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Expression of capsules by haemophilus influenzae in mixed infections
Journal of Infection (1996) 33, 65-67
Letters to the Editor Expression of Capsules by Haemophilus influenzae in Mixed Infections Accepted for publication 22 February 1996 Sir, Brook and Gillmore ~ investigated the capacity of eight Haernophilus influenzae strains (3 non-capsulate and 5 'slightly capsulated') to cause abscess formation in mice, alone and mixed with encapsulated 'helper' strains of other species. No abscesses were formed when the eight H. influenzae strains or non-capsulated strains of the other species were inoculated alone. However, viable cells, non-viable cells or purified capsular material from the encapsulated helper strains resulted in abscess formation in ~:he majority of animals. The inoculation of the three non-capsulate and five slightly capsulated H. influenzae with one of the 'helpers' (viable, non-viable or capsular material) all resulted in abscess formation (i.e. as shown for the substances without H. influenzae). Profusely capsulated (type b) organisms were recovered from the lesions in the mice inoculated with mixtures containing the five originally 'slightly capsulated' strains but not those containing the non-capsulated strains. The newly profusely capsulated strains induced abscesses alone but encapsulation and the ability to form abscesses alone was lost after in vitro passage. The authors suggest that the recovery of the profusely type b capsulated H. influenzae is due to transformation or selection, where the protection afforded by the capsules of the helpers enables the survival and selection of encapsulated H. influenzae. The authors state that the five strains may have been genetically type b but not expressing sufficient material to react in the serological me~hods used and that gene probes are needed to determine whether these were type b strains or mutants. The in vitro loss of 1t. influenzae type b capsule has been shown to be a frequent event (0.1-0.3%) 2 which is due to the loss of the gene for export which occurs when the usually duplicated locus recombines to give a single copy. 3 It is extremely unlikely that these Hib- strains, which are non-serotypable mutants of type b, could acquire the gene(s) necessary to express type b capsule by transformation. It seems much more likely that the remaining genetically intact organisms would expand if conditions permitted. Animal passage has long been recognized to restore bacterial virulence and a method 0163~i453/96/040065 + 03 $12.00/0
for selecting encapsulated, virulent strains is probably what Brook and Gillmore I have demonstrated. Capsule gene probes are easily prepared and have been used widely since 1991.4-8 In addition, recently described PCRbased methods enable the unequivocal determination of capsule genotype and detection of mutant capsule genetic structures, s-10 Brook and Gillmore I have illustrated the problem of using in vitro passaged organisms for virulence studies and do not clearly demonstrate any 'helper' effect of capsules from other organisms in the in vivo situation with 'slightly capsulated' 1-1.influenzae strains. In addition, the ability of the non-capsulate strains to form abscesses only in the presence of the helpers may suggest that 'helpers' aid survival of non-capsulated organisms but an increase in virulence cannot be attributed as abscesses were also formed in the absence of the non-capsulate strains. The study of the role of individual components in a complex biological situation, such as the biofilm-like environment created in this study, remains extremely difficult and it is especially important to define the genetic characteristics of organisms used in such studies. With the ease and availability of probe- and PeR-based capsular genotyping, it is a pity that the authors did not ascertain the genetic status of encapsulation in their strains of H.
influenzae. J. Z ]ordens and M. P. E. Slack
PI-1LS, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.
References 1 Brook I, Gilhnore JD. Expression of capsules by Haemophilus influenzae in mixed infections. J Infect 1995; 30: 219-222. 2 Hoiseth SK, Connelly CJ, Moxon ER. Genetics of spontaneous highfrequency loss of b capsule in Haemophilus influenzae. Infect Immun 1985; 49: 389-395. 3 Kroll JS, Hopkins I, Moxon ER. Capsule loss in It. influenzae type b occurs by recombination-mediated disruption of a gene essential for polysaccharide export. Cell 1988; 53: 347-356. 4 Kroll JS, Ely S, Moxon ER. Capsular typing of Haemophilus influenzae with a DNA probe. Molec Cell Probe 1991; 5: 375-379. 5 Falla TJ, Dobson SRM, Crook DWM et al. Population-based study of non-typable Haernophilus ir~uenzae invasive disease in children and neonates. Lancet 1993; 341: 851-854. 6 St. Geme JW, Takala A, Esko E, Falkow S. Evidence for capsule gene sequences among pharyngeal isolates of nontypeable Haemophilus influenzae. J Infect Dis 1994; 169: 337-342. 7 Falla TI, Crook DWM, Anderson EC, et al. Characterization of capsular genes in HaemophiIus influenzae isolates from H. influenzae type b vaccine recipients. ] lnfect Dis 1995; 171: 1075-1076. 8 Leaves NI, Falla TJ, Crook DWM. The elucidation of novel capsular © 1996 The British Society for the Study of Infection
66
Letters to the Editor
genotypes of Haemophilus influenzae type b using the polymerase chain reaction. ] Med Microbiol 1995; 43: 120-124. 9 Jordens J2, Falla T, Slack MPE. Haemophilus influenzae--the past, the present and the future. In: Duerden BI, ed. 25 9ears on - an update on topics selectedfrom the first volume of the Journal. J Med Microbiol 1994; 41: 149-167. 10 Falla TI, Crook DWM, Brophy LN, Maskell D, Kroll JS, Moxon-ER. PeR for capsular typing of ttaemophilus influenzae. J Clin MicrobJol 1994; 32: 2382-2386.
Rotavirus Infection in AIDS-associated Diarrhoea Accepted for publication 22 March 1 9 9 6 Sir, Diarrhoea is a c o m m o n complication of h u m a n immunodeficiency virus (HIV) infection} Many infectious agents have been found to cause diarrhoea in AIDS patients including bacterial, protozoan and helminthic pathogens. 2 The role of viral infections, chiefly rotavirus, is still unclear. 3 The aim of the present study was to evaluate the role of rotavirus in AIDS-associated diarrhoea. We examined stool samples collected from 18 AIDS patients who presented with acute diarrhoea to the Institute of Infectious Diseases of Sassari University between October 1991 and April 1992. Diarrhoea, defined as more t h a n three fluid stools per day, resolved over few days and faecal specimens were collected early during acute illness. In addition, we studied stool samples obtained from 17 AIDS-patients without diarrhoea, admitted at our Institute during the same period. The two groups were comparable for age, sex, risk factors, AIDS index diseases, time of permanence in the CDC Stage IV, and CD4 cell count ( m e a n + S.D.: 125 + 86 vs. 116 + 78; Student's t-test; Ms). Stool specimens collected from 16 sex- and age-matched HIV-negative patients with acute diarrhoea were also studied. Faecal samples from 20 normal subjects served as controls. The specimens were examined by microscopy and culture, using standard methods, to exclude potentially pathogenic bacteria and parasites occurring in these patients (including Salmonella spp., Shigella spp., Campylobacter jejuni, Yersinia enterocolitica, Aeromonas hydrophila, Vibrio parahaemoliticus, atypical mycobacteria, Cryptosporidium spp., Isopora belli, Giardia lamblia, Entamoeba hystolitica, Strongiloides stercoralis). Chromotrope stains were used for microsporidia. Clostridium difficile toxin was detected by a standard cytotoxic assay. Rotavirus antigen detection in faeces was performed by an available enzymelinked immunosorbent assay (Rotazyme II, Abbott
Table I. Prevalence of antibody to rotavirus in different sera-groups. Group
n
No. positive
(%)
Healthy subjects HIV-negativeindividuals with diarrhoea AIDS patients without diarrhoea AIDS patients with diarrhoea
40
1
(2.5)
27
1
(3.7)
57
11
(19.3)
18
8
(44.4)
Laboratories, North Chicago, Ill, U.S.A.). The rapid resolution of diarrhoea induced us to exclude an endoscopic evaluation with biopsies (for the CMV detection). Rotavirus antigen was found in six (33.3%) out of 18 AIDS patients with diarrhoea. There was no difference in CD4 cell count between rotavirus-positive and rotavirusnegative patients (136 + 108 vs. 120 + 105; Student's ttest; Ms). No positive result was seen in other groups. In addition we retrospectively looked for anti-rotavirus antibodies in serum samples collected from further 18 AIDS patients with and from 57 AIDS patients without acute diarrhoea. Circulating anti-rotavirus immunoglobulins were also measured in 27 HIV-negative patients with acute diarrhoea and in 40 healthy subjects by using complement fixation test. As can be seen in Table I, the prevalence of rotavirus infection differed significantly a m o n g the groups we examined (Chi-squared test; P = 0.0011). In HIV-negative individuals with diarrhoea the exposure to the virus was of the same order of that in normal subjects; on the contrary, a higher frequency of anti-rotavirus antibodies was present in AIDS-patients who did (Fisher's exact test; P = 0 . 0 0 1 6 ) or did not (Fisher's exact test; P = 0 . 0 1 3 ) have abdominal complaints; a significant difference was also found between the two groups of AIDS patients (Fisher's exact test; P = 0.037). The present data would suggest a role for rotavirus in diarrhoea of our AIDS patients. Although rotavirus infections are primarily confined to infants and small children, they are frequently associated with diarrhoea in adults, particularly family members of affected infants, geriatric patients, and immunocompromized hosts} It is still unclear whether this virus m a y be an important aetiologic agent of diarrhoea in HIV-infected patients. Indeed, conflicting results have been reported in the studies dealing with rotavirus infections in AIDS. Smith et aI. were unable to detect rotavirus antigen in any patients' stools~; in contrast, rotavirus was the predominant pathogen detected in the stools of homosexual men with AIDS and diarrhoea in Sydney. 6 Such disparate results suggest marked geographic variations in gastrointestinal viral infections in these patients. In the
Letters to the Editor Expression of Capsules by Haemophilus influenzae in Mixed Infections Accepted for publication 22 February 1996 Sir, Brook and Gillmore ~ investigated the capacity of eight Haernophilus influenzae strains (3 non-capsulate and 5 'slightly capsulated') to cause abscess formation in mice, alone and mixed with encapsulated 'helper' strains of other species. No abscesses were formed when the eight H. influenzae strains or non-capsulated strains of the other species were inoculated alone. However, viable cells, non-viable cells or purified capsular material from the encapsulated helper strains resulted in abscess formation in ~:he majority of animals. The inoculation of the three non-capsulate and five slightly capsulated H. influenzae with one of the 'helpers' (viable, non-viable or capsular material) all resulted in abscess formation (i.e. as shown for the substances without H. influenzae). Profusely capsulated (type b) organisms were recovered from the lesions in the mice inoculated with mixtures containing the five originally 'slightly capsulated' strains but not those containing the non-capsulated strains. The newly profusely capsulated strains induced abscesses alone but encapsulation and the ability to form abscesses alone was lost after in vitro passage. The authors suggest that the recovery of the profusely type b capsulated H. influenzae is due to transformation or selection, where the protection afforded by the capsules of the helpers enables the survival and selection of encapsulated H. influenzae. The authors state that the five strains may have been genetically type b but not expressing sufficient material to react in the serological me~hods used and that gene probes are needed to determine whether these were type b strains or mutants. The in vitro loss of 1t. influenzae type b capsule has been shown to be a frequent event (0.1-0.3%) 2 which is due to the loss of the gene for export which occurs when the usually duplicated locus recombines to give a single copy. 3 It is extremely unlikely that these Hib- strains, which are non-serotypable mutants of type b, could acquire the gene(s) necessary to express type b capsule by transformation. It seems much more likely that the remaining genetically intact organisms would expand if conditions permitted. Animal passage has long been recognized to restore bacterial virulence and a method 0163~i453/96/040065 + 03 $12.00/0
for selecting encapsulated, virulent strains is probably what Brook and Gillmore I have demonstrated. Capsule gene probes are easily prepared and have been used widely since 1991.4-8 In addition, recently described PCRbased methods enable the unequivocal determination of capsule genotype and detection of mutant capsule genetic structures, s-10 Brook and Gillmore I have illustrated the problem of using in vitro passaged organisms for virulence studies and do not clearly demonstrate any 'helper' effect of capsules from other organisms in the in vivo situation with 'slightly capsulated' 1-1.influenzae strains. In addition, the ability of the non-capsulate strains to form abscesses only in the presence of the helpers may suggest that 'helpers' aid survival of non-capsulated organisms but an increase in virulence cannot be attributed as abscesses were also formed in the absence of the non-capsulate strains. The study of the role of individual components in a complex biological situation, such as the biofilm-like environment created in this study, remains extremely difficult and it is especially important to define the genetic characteristics of organisms used in such studies. With the ease and availability of probe- and PeR-based capsular genotyping, it is a pity that the authors did not ascertain the genetic status of encapsulation in their strains of H.
influenzae. J. Z ]ordens and M. P. E. Slack
PI-1LS, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.
References 1 Brook I, Gilhnore JD. Expression of capsules by Haemophilus influenzae in mixed infections. J Infect 1995; 30: 219-222. 2 Hoiseth SK, Connelly CJ, Moxon ER. Genetics of spontaneous highfrequency loss of b capsule in Haemophilus influenzae. Infect Immun 1985; 49: 389-395. 3 Kroll JS, Hopkins I, Moxon ER. Capsule loss in It. influenzae type b occurs by recombination-mediated disruption of a gene essential for polysaccharide export. Cell 1988; 53: 347-356. 4 Kroll JS, Ely S, Moxon ER. Capsular typing of Haemophilus influenzae with a DNA probe. Molec Cell Probe 1991; 5: 375-379. 5 Falla TJ, Dobson SRM, Crook DWM et al. Population-based study of non-typable Haernophilus ir~uenzae invasive disease in children and neonates. Lancet 1993; 341: 851-854. 6 St. Geme JW, Takala A, Esko E, Falkow S. Evidence for capsule gene sequences among pharyngeal isolates of nontypeable Haemophilus influenzae. J Infect Dis 1994; 169: 337-342. 7 Falla TI, Crook DWM, Anderson EC, et al. Characterization of capsular genes in HaemophiIus influenzae isolates from H. influenzae type b vaccine recipients. ] lnfect Dis 1995; 171: 1075-1076. 8 Leaves NI, Falla TJ, Crook DWM. The elucidation of novel capsular © 1996 The British Society for the Study of Infection
66
Letters to the Editor
genotypes of Haemophilus influenzae type b using the polymerase chain reaction. ] Med Microbiol 1995; 43: 120-124. 9 Jordens J2, Falla T, Slack MPE. Haemophilus influenzae--the past, the present and the future. In: Duerden BI, ed. 25 9ears on - an update on topics selectedfrom the first volume of the Journal. J Med Microbiol 1994; 41: 149-167. 10 Falla TI, Crook DWM, Brophy LN, Maskell D, Kroll JS, Moxon-ER. PeR for capsular typing of ttaemophilus influenzae. J Clin MicrobJol 1994; 32: 2382-2386.
Rotavirus Infection in AIDS-associated Diarrhoea Accepted for publication 22 March 1 9 9 6 Sir, Diarrhoea is a c o m m o n complication of h u m a n immunodeficiency virus (HIV) infection} Many infectious agents have been found to cause diarrhoea in AIDS patients including bacterial, protozoan and helminthic pathogens. 2 The role of viral infections, chiefly rotavirus, is still unclear. 3 The aim of the present study was to evaluate the role of rotavirus in AIDS-associated diarrhoea. We examined stool samples collected from 18 AIDS patients who presented with acute diarrhoea to the Institute of Infectious Diseases of Sassari University between October 1991 and April 1992. Diarrhoea, defined as more t h a n three fluid stools per day, resolved over few days and faecal specimens were collected early during acute illness. In addition, we studied stool samples obtained from 17 AIDS-patients without diarrhoea, admitted at our Institute during the same period. The two groups were comparable for age, sex, risk factors, AIDS index diseases, time of permanence in the CDC Stage IV, and CD4 cell count ( m e a n + S.D.: 125 + 86 vs. 116 + 78; Student's t-test; Ms). Stool specimens collected from 16 sex- and age-matched HIV-negative patients with acute diarrhoea were also studied. Faecal samples from 20 normal subjects served as controls. The specimens were examined by microscopy and culture, using standard methods, to exclude potentially pathogenic bacteria and parasites occurring in these patients (including Salmonella spp., Shigella spp., Campylobacter jejuni, Yersinia enterocolitica, Aeromonas hydrophila, Vibrio parahaemoliticus, atypical mycobacteria, Cryptosporidium spp., Isopora belli, Giardia lamblia, Entamoeba hystolitica, Strongiloides stercoralis). Chromotrope stains were used for microsporidia. Clostridium difficile toxin was detected by a standard cytotoxic assay. Rotavirus antigen detection in faeces was performed by an available enzymelinked immunosorbent assay (Rotazyme II, Abbott
Table I. Prevalence of antibody to rotavirus in different sera-groups. Group
n
No. positive
(%)
Healthy subjects HIV-negativeindividuals with diarrhoea AIDS patients without diarrhoea AIDS patients with diarrhoea
40
1
(2.5)
27
1
(3.7)
57
11
(19.3)
18
8
(44.4)
Laboratories, North Chicago, Ill, U.S.A.). The rapid resolution of diarrhoea induced us to exclude an endoscopic evaluation with biopsies (for the CMV detection). Rotavirus antigen was found in six (33.3%) out of 18 AIDS patients with diarrhoea. There was no difference in CD4 cell count between rotavirus-positive and rotavirusnegative patients (136 + 108 vs. 120 + 105; Student's ttest; Ms). No positive result was seen in other groups. In addition we retrospectively looked for anti-rotavirus antibodies in serum samples collected from further 18 AIDS patients with and from 57 AIDS patients without acute diarrhoea. Circulating anti-rotavirus immunoglobulins were also measured in 27 HIV-negative patients with acute diarrhoea and in 40 healthy subjects by using complement fixation test. As can be seen in Table I, the prevalence of rotavirus infection differed significantly a m o n g the groups we examined (Chi-squared test; P = 0.0011). In HIV-negative individuals with diarrhoea the exposure to the virus was of the same order of that in normal subjects; on the contrary, a higher frequency of anti-rotavirus antibodies was present in AIDS-patients who did (Fisher's exact test; P = 0 . 0 0 1 6 ) or did not (Fisher's exact test; P = 0 . 0 1 3 ) have abdominal complaints; a significant difference was also found between the two groups of AIDS patients (Fisher's exact test; P = 0.037). The present data would suggest a role for rotavirus in diarrhoea of our AIDS patients. Although rotavirus infections are primarily confined to infants and small children, they are frequently associated with diarrhoea in adults, particularly family members of affected infants, geriatric patients, and immunocompromized hosts} It is still unclear whether this virus m a y be an important aetiologic agent of diarrhoea in HIV-infected patients. Indeed, conflicting results have been reported in the studies dealing with rotavirus infections in AIDS. Smith et aI. were unable to detect rotavirus antigen in any patients' stools~; in contrast, rotavirus was the predominant pathogen detected in the stools of homosexual men with AIDS and diarrhoea in Sydney. 6 Such disparate results suggest marked geographic variations in gastrointestinal viral infections in these patients. In the