- Email: [email protected]
MYCOBACTERIAL CULTURE: WHAT TEMPERATURE?
534
Fig 1-Barium swallow, pharyngeal pouch.
Fig 2-Diagram of
demonstrating
1960
operation,’
which differs from ours only in that we divided the azygos vein.
barium Fig 3-Postoperative swallow, showing pouch draining into oesophagus.
The chest was opened along the upper border of the fifth rib. The the mediastinal pleura and down to the level of the azygos vein. The pleura over it was incised and the apex of the pouch dissected. The azygos vein was divided between ligatures and the oesophagus in this area exposed. When a good view of the pouch was obtained a pursestring was inserted in the apex and its contents sucked out. The area was packed with swabs soaked in mercuric chloride and the pouch was washed out with chlorhexidine. The oesophagus was incised longitudinally and the two openings, about 5 cm long, were sutured together with a single layer, taking full thickness of both pouch and oesophagus. The mediastinal pleura was closed over the anastomosis. A drain was taken up to the site and the chest was closed in layers. Contrast studies on postoperative day 6 revealed free drainage of the pouch and an intact anastomosis (fig 3). After treatment for a chest infection the patient was discharged 3 weeks after the operation with his dysphagia much improved. He remains well and has put on 6kg in 2 months.
By contrast, 7 patients in our unit had neutropenic enterocolitis due to C septicum. 5 had bowel resected and 3 of them survived. The other 2 died within 12 h of presentation without surgical intervention. In 1 patient who underwent laparotomy and was receiving intravenous vancomycin (which is active against C septicum) C difficile was initially thought to be implicated, on the basis of isolation from faeces and detection of cytotoxin. Blood cultures were negative but C septicum was demonstrated in the bowel wall by direct ffi=unofluorescence.1 We have found that neutropenic enterocolitis associated with C septicum is an overwhelming disease causing extensive and irreversible gut necrosis. Patients do not recover without urgent surgical intervention. Whilst agreeing with our colleagues Dr King and Dr Wight (Nov 28, p 1279) that neutropenic patients with clinical signs of ileitis or caecitis may recover with antibiotic therapy alone, we have seen this associated with C tertium bacteraemia only. Neutropenic enterocolitis with gut necrosis has not been demonstrated in such patients.
We thank Mr Tom Treasure, Dr G. Beynon, permission to report this case.
Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge CB2 2QW
pharyngeal pouch protruded through
Middlesex Hospital, London W1N 8AA
and Dr
J. Croker for
J. L. MORRIS
ANITA RAMPLING GAIL SPEIRS R. E. WARREN
S. A. RAY spore-bearing bacteria of the human intestine in health and in diseases J Infect Dis 1924: 35: 423. King A, Rampling A, Wight DGD Neutropemc enterocolitis due to Closridium septicum infection J Clin Pathol 1984, 37: 335-43.
1. Kahn MC. Anaerobic 1.
Jackson JW, Slack WW, Williams RA. Pharyngeal pouch treated by diverticulooesophagostomy. Lancet 1960; i: 470-71.
CLOSTRIDIUM TERTIUM AND THE GUT
SIR,-We were interested to read the account by Dr Yates and colleagues (Jan 23, p 185) of a patient with neutropenic enterocolitis associated with Clostridium tertium. We wonder whether the organism might have been seen within the submucosal tissue of the caecum, by a specific immunofluorescent antibody to C tertium, or whether C septicum might have been similarly demonstrated. C tertium is a common gut commensal’ and its significance except in blood cultures is difficult to assess. In recent years 18 of our neutropenic patients have had C tertium bacteraemia accompanied by fever, abdominal pain, colonic bleeding, and diarrhoea. The infection was mild and 15of these patients recovered on antibiotic therapy alone.
certain
2
MYCOBACTERIAL CULTURE: WHAT TEMPERATURE?
SIR,-Dr Ispahani and Dr Baker (Feb 6, p 305) show the value of prolonging the incubation time to twelve weeks for the isolation of "atypical" mycobacteria, especially Mycobacterzum malmoense. But they do not state the temperature of incubation. M tuberculosis, an obligate pathogen, is well adapted to growth at 37°C but this is not necessarily the optimum temperature for other species which, essentially, are environmental saprophytes. Although some species (eg, M xerwPl) are thermophilic, for others 3TC is at or near the upper limit at which they will grow. In particular, the skin pathogens M marinum, M haemophilum, and M ulcerans fail to grow at this temperature, especially on primary isolation. Furthermore,
535
although strains of M chelonei from clinical specimens appear to have adapted to growth at body temperature, we have found that
specific IgM could not be detected. The fetus died in utero 1 week later. It weighed 230 g and was hydropic and macerated, but had no
strains fail to grow at this temperature on subculture. we recommend that material for mycobacterial culture should be inoculated at 35°C rather than 37°C, and that biopsy specimens from skin lesions should, in addition, be incubated at 30-33°C.
structural abnormalities. In the other case non-immune hydrops of a 19-week fetus was diagnosed by ultrasound scan in a 19-year-old woman. The karyotype was 46, XX but fetal haemoglobin was low at 3 g/dl. B 19 DNA was detected. 3 weeks later the fetal haemoglobin had fallen to 2-1g/dl and B19 DNA was still present. The fetus died in utero 1 week later at 23 weeks. It weighed 600 g and was hydropic and macerated, but showed no gross structural abnormality. In the spleen, liver, kidney, heart, and lung of both fetuses there were cells with swollen pale nuclei containing inclusion bodies, suggestive of virus infection. Formalin-fixed, paraffin-embedded fetal tissues were examined for B 19 DNA by in situ hybridisation.1 Sections were incubated with a biotinylated recombinant DNA probe (pVTMl) which encodes a 700 base portion of the B19 genome. The probe hybridised to the nuclei of some cells in the spleen (figure), liver, heart, and kidney but control biotinylated vector (pAT 153) DNA did not. Only one of the two women had atypical B19 clinical illness. Both had abnormal scans which led to the detection of fetal anaemia. In the absence of blood-group incompatibility and fetomaternal transfusion, hydrops and fetal anaemia should alert the obstetrician to the liklihood of intrauterine B19 infection. The viral diagnosis required detection of B19 in fetal blood, since specific IgM was not detectable in maternal or fetal blood after the hydrops developed. Autolysis may make histological appearances hard to interpret. The intranuclear inclusions suggested viral infection but in situ hybridisation permits virus-specific diagnosis. The biotinylated B19 DNA probe technique can be used on formalin fixed, paraffin-embedded tissues, so retrospective studies are possible.
some
Accordingly
Cardiothoracic Institute, London SW3 6HP
JOHN M. GRANGE
PHLS Regional Centre for Tuberculosis
Bacteriology, Public Health Laboratory, Dulwich Hospital, London
MALCOLM D. YATES
HUMAN PARVOVIRUS B19 AND HYDROPS FETALIS
SiR,—Maternal human parvovirus B 19 virus infection may lead
hydrops fetalis.1’ Five cases have been published: we decribe a sixth in detail, note a seventh, and discuss diagnostic approaches. A 27-year-old woman presented to an antenatal booking clinic on July 3, 1987, with a history of sore throat, arthralgia, and flushed face lasting for a week in the middle of June. Her blood containeds 6-8 arbitrary units of B 19-specific IgM and 29 units of specific IgG. An ultrasound scan revealed a normal singleton fetus of 15 weeks and a second scan on July 21 revealed an 18 week fetus with abdominal ascites and pleural and pericardial effusions. Fetal heart size and rate were normal and there were no other abnormalities. The patient is rhesus positive with no atypical antibodies and there was no serological evidence of rubella or toxoplasmosis. On Aug 11 cordocentesis was done. The haemoglobin of the fetus was 1 ’7 g/dl. The karyotype was 46, XY. This fetal serum contained B 19 DNA by dot-blot hybridisation,6 and 8 units of B19 specific IgG but
to
Departments of Medical Microbiology Obstetrics and Gynaecology, and Histopathology University College and Middlesex Hospital School of Medicine, London WC1E 6JJ
M. J. ANDERSON M. N. KHOUSAM D. J. MAXWELL S. J. GOULD L. C. HAPPERFIELD W. J. SMITH
1. Brown T, Anand A, Ritchie LD, et al. Intrauterine parvovirus infection associated with hydrops fetalis. Lancet 1984; ii: 1033-34 2. Gray ES, Anand A, Brown T. Parvovirus infection in pregnancy Lancet 1986; i. 208. 3. Bond PR, Caul EO, Usher J, et al. Intrauterine infection with human parvovirus. Lancet 1986; i. 448-49. 4. Carrington D, Gilmore DH, Whittle MJ, et al. Maternal serum alpha-fetoprotein: a marker of fetal aplastic crisis during intrauterine parvovirus infection. Lancet 1987; i: 433-35. 5 Cohen BJ, Mortimer PP, Pereira MS Diagnostic assays with monoclonal antibodies for the human serum parvovirus-like virus. (SPLV) J Hyg (Camb) 1983; 91: 113-30. 6. Anderson MJ, Jones SE, Minson AC Diagnosis of human parvovirus infection by dot-blot hybridisation using cloned viral DNA. J. Med Virol 1985; 15: 163-72. 7 Garson JA. van den Berghe JA, Kemshead JT. Novel non-isotopic in situ hybridisation technique detects small (1kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and B-NGF genes. Nucl Acids Res 1987; 15: 4761-70.
B19 PARVOVIRUS INFECTION OF MYOCARDIAL CELLS
In situ
hybridisation of
B19 DNA
probe to fetal spleen
cells.
Positive reaction (upper) indicated by dense precipitate; no hybridisation to vector alone (lower, control). Counterstain methyl-green.
SIR,-Parvoviruses infect many organs in animals.l In contrast, human parvovirus (B 19) had been thought to infect only nucleated red blood-cells. Hence it causes aplastic crises in patients with chronic haemolytic anaemia,and severe anaemia in fetuses resulting in hydrops fetalis.3 Other cell types may be involved: white cells and platelets,’ and myocardium and muscle.s Weiland et aP also described developmental eye abnormalities. However, while pathological conditions associated with parvovirus infection were described in these studies, viral DNA was not identified in the abnormal cells. Using a combination of sequential immunocytochemistry and in-situ DNA hybridisation on the same sections, we have been able to identify viral DNA in some cell types. The material was from a previous in-situ hybridisation study6 which showed that B19 parvovirus infection was the cause of unexplained hydrops fetalis in 4 of 13 cases. To determine the site and cell type infected we studied lung, heart, brain, spleen, adrenal,
Fig 1-Barium swallow, pharyngeal pouch.
Fig 2-Diagram of
demonstrating
1960
operation,’
which differs from ours only in that we divided the azygos vein.
barium Fig 3-Postoperative swallow, showing pouch draining into oesophagus.
The chest was opened along the upper border of the fifth rib. The the mediastinal pleura and down to the level of the azygos vein. The pleura over it was incised and the apex of the pouch dissected. The azygos vein was divided between ligatures and the oesophagus in this area exposed. When a good view of the pouch was obtained a pursestring was inserted in the apex and its contents sucked out. The area was packed with swabs soaked in mercuric chloride and the pouch was washed out with chlorhexidine. The oesophagus was incised longitudinally and the two openings, about 5 cm long, were sutured together with a single layer, taking full thickness of both pouch and oesophagus. The mediastinal pleura was closed over the anastomosis. A drain was taken up to the site and the chest was closed in layers. Contrast studies on postoperative day 6 revealed free drainage of the pouch and an intact anastomosis (fig 3). After treatment for a chest infection the patient was discharged 3 weeks after the operation with his dysphagia much improved. He remains well and has put on 6kg in 2 months.
By contrast, 7 patients in our unit had neutropenic enterocolitis due to C septicum. 5 had bowel resected and 3 of them survived. The other 2 died within 12 h of presentation without surgical intervention. In 1 patient who underwent laparotomy and was receiving intravenous vancomycin (which is active against C septicum) C difficile was initially thought to be implicated, on the basis of isolation from faeces and detection of cytotoxin. Blood cultures were negative but C septicum was demonstrated in the bowel wall by direct ffi=unofluorescence.1 We have found that neutropenic enterocolitis associated with C septicum is an overwhelming disease causing extensive and irreversible gut necrosis. Patients do not recover without urgent surgical intervention. Whilst agreeing with our colleagues Dr King and Dr Wight (Nov 28, p 1279) that neutropenic patients with clinical signs of ileitis or caecitis may recover with antibiotic therapy alone, we have seen this associated with C tertium bacteraemia only. Neutropenic enterocolitis with gut necrosis has not been demonstrated in such patients.
We thank Mr Tom Treasure, Dr G. Beynon, permission to report this case.
Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge CB2 2QW
pharyngeal pouch protruded through
Middlesex Hospital, London W1N 8AA
and Dr
J. Croker for
J. L. MORRIS
ANITA RAMPLING GAIL SPEIRS R. E. WARREN
S. A. RAY spore-bearing bacteria of the human intestine in health and in diseases J Infect Dis 1924: 35: 423. King A, Rampling A, Wight DGD Neutropemc enterocolitis due to Closridium septicum infection J Clin Pathol 1984, 37: 335-43.
1. Kahn MC. Anaerobic 1.
Jackson JW, Slack WW, Williams RA. Pharyngeal pouch treated by diverticulooesophagostomy. Lancet 1960; i: 470-71.
CLOSTRIDIUM TERTIUM AND THE GUT
SIR,-We were interested to read the account by Dr Yates and colleagues (Jan 23, p 185) of a patient with neutropenic enterocolitis associated with Clostridium tertium. We wonder whether the organism might have been seen within the submucosal tissue of the caecum, by a specific immunofluorescent antibody to C tertium, or whether C septicum might have been similarly demonstrated. C tertium is a common gut commensal’ and its significance except in blood cultures is difficult to assess. In recent years 18 of our neutropenic patients have had C tertium bacteraemia accompanied by fever, abdominal pain, colonic bleeding, and diarrhoea. The infection was mild and 15of these patients recovered on antibiotic therapy alone.
certain
2
MYCOBACTERIAL CULTURE: WHAT TEMPERATURE?
SIR,-Dr Ispahani and Dr Baker (Feb 6, p 305) show the value of prolonging the incubation time to twelve weeks for the isolation of "atypical" mycobacteria, especially Mycobacterzum malmoense. But they do not state the temperature of incubation. M tuberculosis, an obligate pathogen, is well adapted to growth at 37°C but this is not necessarily the optimum temperature for other species which, essentially, are environmental saprophytes. Although some species (eg, M xerwPl) are thermophilic, for others 3TC is at or near the upper limit at which they will grow. In particular, the skin pathogens M marinum, M haemophilum, and M ulcerans fail to grow at this temperature, especially on primary isolation. Furthermore,
535
although strains of M chelonei from clinical specimens appear to have adapted to growth at body temperature, we have found that
specific IgM could not be detected. The fetus died in utero 1 week later. It weighed 230 g and was hydropic and macerated, but had no
strains fail to grow at this temperature on subculture. we recommend that material for mycobacterial culture should be inoculated at 35°C rather than 37°C, and that biopsy specimens from skin lesions should, in addition, be incubated at 30-33°C.
structural abnormalities. In the other case non-immune hydrops of a 19-week fetus was diagnosed by ultrasound scan in a 19-year-old woman. The karyotype was 46, XX but fetal haemoglobin was low at 3 g/dl. B 19 DNA was detected. 3 weeks later the fetal haemoglobin had fallen to 2-1g/dl and B19 DNA was still present. The fetus died in utero 1 week later at 23 weeks. It weighed 600 g and was hydropic and macerated, but showed no gross structural abnormality. In the spleen, liver, kidney, heart, and lung of both fetuses there were cells with swollen pale nuclei containing inclusion bodies, suggestive of virus infection. Formalin-fixed, paraffin-embedded fetal tissues were examined for B 19 DNA by in situ hybridisation.1 Sections were incubated with a biotinylated recombinant DNA probe (pVTMl) which encodes a 700 base portion of the B19 genome. The probe hybridised to the nuclei of some cells in the spleen (figure), liver, heart, and kidney but control biotinylated vector (pAT 153) DNA did not. Only one of the two women had atypical B19 clinical illness. Both had abnormal scans which led to the detection of fetal anaemia. In the absence of blood-group incompatibility and fetomaternal transfusion, hydrops and fetal anaemia should alert the obstetrician to the liklihood of intrauterine B19 infection. The viral diagnosis required detection of B19 in fetal blood, since specific IgM was not detectable in maternal or fetal blood after the hydrops developed. Autolysis may make histological appearances hard to interpret. The intranuclear inclusions suggested viral infection but in situ hybridisation permits virus-specific diagnosis. The biotinylated B19 DNA probe technique can be used on formalin fixed, paraffin-embedded tissues, so retrospective studies are possible.
some
Accordingly
Cardiothoracic Institute, London SW3 6HP
JOHN M. GRANGE
PHLS Regional Centre for Tuberculosis
Bacteriology, Public Health Laboratory, Dulwich Hospital, London
MALCOLM D. YATES
HUMAN PARVOVIRUS B19 AND HYDROPS FETALIS
SiR,—Maternal human parvovirus B 19 virus infection may lead
hydrops fetalis.1’ Five cases have been published: we decribe a sixth in detail, note a seventh, and discuss diagnostic approaches. A 27-year-old woman presented to an antenatal booking clinic on July 3, 1987, with a history of sore throat, arthralgia, and flushed face lasting for a week in the middle of June. Her blood containeds 6-8 arbitrary units of B 19-specific IgM and 29 units of specific IgG. An ultrasound scan revealed a normal singleton fetus of 15 weeks and a second scan on July 21 revealed an 18 week fetus with abdominal ascites and pleural and pericardial effusions. Fetal heart size and rate were normal and there were no other abnormalities. The patient is rhesus positive with no atypical antibodies and there was no serological evidence of rubella or toxoplasmosis. On Aug 11 cordocentesis was done. The haemoglobin of the fetus was 1 ’7 g/dl. The karyotype was 46, XY. This fetal serum contained B 19 DNA by dot-blot hybridisation,6 and 8 units of B19 specific IgG but
to
Departments of Medical Microbiology Obstetrics and Gynaecology, and Histopathology University College and Middlesex Hospital School of Medicine, London WC1E 6JJ
M. J. ANDERSON M. N. KHOUSAM D. J. MAXWELL S. J. GOULD L. C. HAPPERFIELD W. J. SMITH
1. Brown T, Anand A, Ritchie LD, et al. Intrauterine parvovirus infection associated with hydrops fetalis. Lancet 1984; ii: 1033-34 2. Gray ES, Anand A, Brown T. Parvovirus infection in pregnancy Lancet 1986; i. 208. 3. Bond PR, Caul EO, Usher J, et al. Intrauterine infection with human parvovirus. Lancet 1986; i. 448-49. 4. Carrington D, Gilmore DH, Whittle MJ, et al. Maternal serum alpha-fetoprotein: a marker of fetal aplastic crisis during intrauterine parvovirus infection. Lancet 1987; i: 433-35. 5 Cohen BJ, Mortimer PP, Pereira MS Diagnostic assays with monoclonal antibodies for the human serum parvovirus-like virus. (SPLV) J Hyg (Camb) 1983; 91: 113-30. 6. Anderson MJ, Jones SE, Minson AC Diagnosis of human parvovirus infection by dot-blot hybridisation using cloned viral DNA. J. Med Virol 1985; 15: 163-72. 7 Garson JA. van den Berghe JA, Kemshead JT. Novel non-isotopic in situ hybridisation technique detects small (1kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and B-NGF genes. Nucl Acids Res 1987; 15: 4761-70.
B19 PARVOVIRUS INFECTION OF MYOCARDIAL CELLS
In situ
hybridisation of
B19 DNA
probe to fetal spleen
cells.
Positive reaction (upper) indicated by dense precipitate; no hybridisation to vector alone (lower, control). Counterstain methyl-green.
SIR,-Parvoviruses infect many organs in animals.l In contrast, human parvovirus (B 19) had been thought to infect only nucleated red blood-cells. Hence it causes aplastic crises in patients with chronic haemolytic anaemia,and severe anaemia in fetuses resulting in hydrops fetalis.3 Other cell types may be involved: white cells and platelets,’ and myocardium and muscle.s Weiland et aP also described developmental eye abnormalities. However, while pathological conditions associated with parvovirus infection were described in these studies, viral DNA was not identified in the abnormal cells. Using a combination of sequential immunocytochemistry and in-situ DNA hybridisation on the same sections, we have been able to identify viral DNA in some cell types. The material was from a previous in-situ hybridisation study6 which showed that B19 parvovirus infection was the cause of unexplained hydrops fetalis in 4 of 13 cases. To determine the site and cell type infected we studied lung, heart, brain, spleen, adrenal,