1462
Identification of the Rochalimaea henselae 16S rRNA Sequence in the Liver of a French Patient with Bacillary Peliosis Hepatis
Reprints or correspondence: Dr. Stefano Marullo, Laboratoire d'Imrnunopharmacologie Moleculaire, ICGM. 22 rue Mechain, 75013 Paris, France. The Journal of Infectious Diseases 1992;166:1462 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0047$01.00
Inhibition of the Salmonella typhi Oral Vaccine Strain, Ty21a, by Mefloquine and Chloroquine Colleagues-Travelers who visit a travelers' health service within weeks of departure may be prescribed the oral typhoid vaccine and antimalarial prophylaxis concurrently. We investigated whether mefloquine or chloroquine has activity against the Salmonella typhi Ty21 a vaccine strain to assess whether simultaneous administration might lead to decreased efficacy of the vaccine. Ty21a (Vivotif Berna, Swiss Serum and Vaccine Institute, Berne, Switzerland), lot 115371A, was used. Salmonella group
Reprints or correspondence: Dr. Harold Horowitz, Division oflnfectious Diseases. Westchester County Medical Center. Room 209, Macy Pavilion SE, Valhalla, NY 10595.
identical to that reported for R. henselae [I, 3, 7]. Therefore, while less common in Europe than in the United States, bacillary peliosis hepatis seems to be caused by the same microorganism. Three months before developing peliosis hepatis, our patient was given a cat. This suggests, as in many US cases, that cat scratches or bites from arthropods living on cats may be one possible inoculation mode of the pathogen. Stefano Marullo, Arnauld Jaccard, Dominique Roulot, Claire Mainguene, and Jean Pierre Clauvel Departenient d'Immunohematologle. Hiipital Saint Louis, Paris; Service d'Hepatogastroenterologie and Service d'Anatomie et de Cytologie Pathologiques, Hiipital Avicenne, Bobigny. France References
I. Welch DF. Pickett DA. Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae sp. nov.. a cause of septicemia, bacillary angiomatosis and parenchymal bacillary peliosis. J Clin Microbiol 1992;30:27580. 2. Slater LN. Welch DF. Min KW. Rochalimaea henselae causes bacillary angiomatosis and peliosis hepatis. Arch Intern Med 1992; 152:602-6. 3. Reiman DA, Loutit JS. Schmidt TM. Falkow S, Tompkins LS. The agent of bacillary angiomatosis-an approach to the identification of uncultured pathogens. N Engl J Med 1990;323: 1573-80. 4. Perkocha LA, Geaghan SM. Yen TSB, et al. Clinical and pathological features of bacillary peliosis hepatis in association with human immunodeficiency virus infection. N Engl J Med 1990;323:1581-6. 5. Slater LN. Welch DF. Hensel D. Coody DW. A newly recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N Engl J Med 1990;323: 1587-93. 6. Gyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Nat! Acad Sci USA 1988;85:7652-6. 7. Reiman DA. Falkow S, LeBoit PE. et al. The organism causing bacillary angiomatosis, peliosis hepatis, and fever and bacteremia in immunocompromised patients. N Engl J Med 1991;324:1514.
D, CCIOI, was isolated from a patient at Westchester County Medical Center (Valhalla, NY), identified with standard biochemical techniques, and used as a control strain. Stock solutions of mefloquine hydrochloride (Hoffmann-La Roche, Nutley, NJ) and chloroquine phosphate (Sterling Drug, Rensselaer, NY) were prepared in 10 mm of ethanol and distilled water, respectively, then sterile filtered. MICs ofantibiotics were determined by twofold dilutions ofmefloquine hydrochloride orchloroquine phosphate in brain-heart infusion (BHI) broth at an inoculum size of 105, 106 , and 107 cfu/ml., Control tubes contained BHI broth alone. All tubes were incubated at 35°C for 18 h. Time-kill experiments were done as follows. BHI broth containing mefloquine hydrochloride at 16 JLg/mL was inoculated with 106-10 7 cfu/ml. and incubated at 35°C. At each time point the tubes were vortexed and aliquots removed. Each aliquot was diluted in BHI broth, then plated onto trypticase soy agar (BBL, Cockeysville, MD) containing 5% sheep's blood and incubated at 35°C overnight to obtain viable cell counts. At later time points, when dilutions were not necessary, aliquots were spun
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 2, 2015
Colleagues-Rochalimaea henselae, a recently identified pathogen [1, 2], is the causative agent of bacillary angiomatosis [3], bacillary peliosis hepatis [4], and prolonged fever with persistent bacteremia [5], diseases that occur primarily in immunocompromised patients. Because these conditions have been found almost exclusively in patients living in the United States, the existence of R. henselae outside North America has been questioned. A 36-year-old French patient with AIDS, who had never been in North America, was seen in our department for fever, weight loss, abdominal pain, hepatosplenomegaly, pancytopenia, and moderately elevated levels of aminotransferase, -y-glutamyltransferase, and alkaline phosphatase. Transvenous liver biopsy yielded a tissue specimen with multiple peliotic spaces and microabscesses. A liver section stained by the Warthin-Starry technique showed clumps of bacilli, confirming the diagnosis ofbacillary peliosis hepatis. DNA was extracted from a fragment of the liver biopsy specimen and submitted to 30 cycles of polymerase chain reaction amplification, using primers p93E and p 13B as described by Reiman et al. [3]. A 480-bp fragment, corresponding to a eubacterial 16S rRNA gene region, was enzymatically amplified and submitted to an additional 30 cycles of amplification with unequal molar amounts of two amplification primers to generate ssDNA for sequence analysis [6]. The sequence obtained was
The Journal of Infectious Diseases 1992;166:1462-4 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0048$01.00
JID 1992; 166 (December)
Correspondence
Correspondence
lID 1992; 166 (December)
10'
Figure 1. In vitro time-kill curve of S. typhi Ty2la by mefloquine hydrochloride. At time O. Ty21 a was inoculated into brain-heart infusion broth alone (0) or containing 16 Itg/mL mefloquine (.).
following accumulation of toxic metabolic products [I]. Although the vaccine strain is rarely found in stool samples after the second day after vaccination and is not found in small intestinal aspirates after I day of vaccination [2], replication of the vaccine strain appears to be necessary for efficacy, since orally administered whole killed S. typhi vaccines have provided poor levels of protection even after 12 vaccine doses [3, 4]. The enteric-coated vaccine preparation has provided from 33%to 67%efficacy in field trials [5-7]. The degree ofprotection in vaccine trials has depended on multiple factors [3, 5-7], including inoculum size [3], dosing interval [5], frequency of dosing [8], age of the vaccinee [5, 6], and vaccine preparation (enteric-coated, non-enteric-coated, or liquid) [3, 5-7]. Four doses of 2-4 X 109 viable organisms in the enteric-coated preparation have been demonstrated to be superior to 3 doses [8]. At present, 4 doses of this preparation over 7 days (given on alternating days) are recommended for travelers. The clinical relevance of our findings depends on the complex relationship of the many factors that contribute to enhancing protective immunity induced by the vaccine as well as on the pharmacokinetics and bioavailability of chloroquine and mefioquine. Mefioquine appears to be much more likely than chloroquine to have a deleterious effect on immunogenicity of Ty21a because of the low MICs ofmefioquine for Ty21a and because of its rapid effect in time-kill studies. The present preparation of mefioquine hydrochloride is well absorbed and has an absorption t l / 2 of 1-4 h [9]. Despite excellent absorption, the recommended prophylactic dose of 250 mg of mefioquine weekly most probably leads to gastric concentrations considerably> 16 ~g/mL for many hours. Most ofthe data on mefioquine metabolism are from studies in animals in which the primary route of excretion is bile and feces [9]. If metabolism is similar in humans, vaccine organisms may also be inhibited in the small intestine by mefioquine that has been excreted by the enterohepatic circulation. Although peak plasma concentrations of mefioquine after a 250-mg oral dose are only in the nanogram range, the plasma levels are of uncertain relevance since it is unlikely that bloodstream invasion is necessary for vaccine efficacy. The data presented here raise the question of whether mefioquine administration should be separated from that of the Ty21a vaccine. It is not clear that the same considerations are necessary for chloroquine, given its higher MICs. However, chloroquine has been shown to decrease the immunogenicity of the rabies vaccine when the two were given concurrently and may have similar immunosuppressive effects on the Ty21a vaccine [10]. The exact timing of the dosage separation cannot be determined from these data. However, the inhibition of even I vaccine dose or the lowering of viable bacteria could potentially decrease vaccine efficacy. Clinical studies to determine the humoral and intestinal response to the Ty21 a vaccine in patients given mefioquine or chloroquine simultaneously would help shed light on this problem.
Harold Horowitz and Carol A. Carbonaro 45
90 135 TIME (minutes)
180
Department of Medicine, Division of Infectious Diseases and Microbiology, Westchester County Medical Center, Valhalla, New York
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 2, 2015
and resuspended in an equal volume of BHI broth before plating. Control tubes contained no antibiotic. The MICs ofchloroquine and mefioquine for Ty21 a were 512 and 16 ~g/mL, respectively. The results obtained with chloroquine for the control strain, CC I0 I, are identical to those for Ty21 a. However, the MIC of mefioquine was threefold greater for strain CCIO I than for Ty2la. At these concentrations, mefioquine is bactericidal for both Ty21 a and CC I0 I. Since stock solutions of mefioquine are prepared in 10 mm of ethanol, the possibility was considered that the observed effects of mefioquine were due to the presence of inhibitory concentrations of ethanol. Growth inhibition was not observed in mock MIC experiments in which ethanol was present at concentrations equivalent to those present in actual antibiotic-containing tubes. To determine the exposure time required for killing ofTy21a by mefioquine, time-kill curves were generated. Figure I depicts the results of one such experiment, showing that killing began quickly. Within 3 h, viable counts were decreased by nearly 3 logs. The oral live attenuated typhoid vaccine S. typhi Ty21a provides a relatively effective, safe, and more expeditious alternative to the traditional parenterally administered killed S. typhi vaccine. The oral vaccine strain is a mutant that lacks the enzyme UDP-galactose-4-epimerase activity and displays -20% of the activity ofthe parenteral levels ofgalactose-I-phosphate-uridyl transferase and galactokinase [I]. As a result, the vaccine strain replicates for a brief period before galactose-induced lysis
1463
Identification of the Rochalimaea henselae 16S rRNA Sequence in the Liver of a French Patient with Bacillary Peliosis Hepatis
Reprints or correspondence: Dr. Stefano Marullo, Laboratoire d'Imrnunopharmacologie Moleculaire, ICGM. 22 rue Mechain, 75013 Paris, France. The Journal of Infectious Diseases 1992;166:1462 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0047$01.00
Inhibition of the Salmonella typhi Oral Vaccine Strain, Ty21a, by Mefloquine and Chloroquine Colleagues-Travelers who visit a travelers' health service within weeks of departure may be prescribed the oral typhoid vaccine and antimalarial prophylaxis concurrently. We investigated whether mefloquine or chloroquine has activity against the Salmonella typhi Ty21 a vaccine strain to assess whether simultaneous administration might lead to decreased efficacy of the vaccine. Ty21a (Vivotif Berna, Swiss Serum and Vaccine Institute, Berne, Switzerland), lot 115371A, was used. Salmonella group
Reprints or correspondence: Dr. Harold Horowitz, Division oflnfectious Diseases. Westchester County Medical Center. Room 209, Macy Pavilion SE, Valhalla, NY 10595.
identical to that reported for R. henselae [I, 3, 7]. Therefore, while less common in Europe than in the United States, bacillary peliosis hepatis seems to be caused by the same microorganism. Three months before developing peliosis hepatis, our patient was given a cat. This suggests, as in many US cases, that cat scratches or bites from arthropods living on cats may be one possible inoculation mode of the pathogen. Stefano Marullo, Arnauld Jaccard, Dominique Roulot, Claire Mainguene, and Jean Pierre Clauvel Departenient d'Immunohematologle. Hiipital Saint Louis, Paris; Service d'Hepatogastroenterologie and Service d'Anatomie et de Cytologie Pathologiques, Hiipital Avicenne, Bobigny. France References
I. Welch DF. Pickett DA. Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae sp. nov.. a cause of septicemia, bacillary angiomatosis and parenchymal bacillary peliosis. J Clin Microbiol 1992;30:27580. 2. Slater LN. Welch DF. Min KW. Rochalimaea henselae causes bacillary angiomatosis and peliosis hepatis. Arch Intern Med 1992; 152:602-6. 3. Reiman DA, Loutit JS. Schmidt TM. Falkow S, Tompkins LS. The agent of bacillary angiomatosis-an approach to the identification of uncultured pathogens. N Engl J Med 1990;323: 1573-80. 4. Perkocha LA, Geaghan SM. Yen TSB, et al. Clinical and pathological features of bacillary peliosis hepatis in association with human immunodeficiency virus infection. N Engl J Med 1990;323:1581-6. 5. Slater LN. Welch DF. Hensel D. Coody DW. A newly recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N Engl J Med 1990;323: 1587-93. 6. Gyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Nat! Acad Sci USA 1988;85:7652-6. 7. Reiman DA. Falkow S, LeBoit PE. et al. The organism causing bacillary angiomatosis, peliosis hepatis, and fever and bacteremia in immunocompromised patients. N Engl J Med 1991;324:1514.
D, CCIOI, was isolated from a patient at Westchester County Medical Center (Valhalla, NY), identified with standard biochemical techniques, and used as a control strain. Stock solutions of mefloquine hydrochloride (Hoffmann-La Roche, Nutley, NJ) and chloroquine phosphate (Sterling Drug, Rensselaer, NY) were prepared in 10 mm of ethanol and distilled water, respectively, then sterile filtered. MICs ofantibiotics were determined by twofold dilutions ofmefloquine hydrochloride orchloroquine phosphate in brain-heart infusion (BHI) broth at an inoculum size of 105, 106 , and 107 cfu/ml., Control tubes contained BHI broth alone. All tubes were incubated at 35°C for 18 h. Time-kill experiments were done as follows. BHI broth containing mefloquine hydrochloride at 16 JLg/mL was inoculated with 106-10 7 cfu/ml. and incubated at 35°C. At each time point the tubes were vortexed and aliquots removed. Each aliquot was diluted in BHI broth, then plated onto trypticase soy agar (BBL, Cockeysville, MD) containing 5% sheep's blood and incubated at 35°C overnight to obtain viable cell counts. At later time points, when dilutions were not necessary, aliquots were spun
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 2, 2015
Colleagues-Rochalimaea henselae, a recently identified pathogen [1, 2], is the causative agent of bacillary angiomatosis [3], bacillary peliosis hepatis [4], and prolonged fever with persistent bacteremia [5], diseases that occur primarily in immunocompromised patients. Because these conditions have been found almost exclusively in patients living in the United States, the existence of R. henselae outside North America has been questioned. A 36-year-old French patient with AIDS, who had never been in North America, was seen in our department for fever, weight loss, abdominal pain, hepatosplenomegaly, pancytopenia, and moderately elevated levels of aminotransferase, -y-glutamyltransferase, and alkaline phosphatase. Transvenous liver biopsy yielded a tissue specimen with multiple peliotic spaces and microabscesses. A liver section stained by the Warthin-Starry technique showed clumps of bacilli, confirming the diagnosis ofbacillary peliosis hepatis. DNA was extracted from a fragment of the liver biopsy specimen and submitted to 30 cycles of polymerase chain reaction amplification, using primers p93E and p 13B as described by Reiman et al. [3]. A 480-bp fragment, corresponding to a eubacterial 16S rRNA gene region, was enzymatically amplified and submitted to an additional 30 cycles of amplification with unequal molar amounts of two amplification primers to generate ssDNA for sequence analysis [6]. The sequence obtained was
The Journal of Infectious Diseases 1992;166:1462-4 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0048$01.00
JID 1992; 166 (December)
Correspondence
Correspondence
lID 1992; 166 (December)
10'
Figure 1. In vitro time-kill curve of S. typhi Ty2la by mefloquine hydrochloride. At time O. Ty21 a was inoculated into brain-heart infusion broth alone (0) or containing 16 Itg/mL mefloquine (.).
following accumulation of toxic metabolic products [I]. Although the vaccine strain is rarely found in stool samples after the second day after vaccination and is not found in small intestinal aspirates after I day of vaccination [2], replication of the vaccine strain appears to be necessary for efficacy, since orally administered whole killed S. typhi vaccines have provided poor levels of protection even after 12 vaccine doses [3, 4]. The enteric-coated vaccine preparation has provided from 33%to 67%efficacy in field trials [5-7]. The degree ofprotection in vaccine trials has depended on multiple factors [3, 5-7], including inoculum size [3], dosing interval [5], frequency of dosing [8], age of the vaccinee [5, 6], and vaccine preparation (enteric-coated, non-enteric-coated, or liquid) [3, 5-7]. Four doses of 2-4 X 109 viable organisms in the enteric-coated preparation have been demonstrated to be superior to 3 doses [8]. At present, 4 doses of this preparation over 7 days (given on alternating days) are recommended for travelers. The clinical relevance of our findings depends on the complex relationship of the many factors that contribute to enhancing protective immunity induced by the vaccine as well as on the pharmacokinetics and bioavailability of chloroquine and mefioquine. Mefioquine appears to be much more likely than chloroquine to have a deleterious effect on immunogenicity of Ty21a because of the low MICs ofmefioquine for Ty21a and because of its rapid effect in time-kill studies. The present preparation of mefioquine hydrochloride is well absorbed and has an absorption t l / 2 of 1-4 h [9]. Despite excellent absorption, the recommended prophylactic dose of 250 mg of mefioquine weekly most probably leads to gastric concentrations considerably> 16 ~g/mL for many hours. Most ofthe data on mefioquine metabolism are from studies in animals in which the primary route of excretion is bile and feces [9]. If metabolism is similar in humans, vaccine organisms may also be inhibited in the small intestine by mefioquine that has been excreted by the enterohepatic circulation. Although peak plasma concentrations of mefioquine after a 250-mg oral dose are only in the nanogram range, the plasma levels are of uncertain relevance since it is unlikely that bloodstream invasion is necessary for vaccine efficacy. The data presented here raise the question of whether mefioquine administration should be separated from that of the Ty21a vaccine. It is not clear that the same considerations are necessary for chloroquine, given its higher MICs. However, chloroquine has been shown to decrease the immunogenicity of the rabies vaccine when the two were given concurrently and may have similar immunosuppressive effects on the Ty21a vaccine [10]. The exact timing of the dosage separation cannot be determined from these data. However, the inhibition of even I vaccine dose or the lowering of viable bacteria could potentially decrease vaccine efficacy. Clinical studies to determine the humoral and intestinal response to the Ty21 a vaccine in patients given mefioquine or chloroquine simultaneously would help shed light on this problem.
Harold Horowitz and Carol A. Carbonaro 45
90 135 TIME (minutes)
180
Department of Medicine, Division of Infectious Diseases and Microbiology, Westchester County Medical Center, Valhalla, New York
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on July 2, 2015
and resuspended in an equal volume of BHI broth before plating. Control tubes contained no antibiotic. The MICs ofchloroquine and mefioquine for Ty21 a were 512 and 16 ~g/mL, respectively. The results obtained with chloroquine for the control strain, CC I0 I, are identical to those for Ty21 a. However, the MIC of mefioquine was threefold greater for strain CCIO I than for Ty2la. At these concentrations, mefioquine is bactericidal for both Ty21 a and CC I0 I. Since stock solutions of mefioquine are prepared in 10 mm of ethanol, the possibility was considered that the observed effects of mefioquine were due to the presence of inhibitory concentrations of ethanol. Growth inhibition was not observed in mock MIC experiments in which ethanol was present at concentrations equivalent to those present in actual antibiotic-containing tubes. To determine the exposure time required for killing ofTy21a by mefioquine, time-kill curves were generated. Figure I depicts the results of one such experiment, showing that killing began quickly. Within 3 h, viable counts were decreased by nearly 3 logs. The oral live attenuated typhoid vaccine S. typhi Ty21a provides a relatively effective, safe, and more expeditious alternative to the traditional parenterally administered killed S. typhi vaccine. The oral vaccine strain is a mutant that lacks the enzyme UDP-galactose-4-epimerase activity and displays -20% of the activity ofthe parenteral levels ofgalactose-I-phosphate-uridyl transferase and galactokinase [I]. As a result, the vaccine strain replicates for a brief period before galactose-induced lysis
1463
