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selection of HLA matched unrelated donors. Lancet 1991; 337: 1049-52. 10. Clement LT, Vinta PE, Bradley GE, et al. Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate. J Immunol 1990; 145: 102-08. 11. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977; 91: 335-44. 12. Gartner S, Kaplan HS. Long-term cultures of human bone marrow cells.

Proc Natl Acad Sci USA 1980; 77: 4756-59. HJ, Lansdorp PM, Henkelman DH, et al. Functional characterization of individual human hematopoietic stem at limiting dilution on supportive marrow stromal cells. Proc Natl Acad Sci USA 1990; 87: 3584-88.

13. Sutherland

14. Fauser AA, Messner HA. Granuloerythropoietic colonies in human bone marrow. Blood 1978; 52: 1243-48.

15. Messner HA, Jarmal N, Izajuime C. The growth of large megakaryocyte colonies from human bone marrow. J Cell Physiol 1982; 1 (suppl 1): 45-51.

LH, Geary CG, Chang J, et al. Functional studies of bone haemopoietic and stromal cells in the myelodysplastic syndromes (MDS). Br J Haematol 1990; 75: 16-25. 17. Donay L, Gorin NC, Jean-Yves M, et al. Recovery of CFU-GM from cryopreserved marrow and in vivo evaluation after autologous bone marrow transplantation are predictive of engraftment. Exp Hematol 1986; 14: 358-65. 18. Ma DDF, Varga DE, Biggs JC. Donor marrow progenitors (CFI-Mix, BFU-E and CFU-GM) and haemopoietic engraftment following HLA matched sibling bone marrow transplantation. Leukaemia Res 1987; 11: 141-47. 19. Gorin NC. Collection, manipulation and freezing of haemopoietic stem cells. Clin Haematol 1986; 15: 19-48. 20. Simmons PJ, Przepiorka D, Thomas ED, Torok-Storb B. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature 1987; 328: 429-32. 21. Linch DC, Brent L. Can cord blood be used? Nature 1989; 340: 676. 22. Auerbach AD, Lin Q, Ghosh R, et al. Prenatal identificaton of potential donors for umbilical cord blood transplantation for Fanconi anaemia. Transfusion 1990; 30: 682-87. 16. Coutinho

marrow

Disseminated "Mycobacterium genavense" infection in patients with AIDS

We describe 18 patients with advanced HIV most of whom had a chronic illness characterised by fever, diarrhoea, and massive loss of weight. Biopsy and necropsy samples revealed abundant acid-fast microorganisms in intestines, liver, spleen, lymph nodes, and many other tissues, which did not grow on solid media, although limited growth was observed in liquid blood cultures. Using primers complementary to bacterial 16S rRNA we amplified DNA sequences from tissue and leucocyte extracts and from blood-culture bottles. The sequences obtained were unique and suggest that the microorganism is a new member of the genus Mycobacterium, for which we propose the name Disseminated "Mycobacterium genavense". infection with "M genavense" should be considered in the differential diagnosis of HIV-infected patients with extreme immunosuppression, wasting, and

infection,

fever. Introduction infections are the hallmark of AIDS. of these, such as Pneumocystis carinii pneumonia, may be prevented,l and as highly immunosuppressed patients with AIDS survive longer, unusual diseases will become more frequent and previously unknown infections will emerge. In 1990 we described a patient with HIV infection who died from an overwhelming infection with an acid-fast microorganism that could be transferred to immunosuppressed nude mice. Limited growth was observed in liquid (Middlebrook 13A2) culture, but not on solid media and the nature of this microorganism remained undetermined.3 To characterise bacteria that cannot be cultured, new methods have lately become available. Sequencing of ribosomal RNA is the most promising because part of these sequences are widely conserved among bacterial species

Opportunistic

Because

some

while other,

neighbouring sequences vary. 4 When primers complementary to conserved sequences, DNA of unknown microorganisms can be amplified5,6 and comparison with known sequences permits precise phylogenetic and taxonomic classification.4,7 are

Here

we

report disseminated infection with

an

acid-fast

microorganism in 18 patients with fever, diarrhoea, and loss of weight. The organism could not be grown on solid media but amplification of ribosomal RNA genes yielded sequences which were identical in every case and differed from previously known mycobacterial sequences, suggesting a new species, here tentatively named

"Mycobacterium genavense". Methods DNA was extracted from liquid Middlebrook 13A blood cultures’ of 11 patients, and liver, spleen, lymph nodes, and/or polymorphonuclear leucocytes (buffy coats) of 7.11 The primers have been described,’*’*’ or were synthesised on the basis of published sequences7 10 DNA sequences were amplified9 (further details from E. C. B.). Negative controls were interspersed with clinical samples during every amplification and consisted of reaction mixtures with DNA extracted from blank samples (to control for contamination during DNA extraction) and of polymerase chain reaction (PCR) mixtures without added DNA. Additional controls contained DNA extracted from uninfected human liver and lymph nodes. The adequacy of DNA extraction and amplification from human tissue was ascertained by amplifying the &bgr;-globin gene. The amplified

samples (10 µ1) were subjected to electrophoresis through 0-8% gels, and the DNA was visualised after staining with

agarose

ethidium bromide. ADDRESSES: Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, 3000 Hannover 61, Germany (E. C. Bottger, MD, A. Teske, BS, P. Kirschner, MD); Division of Infectious Diseases, Hôpital Cantonal Universitaire, 1211 Geneva 4, Switzerland (S. Bost, MD, B. Hirschel, MD); Department of Medical Genetics and Microbiology, Geneva University Medical School (H. R Chang, MD); and Laboratoire de Microbiologie, CHUV, Lausanne (V. Beer, MD). Correspondence to Dr Bernard Hirschel or Dr Erik C. Böttger.

77

TABLE I-CLINICAL DATA ON PATIENTS WITH "M GENAVENSE"

CMV=cytomegalovirus, IVDA intravenous drug addict, PCP=Pcaf//?//pneumonia Survival is time from first observation of acid-fast bacteria in biopsies or blood cultures to death CD4 count is one closest to time of positive blood cultures and is in CD4+ + lymphocytes/ill. (Case 1 has been published’.) *Matenal from which DNA was extracted and 16S rRNA determined, talive at the time of writing

The amplified gene fragments were sequenced directly.s A contiguous stretch of 1449 nucleotide positions covering 95 % of the 16S rRNA gene was obtained from patients 1 and 2 (European Molecular Biology Laboratory sequence accession number X 60070). For patients 3-18 the nucleic acid sequence of the characteristic hypervariable regions was determined" (positions 120-280 and 400-450). For the phylogenetic analysis regions of uncertain alignment were omitted. The algorithms used for alignment, the calculation of pairwise distances, and the construction of the phylogenetic tree by the neighbourliness method have been described.7

Results Clinical details are given in table I. 16 of 18 patients were from Switzerland; we have no evidence that they were in contact with each other. All were HIV seropositive and had very low CD4 counts, and most had fever, diarrhoea, and weight loss. 5 (nos 1, 9, 11, 13, and 14) had no

life-threatening opportunistic infections (apart "M genavense"), as illustrated by case 1.3

from

A 28-year-old drug addict had diarrhoea, anorexia, nausea, fever, a 5 kg weight loss, and oral thrush; his CD4 lymphocyte count was 45/µl. Acid-fast rods were found on duodenal and bone marrow biopsies, but cultures remained negative. During the ensuing year, he lost more than 20 kg. He was readmitted with fever, anaemia, hepatosplenomegaly, ascites, and renal failure. Acid-fast

rods were seen in ascites, urine, and buffy coat. He died and necropsy several organs contained acid-fast rods. No other opportunistic infection was found. DNA was extracted from liver, spleen, and polymorphonuclear lymphocytes, and the 16S rRNA gene was amplified (fig 1). 11 other patients had multiple diseases together with "Mgenavense" infection, which may have been responsible for some of the symptoms and signs. In the 2 remaining patients, the link of the mycobacterial infection to signs and symptoms was tenuous, as shown by case 8. A 26-year-old drug addict had behaviour problems and early signs of dementia in 1989. These were ascribed to multifocal progressive leukoencephalopathy on the basis of magnetic resonance imaging. He was given zidovudine and achieved remission, although his CD4 count fell below 10/µl. In June, 1991, two blood cultures were drawn because of intermittent fever to 38-5°C. These cultures were positive in September, showing acid-fast bacteria that failed to grow on solid media. However, he was not treated with antimycobacterial drugs because his fever had disappeared and because he had neither digestive symptoms nor loss of weight. In November he had high fever with rapidly progressive respiratory distress. Bronchoalveolar lavage revealed pulomonary toxoplasmosis but no mycobacteria. He died from respiratory failure. No necropsy was done. 11patients were treated with various drugs directed against M avium-intracellulare. Response was difficult to at

78

1

2

3 4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20

Fig 1-Agarose-gel electrophoresis of amplified DNA. DNA amplified with primers specific for mycobacteria in combination with universal primers. Lanes 1-8 and 15-20 show amplification of 5’ end, and lanes 9-14 amplification of 3’end of 165 rRNA gene. Sources of amplified DNAs were urine (lane 1 ), supernatant serum after centrifugation (lane 2), polymorphonuclear leucocytes (lanes 3 and 9), spleen (lanes 4 and 10), liver (lanes 5 and 11 ) of patient 1; liver (lane 17), lymph node (lane 18), and spleen (lane 19) of patient 2. Lanes 8,13, and 15 are positive controls and contain DNA isolated from cultures of M tuberculosis. Other six lanes are negative controls: in 6,12, and 16, extractions from blank samples were added to PCR reagents; in 7,14, and 20, PCR reagents were processed without additions.

judge retrospectively, but 7 seemed to have had a temporary decrease in fever and stabilisation of weight. 12 patients have died, 5-16 months after diagnosis. Cultures of blood or of tissue samples in liquid Middlebrook 13A medium (Bactec System, Becton Dickinson) showed limited growth in 16 cases but subculture on solid media was unsuccessful. Blood-culture bottles apart, acid-fast microorganisms were seen in bone marrow (in all 4 of the patients who had this examination), intestinal tissue (4/4), lymph nodes (6/6), liver (2/2), and multiple other tissues (2/2), but again culture-based attempts at identification failed. Tissue from liver and spleen and polymorphonuclear leucocytes (PMNs) were chosen for study in cases 1 and 2 because microscopic examination had revealed numerous acid-fast organisms. Universal and mycobacteria-specific primers were used for amplification. These primers can detect fewer than ten mycobacteria.9 After thirty-nine cycles of PCR with two different primer pairs, amplified products of the appropriate size were detected with liver, spleen, or PMNs of patient 1 (fig 1). Urine and centrifuged serum were amplification negative; nor did they reveal acid-fast organisms on microscopy. DNA from spleen, lymph node, and liver from patient 2 was also extracted and amplified (fig 1). The amplified PCR products were sequenced, revealing an identical nucleotide composition for DNA extracted TABLE II-ALIGNMENT OF MYCOBACTERIAL 16S rRNA

SEQUENCES

from liver, spleen, and PMNs from patient 1, which was in turn identical to that of DNA extracted from spleen, lymph node, and liver from patient 2. It was different from any known sequence in the EMBL database, and showed a high degree of homology to 16S sequences from other mycobacteria (93-2-99-2%). Inspection of the sequence revealed the presence of regions that are characteristic for the genus Mycobacterium9 as well as unique sequences (table II). The alignment with published mycobacterial16S rRNA sequences7.10,12 was used to construct a phylogenetic tree. As shown in fig 2 the organism, for which we propose the name "Mycobacterium genavense" (the Latin name for Geneva is Genava), falls within the confines of the genus Mycobacterium, its closest relative being M simiae. These findings were applied to cases 3-18. DNA was amplified from the blood culture bottles (11cases), lymph nodes (4), and bone marrow (1). The characteristic signature sequences of "M genavense" were present (table II). Controls included DNA extracted from samples of frozen liver and lymph nodes of patients with and without AIDS, from the buffers and enzymes used for PCR, and from the Middlebrook 13A medium used for mycobacterial blood cultures: in all of these, no amplified DNA fragment was observed. Numerous culture-positive Bactec 12B bottles from the mycobacterial laboratory at the Medical School, Hannover, served as positive controls and to exclude sample-to-sample contamination; they yielded the expected DNA sequences including M tuberculosis, M xenopi, M gordonae, M avium, M simiae, and M intracellulare.l’ From sputum of a patient whose cultures grew M avium-intracellulare, a rRNA sequence characteristic of M aviun-intracellulare was amplified (data not

shown). Discussion First nucleotide corresponds to position 176 of Eschenchia co 16S rRNA. M tuberculosis used as source of reference sequence Only nucleotides different from M tuberculosis are shown; dashes mdicate deletions. (Signature sequences of more than thirty other mycobactenal species including all described slow-growing species are available from E. C B.; they all differ from "Mgenavense".)

Bacterial species that cannot be grown on artificial media may nonetheless be associated with a distinctive diseaseeg, leprosy M leprae) and syphilis (Treporiema pallidum). The clinical course in most of our 18 patients was strikingly

79

Fig 2-Phylogenetic tree of selected mycobacteria as established by 16S rRNA sequencing. Tree was "rooted" using Nasteroides as an"outgroup". Bar indicates difference of ten nucleotides.

were relentless loss of weight, and diarrhoea. These symptoms were probably due to fever, massive infection of the intestine and the liver, but necropsy revealed that other organs such as lungs, bone marrow, and lymph nodes were also involved. In 5 patients (table i), no other major opportunistic infection was evident. In 2 cases, absence of other infections was confirmed by necropsy, strongly suggesting that "M genavense" may have contributed to the deaths. At the other extreme, 2 patients (cases 7 and 8) had few digestive symptoms and little weight loss when "Mgenavense" was discovered in blood cultures drawn because of intermittent fever. Our experience suggests that the pathogenicity of "M genavense" is similar to that of M avium-intracellulare.13 Traditional methods, such as staining or culture, constrain microbiologists and physicians in their efforts to detect and describe microorganisms. Few are recognisable on morphology alone, and culture is only possible after the physiological niche of the microorganism has been detected and duplicated experimentally. Without culture, however, biochemical analysis and identification is usually impossibleJ 14,15 because the quantity and quality of pathogens extracted directly from human tissue is insufficient. In our case, culture was unsuccessful after 6 months’ incubation in the following media: Coletsos, Ogawa, Stonebrink, Middlebrook 12B, 13A and 12B enriched with iron citrate, ammonium, haemin, and mycobactin, at 30°, 37°, and 45°C.33 When abundant acid-fast rods are seen in tissue but do not grow well in culture, leprosy comes to mind but the signs and symptoms, the absence of growth in the footpads of NMRI mice (not shown), and the rapid growth of M leprae in immunosuppressed mice3argue against this. Chemical analysis of cell walls can be used to classify mycobacteria. Thin-layer chromatography of mycolic acids extracted from necropsy tissues in case 1 revealed three spots corresponding to a, a’, and keto mycolates, and gas chromatography revealed tuberculostearic acid and hexadecanoic acid.3 These patterns are similar to those of M simiae and M malmoense. However, M simiae and M malmoense are phylogenetically different from one another (fig 2). Analysis of 16S rRNA can resolve these ambiguities and is especially suited to microorganisms that are difficult to grow in culture. Universal primers (ie, oligonucleotides shared by all bacteria) theoretically permit amplification of any bacterial 16S rRNA,5.6 whereas more specific primers will restrict amplification to certain genera

similar; the main features

of microorganism.9 Amplification with universal primers is hampered by problems due to contamination and lack of specificity.6,16 Use of a genus-specific primer was suggested by the acid-fastness of the microorganism, so we used oligonucleotides specific for mycobacteria, in combination with primer sequences common to all bacteria. Thus, we avoided problems with contaminating DNA in Taq polymerase ’16 " and with contaminating enterobacteriaceae that must have been present in necropsy samples taken without use of sterile technique more than 12 h after death (as in patient 2). The sequence data show that "M genavense" is a mycobacterium, that it differs from known mycobacteria, and that it is related to M simiae. Infection with "Mgenavense" may be more frequent than the 18 cases described here suggest. Several unlikely events had to happen for us to identify an isolate-namely microscopical detection of acid-fast rods in normally sterile tissue; some growth in blood cultures; and the availability, months later, after efforts at identification have failed, of the original culture. Furthermore this organism is likely to be found more frequently in future now that zidovudine18 and Pneumocystis carinii pneumonia prophylaxis1 have changed the course of HIV infection,19 so that more patients with very low CD4 counts survive without pneumocystis pneumonia. These patients are vulnerable to infections with non-tuberculous mycobacteria such as M aviumintracellulare 13 and, we suggest, "M genavense". This organism should be considered seriously in the differential diagnosis of patients with CD4 counts below 100/)J,1 and diarrhoea, weight loss, and fever. Biopsy of liver, duodenum, bone marrow, or lymph nodes may reveal acid-fast microorganisms that cannot be cultured on solid media, although blood cultures in Middlebrook 13A medium may show some growth. We are unable to make firm treatment recommendations but, by analogy with M avium-intracellulare, amikacin, clofazimine, ethambutol, rifambutin, clarithromycin, azithromycin, and ciprofloxacin (and other fluoroquinolones) may be tried. The natural reservoir and the epidemiology of "M genavense" remain to be established. The striking gastrointestinal symptoms and signs suggest the gut as the reservoir from which "M genavense" invades other tissues. The use of species-specific sequences (table II) in combination with PCR driven by mycobacterial primers will permit rapid, sensitive and specific detection of "M genavense", and an assessment of its prevalence in symptomatic and symtomless HIV infection and in HIVseronegative patients with depressed or normal immunity. We thank Dr P. Francioli and Dr J.-Ph. Chave, Lausanne; Dr W. Zimmerli, Basel; Dr M. Hausennann, Liestal; Dr J. Wintsch, Geneva; Dr R. Malinvemi and Dr U. Schaad, Bern; Dr D. Nadal, Dr M. Flepp, and Dr R. Weber, Zurich; Prof R. Schmidt, Hannover, and Dr M. Fille, Innsbruck, for referral and information on patients 2-18; Prof W. Wegmann, Institute of Pathology, Liestal, for tissue samples and necropsy data for patient 2; Dr J.-D. Piguet, Institut d’Hygiene, Geneva, and Dr M. Salfinger, Zurich, for attempts to culture "M genavense" and for submitting blood cultures for analysis; and Dr J. Wolters, Institut fur Allgemiene Mikrobiologie, University of Kiel, for help with the computer analysis. E. C. B. is supported by a grant from the Bundesministerium fiir Forschung und Technologie (no OlKI89117) and R. C. by a grant from the Swiss National Science Foundation.

REFERENCES 1. Hirschel B,

Chopard P, Chave JP, et al. Primary prevention of Pneumocystis carinii pneumonia by inhalation of pentamidine: preliminary results from a placebo-controlled randomized trial. Med

Klin 1990; 85: 268-70 2. Middlebrook G. Automated radiometric detection of Mycobacterium tuberculosis in selective media. Am Rev Respir Dis 1977; 115: 1066-69.

80

3. Hirschel

B, Chang HR, Mach N,

et

al. Fatal infection with

a

novel

mycobacterium in a man with the acquired immunodeficiency syndrome. N Engl J Med 1990; 323: 109-13. 4. Woese CR. Bacterial evolution. Microbiol Rev 1987; 51: 221-71. 5. Edwards U, Rogall T, Blocker H, Emde M, Böttger EC. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic unidentified

Acids Res 1989; 17: 7843-53.

Relman DA, Loutit JS, Schmit TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis. N Engl J Med 1990; 323: 1573-80. 7. Rogall T, Wolters J, Flohr T, Böttger EC. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol 1990; 40: 323-30. 8. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning. A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 6.

1982. 9.

Böddinghaus B, Rogall T, Flohr T. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol 1990; 28:

1751-59. 10. Stahl DA, Urbance

JW. The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J Bacteriol 1990; 172: 116-24. 11. Rogall T, Flohr T, Böttger EC. Differentiation of Mycobacterium

species by direct sequencing of amplified DNA. J Gen Microbiol 1990; 136: 1915-20. 12. Teske A, Wolters J,

Böttger EC. The 16S RNA nucleotide sequence of Mycobacterium leprae: phylogenetic position and development of DNA probes. FEMS Microbiol Lett 1991; 64: 231-37. 13. Jacobson MA. Mycobacterial diseases. In: Sande MA, Volberding PA, eds. The medical management of AIDS, 3rd ed. Philadelphia: Saunders, 1990: 291-303. 14. Brenner DJ. Impact of modern taxonomy of clinical microbiology. ASM News 1983; 49: 58-63. 15. Brenner DJ. Taxonomy, classification, and nomenclature of bacteria. In: Lennette EH, Balows A, Hausler WJ, Truant JP, eds. Manual of clinical microbiology, 3rd ed. Washington: American Society for Microbiology, 1980: 1-6. 16. Böttger EC. Frequent contamination of Taq polymerase with DNA. Clin Chem 1990; 36: 1258. 17. Rand KH, Houck H. Taq polymerase contains bacterial DNA of unknown origin. Mol Cell Probes 1990; 4: 445-50. 18. Fischl MA, Richman DD, Causey DM, et al. Prolonged zidovudine therapy in patients with AIDS and advanced AIDS-related complex. JAMA 1989; 262: 2405-10. 19. Graham NM, Zeger SL, Park LP, et al. Effect of zidovudine and Pneumocystis carinii pneumonia prophylaxis on progression of HIV-1 infection to AIDS. Lancet 1991; 338: 265-69.

SHORT REPORTS Low cerebrospinal fluid concentration of free gammaaminobutyric acid in startle disease

The

pathophysiology of startle disease (hyperekplexia) is unknown. Hyperactivity of the brainstem reticular formation has been suggested as a cause. We report a newborn infant with classic features of startle disease in whom cerebrospinal fluid (CSF) concentrations of gamma-aminobutyric acid (GABA) were substantially lower than normal during the first weeks of life. She improved greatly on clonazepam treatment. We suggest that the signs of

this disorder may be due to a genetic defect or to delayed maturation resulting in low CSF GABA.

Startle disease (hyperekplexia), first described in a family who sustained violent falls precipitated by fright, stress, or surprise,1 is inherited as an autosomal dominant trait with incomplete penetrance.2.3 Most patients present in the neonatal period, either with "stiff baby syndrome", or with a seizure disorder4,5 characterised by myoclonic jerks, increased muscle tone, and severe apnoea but without concomitant discharges on electroencephalography (EEG). This disorder is easily mistaken for neonatal convulsion,4 since the EEG changes due to ocular movement or other muscle artifact are frequent,6 so its true incidence is probably underestimated. The pathophysiology is unknown. The startle response is thought to be mediated through the brainstem. Hyperactivity of the brainstem reticular formation, due either to intrinsic hyperexcitability or to insufficient cortical

inhibition could lead to startle disease. Delayed maturation of these systems has also been suggested since the signs abate with age.2 No biochemical abnormality has been found. We report an infant with classic features of startle disease who had an abnormality of cerebrospinal fluid (CSF) gamma-aminobutyric acid (GABA). The baby girl, born at 38 weeks’ gestation, was the first child of non-consanguinous parents. The mother was a 24-year-old Asian primigravida. Pregnancy and labour were uneventful. Birthweight was 2560 g (10th centile), length 48 cm (50th centile), and head circumference 36 cm (97th centile). Apgar scores at 1 and 5 min were 6 and 9, respectively, and cord-blood pH was 7-4. 1 h after birth the infant had generalised jerks which were diagnosed as convulsions. She was given 20 mg/kg phenobarbitone before transfer to the neonatal unit. 8 h later she was very hypotonic. On day 3 she still had pronounced truncal hypotonia but the tone in the limbs was abnormally hgh. She had exaggerated tendon reflexes and became jittery when touched. The limb hypertonia diminished during sleep, but increased when she was touched. She had poor visual fixation and pursuit but responded to auditory stimuli without startle. Physical examination was otherwise normal. On day 5 there were seizure-like episodes, consisting of myoclonic movement of the limbs, rigidity of the trunk and limbs, flexion of the upper limbs, fisting of the hands, and extension of the lower extremities. Some episodes were accompanied by apnoea and upward deviation of the eyes. The heart rate initially increased, but arterial oxygen saturation (SaOz) fell to 60%, so episodes were followed by brief bradycardia, which was corrected by administration of oxygen. The jerks and stiffening could be inhibited by holding the infant tightly or by bending her forward, but were precipitated by handling, tapping, and especially by turning her into the prone position. No seizure discharges could be recorded on standard EEG or on four-channel continuous monitoring. No abnormalities in organicacid or aminoacid measurements and no infections were found. Cranial ultrasound showed a small intraventricular haemorrhage on day 2 but magnetic resonance imaging at 1 week and 3 months was normal, as were proton spectroscopy, auditory brainstem responses, and visual evoked responses. Latency of the somatosensory responses was normal, but the amplitude was larger than expected for age. Empirical treatment with pyridoxine during an attack produced a doubtful response and 10 mg/kg intravenous phenobarbitone did