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probiotic Bacterial overgrowth
Effect of Lactobacillus strains (L. casei and L. acidophillus strains CERELA)
on bacterial overgrowth-related
chronic diarrhea DAVID GAON1, 2, 3, CARMEN
GARMENDIA2, NORBERTO O. MURRIELO3, ALFREDO DE CUCCO GAMES3, ANGEL
CERCHIO3, RICARDO QUINTAS1, SILVIA N. GONZALEZ4, GUILLERMO OLIVER4 San Miguel de Tucumán Abstract Small bowel bacterial overgrowth and related diarrhea is a condition that frequently accompanies anatomic disorders, surgically created blind loops or strictures with partial small bowel obstruction and although it is often controlled with antimicrobial therapy, alternative treatment may be needed. The aim of this study was to evaluate the efficacy of an oral probiotic preparation of 2 viable lyophilized strains of lactobacilli (1.5 g each) compared with placebo.Twenty two patients with proven overgrowth and chronic diarrhea are described. In random order and double-blind fashion, 2 groups of patients received identical capsules with both Lactobacillus casei and L. acidophillus strains CERELA (12 patients) (LC) and placebo (10 patients) (P) during three consecutive periods of 7 days each followed by a similar three periods of control after withdrawal. At the end of each period the mean daily number of stools, glucose breath H2 test, and symptoms were considered. Lactobacillus were investigated in feces in both groups at day 0 (baseline), on day 21 of treatment with LC and P and on day 21 after withdrawal. Compared with P a significant reduction in mean daily number of stools was achieved with LC (p< 0.005) at 15 days, and (p< 0.0005) at 21 days and the effect was sustained at 7 days and 15 days (p< 0.005) after withdrawal. With respect to breath H2 level a significant decrease in H2 concentration was noted at 7 days (p< 0.005) at 15 days, and 21 days (p< 0.0001 ) with LC and only a significant decrease (p< 0.005) was observed at 7 days after withdrawal. No significant changes were observed with respect to symptoms .The Lactobacillus CERELA strains were isolated from the feces in all patients LC (n=12) on day 21, and by contrast no Lactobacillus were observed except in two patients out of seven patients after withdrawal. In summary, this study provides evidence that LC are effective for treatment of bacterial overgrowth - related chronic diarrhea, and suggest that probiotics must be used with continuity. Key words: probiotic, bacterial overgrowth, chronic diarrhea
Efecto de cepas de Lactobacillus (L. casei, L. acidophillus CERELA)
sobre la diarrea crónica debida al sobrecrecimiento bacteriano. La
diarrea crónica debida al sobrecrecimiento bacteriano es una condición
que frecuentemente acompaña a las alteraciones anatómicas, asa ciega o
estrecheces post quirúrgicas del intestino delgado, con parcial
obstrucción, y aunque con frecuencia se controla con antimicrobianos,
tratamientos alternativos pueden ser necesarios. El propósito de este
estudio randomizado, doble ciego, fue determinar si la asociación de
dos cepas liofilizadas de Lactobacillus casei (1.5 g) y Lactobacillus
acidophillus (1.5g) desarrollada por CERELA (Centro de Referencia para
Lactobacilos) es más efectiva que un placebo, en pacientes con diarrea
crónica debida a un sobrecrecimiento bacteriano del intestino delgado.
Fueron estudiados 22 pacientes; 12 recibieron lactobacilos (L) y 10
placebo (P) en cápsulas idénticas, 2 veces por día durante tres
períodos consecutivos de 7 días seguido por 3 períodos similares post
tratamiento. Al final de cada período se evaluó el promedio diario del
número de heces, la presencia de sobrecrecimiento por el empleo de la
prueba de concentración de H2 post carga con glucosa en el aire
espirado y la persistencia de los síntomas clínicos. La presencia de
lactobacilos en las heces también fue investigada en ambos grupos en
el período basal, a los 21 días de tratamiento y 21 días post
tratamiento. Comparado con P una significativa reducción en el número
diario de heces fue observado con L a los 15 días (p< 0.005) y a los
21 días (p< 0.0005) y este efecto se sostuvo a los 7 y 15 días (p<
0.005) post tratamiento. Respecto al H2 una significativa reducción
fue observada a los 7 días (p< 0.005) a los 15 y 21 días (p< 0.0001)
con L durante el tratamiento y solo a los 7 días (p< 0.0005) posterior
al tratamiento. No hubo cambios significativos con respecto a los
síntomas. Las cepas CERELA se aislaron de las heces de todos los
pacientes tratados con L (n=12) recogidas a los 21 días de tratamiento
pero en cambio solo se aislaron de 2 pacientes (n=7) a los 21 días
post tratamiento. Se concluye que el empleo de cepas de lactobacilos
(L. casei, L. acidophillus CERELA) son efectivas en el tratamiento de
la diarrea crónica debida a un sobrecrecimiento bacteriano del
intestino delgado a cuyo fin se sugiere una admistración continuada de
estos probióticos. Palabras clave: probióticos, sobrecrecimiento bacteriano, diarrea crónica
Postal address: Dr. David Gaón, Lavalle 1438, 1022 Buenos Aires, Argentina Fax (54-11) 4373-8025 e-mail: david d.gaon@gsk.com
Received: 8-VIII-2001
Accepted: 28-XII-2001
Bacterial overgrowth is defined as 104 of predominantly Gram negative anaerobes of colonic origin per ml of small bowel fluid1. The syndrome is mainly caused by stasis or an anatomic abnormality and it is characterized by the pre-sence of chronic diarrhea and/or malabsorption and weight loss1-3. The diagnosis of bacterial overgrowth can be adequa-tely screened by demonstrating an early peak in breath hydrogen concentration after administration of an oral glucose load4,5 and a treatment with broad-spectrum antibiotics either for sporadic or on a continuous basis is often necessary6. Beneficial therapy may also involve probiotics agents, components of the natural flora including lactic acid bacteria such as lactobacilli, streptococci and bifidobacteria and new probiotics such as yearst (e.g., Saccharomyces boulardii) and entirely unrelated bacteria (Clostridium, Bacillus subtilis). Defined as living microorganisms, which upon ingestion in certain number exert health benefits beyond inherent basic nutrition, improving the properties of indigenous microbiota7, the clinical importance of probiotics is supported by many observations. Lactobacillus spp have been shown to be able to prevent the development of spontaneous colitis and attenuate established colitis in IL-10 knockout mice8,9. Two controlled studies comparing a nonpathogenic strain of Escherichia coli versus mesalamine in patients with ulcerative colitis registered no significant changes in relapse rates10,11. Also, probiotic preparations with the presence of a mixture of different bacterial species have been shown to be beneficial in the maintenance and treatment of chronic pouchitis12 and have been effective in the prevention of relapses in patients with ulcerative colitis13. Lactobacilli alleviate viral14 or bacterial15 induced diarrhea, especially in infants and with reference to bacte-rial overgrowth, probiotics were evaluated successfully in children with short bowel syndrome16. Saccharomyces boulardii has proved to be effective too, though recent reports have reached an opposite conclusion at least when it has been used for a short time17. Lactobacilli has never been
tested in a controlled study for reduction of small bowel bacterial
overgrowth and related diarrhea. Non controlled trials have been
performed studying long treatments or following those with probiotic
withdrawal. We initiated a double-blind clinical trial to explore
those effects of lactobacilli using Lactobacillus casei (CRL 431) and
Lactobacillus acidophilus (CRL 730) strains, from CERELA, culture
collections. Materials and Methods Patients. Twenty four consecutive ambulatory patients with chronic diarrhea and conditions that predisposed them to bacterial overgrowth of the small bowel, referred to inpatients consultative services were enrolled in our study from March 1993 to June 1997. All patients were dependent on antibiotic therapy as evidenced by exacerbations of disease after withdrawal of medication or reduction in dose. Six were being maintained on indefinite treatment schedules whose doses had been adjusted individually to stabilize the clinical symptoms. Diabetic autonomic neuropathy was present in 3 patients, Billroth II gastrectomy with or without vagotomy in 5 patients; idiopathic intestinal pseudo-obstruction in 1 patients; intestinal pseudo-obstruction after abdominal radiotherapy for gynecologic cancer in 3 patients; atrophic gastritis in 2 patients; prolonged treatment with H2 receptor antagonist in 2 patients; ileocecal resection in 5 patients; jejunoileal bypass in 2 patient and scleroderma in 1 patient. Chronic diarrhea was defined as a change in bowel habits consisting of at least 3 loose watery stools per day for a minimun of 3 months and bacterial overgrowth was based on a positive glucose ingestion hydrogen breath test. Informed written consent was obtained from all patients and the study performed in accordance with the Declaration of Helsinki was approved by the medical ethics committee of Hospital ELMA (Empresas Líneas Marítimas Argentinas) and Hospital Español , Buenos Aires. Study medication. Identical capsules cointaining 3 g of maize starch (placebo) and 2 strains of Lactobacillus CERELA (L. casei, L. acidophillus) containing 1.5 g of viable lyophhilized bacteria each per capsule, were administered orally twice daily for 21 days.The taste and smell of the active drug or placebo were not really identifiable. Sufficient compliance was assumed when patients took more than 90% of the prescribed study medication. Study design. This study was a randomized, double-blind, placebo-control. After 4 weeks without antibiotics or other drugs affecting gastrointestinal function, came three 7 day periods of treatment followed by 3 similar periods of control after withdrawal periods. Prior to treatment and every period thereafter, patients were matched for sypmtoms and degree of symptoms severity, the mean daily number of stools, and a glucose hydrogen breath test between the two groups. Patients were asked to keep a record of stool frequency on daily cards, and the evaluation of symptoms, on the basis of its percived intensity, consisted of a questionnaire concerning the presence and severity of four symptoms: abdominal cramp/colicky, flatulence, borborygmi and bloating. They were graded according to the following scale 0= absent, 1=mild (ocassional symptoms), 2= moderate (frequent discomfort that was not treatment), 3=severe (frequent discomfort with a painful sensation that was treatment). Any medication taken, and any side effects were also, recorded. Patients were allowed to eat their prescription diet including milk (2 cup) and wine (1 cup)/24h. The medications were dispensed by one of the inves-tigators (DG) at each visit, and no concurrent treatments were allowed. Breath H2 testing. Breath H2 concentration was measured after 12 hours fasting, and to minimize basal H2 excretion, the dinner meal on the day before the test consisted of rice and meat. None of the patients smoked and all were sedentary throughout the test. The lactulose test was performed prior to the other test to ensure that all participants harbored a bacterial flora able to generate a significant sustained rise in H2 in end-expiratory air of more than 10 ppm. All participants fulfilled this criterion. End-expiratory breath samples were collected before and at 15 min intervals for 2 hours after ingestion of 50g of D-glucose as previously described5. An increase in breath hydrogen concentration of more than 10 ppm above baseline value after glucose, at any sampling time, was considered a positive result on at least 2 samples. The tests were performed at baseline and on the last day of each 7-day period. End-expiratory breath samples were analyzed using Quintron CM model (Milwaukee WI). Minimal detectable H2 concentration was 1 ppm. Stool culture. Stool specimens were collected and inmediately frozen and stored at -20°C until they were assayed (within 10 days), at baseline, at day 21 with treatment, and at day 21 after withdrawal in both groups of patients. Lactobacillus spp strains CERELA were analysed as described earlier15. Stool specimens or rectal swabs of the patients were collected in Lapt soft agar. Adequate dilutions were plated onto LBS -raffinose agar (LBS agar is a selective medium for lactobacilli). The final pH was 6.5. A solution of glucose, sterilized by filtration,was added at 1% final concentration. After 48 h of incubation at 37 °C under CO2 atmosphere, the number of colony forming units per gram (CFU/g), was determined. Statistical analysis. Population global profile was defined throughout the analysis of the means, the standard deviations and the frequency of distribution. Discrete variables were assessed with the use of the Chi-squared test. Continuous data from both groups of subjects were compared with the use of the two-sided T-test or in case that the data had no normal distribution the Wilcoxon-Mann-Whitney rank-sum test was used. Wilcoxon-rank-sum test or paired T-test were used for paired data. A p value of less than 0.05
was considered as statistically significant. All data were collected
on an Excel spreadsheet and then analysis by the Statistica/W and EPI
info programs. Results Patients. Twenty-eight
patients were screened and twenty-four were eligible; 12 were randomly
assigned to receive Lactobacillus spp strains CERELA and 12 to receive
placebo. Twenty -two patients completed the study because 2 patients
belonging to the placebo groups were excluded during the 1st week of
treatment for non-compliance. Both study groups and their dates with
respect to age, sex, and duration of diarrhea are shown in Table1. Clinical Results Number of stool. Mean daily number of stools (mean + SEM) during each period of 7 days is shown in Fig 1. Compared with placebo a significant reduction (p< 0.005) in mean daily number of stools was achieved with Lactobacillus spp. at 15 days (2.27 ± 1.51) and significant differences (p< 0.0005) at 21 days ( mean 1.55 ± 1.17) and the effect was sustained at 7 days (2.52 ± 1.20 vs 3.70 ± 0.85) and at 15 days (3.18 ± 0.95 vs 3.80 ± 1.16) (p< 0.005 respectively) after withdrawal. No significant changes were
registered in mean scores for abdominal cramps, borborigmi, flatulence
and bloating at all intervals of treatments or withdrawal. Breath H2 testing Treatment periods. Fasting breath H2 level was similar in concentration at the beginning between both groups but a significant decrease in H2 excreted was noted between baseline, and at the end of each period (7,15 and 21 days) when was compared the Lactobacillus spp. group vs placebo (p< 0.005; p< 0.0001; p< 0.0001, respectively). Fig. 2. Withdrawal periods. A significant decrease in H2 excreted was observed only at 7 days when was compared the Lactobacillus spp. group vs placebo (p< 0.005). There were not significant differences observed at 15 and 21 days. Fig. 2.
Bacteriology Prior to beginning the study no fecal samples in the different groups yielded colonial forms like L. casei and L. acidophillus strains CERELA. On day 21 in all patients (n=12) assigned to probiotic therapy the stool cultures allowed the recovery of lactobacilli in the following concentrations: L. acidophillus 107-108 CFU/g, and L. casei 106 -107 CFU/g, indicating that lactobacilli CERELA have the ability to survive the passage through the gastrointestinal tract. By contrast, none of control group (n=10) excreted Lactobacillus strains CERELA. After discontinuing the treatment, persistence of lactobacilli was studied in 7 patients of each group. The organisms could be recovered from fecal specimens in only 2 patients (probiotic therapy group), and the viable fecal counts were 104-105 CFU/g for both Lactobacillus (L. casei, L. acidophillus). Safety. No side effects
were registered in either group of patients. Discussion The administration of Lactobacillus casei and Lactobacillus acidophillus strains CERELA, resulted in the attenuation of bacterial overgrowth related-diarrhea, and reduction in the mean concentration of breath-excreted H2, significantly. As some investigators did before, we choose reduction in the number of stools as an objective and clinically relevant endpoint, also we have used breath-excreted H2 as an indicator of therapeutic efficacy in bacterial overgrowth4, 5. Attending the opinion of Madsen et al8, 21 days of treatment with lactobacilli were selected despite the higher risk of drop-out as a compromise designed to ensure the most efficient length of therapy, the optimal bacterial concentration levels (CFU/g) in the intestine, and to establish the best way to prevent recurrence. The exact mechanism by which Lactobacillus spp. exert protection against the bacterial overgrowth is unknown. However, it is known that certain Lactobacillus spp. adhere to mucosal surfaces, and sterically inhibit the attachment of aerobic gram-negative bacteria15, 18-20. They secrete inhibitory products that have antimicrobial activity, and reduce pH in the colonic lumen which may decrease the growth of pathogenic bacterial species21. In addition to these well-known effects, Lactobacillus spp.also induce proliferation of immune cells22, have nutritional benefits including the induction of growth factor, and enhance synthesis of secretory immunoglobulin A23. Lactobacillus spp. provide protection against increase in intestinal permeability24, and as recent reports have shown, they have a high antagonist effect on intestinal inflammatory disease and prevent the development of colitis25, 26. Also disminishe the severity of alcohol-induced liver disease because they reduce gut-derived endotoxemia27. An obvious prerequisite for such a beneficial activity is that the peroral administration of Lactobacillus strains survives passage through the gastrointestinal tract in humans. Our results showed that L. casei and L. acidophillus strains CERELA have that property, but also, delayed significantly the mouth-caecum transit time28, adhere to a human colon carcinoma cell line, and promote clinical recovery from enteral infection29, demonstrating their ability to modulate complex mecha-nisms of host defense against pathogens30. Such activities are compatible with the notion of a probiotic agent. The data obtained in this study indicated that bacteriotherapy with Lactobacillus strains CERELA was very effective since this treatment reduced the daily stool frequency, showing a relationship between the levels of lactobacilli, and enteric disorders. Also showed that without additional oral bacteriotherapy the diarrhea can be restored, since unfortunately lactobacilli, do not permanently colonize the gastrointestinal tract. Other probiotic therapy (VSL#3), reported to be of benefit in the control of chronic pouchitis, also failed to provide long-term protection as evidenced by recurrence of disease, and returned of fecal lactobacilli and bifidobacterial concentrations to pretreatment levels, 1 month after stopping probiotic treatment13. The analysis of these results also suggests that pro-biotics must be used with continuity because the effects start after a week, so that, the results obtained by Attar et al17, who did not establish any correlation examining different antimicrobial agent and Saccharomyces boulardii in 10 patients, were because their results were based following only 7-day treatment. There is growing interest in detecting candidate probiotic micro-organisms that may provide protection against few gastrointestinal diseases. This study showed that Lactobacillus casei and Lactobacillus acidophillus strains CERELA, significantly reduced both, the number of stools in bacterial overgrowth-related chronic diarrhea, and glucose breath-expired H2 concentration. This study raises the
possibility that protective actions of bacteriotherapy in the
intestinal tract are multifaceted, acting also at least as a
bacteriostatic, unless this concept ought to be clearly demostrated,
yet. Acknowledgement: Support in part by grants from SANCOR Cooperativas Unidas Ltda.Sunchales, Argentina. The authors thank Virginia
Gunther Frers for help in preparing the manuscript and ELMA and
Hospital Español Laboratories for excellent technical assistence. References 1. Toskes PP. Bacterial overgrowth of gastrointestinal tract. Adv Intern Med 1993; 38: 387-407. 2. Gracey M. Contaminated small bowel syndrome patho-genesis, diagnosis and treatment. Am J Clin Nutr 1979; 32: 234-43. 3. Kirch M. Bacterial overgrowth. Am J Gastroenterol 1990; 85: 231-7 4. King C, Toskes PP. Comparison of the 1-gram [14C] xylose 10-gram lactulose H2 and 80-gram glucose H2 breath test in patients with small intestinal bacterial overgrowth. Gastroenterology 1986; 91: 1447-51. 5. Kerlin P, Wong L. Breath hydrogen testing in bacterial overgrowth of the small intestine.Gastroenterology 1988; 95: 982-8. 6. Vanderhoof JA, Langnas AN. Short -bowel syndrome in children and adults. Gastroenterology 1997; 112: 1767-78. 7. Schaafsma G. State of the art concerning probiotic strains in milk products. IDF Nutr Newsl 1996; 5: 23-4. 8. Mudsen KL, Doyle JS, Jewell LD, et al. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 1999; 116: 1107-14. 9. Collins JK, Murphy L. A controlled trial of probiotic treatment of IL-10 knockout mice. Gastroenterology 1999; 116: A2981(Abstract). 10. Kruis W, Schuts E, Fric P, Fixa B, Judmaierg G, Stolte M. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 1997; 11: 853-8. 11. Rembacken BJ, Snelling AM, Haukey PM, Chalmers DM, Axon ART. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomized trial. Lancet 1999; 354: 635-9. 12. Gianchetti P, Rizello F, Venturi A, et al. Oral bacterio-therapy as maintenance treatmen in patients with chronic pouchitis: a double-blind placebo controlled trial Gastroen-terology 2000; 119: 305-9. 13. Venturi A, Gionchetti P, Rizello F, et al. Impact on the fecal flora composition by a new probiotic preparation: prelimi-nary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmac Ther 1999; 13: 1103-8. 14. Isolauri E, Kaila M, Mykkanen M, Ling WH, Salimen S. Oral bacteriotherapy for viral gastroenteritis. Dig Dis Sci 1994; 39: 2595-600. 15. Gonzalez SN, Cardozo R, Apella MC, Oliver G. Biothe-rapeutic role of fermented milk. Biotherapy 1995; 8: 129-34. 16. Young RJ, Vanderhoof JA. Probiotic therapy in children with short bowel syndrome and bacterial overgrowth. Gastroenterology 1997; 112: A916.(Abstract). 17. Attar A, Flourie B, Rambaud et al. Antibiotic efficacy in small intestinal bacterial overgrowth-related chronic diarrhea: a crossover randomized trial. Gastroenterology 1999; 117: 794-7. 18. Kleeman EG, Klaenhammer TR. Adherence of Lacto-bacillus species to human fetal intestinal cell. J Dairy Sci 1982; 65: 2063-9. 19. Dunne C, O´Halloran S, O’Mahony LO, et al. Epithelial adhesion of probiotic microorganism in vitro and in vivo. Gastroenterology 1999; 116: G3058 (Abstract). 20. Wagner ED, Warner T, Roberts L, et al. Colonization of congenitally immunodeficient mice with probiotic bacteria. Infect Immun 1997; 65: 3345-51. 21. Lee YK, Salminen S. The coming age of probiotics. Trends Food Sci Technol 1995; 6: 241-5. 22. Kaila M, Isolauri E, Soppi E, et al. Enhancement of circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr Res 1992; 32: 141-4. 23. Malin M, Suomalainen H, Saxelin, M et al . Promotion of Ig A immune response in patients with Crohn’s disease by oral bacteriotherapy with Lactobacillus GG. Ann Nutr Metab 1996; 40: 137-45. 24. Isolauri E, Majamaa H, Arvola T, Rantala I, Virtanen E, Arvilommi H. Lactobacillus casei strain GG reverses increased intestinal permeability induced by cow milk in suckling rats. Gastroenterology 1993; 105: 1643-50. 25. Campieri M, Gionchetti P. Probiotics in inflammatory bowel disease: New insight to pathogenesis or a possible therapeutic alternative Editorial. Gastroenterology 1999; 116: 1246-60. 26. Fabia R, Ar Rajab A, Johansson ML, et al. The effect of exogenous administration of Lactobacillus reuteri R2LC and oat fiber on acetic acid-induced colitis in the rat. Scan J Gastroenterol 1993; 28: 155-62 27 . Nanji A.A, Khettry U, Sadrzadeh S.M. Lactobacillus feeding reduced endotoxemic unseverity of experimental alcoholic liver. Proc Soc Exp Bio Med 1994; 205: 243-7. 28. Gaón D, Dowek J, Gomez Zavaglia A, Ruiz Holgado AP, Oliver G. Digestión de la lactosa por una leche fermentada con Lactobacillus acidophilus y Lactobacillus casei de origen humano. Medicina (Buenos Aires) 1995; 55: 237-42. 29. Perdigon G, Alvarez S, Ruiz Holgado AP, et al. Immu- noadjuvant activity of oral Lactobacillus casei: Influence of dose on the secretory immune response and protective capacity in intestinal infections. J Dairy Res 1991; 58: 485-96. 30. Perdigon G, Alvarez S. Probiotics and the immune state. In Fuller R, (ed) Probiotic. The Scientific basis. London. Chapman and Hall, 1992 p146-76. Fig. 1.– Lactobacillus strains CERELA vs placebo. Treatment period and post treatment. Number of stools/day during 7 days of each period. Results are mean +/- SEM. Fig.2.– Lactobacillus
strains CERELA vs placebo. Treatment period and post treatment. Breath
excreted H2 concentration (ppm) for 2 hours at the end of each period.
Results are mean +/- SEM. Table 1.– Clinical
characteristics of the 2 patients groups for full data set Characteristics Lactobacillus (Cerela) Placebo p N=12 N=10 AGE (ys)a 50.83 ± 16.76 48.80 ± 16.35 NS SEX (M/F) 7 / 5 6 / 4 NS Duration of 3.08 ± 1.98 2.50 ± 1.58 NS diarrhea before inclusion (ys)a a Mean + / -SEM |
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