- Email: [email protected]
LATENT VITAMIN K DEFICIENCY IN HEALTHY INFANTS?
1421 here since the true peak occurs very shortly after the end of the injection; at 20 min the concentration is falling rapidly because distribution of drug from the blood is still the dominant pharmacokinetic process. Equilibration with extravascular tissue occurs about 60 min after injection and, theoretically, it makes the best sense to monitor then since the concentration observed is a better reflection of that in the tissues, including the more inaccessible sites where infection may well be. Because of the rapidly falling concentrations at 20 min only small variations in timing, as is very likely on the wards, will cause large differences in the observed concentrations which could falsely be taken to warrant changes in therapy. Even a moderate shortening from 20 min might result in a very high level. A further practical point is that it is customary to monitor an approximate peak at 60 min after the intramuscular injection of an aminoglycoside. Constancy of advice in using the "one-hour level" for both modes of administration is less confusing for ward staff and removes a potential source of error. In their discussion on vancomycin the working party should have pointed out that the peak concentration should be measured at the end of the 60 min infusion (a true peak) and not some time after the infusion has been stopped. The report carries no warning about the use of benzylpenicillin at
the dosage recommended (12 megaunits daily) in the presence of reduced renal function, a finding not uncommon in endocarditis. This antibiotic has a potential for serious neurotoxicity if very high blood concentrations occur, and I have witnessed a patient with endocarditis having seizures in these circumstances. High dosage of benzylpenicillin requires empirical modification in renal failure or, better still, blood concentrations should be monitored. Department of Medical Southmead Hospital, Bristol BS10 5NB
Microbiology,
letters have been shown follows.-ED. L.
**These
D. S. REEVES to
Dr Simmons, whose
reply
SiR,—Dr Robinson rightly points out that for most penicillinsensitive streptococci the MIC and MBC of benzylpenicillin are much less than 1 mg/l. However, the working party’s use of the term "fully sensitive" should be interpreted in the context of its report. We recommended that penicillin and gentamicin should normally be used for the treatment of streptococcal endocarditis. The term "fully sensitive" was used to distinguish those organisms causing infections, for which the gentamicin should be given for only two weeks, from those "with reduced sensitivity to penicillin", for which gentamicin should be given for not less than four weeks. In this respect we regard an MBC of 1 mg/l as a suitable dividing line. She fears that some physicians, rendered overoptimistic by our terminology, may treat some patients, especially those with impaired renal function, with inadequate amounts of penicillin alone. "Low doses" of penicillin should never be used for the treatment of endocarditis in patients with normal renal function. If the dosage has to be reduced, provided the reduction is proportionate to the degree of renal failure, the blood levels should still be substantially above 1 mg/1. Dr Reeves says that we should not have used the term "peak" concentration when referring to the level in blood 20 min after an intravenous bolus injection. Although it is common parlance, he is correct. His comments about the relative merits of 20 min and 60 min gentamicin blood levels are ofinterest. When the working party looked at current advice in respect of the timing of the collection of blood after intravenous injections of gentamicin, we found that one data sheet (’Cidomycin’) indicated that peak levels were reached after 15 min and another (’Genticin’) gave no precise advice. The British National Formulary (1985, no 10: 201) recommends that plasma concentrations should be measured about 20 min after an intravenous injection, and since all of the members of the working party followed this recommendation, we included it in our report. However, if one hour levels come to be accepted as being more appropriate, the working party, when it reviews its recommendations, will certainly take note of the fact. Department of Clinical Bacteriology, Guy’s Hospital, London SE1 9RT
N. A. SIMMONS, Chairman, BSAC Endocarditis Working Party
UNTREATED MYELOMENINGOCELE
SIR,-The Newcastle group’s report (Nov 2, p 993) that
a
with extensive myelomeningocele survive if left untreated, but cared for at home, should be seen in context. The conclusion that, mobility apart, such children are ultimately "no more handicapped than the children offered immediate surgical treatment" need not be read as an invitation to return to a nonselective policy of treating babies with myelomeningocele. The 27 infants taken home represent just 11% of those not selected for immediate surgical treatment. How they differed from the 89% who were kept in hospital (and died) is not pursued, but perhaps a clue is that families usually requested discharge home "if the baby was still making satisfactory progress 2-4 weeks after delivery"; those not progressing well (most probably as a result of
proportion of babies
central
nervous system sepsis or rapidly increasing hydrocephalus) presumably kept in hospital. By inference, those babies taken home were largely self-selecting and likely to be inherently superior to the majority with large myelomeningoceles. They seem to have milder hydrocephalus since shunts had been inserted quite late and none had appreciable intellectual impairment. This runs counter to other experience, which is that many patients with lumbar and thoracolumbar myelomeningoceles have intelligence within the educationally subnormal range (or worse) and that, in general, the degree of intellectual impairment relates directly to the extent of the myelomeningocele and to the neurological deficit.l-3 Apart from their superior intelligence, the survivors appear to be just as handicapped as any others with extensive myelomeningocele. All were wheelchair bound, in contrast to only 50% of those selected for immediate surgery and, predictably, all had a neuropathic bladder, whereas around one quarter of the surgically treated cases escaped this affliction. Problems with the upper renal tracts are were
mentioned only en þassant although there is evidence that the risk of these increases with the extent of the myelomeningocele.4,5 Spinal deformities are not described, yet most of these children could be expected to be severely deformed by adolescence, in contrast to those with smaller lesions,3while pressure sores are another 3
probable complications A simple interpretation of this report is that there exists a small group of babies with lumbar and thoracolumbar myelomeningoceles who have mild hydrocephalus and, as a result, are more likely to survive and to have reasonably normal intelligence. In other respects their handicaps are predictably severe. This conclusion would be neither startling nor original and I remain puzzled (even after several readings) as to why the Dean of Durham and his colleagues (Nov 2, p 996) felt moved to agonise, yet agam, over the ethical issues involved. Department of Paediatric Urology, Hey Children’s Hospital, Liverpool L12 2AP. Alder
A. M. K. RICKWOOD
1. Hunt 2.
3
4. 5
GM, Holmes AE. Some factors relating to intelligence in treated children with spma bifida Devl Med Child Neurol 1975, suppl 35: 65-69 Lonton AP Location of the myelomeningocele and its relationship to subsequent physical and intellectual abilities in children with myelomeningocele with hydrocephalus. Z Kinderchir 1977, 22: 510-19. Rickwood AMK, Hodgson J, Lonton AP, Thomas DG. Medical and surgical complications in adolescents and young adults with spina bifida. Health Trends 1984; 16: 91-95. Hunt GM, Bishop MC, Whitaker RH, Doyle PT Sensory level and renal prognosis in myelomeningocele. Z Kinderchir 1981; 34: 384-89. Rickwood AMK, Thomas DG. The upper renal tracts in adolescents and young adults with myelomeningocele. Z Kinderchir 1984, 39: 104-06.
LATENT VITAMIN K DEFICIENCY IN HEALTHY INFANTS?
SIR,-Late onset haemorrhagic disease of the newborn (HDN) is life-threatening. Most cases have been observed in Asia: in Japan the incidence was 1 in 4500 in unselected babies and 81°70 of infants with HDN presented with intracranial haemorrhage.However, 20 cases over four years have been recorded in West Germany, suggesting an incidence of at least 1 per 100 000.2 Latent vitamin K deficiency without clinical evidence of bleeding may be more common. In a Japanese study PIVKA II, a marker of vitamin K 3 status, was not detected in 14% of healthy 1-month-old babies. Data for European infants have not been reported.
1422 A, Nagao T, Satoh Ch, Kondoh A. A study on 152 newborns below 30 days of age by crossed immunoelectrophoresis of prothrombin with capillary blood. Blood Vessel 1982, 13: 202-06 (Japanese). v Knes R, Zenses Ch, Gòbel U Immunoelectrophoretic determination of PIVKA II in capillary plasma. Haemostasis 1985; 15: 42-43. Petrich Ch, Pothmann R, Döhmen A, v Voss H, Gobel U. A haematocrit corrected micro determination of the P&P-test and blood clotting factors in capillary blood Thromb Diathes Haemorrh 1984, 32: 510-19. v Kries R, Göbel U, Maase B Vitamin K deficiency in the newborn. Lancet 1985; ii: 728-29. Haroon Y, Shearer MJ, Gunn WG, McEnery G, Barkhan P. The content of phylloquinone (vitamin K1) in human milk, cow’s milk and infant formula determined by high-performance liquid chromatography. J Nutr 1982; 112: 1105-17. v Kries R, Reifenhauser A, Göbel U, McCarthy P, Shearer MJ, Barkhan P. Late onset haemorrhagic disease of newborn with temporary malabsorption of vitamin Kl. Lancet 1985; i, 1035.
3 Ilzuka
4. 5.
6
7.
8.
Cumulative distribution of factor II clotting time and activity in healthy babies on day 5 or 6 and in week 5 or 6 of life.
We measured PIVKA II in capillary blood4 from 202 non-selected German babies on the 5th or 6th day oflife and during the 5th or 6th week. In 100 babies factor II clotting activities were also measured, to detect the functional relevance of latent vitamin K deficiency. Clotting tests were done on citrated whole blood obtained from a heel prick.5 On day 5 or 6 the frequency of PIVKA II was considerable, especially in babies who were being exclusively breastfed. Beyond the 4th week, however, PIVKA II was observed in only 1 baby (a breast-fed boy) (table); although no vitamin K was given, PIVKA II was no longer detected 3 weeks later. The pattern for factor II was imilar: on days 5 or 6 several babies had prolonged factor II clotting times and 507o had a factor II clotting activity below 10% (figure). Exclusively breastfed newborns showed longer factor II clotting times than babies receiving additional formula or formula only (table). After the 4th week of life no baby showed a factor II clotting activity below 50%, and there was no difference between the different feeding schedules. FREQUENCY OF
PIVKA II AND DATA ON FACTOR II CLOTTING TIMES
Low oral intake and feeding of maternal milk, known to contain little vitamin K, account for the high PIVKA II incidence in babies aged 5 or 6 days.6 Although the vitamin K content of maternal milk is lower than that in formula,7 vitamin K deficiency beyond the 4th week was observed in only 1 out of 113 breastfed infants. There are two possible explanations. Breastfed infants beyond the 4th week of life receive more milk because lactation is by then fully established. Alternatively, vitamin K absorption may mature. Impaired absorption of vitamin K has been recently demonstrated in an infant with late onset HDN,8 and insufficient absorption may play a part in causing late onset of HDN and latent vitamin K deficiency. Latent vitamin K deficiency is less common in Europe than it is in Japan. Even so, it may be more frequent in Europe than is generally thought. Epidemiological studies of the incidence of late onset HDN and latent vitamin K deficiency are required. Department of Neonatology and Gastroenterology and Department of Paediatric Haematology and Oncology, Zentrum fur
Kinderheilkunde,
University of Düsseldorf, 4000 Düsseldorf 1, West Germany
SIR,-Chemical, heat, and pH inactivation of HTLV-III/LAV in vitro have been reported. 1,2 Nonoxynol-9, a non-ionic surfactant, is a chemical ingredient of several spermicides. In vitro, spermicides inhibit the growth of several microbes, including herpes simplex virus.3-7 We describe here the in vitro inactivation of HTLVIII/LAV by nonoxynol-9. Peripheral blood lymphocytes from HTLV-III antibody negative volunteers not in any AIDS risk group were separated by densitygradient centrifugation. The culture medium was RPMI 1640 supplemented with 100 U/ml penicillin, 50 qg/ml streptomycin, 2 mmol/1 L-glutamine, 10% heat-inactivated fetal bovine serum, and 10 g/ml phytohaemagglutinin. Cells were incubated at 37°C in 5% carbon dioxide. After 3 days, the cells were washed twice with phosphate-buffered saline (pH 7-2). The cell pellet was then incubated with HTLV-III (reverse transcriptase activity [RT] 105 per 2 x 106 cells) for 30 min at 37°C in 5 ml of the above medium to which interleukin 2, 0 - 0407o v/v sheep anti-interferon, and 0 - 02 g/ml polybrene had been added, and cells were then suspended in this medium at a concentration of 106/ml and incubated. Supernatant fluids were harvested by sequential centrifugation on days 2, 4, 8, 10, and 12 and stored at -70°C. A microculture system was used to titrate infectious HTLV-111. RT assays8 were done on days 2, 4, 8, 10, and 12 on cells treated with nonoxynol-9 immediately after inoculation. The RT assay used Mg+as the cation and poly(rA) . poly(dT)I2-IS as the template primer. Assays were done in duplicate. Nonoxynol-9, in the concentrations used, did not interfere with the RT assay. Nonoxynol-9 treated and untreated HTLV-111 infected cell cultures were used as negative and positive controls, respectively. Treatment of HTLV-III with nonoxynol-9 at final concentrations of 0-05% (v/v) or greater resulted in undetectable virus by the IDso assay (table). Toxicity controls suggested that 2-5% (v/v) nonoxynol-9 and lower concentrations were appropriate for ID50 determinations. To assess the effect of the cytotoxicity of nonoxynol-9 on the RT assay pure nonoxynol-9 was diluted to 50%, 25%, 10%, 5%, and 1% with RPMI 1640 (v/v) and added to actively growing uninfected PHA-stimulated normal human lymphocytes. A 507o solution of nonoxynol-9 significantly reduced lymphocyte viability and thus IDso ASSAY FOR DETECTION OF HTLV-III
R. V. KRIES B. MAASE A. BECKER U. GOBEL
K. Etiology of vitamin K deficiency in infants. Perinat Med 1982; 12: 1029-34 (Japanese). 2. Sutor AH, Pollmann H, v Knes R, et al. Vitamin K Mangelblutungen bei Sauglingen jenseits der Perinatalperiode. Weitere Beobachtungen Monatsschr Kinderheilk 1985, 132: 608 1.
INACTIVATION OF HTLV-III/LAV-INFECTED CULTURES OF NORMAL HUMAN LYMPHOCYTES BY NONOXYNOL-9 IN VITRO
Nakayama
*< 10 = no
detectable
virus
the dosage recommended (12 megaunits daily) in the presence of reduced renal function, a finding not uncommon in endocarditis. This antibiotic has a potential for serious neurotoxicity if very high blood concentrations occur, and I have witnessed a patient with endocarditis having seizures in these circumstances. High dosage of benzylpenicillin requires empirical modification in renal failure or, better still, blood concentrations should be monitored. Department of Medical Southmead Hospital, Bristol BS10 5NB
Microbiology,
letters have been shown follows.-ED. L.
**These
D. S. REEVES to
Dr Simmons, whose
reply
SiR,—Dr Robinson rightly points out that for most penicillinsensitive streptococci the MIC and MBC of benzylpenicillin are much less than 1 mg/l. However, the working party’s use of the term "fully sensitive" should be interpreted in the context of its report. We recommended that penicillin and gentamicin should normally be used for the treatment of streptococcal endocarditis. The term "fully sensitive" was used to distinguish those organisms causing infections, for which the gentamicin should be given for only two weeks, from those "with reduced sensitivity to penicillin", for which gentamicin should be given for not less than four weeks. In this respect we regard an MBC of 1 mg/l as a suitable dividing line. She fears that some physicians, rendered overoptimistic by our terminology, may treat some patients, especially those with impaired renal function, with inadequate amounts of penicillin alone. "Low doses" of penicillin should never be used for the treatment of endocarditis in patients with normal renal function. If the dosage has to be reduced, provided the reduction is proportionate to the degree of renal failure, the blood levels should still be substantially above 1 mg/1. Dr Reeves says that we should not have used the term "peak" concentration when referring to the level in blood 20 min after an intravenous bolus injection. Although it is common parlance, he is correct. His comments about the relative merits of 20 min and 60 min gentamicin blood levels are ofinterest. When the working party looked at current advice in respect of the timing of the collection of blood after intravenous injections of gentamicin, we found that one data sheet (’Cidomycin’) indicated that peak levels were reached after 15 min and another (’Genticin’) gave no precise advice. The British National Formulary (1985, no 10: 201) recommends that plasma concentrations should be measured about 20 min after an intravenous injection, and since all of the members of the working party followed this recommendation, we included it in our report. However, if one hour levels come to be accepted as being more appropriate, the working party, when it reviews its recommendations, will certainly take note of the fact. Department of Clinical Bacteriology, Guy’s Hospital, London SE1 9RT
N. A. SIMMONS, Chairman, BSAC Endocarditis Working Party
UNTREATED MYELOMENINGOCELE
SIR,-The Newcastle group’s report (Nov 2, p 993) that
a
with extensive myelomeningocele survive if left untreated, but cared for at home, should be seen in context. The conclusion that, mobility apart, such children are ultimately "no more handicapped than the children offered immediate surgical treatment" need not be read as an invitation to return to a nonselective policy of treating babies with myelomeningocele. The 27 infants taken home represent just 11% of those not selected for immediate surgical treatment. How they differed from the 89% who were kept in hospital (and died) is not pursued, but perhaps a clue is that families usually requested discharge home "if the baby was still making satisfactory progress 2-4 weeks after delivery"; those not progressing well (most probably as a result of
proportion of babies
central
nervous system sepsis or rapidly increasing hydrocephalus) presumably kept in hospital. By inference, those babies taken home were largely self-selecting and likely to be inherently superior to the majority with large myelomeningoceles. They seem to have milder hydrocephalus since shunts had been inserted quite late and none had appreciable intellectual impairment. This runs counter to other experience, which is that many patients with lumbar and thoracolumbar myelomeningoceles have intelligence within the educationally subnormal range (or worse) and that, in general, the degree of intellectual impairment relates directly to the extent of the myelomeningocele and to the neurological deficit.l-3 Apart from their superior intelligence, the survivors appear to be just as handicapped as any others with extensive myelomeningocele. All were wheelchair bound, in contrast to only 50% of those selected for immediate surgery and, predictably, all had a neuropathic bladder, whereas around one quarter of the surgically treated cases escaped this affliction. Problems with the upper renal tracts are were
mentioned only en þassant although there is evidence that the risk of these increases with the extent of the myelomeningocele.4,5 Spinal deformities are not described, yet most of these children could be expected to be severely deformed by adolescence, in contrast to those with smaller lesions,3while pressure sores are another 3
probable complications A simple interpretation of this report is that there exists a small group of babies with lumbar and thoracolumbar myelomeningoceles who have mild hydrocephalus and, as a result, are more likely to survive and to have reasonably normal intelligence. In other respects their handicaps are predictably severe. This conclusion would be neither startling nor original and I remain puzzled (even after several readings) as to why the Dean of Durham and his colleagues (Nov 2, p 996) felt moved to agonise, yet agam, over the ethical issues involved. Department of Paediatric Urology, Hey Children’s Hospital, Liverpool L12 2AP. Alder
A. M. K. RICKWOOD
1. Hunt 2.
3
4. 5
GM, Holmes AE. Some factors relating to intelligence in treated children with spma bifida Devl Med Child Neurol 1975, suppl 35: 65-69 Lonton AP Location of the myelomeningocele and its relationship to subsequent physical and intellectual abilities in children with myelomeningocele with hydrocephalus. Z Kinderchir 1977, 22: 510-19. Rickwood AMK, Hodgson J, Lonton AP, Thomas DG. Medical and surgical complications in adolescents and young adults with spina bifida. Health Trends 1984; 16: 91-95. Hunt GM, Bishop MC, Whitaker RH, Doyle PT Sensory level and renal prognosis in myelomeningocele. Z Kinderchir 1981; 34: 384-89. Rickwood AMK, Thomas DG. The upper renal tracts in adolescents and young adults with myelomeningocele. Z Kinderchir 1984, 39: 104-06.
LATENT VITAMIN K DEFICIENCY IN HEALTHY INFANTS?
SIR,-Late onset haemorrhagic disease of the newborn (HDN) is life-threatening. Most cases have been observed in Asia: in Japan the incidence was 1 in 4500 in unselected babies and 81°70 of infants with HDN presented with intracranial haemorrhage.However, 20 cases over four years have been recorded in West Germany, suggesting an incidence of at least 1 per 100 000.2 Latent vitamin K deficiency without clinical evidence of bleeding may be more common. In a Japanese study PIVKA II, a marker of vitamin K 3 status, was not detected in 14% of healthy 1-month-old babies. Data for European infants have not been reported.
1422 A, Nagao T, Satoh Ch, Kondoh A. A study on 152 newborns below 30 days of age by crossed immunoelectrophoresis of prothrombin with capillary blood. Blood Vessel 1982, 13: 202-06 (Japanese). v Knes R, Zenses Ch, Gòbel U Immunoelectrophoretic determination of PIVKA II in capillary plasma. Haemostasis 1985; 15: 42-43. Petrich Ch, Pothmann R, Döhmen A, v Voss H, Gobel U. A haematocrit corrected micro determination of the P&P-test and blood clotting factors in capillary blood Thromb Diathes Haemorrh 1984, 32: 510-19. v Kries R, Göbel U, Maase B Vitamin K deficiency in the newborn. Lancet 1985; ii: 728-29. Haroon Y, Shearer MJ, Gunn WG, McEnery G, Barkhan P. The content of phylloquinone (vitamin K1) in human milk, cow’s milk and infant formula determined by high-performance liquid chromatography. J Nutr 1982; 112: 1105-17. v Kries R, Reifenhauser A, Göbel U, McCarthy P, Shearer MJ, Barkhan P. Late onset haemorrhagic disease of newborn with temporary malabsorption of vitamin Kl. Lancet 1985; i, 1035.
3 Ilzuka
4. 5.
6
7.
8.
Cumulative distribution of factor II clotting time and activity in healthy babies on day 5 or 6 and in week 5 or 6 of life.
We measured PIVKA II in capillary blood4 from 202 non-selected German babies on the 5th or 6th day oflife and during the 5th or 6th week. In 100 babies factor II clotting activities were also measured, to detect the functional relevance of latent vitamin K deficiency. Clotting tests were done on citrated whole blood obtained from a heel prick.5 On day 5 or 6 the frequency of PIVKA II was considerable, especially in babies who were being exclusively breastfed. Beyond the 4th week, however, PIVKA II was observed in only 1 baby (a breast-fed boy) (table); although no vitamin K was given, PIVKA II was no longer detected 3 weeks later. The pattern for factor II was imilar: on days 5 or 6 several babies had prolonged factor II clotting times and 507o had a factor II clotting activity below 10% (figure). Exclusively breastfed newborns showed longer factor II clotting times than babies receiving additional formula or formula only (table). After the 4th week of life no baby showed a factor II clotting activity below 50%, and there was no difference between the different feeding schedules. FREQUENCY OF
PIVKA II AND DATA ON FACTOR II CLOTTING TIMES
Low oral intake and feeding of maternal milk, known to contain little vitamin K, account for the high PIVKA II incidence in babies aged 5 or 6 days.6 Although the vitamin K content of maternal milk is lower than that in formula,7 vitamin K deficiency beyond the 4th week was observed in only 1 out of 113 breastfed infants. There are two possible explanations. Breastfed infants beyond the 4th week of life receive more milk because lactation is by then fully established. Alternatively, vitamin K absorption may mature. Impaired absorption of vitamin K has been recently demonstrated in an infant with late onset HDN,8 and insufficient absorption may play a part in causing late onset of HDN and latent vitamin K deficiency. Latent vitamin K deficiency is less common in Europe than it is in Japan. Even so, it may be more frequent in Europe than is generally thought. Epidemiological studies of the incidence of late onset HDN and latent vitamin K deficiency are required. Department of Neonatology and Gastroenterology and Department of Paediatric Haematology and Oncology, Zentrum fur
Kinderheilkunde,
University of Düsseldorf, 4000 Düsseldorf 1, West Germany
SIR,-Chemical, heat, and pH inactivation of HTLV-III/LAV in vitro have been reported. 1,2 Nonoxynol-9, a non-ionic surfactant, is a chemical ingredient of several spermicides. In vitro, spermicides inhibit the growth of several microbes, including herpes simplex virus.3-7 We describe here the in vitro inactivation of HTLVIII/LAV by nonoxynol-9. Peripheral blood lymphocytes from HTLV-III antibody negative volunteers not in any AIDS risk group were separated by densitygradient centrifugation. The culture medium was RPMI 1640 supplemented with 100 U/ml penicillin, 50 qg/ml streptomycin, 2 mmol/1 L-glutamine, 10% heat-inactivated fetal bovine serum, and 10 g/ml phytohaemagglutinin. Cells were incubated at 37°C in 5% carbon dioxide. After 3 days, the cells were washed twice with phosphate-buffered saline (pH 7-2). The cell pellet was then incubated with HTLV-III (reverse transcriptase activity [RT] 105 per 2 x 106 cells) for 30 min at 37°C in 5 ml of the above medium to which interleukin 2, 0 - 0407o v/v sheep anti-interferon, and 0 - 02 g/ml polybrene had been added, and cells were then suspended in this medium at a concentration of 106/ml and incubated. Supernatant fluids were harvested by sequential centrifugation on days 2, 4, 8, 10, and 12 and stored at -70°C. A microculture system was used to titrate infectious HTLV-111. RT assays8 were done on days 2, 4, 8, 10, and 12 on cells treated with nonoxynol-9 immediately after inoculation. The RT assay used Mg+as the cation and poly(rA) . poly(dT)I2-IS as the template primer. Assays were done in duplicate. Nonoxynol-9, in the concentrations used, did not interfere with the RT assay. Nonoxynol-9 treated and untreated HTLV-111 infected cell cultures were used as negative and positive controls, respectively. Treatment of HTLV-III with nonoxynol-9 at final concentrations of 0-05% (v/v) or greater resulted in undetectable virus by the IDso assay (table). Toxicity controls suggested that 2-5% (v/v) nonoxynol-9 and lower concentrations were appropriate for ID50 determinations. To assess the effect of the cytotoxicity of nonoxynol-9 on the RT assay pure nonoxynol-9 was diluted to 50%, 25%, 10%, 5%, and 1% with RPMI 1640 (v/v) and added to actively growing uninfected PHA-stimulated normal human lymphocytes. A 507o solution of nonoxynol-9 significantly reduced lymphocyte viability and thus IDso ASSAY FOR DETECTION OF HTLV-III
R. V. KRIES B. MAASE A. BECKER U. GOBEL
K. Etiology of vitamin K deficiency in infants. Perinat Med 1982; 12: 1029-34 (Japanese). 2. Sutor AH, Pollmann H, v Knes R, et al. Vitamin K Mangelblutungen bei Sauglingen jenseits der Perinatalperiode. Weitere Beobachtungen Monatsschr Kinderheilk 1985, 132: 608 1.
INACTIVATION OF HTLV-III/LAV-INFECTED CULTURES OF NORMAL HUMAN LYMPHOCYTES BY NONOXYNOL-9 IN VITRO
Nakayama
*< 10 = no
detectable
virus