440
probably patients.
facilitate extension of the
technique
to more
Our method of cannula insertion was more simple and rapid than that proposed by the pioneers in the clinical use of the hemopump3 in which the cannula was implanted through a prosthetic graft, sutured end to side to the iliac or femoral artery. That method is much too complex and time-consuming for a very temporary implantation, as for coronary angioplasty. The design of the pump occluder, which prevents retrograde bleeding during implantation, made our method possible. The introduction of the hemopump method into intensive care units will probably change treatment strategies in the most critically ill patients. The technique should allow very early decompression of the left ventricle in massive myocardial infarction, so enhancing the chances of recovery of the jeopardised ischaemic non-infarcted areas.
Left-ventricular unloading and arterial revascularisation are important factors in determining the extent of myocardial recovery in such circumstances.5 A reduction in the need for urgent cardiac replacement or invasive methods of support for the failing heart might be seen as a result. REFERENCES D, Dubois-Randé JL, Geschwind H, et al. Left-ventricular support by intraventricular blood pump during high-risk coronary angioplasty. Lancet 1989; i: 561. Wampler RK, Moise JC, Frazier OH, Olsen DB. In vivo evaluation of a peripheral vascular access axial flow blood pump. ASAIO Trans 1988;
1. Loisance
2.
34: 450-54. 3. Frazier OH, Duncan J, Nakatani T, Parnis S, Fuga J. Clinical experience with the Hemopump. Trans Am Soc Int Art Organs (in press). 4. Hinglais J, Gourgon R, Weiss M. Les assistances cardio circulatoires. Coeur Med Int 1965; 4: 85-91. 5. Laks H, Rosenbranz ER, Buckberg GD. Surgical treatment of cardiogenic shock after myocardial infarction. Circulation 1986; 74
(suppl III): 15-22.
Rapid detection of herpes-simplex-virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis
Herpes-simplex-virus (HSV) DNA in cerebrospinal fluid was amplified by use of the polymerase chain reaction and identified by hybridisation to a specific oligonucleotide probe. Specimens of cerebrospinal fluid (CSF) from 4 of 4 patients with herpes simplex encephalitis were positive for HSV DNA, whereas CSF specimens from 6 patients with other central-nervous-system infections were negative. This technique may expedite diagnosis of herpes simplex encephalitis.
antigens and antibodies in CSF have been developed, they are rarely positive in early stages of the
for HSV
infection. The polymerase chain reaction (PCR) is a method by which very small quantities of DNA can be enzymatically amplified for detection by conventional methods such as DNA hybridisation9-a technique used successfully for infectious detection of several agents, including and human immunodeficiency virus.10-12 papillomaviruses We have used PCR to detect HSV DNA in CSF from patients with herpes simplex encephalitis.
Introduction
Methods
(HSV) is the commonest cause of in man,l with an approximate annual incidence, in the USA, of 1 in 250 000.2 HSV causes progressive cerebral necrosis and oedema, with up to 70% mortality if untreated; of the survivors of untreated herpes simplex encephalitis about two-thirds have neurological deficits, sometimes severe.3 Effective antiviral drugs, such as vidarabine and acyclovir, are available but are most effective when given early.2 However, diagnosis cannot be based solely on clinical criteria because other diseases (eg, Epstein-Barr virus encephalitis, togavirus encephalitis, and tuberculous meningitis) may give similar symptoms and signs.4 An accurate diagnostic test for early diagnosis of herpes simplex encephalitis is therefore needed but, at present, examination of brain-biopsy specimens remains the only conclusive way to diagnose early herpes simplex encephalitis.s Viral culture of cerebrospinal fluid (CSF) is positive in only about 4% of patients with herpes simplex encephalitis confirmed by brain biopsy,’ and although tests
Approximately 300 µl CSF was obtained from 4 patients with herpes simplex encephalitis proven by brain biopsy or at autopsy
Herpes simplex severe
focal
virus
encephalitis
and from 6 controls who had other central-nervous-system infections or systemic lupus erythematosus. CSF was ultracentrifuged at 37 000 rpm for 1 h, and pellets and supernatants saved. Pellets were lysed (total volume 50 µl) in 50 mmol/1 KCI, 10 mmol/1 "tris"-HC1 (pH 8-3), 2-5 mmol/1 MgC1z’ 0-45% ’Nonidet P-40’, and 0.45% ’Tween 20’ that contained 60 µg/ml proteinase K for 1 h at 55°C, followed by phenol/chloroform extraction (1/1 by volume) and ethanol precipitation. Supernatants underwent direct phenol/chloroform extraction and ethanol precipitation. DNA was amplified in a 100pi reaction mixture that contained 50 mmol/1 KCI, 10 mmol/1 "tris"-HC1 (pH 8-3), 2-5 mmol/1 MgCl2, ADDRESSES. Departments of Pediatrics (A. H. Rowley, MD) and Medicine (S. M. Wolinsky, MD), Northwestern University, Chicago, Illinois; and Departments of Pediatrics (Prof R. J Whitley, MD) and Clinical Virology (F. D. Lakeman, MD), University of Alabama, Birmingham, Alabama, USA. Correspondence to Dr A. H. Rowley, Division of Infectious Diseases, Children’s Memorial Hospital, 2300 Children’s Plaza, Box 20, Chicago, IL 60614, USA.
441
CLINICAL DATA
because the technique does not require the presence of intact non-antibody-bound viral particles, and may avoid the need for brain biopsy. Measurement of HSV DNA by this technique may also be a guide to the effect of treatment with antiviral agents such as acyclovir. Further studies of the sensitivity and specificity of this technique in the early diagnosis of herpes simplex encephalitis, and into a possible correlation of HSV DNA concentrations in the CSF with antiviral therapy and clinical outcome, are under way. REFERENCES
AJ, Whitley RJ, Visintine AN, et al. Herpes simplex virus encephalitis: laboratory evaluations and their diagnostic significance. J Infect Dis 1982; 145: 829-36. 2. Whitley RJ. Herpes simplex virus infections of the central nervous system. Am J Med 1988; 85 (S 2A): 61-67. 3. Whitley RJ, Soong SJ, Dolin R, et al. Adenosine arabinoside therapy of biopsy-proven herpes simplex encephalitis. N Engl J Med 1977; 297: 1. Nahmias
HSE= herpes simplex encephalitis, CMV = cytomegaloVIrus, SLE=systemic lupus erythematosus, NA=not available
289-94.
200 pmol each of all four deoxyribonucleoside triphosphates, 2-5 units of Taq polymerase, and 50 pmol each of two flanking oligonucleotide primers derived from a highly conserved region of the DNA polymerase gene of HSV.13 Amplification (in an automated DNA thermal cycler [Perkin Elmer-Cetus, Norwalk, CT, USA]) consisted of 35 cycles at 94°C for 1 min (denaturation), 55 °C for 1 min (annealing), and 72°C for 90 s (extension). Amplified product was cross-linked under ultraviolet light to a nylon membrane (’Gentran’, Plasco, Wuboin, MA, USA) and prehybridised in 10 x Denhardt’s solution (1 x Denhardt’s solution=002% polyvinylpyrrolidone, 0-02% Ficoll, and 0-02% bovine serum albumin), 0-1% sodium dodecyl sulphate (SDS), 2 x SSPE (1 x SSPE = 0.18 moljl sodium chloride, 10 mmoljl sodium phosphate, 1 mmol/1 edetic acid [pH 74]), and 0.mg/ml salmon sperm DNA at 68°C for 45 min. The blot was hybridised with 500 000 disintegrations per min/ml 3zP-end-labelled oligonucleotide probe derived from a conserved internal sequence13 at 55 °C for 3 h. Excess probe was removed by two washes with 2 x SSC, 0-5% SDS at 55°C (lxSSC-150 mmol/1 sodium chloride, 15 mmol/1 sodium citrate). The nylon filter was then air-dried and autoradiographed by exposure to ’XAR’ film (Kodak, Rochester, NY, USA) with an intensifying screen for 16 h at 70°C. -
Results 4 of 4 CSF supernatants from patients with herpes simplex encephalitis were positive for HSV DNA by PCR, compared with 0 of 6 patients with other central-nervoussystem infections or systemic lupus erythematosus (see table and figure). None of the ultracentrifuged pellets yielded a positive result.
Discussion Our
preliminary results indicate that enzymatic amplification of HSV DNA in CSF may enable accurate diagnosis of herpes simplex encephalitis within 2-3 days. The presence of HSV DNA in the CSF supernatant, but ultracentrifuged pellets, may indicate the presence of free viral DNA rather than intact viral particles. Enzymatic amplification offers an advantage over culture not in the
Dot-blot
amplification and hybridisation of HSV DNA in CSF. 1-10=patients 1-10 as in table, 11 = negative control. (Sample 4 is clearly positive on original autoradiograph )
Whitley RJ, Cobbs CG, Alford CA, et al. Diseases that mimic herpes simplex encephalitis. JAMA 1989; 262: 234-39. 5. Johnson RT, Olson LC, Buescher EL. Herpes simplex virus infections of the nervous system. Arch Neurol 1968; 18: 260-63. 6. Lakeman FD, Koga J, Whitley RJ. Detection of antigen to herpes simplex virus in cerebrospinal fluid from patients with herpes simplex encephalitis. J Infect Dis 1987; 155: 1172-78. 7. Skoldenberg B, Forsgren M, Alestig K, et al. Acyclovir versus vidarabine in herpes simplex encephalitis. Lancet 1984; ii: 707-11. 8. Kahlon J, Chatterjee S, Lakeman FD, Lee F, Nahmias AJ, Whitley RJ. Detection of antibodies to herpes simplex virus in the cerebrospinal fluid of patients with herpes simplex encephalitis. J Infect Dis 1987; 4.
155: 38-44. 9. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-54. 10. Kwok S, Mack DH, Mullis K, et al. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J Virol 1987; 61: 1690-94. 11. Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM. Molecular diagnostics of human cancer. Use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells 1989; 7: 209-14. 12. Kwok S, Ehrlich G, Poiesz B, Kalish R, Sninsky JJ. Enzymatic amplification of HTLV-1 viral sequences from peripheral blood mononuclear cells and infected tissues. Blood 1988; 72: 1117-23. 13. Rowley AH, Wolinsky SM. Direct detection of herpesvirus DNA sequences in clinical samples by in vitro enzymatic amplification. Pediatr Res 1989; 25: 189A.
From The Lancet Method of making
gelatinous capsules
The following method is given by M. Desfontenelles, in a recent number of the Journal de ChimieTake the swimming-bag of a tench, or any fish about five to seven inches in length; fix the bag to the end of a copper tube by means of a ligature, and cover the ligature with another tube, which contains at its middle part a small valve; below the latter is a small opening, closed by a key. On blowing through the extremity of the tube the bladder is inflated, and the air retained by the valve; a solution of gelatine is then prepared after M. Garot’s formula (Journal de Chimie, March, 1838). The bladder is greased with some lard, and then dipped in the gelatine; on being withdrawn, the tube is rolled by the fingers, in order to diffuse the gelatine equally over the bladder, and the mass is allowed to cool. When the gelatine is quite cold the capsule is separated, the little key turned, and the air allowed to escape; the mould is then easily withdrawn, as the grease prevents it from sticking to the gelatine. (7 March 1840)
probably patients.
facilitate extension of the
technique
to more
Our method of cannula insertion was more simple and rapid than that proposed by the pioneers in the clinical use of the hemopump3 in which the cannula was implanted through a prosthetic graft, sutured end to side to the iliac or femoral artery. That method is much too complex and time-consuming for a very temporary implantation, as for coronary angioplasty. The design of the pump occluder, which prevents retrograde bleeding during implantation, made our method possible. The introduction of the hemopump method into intensive care units will probably change treatment strategies in the most critically ill patients. The technique should allow very early decompression of the left ventricle in massive myocardial infarction, so enhancing the chances of recovery of the jeopardised ischaemic non-infarcted areas.
Left-ventricular unloading and arterial revascularisation are important factors in determining the extent of myocardial recovery in such circumstances.5 A reduction in the need for urgent cardiac replacement or invasive methods of support for the failing heart might be seen as a result. REFERENCES D, Dubois-Randé JL, Geschwind H, et al. Left-ventricular support by intraventricular blood pump during high-risk coronary angioplasty. Lancet 1989; i: 561. Wampler RK, Moise JC, Frazier OH, Olsen DB. In vivo evaluation of a peripheral vascular access axial flow blood pump. ASAIO Trans 1988;
1. Loisance
2.
34: 450-54. 3. Frazier OH, Duncan J, Nakatani T, Parnis S, Fuga J. Clinical experience with the Hemopump. Trans Am Soc Int Art Organs (in press). 4. Hinglais J, Gourgon R, Weiss M. Les assistances cardio circulatoires. Coeur Med Int 1965; 4: 85-91. 5. Laks H, Rosenbranz ER, Buckberg GD. Surgical treatment of cardiogenic shock after myocardial infarction. Circulation 1986; 74
(suppl III): 15-22.
Rapid detection of herpes-simplex-virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis
Herpes-simplex-virus (HSV) DNA in cerebrospinal fluid was amplified by use of the polymerase chain reaction and identified by hybridisation to a specific oligonucleotide probe. Specimens of cerebrospinal fluid (CSF) from 4 of 4 patients with herpes simplex encephalitis were positive for HSV DNA, whereas CSF specimens from 6 patients with other central-nervous-system infections were negative. This technique may expedite diagnosis of herpes simplex encephalitis.
antigens and antibodies in CSF have been developed, they are rarely positive in early stages of the
for HSV
infection. The polymerase chain reaction (PCR) is a method by which very small quantities of DNA can be enzymatically amplified for detection by conventional methods such as DNA hybridisation9-a technique used successfully for infectious detection of several agents, including and human immunodeficiency virus.10-12 papillomaviruses We have used PCR to detect HSV DNA in CSF from patients with herpes simplex encephalitis.
Introduction
Methods
(HSV) is the commonest cause of in man,l with an approximate annual incidence, in the USA, of 1 in 250 000.2 HSV causes progressive cerebral necrosis and oedema, with up to 70% mortality if untreated; of the survivors of untreated herpes simplex encephalitis about two-thirds have neurological deficits, sometimes severe.3 Effective antiviral drugs, such as vidarabine and acyclovir, are available but are most effective when given early.2 However, diagnosis cannot be based solely on clinical criteria because other diseases (eg, Epstein-Barr virus encephalitis, togavirus encephalitis, and tuberculous meningitis) may give similar symptoms and signs.4 An accurate diagnostic test for early diagnosis of herpes simplex encephalitis is therefore needed but, at present, examination of brain-biopsy specimens remains the only conclusive way to diagnose early herpes simplex encephalitis.s Viral culture of cerebrospinal fluid (CSF) is positive in only about 4% of patients with herpes simplex encephalitis confirmed by brain biopsy,’ and although tests
Approximately 300 µl CSF was obtained from 4 patients with herpes simplex encephalitis proven by brain biopsy or at autopsy
Herpes simplex severe
focal
virus
encephalitis
and from 6 controls who had other central-nervous-system infections or systemic lupus erythematosus. CSF was ultracentrifuged at 37 000 rpm for 1 h, and pellets and supernatants saved. Pellets were lysed (total volume 50 µl) in 50 mmol/1 KCI, 10 mmol/1 "tris"-HC1 (pH 8-3), 2-5 mmol/1 MgC1z’ 0-45% ’Nonidet P-40’, and 0.45% ’Tween 20’ that contained 60 µg/ml proteinase K for 1 h at 55°C, followed by phenol/chloroform extraction (1/1 by volume) and ethanol precipitation. Supernatants underwent direct phenol/chloroform extraction and ethanol precipitation. DNA was amplified in a 100pi reaction mixture that contained 50 mmol/1 KCI, 10 mmol/1 "tris"-HC1 (pH 8-3), 2-5 mmol/1 MgCl2, ADDRESSES. Departments of Pediatrics (A. H. Rowley, MD) and Medicine (S. M. Wolinsky, MD), Northwestern University, Chicago, Illinois; and Departments of Pediatrics (Prof R. J Whitley, MD) and Clinical Virology (F. D. Lakeman, MD), University of Alabama, Birmingham, Alabama, USA. Correspondence to Dr A. H. Rowley, Division of Infectious Diseases, Children’s Memorial Hospital, 2300 Children’s Plaza, Box 20, Chicago, IL 60614, USA.
441
CLINICAL DATA
because the technique does not require the presence of intact non-antibody-bound viral particles, and may avoid the need for brain biopsy. Measurement of HSV DNA by this technique may also be a guide to the effect of treatment with antiviral agents such as acyclovir. Further studies of the sensitivity and specificity of this technique in the early diagnosis of herpes simplex encephalitis, and into a possible correlation of HSV DNA concentrations in the CSF with antiviral therapy and clinical outcome, are under way. REFERENCES
AJ, Whitley RJ, Visintine AN, et al. Herpes simplex virus encephalitis: laboratory evaluations and their diagnostic significance. J Infect Dis 1982; 145: 829-36. 2. Whitley RJ. Herpes simplex virus infections of the central nervous system. Am J Med 1988; 85 (S 2A): 61-67. 3. Whitley RJ, Soong SJ, Dolin R, et al. Adenosine arabinoside therapy of biopsy-proven herpes simplex encephalitis. N Engl J Med 1977; 297: 1. Nahmias
HSE= herpes simplex encephalitis, CMV = cytomegaloVIrus, SLE=systemic lupus erythematosus, NA=not available
289-94.
200 pmol each of all four deoxyribonucleoside triphosphates, 2-5 units of Taq polymerase, and 50 pmol each of two flanking oligonucleotide primers derived from a highly conserved region of the DNA polymerase gene of HSV.13 Amplification (in an automated DNA thermal cycler [Perkin Elmer-Cetus, Norwalk, CT, USA]) consisted of 35 cycles at 94°C for 1 min (denaturation), 55 °C for 1 min (annealing), and 72°C for 90 s (extension). Amplified product was cross-linked under ultraviolet light to a nylon membrane (’Gentran’, Plasco, Wuboin, MA, USA) and prehybridised in 10 x Denhardt’s solution (1 x Denhardt’s solution=002% polyvinylpyrrolidone, 0-02% Ficoll, and 0-02% bovine serum albumin), 0-1% sodium dodecyl sulphate (SDS), 2 x SSPE (1 x SSPE = 0.18 moljl sodium chloride, 10 mmoljl sodium phosphate, 1 mmol/1 edetic acid [pH 74]), and 0.mg/ml salmon sperm DNA at 68°C for 45 min. The blot was hybridised with 500 000 disintegrations per min/ml 3zP-end-labelled oligonucleotide probe derived from a conserved internal sequence13 at 55 °C for 3 h. Excess probe was removed by two washes with 2 x SSC, 0-5% SDS at 55°C (lxSSC-150 mmol/1 sodium chloride, 15 mmol/1 sodium citrate). The nylon filter was then air-dried and autoradiographed by exposure to ’XAR’ film (Kodak, Rochester, NY, USA) with an intensifying screen for 16 h at 70°C. -
Results 4 of 4 CSF supernatants from patients with herpes simplex encephalitis were positive for HSV DNA by PCR, compared with 0 of 6 patients with other central-nervoussystem infections or systemic lupus erythematosus (see table and figure). None of the ultracentrifuged pellets yielded a positive result.
Discussion Our
preliminary results indicate that enzymatic amplification of HSV DNA in CSF may enable accurate diagnosis of herpes simplex encephalitis within 2-3 days. The presence of HSV DNA in the CSF supernatant, but ultracentrifuged pellets, may indicate the presence of free viral DNA rather than intact viral particles. Enzymatic amplification offers an advantage over culture not in the
Dot-blot
amplification and hybridisation of HSV DNA in CSF. 1-10=patients 1-10 as in table, 11 = negative control. (Sample 4 is clearly positive on original autoradiograph )
Whitley RJ, Cobbs CG, Alford CA, et al. Diseases that mimic herpes simplex encephalitis. JAMA 1989; 262: 234-39. 5. Johnson RT, Olson LC, Buescher EL. Herpes simplex virus infections of the nervous system. Arch Neurol 1968; 18: 260-63. 6. Lakeman FD, Koga J, Whitley RJ. Detection of antigen to herpes simplex virus in cerebrospinal fluid from patients with herpes simplex encephalitis. J Infect Dis 1987; 155: 1172-78. 7. Skoldenberg B, Forsgren M, Alestig K, et al. Acyclovir versus vidarabine in herpes simplex encephalitis. Lancet 1984; ii: 707-11. 8. Kahlon J, Chatterjee S, Lakeman FD, Lee F, Nahmias AJ, Whitley RJ. Detection of antibodies to herpes simplex virus in the cerebrospinal fluid of patients with herpes simplex encephalitis. J Infect Dis 1987; 4.
155: 38-44. 9. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-54. 10. Kwok S, Mack DH, Mullis K, et al. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J Virol 1987; 61: 1690-94. 11. Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM. Molecular diagnostics of human cancer. Use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells 1989; 7: 209-14. 12. Kwok S, Ehrlich G, Poiesz B, Kalish R, Sninsky JJ. Enzymatic amplification of HTLV-1 viral sequences from peripheral blood mononuclear cells and infected tissues. Blood 1988; 72: 1117-23. 13. Rowley AH, Wolinsky SM. Direct detection of herpesvirus DNA sequences in clinical samples by in vitro enzymatic amplification. Pediatr Res 1989; 25: 189A.
From The Lancet Method of making
gelatinous capsules
The following method is given by M. Desfontenelles, in a recent number of the Journal de ChimieTake the swimming-bag of a tench, or any fish about five to seven inches in length; fix the bag to the end of a copper tube by means of a ligature, and cover the ligature with another tube, which contains at its middle part a small valve; below the latter is a small opening, closed by a key. On blowing through the extremity of the tube the bladder is inflated, and the air retained by the valve; a solution of gelatine is then prepared after M. Garot’s formula (Journal de Chimie, March, 1838). The bladder is greased with some lard, and then dipped in the gelatine; on being withdrawn, the tube is rolled by the fingers, in order to diffuse the gelatine equally over the bladder, and the mass is allowed to cool. When the gelatine is quite cold the capsule is separated, the little key turned, and the air allowed to escape; the mould is then easily withdrawn, as the grease prevents it from sticking to the gelatine. (7 March 1840)
