620

A bolus of corticosteroids should be given before starting OKT3 treatment, to reduce the release of cytokines which could have coagulation-promoting activity.3 Before starting OKT3 treatment the graft perfusion should be evaluated either by doppler sonography or by nuclear scan. Department of Nephrology, Heinrich-Heine Universitat Dusseldorf, W4000 Dusseldorf,

Germany

PRENATAL EXPOSURE TO FOLIC ACID IN MALFORMED CHILDREN

MARKUS HOLLENBECK ANDREAS WESTHOFF DIETER BACH BERND GRABENSEE *Children with causal syndromes

Department of Vascular Surgery and Kidney Transplantation, Heinrich-Heine Universitat Dusseldorf

RALF KOLVENBACH HORST-WILHELM KNIEMEYER

and

were

excluded

very large population study with good clinical definition of would be needed to detect an association. This result should be confirmed, but nonetheless the observed negative (ie, protective) statistical association between hydranencephaly and prenatal folic acid supplementation also accords with Hook’s statement about the effectiveness of postconceptional folic acid supplementation. Since we investigated malformed children with no family history of NTD, our results also support the assumption that folic acid supplementation could prevent not only the recurrence of NTD, but also the first occurrence of this defect in a family. However, if we take into account that the prenatal mortality rates of conceptuses with NTD is about 98%,3 and that most occurred during the early stage of gestation including the prerecognition period, then there may have been a previously affected conceptus in the family that no one would have known about. a

cases

1. Hollenbeck M, Stuhrmann M, Trapp R, Grabensee B. Colour-coded doppler ultrasonography for early detection of rejection after allogenic renal transplantation. Dtsch Med Wochenschr 1991; 116: 921-27. 2. Warshauer DM, Taylor KJW, Bia MJ, et al. Unusual causes of increased vascular impedance in renal transplants: Duplex doppler evaluation. Radiology 1988; 169: 367-70. 3. Goldmann M, Abramowicz D, Depauw L, et al. OKT3-induced cytokine release attenuation by high-dose methylprednisolone. Lancet 1989; ii: 802-03.

Folic acid

supplementation

and neural tube

defects SIR,-We would like to add our experience to that of Professor (April 18, p 1000). The Spanish Collaborative Study of Congenital Malformations (ECEMC) is a hospital-based casecontrol study and surveillance system. Malformed infants are ascertained in each of the participating hospitals by examination of all livebom babies by a physician within the first three days of life. For each malformed baby, the next non-malformed infant of the same sex born in the same hospital was the control. Mothers of cases and controls were interviewed about prenatal, obstetric, and family histories, and exposures during pregnancy.l,2 From April, 1976, to September, 1990, the ECEMC surveyed 830 882 livebom infants. Of those, 16 736 were malformed and 16 574 were selected as

Hook

controls. In a continuing case-control study on multivitamins and folic acid supplementation at any time during the first trimester of pregnancy, we identified a protective effect for neural tube defects (NTD) after post conceptional folic acid (with or without other vitamins) supplementation. Cases were children with NTD and controls were those with defects other than NTD, none of whom had a previously affected individual with NDT in the family (table). The overall odds ratio (OR) was 0-69 (95 % CI 0-51-0-94; p = 0-01). 22 of 54 mothers of infants with NTD, and 1002 of 2163 mothers of children with other defects, used daily doses of folic acid 03 mg or over (OR 0-61 [0-38-ü.96]; p 0-02). The OR for those exposed to less than 0-3 mg folic acid daily was 0 76 (p = 0-2). To control for possible confounders such as voluntary interruption of pregnancy (VIP) which was legalised in Spain in December, 1985, we examined the period before VIP and that afterwards; the respective ORs were 0-66 (p=0’04) and 0.87 (p = 0-6). The OR for the second period could be biased by VIP and by raised awareness of the preventive effect of folic acid. Hook discusses neural tube closure and post-closure rupture of the neural tube in man, and he states that one should not assume that preventive strategies such as maternal folic acid supplementation will only be effective in the periconceptional period. Our results seem to support his point since our data on exposure to folic acid were postconceptional. However, Shiota3 showed that there is wide variation in development of human embryos at any gestational age, which he judged was at least in part normal biological variability that should be taken into account when estimating the teratogenic risk of environmental agents. We therefore cannot totally exclude the possibility that postconceptional folic acid supplementation could also be effective in the prevention of defects of neural tube closure. We also noted that among 15 cases with isolated hydranencephaly, none of the mothers had folic acid supplementation, whereas 28-6% (4 of 14) of controls were exposed at any time during the first trimester (p 0-04). This observation has not been previously reported, perhaps because this defect is rare =

=

ECEMC, Hospital Universitario San Carlos, Faculty of Medicine, Universidad Complutense, 28040 Madrid, Spain

MARÍA-LUÍSA MARTÍNEZ-FRÍAS ELVIRA RODRÍGUEZ-PINILLA

1. Martinez-Frías ML, Salvador J. Epidemiological aspects of prenatal exposure to high doses of vitamin A in Spain. Eur J Epidemiol 1990; 6: 118-23. 2. Martinez-Frías ML. Valproic acid and spina bifida. Lancet 1991; 338: 196-97. 3. Shiota K. Development and intrauterine fate of normal and abnormal human

conceptuses.

Cong Anom 1991; 31: 67-80.

Semi-quantitative detection of syndrome with PCR

Down’s

SiR,—Fetal cells can be retrieved from maternal blood samples.’ Although this material cannot be cultured, it can be analysed by fluorescence in-situ hybridisation (FISH) with gene-specific probes.2 This approach could achieve higher rates of detection than existing methods but it is time-consuming and unsuitable for mass screening. We have pioneered a rapid, semi-quantitative, gene amplification technique. Blood samples were taken from 4 patients with Down’s syndrome and 4 controls, and DNA was isolated by standard methods. The investigators were unaware of the identity of the samples. Two sets of primers3,4 flanking a short 337 bp region of the amyloid precursor protein gene (AP) on chromosome 21 and a 330 bp region linked to the cystic fibrosis gene (CS-7) on chromosome 7 were synthesised, and the two sequences were simultaneously amplified by standard methods except for the addition of [35S]-dATP to the reaction. All reactions were in triplicate and products were resolved on thin 4% polyacrylamide gels. Product bands were visualised by silver staining, excised from the dried gels, and counted by liquid scintillation.

Expressing the results as a ratio of the counts from AP and CS-7

products allowed us to discriminate accurately between the Down’s and control samples (figure). Mean ratios were 1-69 and 1-14, respectively (p < 0-05, U test), and close to the expected values of 15 and 1 ’0. Because inter-replicate variability, especially in the Down’s group, was considerable the mean of the replicates would in practice be used. These ranged from 0-99 to 1-23 (controls) and from 1-48 to 1’84 (Down’s syndrome), respectively. The assay’s speed (one day, including DNA isolation and amplification) makes it well suited to rapid processing. The AP locus is close to the translocation site on chromosome 21 and may therefore be unreliable for detecting Down’s syndrome caused by unbalanced translocations, which could be overcome by amplifying telomeric gene sequences. The method is, however,

621

unsuitable for the detection of the

rarer

mosaic forms of Down’s

syndrome. More significantly, we acknowledge that predictive accuracy will also be dependent on the degree of maternal contamination that can be tolerated. The best technique for separating fetal cells from maternal blood5,6 will need to be followed by a larger study, in which the availability of a simple PCR-based method for DNA measurement in Down’s syndrome should prove valuable. Unit of Pathology, University of Leeds,

Leeds LS2 9JT, UK, Unit of Obstetrics and Gynaecology, St James’s University Hospital, Leeds, and Institute of Epidemiology and Health Services Research, University of Leeds

D. MILLER P.-Z. TANG R. S. V. CARTMILL M. D. GRIFFITH-JONES R. J. LILFORD H. S. CUCKLE

1. Admolfi M. On a non-invasive approach to prenatal diagnosis based on the detection of fetal nucleated cells in maternal blood. Prenat Diagn 1991; 11: 799-804. 2. Lichter P, Boyle AL, Cremer T, Ward DC. Analysis of genes by nonisotopic in situ hybridisation. Genet Anal 1991; 8: 24-35. 3 Salbaum JM, Weidemann A, Lemaire H-G, Masters CL, Beyreuther K. The promoter of Alzheimer’s disease amyloid A4 precursor gene. EMBO J 1988; 7: 2807-13. 4 Lench N, Stanier P, Williamson R Simple non-invasive method to obtain DNA for gene analysis. Lancet 1988; i: 1356-58. 5 Bruch JF, Metezeas P, Garcia-Fonknechten N, et al. Trophoblast like cells sorted from maternal blood using flow cytometry. A multiparametric study involving transmission electron microscopy and fetal DNA amplification. Prenat Diagn 1991; 11: 787-98. 6 Mueller UW, Hawed CS, Wright AE. Isolation of fetal trophoblast cells from penpheral blood of pregnant women. Lancet 1990; 336: 197-201.

Molecular analysis of nosocomial infection by oxacillin-resistant Staphylococcus aureus lacking protein A and clumping factor SIR,—An outbreak ot oxacillm-resistant Staphylococcus

aureus

’ORSA) in 45 patients occurred in an 1800-bed university-affiliated hospital in south Germany in November, 1991. The strains were clumping-factor and protein-A negative by usual agglutination tests. No clumping-factor-negative ORSA had been recorded in the hospital during the 6 months before the identification of the epidemic strains. The outbreak started in the intensive-care unit of the department of surgery, and spread to the intensive-care unit of the department of anaesthesiology and to nearly all surgical wards in the hospital. 4 patients had septicaemia, 1 of whom died with acute endocarditis. Another 2 patients had pneumonia and 1 died. This ORSA caused wound infection in 28 patients, urinary-tract

infections in another 3 patients. 7 patients had the strain in tracheal secretion without signs of pneumonia or other infection. In 1 case an outbreak strain was found at necropsy in ethmoidal cells without signs of staphylococcal infection. All the patients had the typical risk criteria described by Brumfitt et al:1 old age and/or surgical wounds, venous access sites, and/or serious illness such as carcinoma. The 58 nursing and medical staff involved were examined repeatedly for nasal carriage. Only 2 harboured the outbreak strain. This low frequency concurs with Brumfitt et al.l Tube coagulase-test and biochemical analysis of the isolates with the API-System (Bio Merieux) resulted in definitive identification (99-9%) of S aureus. Susceptibility testing proved the ORSA to be sensitive only to novobiocine, tetracycline, co-trimoxazole, vancomycin, rifampicin, and fusidic acid. All 47 ORSA uniformly contained a single plasmid of 32 kb with identical restriction patterns. After digestion of chromosomal DNA with Sma I and separation of the DNA by pulsed-field gel electrophoresis,’ fragment bands of identical size were observed, which indicates clonal identity of the isolates. During the oubreak period, we obtained ORSA suspected to cause smaller outbreaks from three other hospitals in south Germany. DNA fingerprinting showed the same patterns as our strains. The following effective measures were adopted to terminate the series of infections: strict isolation of the infected patients, discharge of carriers as soon as clinically feasible; and treatment of nasal carriers with mupirocin and their exclusion from nursing in intensive-care units until they were shown to be cured. Staphylococci with yellow pigmentation that are negative by slide agglutination tests for clumping factor and protein A should be tested for DNase and free coagulase. Even DNase, clumping-factor, and protein-A negative ORSA have been described in a few sporadic cases.3 In addition to biochemical typing and antibiotic susceptibility patterns, molecular methods for demonstrating clonality are necessary for specifying strains presumed responsible for outbreaks. Institute for Hygiene and Microbiology, University of Wurzburg, D-8700 Wurzburg, Germany, and Department of Surgery,

University Hospital, Wurzburg

ANDREAS SCHWARZKOPF HANNELIESE SCHMIDT-ROTTE HERBERT SCHMIDT ELMAR KUNZ HELGE KARCH JÜRGEN HEESEMANN

Methicillin-presistant Staphylococcus aureus. N Engl J Med 1989; 320: 1188-96. 2. Ichiyama S, Ohta M, Shimokata K, Kato N, Takeuchi J. Genomic DNA fingerprinting by pulsed field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin resistant Staphylococcus aureus. J Clin Microbiol 1991; 29: 2690-95. 3. Neville LO, Billington OJ, Kibbler CC, Gillespie SH. Methicillin resistant Staphylococcus aureus without clumping factor, protein A and DNase. Lancet 1991; 1. Brumfitt W, Hamilton-Miller J.

338: 518.

Magnesium for hyperventilation syndrome

in Rett’s

SIR,—Rett’s syndrome is characterised by irregular cycles of hyperventilation with hypocapnia alternating with apnoea for up to 120 s with reduced arterial oxygen saturation, cyanosis, and unconsciousness.1,2The hypocapnic alkalaemia and hypoxaemia may impair amine production,3 disturb cerebral perfusion,4 and contribute to progressive neurological impairmentWe gave a girl with Rett’s syndrome and epilepsy magnesium as an anticonvulsant after other anticonvulsants had failed. To our surprise hyperventilation greatly improved. So we studied the effects in six other cases. All seven girls had characteristic stage 2 Rett’s syndrome (intellectual regression, hand stereotypies, microcephaly, hyperventilation with hypocapnic alkalaemia, apnoeic episodes sometimes with loss of consciousness, seizures and autistic features’). Magnesium orotate or citrate, initially 4 mg/kg per day in three doses, was given orally. The dose was gradually increased until diarrhoea occurred (usually at 10 mg/kg per day). Six girls had severe hyperventilation and deep cyanosis during apnoeic episodes. The parents were asked to record these episodes before and 1 month after starting magnesium. The daily episodes of