSG courses), cephalexin (33), and amoxicillin-clavulanate (123). Statistical analysis found Mean Age (yr) (SD) 28.06 (7.5) 27.67 (7.8) 0.49 Antibiotic received post-presentation groups, this difference was not statistically significant, and by the 6-month follow-up any observable no significant difference between the pretreatment groups (antibiotics, no antibiotics) in SSG regimen 0.12 (n=58) differences had disappeared. Healing is generally seen without therapy in L. major infection by 6-12 clinical presentation, and no significant difference between the post-treatment groups in months so the 2 month timepoint would be most illustrative of antibiotic effect. Of note, there was 10 day 59 (30%) 63 (38%) Follow-up treatment outcomes. significant loss to follow up at the 2 month visit as subjects had returned to a combat zone and were 20 day 136 (70%) 102 (62%) Lesion size change lost to follow-up (n=0) Lesion size change lost to follow-up difficult to reach, however most redeployed to the US by the 6 month follow-up visit. Background Median Duration of Illness (n=2) Amoxicillin-clavulanate administration with its’ attendant anaerobic spectrum was not associated with any Days (IQR) 112 (55) 121 (58) 0.49 Analysis statistically significant change in clinical outcome. Strains of Staphylococcus and Streptococcus developed ♦ Cutaneous leishmaniasis (CL) presents a risk to non-immune populations in endemic CO3 Analyzed (n=195) Analyzed (n=163) in the cutaneous dysbiosis associated with leishmanial infection may exhibit resistance to treatment with regions, with notable recent outbreaks associated with conflict. Patients beginning antibiotics (Abx) during treatment are included in the Abx+ group amoxicillin-clavulanate [9]. ♦ Leishmanial infection alters the host immune response, and causes a chronic wound, Amoxicillin-clavulanate and doxycycline were the most commonly reported antibiotics contributing to secondary infection of ulcers [1-3] with indigenous microbiota affecting Figure 2a-d. Clinical presentation used. resistance to healing and susceptibility to leishmanial treatment [4,5]. comparison in antibiotic vs. no Conclusions antibiotic groups ♦ CL itself may restrict lesion microbiota, skewed toward anaerobes or coagulase • Despite the interplay between systemic antibiotic usage, leishmanial infection, local negative Staphylococcus [6], leading to substantial secondary infection and even

) immunomodulation, and the skin microbiome, no significant effects associated with sepsis [7]. 2 188 119 antibiotic use were observed in CL lesion healing outcomes or clinical presentation prior to 664, n=223 702, n=137 4, n=223 ♦ In mouse models, the treatment of secondary bacterial infection in leishmaniasis was specific antileishmanial treatment with SSG in this study. associated with an increased cure rates [7] as well as reduced splenic and hepatic 4, n=137 0.54 >0.99 disease burden [8]. • Prior antibiotic use was considered random, since most given as presumptive initial Percent ♦ Limited diagnostic capability early in Operation Iraqi Freedom (OIF) led to a policy of Percent therapy of chronic skin lesions under a command policy. treating leishmaniasis-like sores with a 5-7 day course of antibiotics for pyoderma, >0.99 35 18 • Limitations of this study included that this was a retrospective ad hoc analysis of clinically 0.81 Numberof lesions resulting in patients receiving broad-spectrum antibiotics before transfer to the US for 2d collected data; there was much variability in the antibiotic administered, their dosages, leishmaniasis treatment. (mm size/subject Aggregate 2c duration, some inter-observer differences in lesion measurement and appearance ♦ We sought to determine if any significant differences could be found in clinical 2a 2b descriptions, and challenges with early outcome follow-up due to return to combat zone. presentation and treatment outcome between patients who had received antibiotics Table 2. Median Cure Rates (range) by month of follow up • Future work exploring the effects of antibiotic use on CL and treatment outcomes may before or during their CL treatment and those who had not received antibiotics. benefit from a more systematic approach including serial lesion microbiologic assessment, Figure 3 a,b. Change in lesion size with 10 days SSG 2 mo.(n=63) 6 mo.(n=91) 12 mo.(n=87) Consolidated (n=122) Methods a standardized antibiotic regimen targeted to predominant organisms in secondary SSG treatment compared with and Antibiotic prior 85 (65, 94) 94 (80, 98) 100 (89) • Data were obtained from records of a 418-patient open label study of 10 or 20 days of sodium without antibiotic use 84 (71, 92) infection, and investigation of systemic versus topical antimicrobial use. stibogluconate (SSG) therapy conducted at Walter Reed Army Medical Center among US service Antibiotic during 60 (23, 92) 92 (67, 99) 100 (68) 87 (62, 98) members who had acquired CL in Iraq or Afghanistan (mainly L. major). 3a References NA20 • Subjects with parasitologically confirmed leishmaniasis were included in this analysis if this was No antibiotic 58 (41, 73) 85 (74, 92) 92 (80, 97) 79 (68, 88) 1. Belkaid Y, Segre JA. Dialogue Between Skin Microbiota and Immunity. Science. 2014;346(6212):954-959. their first CL treatment course. 0.89 doi:10.1126/science.1260144. p 0.09 0.43 0.18 0.72 2. Naik S, Bouladoux N, Wilhelm C, et al. Compartmentalized Control of Skin Immunity by Resident Commensals. Science. • We assessed clinical presentation using number of and aggregate size of lesions, appearance, 20 days SSG 2 mo.(n=129) 6 mo.(n=189) 12 mo.(n=179) Consolidated (n=238) 2012;337(6098):1115-1119. doi:10.1126/science.1225152. and lymphatic involvement. Lesions were described and measured on presentation and upon 3. Gimblet C, Loesche MA, Carvalho L, et al. IL-22 Protects Against Tissue Damage During Cutaneous Leishmaniasis. Stäger S, ed. completion of therapy. Antibiotic prior 81 (69, 89) 93 (85, 97) 100 (95) PLOS ONE. 2015;10(8):e0134698. doi:10.1371/journal.pone.0134698. • Outcome status was ascertained by phone, email, or letter at 2, 6, and 12 months after treatment. 88 (80, 93) 4. Lopes MEM, Carneiro MBH, dos Santos LM, Vieira LQ. Indigenous Microbiota and Leishmaniasis. Parasite Immunology. • Patients who received antibiotics after beginning their course of SSG treatment were categorized Percent Change Antibiotic during 56 (33, 76) 91 (76, 96) 97 (84, 100) 81 (67, 90) 2016;38(1):37-44. doi:10.1111/pim.12279. into the non-antibiotic group for initial presentation and moved to the “during” antibiotic group for 5. Lamour SD, Veselkov KA, Posma JM, et al. Metabolic, Immune, and Gut Microbial Signals Mount a Systems Response to outcome evaluation. No antibiotic 72 (59, 82) 90 (82, 95) 97 (91, 99) 87 (79, 92) Leishmania major Infection. Journal of Proteome Research. 2015;14(1):318-329. doi:10.1021/pr5008202. 6. Salgado VR, Queiroz AT de, Sanabani SS, et al. The Microbiological Signature of Human Cutaneous Leishmaniasis Lesions • Outcome evaluation groups were further subdivided in analysis based on whether subjects had p 0.11 0.81 0.39 0.54 Exhibits Restricted Bacterial Diversity Compared to Healthy Skin. Memórias do Instituto Oswaldo Cruz. 2016;111(4):241-251. received a 10-day course of SSG or a 20-day course of SSG. 3b doi:10.1590/0074-02760150436. • Patients missing follow-up lesion measurements were not included in the evaluation of changes in 7. Yehia HM, Al-Olayan EM, El-Khadragy MF, Metwally DM. In Vitro and In Vivo Control of Secondary Bacterial Infection Caused by lesion size or number. 0.63 Figure 4 a-c. Clinical outcome evaluated by SSG duration and by use of amoxicillin-clavulanate Leishmania major. Int J Environ Res Public Health. 2017;14(7). doi:10.3390/ijerph14070777. • Consolidated outcome was combined follow up obtained at either 6 or 12 months. 8. Hendrickx S, Van den Kerkhof M, Mabille D, et al. Combined Treatment of Miltefosine and Paromomycin Delays the Onset of Experimental Drug Resistance in Leishmania Infantum. PLoS Negl Trop Dis. 2017;11(5):e0005620. doi:10.1371/journal.pntd.0005620. Statistical Analysis 9. Gimblet C, Meisel JS, Loesche MA, et al. Cutaneous Leishmaniasis Induces a Transmissible Dysbiotic Skin Microbiota that Promotes Skin Inflammation. Cell Host Microbe. 2017;22(1):13-24.e4. doi:10.1016/j.chom.2017.06.006. • We compared baseline lesion size, number of skin lesions, duration of illness, and change in lesion size using a non-parametric t-test (Mann-Whitney U Test). Percent Change 0.54 0.72 Acknowledgements • Presentation with lymphadenopathy and percent of ulcerated lesions were evaluated with Fisher’s 0.87 We would like to thank the Walter Reed Army Medical Center investigators, Drs. Ananthakrisnan, Bernstein, Byrne, Exact Test. Percentage Percentage Percentage Cephas, Hawkes, Marovich, Polhemus, Wortmann, Yoon, and Ms. Hummer; also Dr. Philip Scott for consultation in • Weused χ2 teststo compare2 month, 6 month, 12 month outcomes. our analysis planning; and the U.S. service members who participated in this treatment trial. We would also like to • Consolidated outcomes were analyzed with Fisher’s Exact Test. Changes in lesion size after 10- thank the IDSA Medical Scholars Program for their funding and support. and 20-day treatment courses were analyzed with the Kruskal-Wallis Test. The opinions and assertions expressed herein are those of the author(s) and do not reflect the official policy or the • Patients with no recorded outcome were not included in the outcome analysis. Uniformed Services University,NA21 the Department of the Army/Navy/Air Force the Department of Defense or the US 4a 4b 4c government. Slide 1

CO3 Add range or interquartile range. CARA OLSEN, 8/21/2017 NA20 Be consistent with the titles, either capitalize all or only the first word and proper names. NAOMI ARONSON, 8/23/2017 NA21 Lets go ahed and credit the IDSA Medical Scholars Program (check the precise name) for your funding support . If needs be I think you could cover over the USU right lower corner with white box and type over. NAOMI ARONSON, 8/23/2017