429
characteristics, and medication in the previous 2 weeks. Stools were examined for ova and parasites by direct wet preparation and specimens with faecal leucocytes were cultured on selective media for shigella and salmonella. 248 specimens (25%) contained B hominis, 211 contained Entamoeba histolytica, and 62 were positive for Giardia intestinalis;
Shigella and Salmonella spp accounted for 9% of specimens. No previous medication was reported for 747 (75%) samples. Tinidazole/metronidazole was reported for 107, tetracycline for 56, and co-trimoxazole for 47. Use of ampicillin, dicloxacillin, erythromycin, diiodohydroxyquine, diloxanide furoate, quinacrine, or mebendazole was reported for small numbers. Previous treatment with tinidazole/metronidazole did not affect the positivity rate for B hominis; as expected, the percentages of E histolytica and G intestinalis recovered were lower. Tetracycline use decreased the recovery rate of B hominis (25% vs 14%; p 0-08) but the striking result was for co-trimoxazole and B hominis when only 1 sample of 47 was positive (p 00004): =
=
Co-t
Organism
(n = 47)
Previous treatment Tet Tin/Met
(n = 56)
B hominis 2% 14% E histolytica 26% 21% Gmmunafu 4% 5% Co-t=co-tnmoxazo!e, Tet = tetracycline,
None
(n=107) (n=747) 23% 10%
27% 22% 3% 7% Tin/Met = tlnldazole/
metronidazole
Several antiparasitic therapies (eg, metronidazole) have been recommended for B hominis infection but their efficacy is unclear and no controlled trials have been done.1 In-vitro studies have shown an inhibitory effect against B hominis for the following drugs: emetine > metronidazole > furazolidone > co-trimoxazole > iodochlorhydroxyquine > pentamidine isethionate.2 In our study, previous use of tinidazole or metronidazole had no effect on recovery of B hominis but co-trimozazole almost completely eliminated it, while not effecting the other pathogens (ie, this result cannot be attributed to elimination of a coexisting organism). Kain et aP demonstrated a significant reduction of B hominis in patients who had recently taken salazopyrine, another sulphate-containing drug. Our findings suggest that prospective studies on the efficacy of co-trimoxazole may be warranted should B hominis be more clearly shown to be a pathogen. CIWEC Clinic,
Kathmandu, Nepal American Peace Kathmandu
ELI SCHWARTZ*
Corps Clinic,
ROBIN HOUSTON†
*,†Present addresses. *Medical Centre for the Traveler, Misgav Ladach Hospital, Jerusalem 93190, Israel; t Emory School of Public Health, Atlanta, Georgia, USA. 1. Markell EK, Udkow MP. Blastocystis hominis: pathogen or fellow traveler? Am J Trop Med Hyg 1986; 35: 1023-26 2. Zierdt CH, Swan JC, Hosseini J. In vitro response of Blastocystis hominis to antiprotozoal drugs. J Protozool 1983; 30: 332-34. 3 Kain KC, Noble MA, Freeman HKJ, Barteluk RL. Epidemiology and clinical features associated with Blastocystis hominis infection. Diagn Microbiol Infect Dis
1987; 8: 235-44.
False-positive legionella serology in campylobacter infection SIR,-Dr Boswell and Dr Kudesia (Jan 18, p 191) report the frequent detection of antilegionella antibodies in patients with campylobacter infection. We report findings that corroborate and extend their observations. In February, 1991, a 57-year-old man with microbiologically confirmed Campylobacterjejuni diarrhoea was incidentally found to have an antibody titre of 512 to Legionella pneumophila serogroup 1 by the indirect immunofluorescent antibody test (IFAT) with formalised yolk-sac antigen. The reactivity in IFAT was abolished by absorption with heat-killed Cjejuni. This absorption was specific since it had no effect on the titre of other antibodies present in the patient’s serum. IFAT-positive serum samples from patients with legionnaires’ disease were also unaffected. Since then we have obtained serum samples from 16 further patients with campylobacter infection in the acute and convalescent stages-12 with Cjejuni, 3 with C coli, and 1 untyped. None was
positive by IFAT in acute-phase samples but 4 patients were positive in samples collected 4-10 days later. Positive samples had titres of 16, 32, 128, and 1024. 2 of these were from patients with C coli, 1 from C jejuni, and 1 untyped infection. Preliminary immunoblotting with these serum samples shows that the main increase in antilegionella antibody appears as a broad lowmolecular-weight band, suggesting that lipopolysaccharide is the principal cross-reactive antigen. These epitopes elicit IgM and IgG antibody responses. Although the frequency of false-positive legionella IFAT tests in campylobacter infection (25%) was significantly lower than that recorded by Boswell and Kudesia (71 %), this nevertheless remains a potentially important source of diagnostic error, which may result in both inappropriate clinical management and epidemological investigation. We recommend that any patient with a positive legionella IFAT after diarrhoeal illness that is not associated with pneumonia has, in addition to a stool culture, a repeat IFAT test after absorption of their serum samples with heat-killed C jejuni. Formalin yolk-sac antigen was supplied by the Public Health Laboratory Service, London.
Department of Medical Microbiology, Royal Liverpool University Hospital, Liverpool L7 8XP, UK Infectious Diseases Unit, Public Health Laboratory,
J. S. CHEESBROUGH T. MAKIN B. C. TAXMAN
Regional
Fazakerley Hospital, Liverpool
N. K.
J. BEECHING J. MUTTON
Immunofluorescence technique for the identification of polioviruses SiR,—To investigate the efficacy of oral poliomyelitis vaccine or the inactivated vaccine in the induction of gut immunity to polioviruses in Gambian infants, an immunofluorescence technique with monoclonal antibodies has been developed to detect
polioviruses in stool specimens. 435 stool specimens were obtained from immunised infants one week after challenge with a standard dose of trivalent or monovalent type-1oral poliomyelitis vaccine. Poliovirus was isolated on HEP-2 Cincinnati cells and identified by the virus neutralisation technique by standard methods1 and by a new indirect immunofluorescence technique. Briefly, the Hep-2 cells were infected with a 1 in 10 dilution of a second passage of the virus in question. When 25-50% of the cells showed a cytopathic effect, they were removed from the culture vessel with phosphate-buffered saline and edetic acid (EDTA), washed and pelleted by centrifugation, and resuspended at 2 x 106 cells/ml in phosphate-buffered saline. 25 at samples of this suspension were placed on each spot of a multispot slide, air-dried, fixed in cold acetone for 5 min, and then stored desiccated in silica gel at -20°C until used. Mouse monoclonal antibodies 1588 (anti-polio type 1), 267 (anti-polio type 2), and 204 (anti-polio type 3) were used at a dilution of 1 in 300 and bound antibodies were detected by ultraviolet-light microscopy with fluorosceinisothiocyanate-labelled F(ab’)2 fragment of goat anti-mouse IgG antibody (full details available from the authors). Preliminary experiments with reference virus strains showed that the monoclonal antibodies were poliovirus type-specific and did not crossreact with the 2 coxsackie viruses (types A9 and B2) or the 2 echoviruses (types 7 and 30) tested. To see if the antibodies reacted type-specifically against both vaccine and wild-type polioviruses, we tested them by immunofluorescence against several intratypic strains of virus that had been characterised by molecular sequencing and by neutralisation with monoclonal antibodies. Monoclonal 1588 detected all 16 wild and 15 vaccine type-1strains. Antibody 267 detected 9 of 10 wild and 8 of 10 vaccine type-2 strains, and antibody 204 detected 6 of 8 wild and 10 of 10 vaccine type-3 strains. Each antibody proved type-specific and did not crossreact with heterologous wild or vaccine strains. Cytopathic effect was noted in 105 of the 435 cultures of stool, and the results of examination of these positive cultures by virus neutralisation or immunofluorescence were compared. Overall, immunofluorescence identified 64 polioviruses (39 type 1,16 type 2,
characteristics, and medication in the previous 2 weeks. Stools were examined for ova and parasites by direct wet preparation and specimens with faecal leucocytes were cultured on selective media for shigella and salmonella. 248 specimens (25%) contained B hominis, 211 contained Entamoeba histolytica, and 62 were positive for Giardia intestinalis;
Shigella and Salmonella spp accounted for 9% of specimens. No previous medication was reported for 747 (75%) samples. Tinidazole/metronidazole was reported for 107, tetracycline for 56, and co-trimoxazole for 47. Use of ampicillin, dicloxacillin, erythromycin, diiodohydroxyquine, diloxanide furoate, quinacrine, or mebendazole was reported for small numbers. Previous treatment with tinidazole/metronidazole did not affect the positivity rate for B hominis; as expected, the percentages of E histolytica and G intestinalis recovered were lower. Tetracycline use decreased the recovery rate of B hominis (25% vs 14%; p 0-08) but the striking result was for co-trimoxazole and B hominis when only 1 sample of 47 was positive (p 00004): =
=
Co-t
Organism
(n = 47)
Previous treatment Tet Tin/Met
(n = 56)
B hominis 2% 14% E histolytica 26% 21% Gmmunafu 4% 5% Co-t=co-tnmoxazo!e, Tet = tetracycline,
None
(n=107) (n=747) 23% 10%
27% 22% 3% 7% Tin/Met = tlnldazole/
metronidazole
Several antiparasitic therapies (eg, metronidazole) have been recommended for B hominis infection but their efficacy is unclear and no controlled trials have been done.1 In-vitro studies have shown an inhibitory effect against B hominis for the following drugs: emetine > metronidazole > furazolidone > co-trimoxazole > iodochlorhydroxyquine > pentamidine isethionate.2 In our study, previous use of tinidazole or metronidazole had no effect on recovery of B hominis but co-trimozazole almost completely eliminated it, while not effecting the other pathogens (ie, this result cannot be attributed to elimination of a coexisting organism). Kain et aP demonstrated a significant reduction of B hominis in patients who had recently taken salazopyrine, another sulphate-containing drug. Our findings suggest that prospective studies on the efficacy of co-trimoxazole may be warranted should B hominis be more clearly shown to be a pathogen. CIWEC Clinic,
Kathmandu, Nepal American Peace Kathmandu
ELI SCHWARTZ*
Corps Clinic,
ROBIN HOUSTON†
*,†Present addresses. *Medical Centre for the Traveler, Misgav Ladach Hospital, Jerusalem 93190, Israel; t Emory School of Public Health, Atlanta, Georgia, USA. 1. Markell EK, Udkow MP. Blastocystis hominis: pathogen or fellow traveler? Am J Trop Med Hyg 1986; 35: 1023-26 2. Zierdt CH, Swan JC, Hosseini J. In vitro response of Blastocystis hominis to antiprotozoal drugs. J Protozool 1983; 30: 332-34. 3 Kain KC, Noble MA, Freeman HKJ, Barteluk RL. Epidemiology and clinical features associated with Blastocystis hominis infection. Diagn Microbiol Infect Dis
1987; 8: 235-44.
False-positive legionella serology in campylobacter infection SIR,-Dr Boswell and Dr Kudesia (Jan 18, p 191) report the frequent detection of antilegionella antibodies in patients with campylobacter infection. We report findings that corroborate and extend their observations. In February, 1991, a 57-year-old man with microbiologically confirmed Campylobacterjejuni diarrhoea was incidentally found to have an antibody titre of 512 to Legionella pneumophila serogroup 1 by the indirect immunofluorescent antibody test (IFAT) with formalised yolk-sac antigen. The reactivity in IFAT was abolished by absorption with heat-killed Cjejuni. This absorption was specific since it had no effect on the titre of other antibodies present in the patient’s serum. IFAT-positive serum samples from patients with legionnaires’ disease were also unaffected. Since then we have obtained serum samples from 16 further patients with campylobacter infection in the acute and convalescent stages-12 with Cjejuni, 3 with C coli, and 1 untyped. None was
positive by IFAT in acute-phase samples but 4 patients were positive in samples collected 4-10 days later. Positive samples had titres of 16, 32, 128, and 1024. 2 of these were from patients with C coli, 1 from C jejuni, and 1 untyped infection. Preliminary immunoblotting with these serum samples shows that the main increase in antilegionella antibody appears as a broad lowmolecular-weight band, suggesting that lipopolysaccharide is the principal cross-reactive antigen. These epitopes elicit IgM and IgG antibody responses. Although the frequency of false-positive legionella IFAT tests in campylobacter infection (25%) was significantly lower than that recorded by Boswell and Kudesia (71 %), this nevertheless remains a potentially important source of diagnostic error, which may result in both inappropriate clinical management and epidemological investigation. We recommend that any patient with a positive legionella IFAT after diarrhoeal illness that is not associated with pneumonia has, in addition to a stool culture, a repeat IFAT test after absorption of their serum samples with heat-killed C jejuni. Formalin yolk-sac antigen was supplied by the Public Health Laboratory Service, London.
Department of Medical Microbiology, Royal Liverpool University Hospital, Liverpool L7 8XP, UK Infectious Diseases Unit, Public Health Laboratory,
J. S. CHEESBROUGH T. MAKIN B. C. TAXMAN
Regional
Fazakerley Hospital, Liverpool
N. K.
J. BEECHING J. MUTTON
Immunofluorescence technique for the identification of polioviruses SiR,—To investigate the efficacy of oral poliomyelitis vaccine or the inactivated vaccine in the induction of gut immunity to polioviruses in Gambian infants, an immunofluorescence technique with monoclonal antibodies has been developed to detect
polioviruses in stool specimens. 435 stool specimens were obtained from immunised infants one week after challenge with a standard dose of trivalent or monovalent type-1oral poliomyelitis vaccine. Poliovirus was isolated on HEP-2 Cincinnati cells and identified by the virus neutralisation technique by standard methods1 and by a new indirect immunofluorescence technique. Briefly, the Hep-2 cells were infected with a 1 in 10 dilution of a second passage of the virus in question. When 25-50% of the cells showed a cytopathic effect, they were removed from the culture vessel with phosphate-buffered saline and edetic acid (EDTA), washed and pelleted by centrifugation, and resuspended at 2 x 106 cells/ml in phosphate-buffered saline. 25 at samples of this suspension were placed on each spot of a multispot slide, air-dried, fixed in cold acetone for 5 min, and then stored desiccated in silica gel at -20°C until used. Mouse monoclonal antibodies 1588 (anti-polio type 1), 267 (anti-polio type 2), and 204 (anti-polio type 3) were used at a dilution of 1 in 300 and bound antibodies were detected by ultraviolet-light microscopy with fluorosceinisothiocyanate-labelled F(ab’)2 fragment of goat anti-mouse IgG antibody (full details available from the authors). Preliminary experiments with reference virus strains showed that the monoclonal antibodies were poliovirus type-specific and did not crossreact with the 2 coxsackie viruses (types A9 and B2) or the 2 echoviruses (types 7 and 30) tested. To see if the antibodies reacted type-specifically against both vaccine and wild-type polioviruses, we tested them by immunofluorescence against several intratypic strains of virus that had been characterised by molecular sequencing and by neutralisation with monoclonal antibodies. Monoclonal 1588 detected all 16 wild and 15 vaccine type-1strains. Antibody 267 detected 9 of 10 wild and 8 of 10 vaccine type-2 strains, and antibody 204 detected 6 of 8 wild and 10 of 10 vaccine type-3 strains. Each antibody proved type-specific and did not crossreact with heterologous wild or vaccine strains. Cytopathic effect was noted in 105 of the 435 cultures of stool, and the results of examination of these positive cultures by virus neutralisation or immunofluorescence were compared. Overall, immunofluorescence identified 64 polioviruses (39 type 1,16 type 2,
