Recombinant B-domain-deleted porcine sequence factor VIII (r-pFVIII) for the treatment of bleeding in patients with congenital haemophilia A and inhibitors.

INTRODUCTION: Development of inhibitors to human FVIII (hFVIII) significantly complicates the control of bleeding events in patients with haemophilia A. AIM: This prospective, multicentre, open-label, non-comparative, Phase II study evaluated the haemostatic activity of a recombinant B-domain-deleted porcine FVIII (r-pFVIII), in the treatment of non-life/non-limb-threatening bleeding in individuals with haemophilia A and FVIII inhibitors. METHODS: Acute bleeding episodes in patients with pFVIII inhibitor titres <0.8 BU mL-1 were treated with 50 U kg-1 body weight r-pFVIII. Those with pFVIII inhibitor titres of >0.8 BU mL-1 received an initial calculated r-pFVIII loading dose followed by 50 U kg-1 treatment dose. Treatment continued at 6-hourly intervals until bleeding was determined, controlled or till a maximum of eight doses was reached. RESULTS: All 25 bleeding episodes in nine patients (mean age: 23.7 years; range: 14-34 years) were controlled successfully with eight or fewer injections of r-pFVIII. The median time from bleeding onset to the administration of r-pFVIII was 5.7 h (range: 1.5-20.0 h). Twenty of the bleeding episodes (80%) were controlled with one treatment dose of r-pFVIII (with or without a loading dose, median dose: 200.8 U kg-1 ; range: 50-576 U kg-1 ) regardless of pFVIII level. r-pFVIII was well tolerated and no treatment-emergent serious adverse events were considered by the investigator to be related to r-pFVIII administration. CONCLUSION: The results suggest that FVIII replacement therapy with r-pFVIII could be a viable alternative to bypassing agents for the treatment of bleeding episodes in individuals with haemophilia A and FVIII inhibitors.

Pharmacokinetics and safety of OBI-1, a recombinant B domain-deleted porcine factor VIII, in subjects with haemophilia A.

OBI-1 is a recombinant B-domain deleted porcine factor VIII (FVIII). FVIII treatment in those with haemophilia A may be complicated by the development of anti-FVIII antibodies (inhibitors) leading to a failure to respond to treatment with human FVIII. To compare the pharmacokinetics and safety of a single dose of OBI-1 with Hyate:C in subjects with haemophilia A and inhibitors, subjects were randomized to receive either Hyate:C followed by placebo or placebo followed by OBI-1 in a double-blind fashion. FVIII levels were assayed using both a one-stage coagulation assay (OSCA) and chromogenic assay. Pharmacokinetic parameters for FVIII were calculated for 6/9 subjects randomized; in three subjects baseline anti-porcine FVIII inhibitors led to a lack of measurable FVIII activity. Mean C(max) appeared higher for OBI-1 (OSCA: 176.00 U dL(-1), standard deviation ± 88.00; chromogenic: 151.00 ± 31.51 U dL(-1)) than Hyate:C (OSCA: 82.3 ± 19.22 U dL(-1); chromogenic: 52.67 ± 13.8 U dL(-1)). Mean AUC also appeared higher for OBI-1 (OSCA: 2082.87 ± 1323.43 U h(-1) dL(-1) ; chromogenic: 1817.28 ± 625.14 U h(-1) dL(-1)) than Hyate:C (OSCA: 1177.8 ± 469.49 U h(-1) dL(-1); chromogenic: 707.61 ± 420.05 U h(-1) dL(-1)). Two infusion-related events occurred: one with Hyate:C, one with placebo. Four of five subjects without anti-porcine FVIII inhibitors at baseline remained porcine FVIII inhibitor negative 29 days after infusion. A single dose of OBI-1 appears to have higher bioavailability than Hyate:C in subjects with haemophilia A without measurable anti-porcine FVIII inhibitors, and is well tolerated. These results should be confirmed in a larger phase 2/3 study.

2-Methylthioadenosine[beta-32P]diphosphate. An agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets.

2-Methylthio-ADP and its radioactive analogue [beta-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin E1. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [beta-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was approximately 3.5 X 10(6) M-1 S-1, and the first order dissociation rate was 0.024 s-1, both measured at 23 degrees C. The dissociation equilibrium constant (approximately 15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin E1-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Ki = 3.5 microM), ATP (7 microM), 2-azido-ADP (0.12 microM), inosine diphosphate (IDP, 150 microM), guanosine diphosphate (GDP, 350 microM), and AMP (800 microM). Binding of 2-methylthio-ADP was also blocked by the non-cell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 32 degrees -40 degrees C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was approximately 15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket.

Identification of phosphoprotein NP33 as a nucleus-associated ribosomal S6 protein and its phosphorylation in hematopoietic cells.

Exposure of HL-60 promyelocytes to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased incorporation of 32P into a Mr approximately 33,000 protein (NP33) found in the nuclear matrices prepared by treating cells with Triton X-100, nucleases, and 2 M NaCl (D. E. Macfarlane, J. Biol. Chem., 261: 6947-6953, 1986). We now report that 12-O-tetradecanoylphorbol-13-acetate causes phosphorylation of NP33 in U937, K562, HEL, Molt-3, and Raji cell lines, all of which are rapidly proliferating cells of hematopoietic origin. 12-O-Tetradecanoylphorbol-13-acetate caused a lesser degree of NP33 phosphorylation in peripheral blood lymphocytes and monocytes and none in granulocytes or platelets. The incorporation of 32P into NP33 was complete in about 10 min, and it was prevented or reversed by staurosporin, indicating that NP33 is continuously phosphorylated and dephosphorylated. NP33 was purified to homogeneity from Triton X-100-washed nuclei or whole cells by extraction with H2SO4, acetone precipitation, and preparative two-dimensional gel electrophoresis. The amino-terminal amino acid sequence of NP33 appears to be the same as that of ribosomal S6 protein. NP33 appears to be S6 protein copurifying with the nuclear matrix.

Absence of phorbol ester-induced down-regulation of myc protein in the phorbol ester-tolerant mutant of HL-60 promyelocytes.

The human promyelocytic leukemia cell line HL-60 has an amplified number of copies of the protooncogene c-myc. It is induced to differentiate by exposure to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have developed a mutant phorbol ester-tolerant (PET) line of HL-60 which undergoes a transient growth arrest but does not differentiate when exposed to TPA (Macfarlane et al., Br. J. Haematol., 68: 291-302, 1988). The defect is not due to a general failure of TPA-induced phosphorylation. In this paper, we show that exposing phorbol ester-sensitive (S) HL-60 cells to TPA caused the disappearance of the c-myc protein antigen (detected on Western blots) in 4 h, whereas TPA had no effect on the c-myc protein content of PET cells. Dimethyl sulfoxide caused the rapid disappearance of the myc antigen in both cells. PET cells had slightly more copies of the c-myc gene detected on Southern blots than S cells. c-myc mRNA was equally unstable in both cells, as determined by Northern blots following actinomycin D. TPA induced the down-regulation of c-myc mRNA in S cells to a greater extent than in PET cells. Dimethyl sulfoxide caused a rapid down-regulation of c-myc mRNA in both cell lines. This shows that PET cells have a defect in the mechanism by which protein kinase C regulates c-myc transcription. Our results provide further evidence that reduction in c-myc expression is necessary for differentiation to occur in HL-60 cells.

Phorbol ester induces apoptosis in HL-60 promyelocytic leukemia cells but not in HL-60 PET mutant.

One of the factors regulating the population size of a clone of proliferating cells is the induction of a physiological suicide mechanism known as apoptosis. We studied apoptosis in the HL-60 human promyelocytic leukemia cell line which differentiates when exposed to phorbol ester (S-cell), and in the PET-cell mutant of HL-60 which is defective in its response to phorbol ester. Exposing S-cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) (3 nM and above) induced morphological changes characteristic of apoptosis (visualized by light microscopy), and induced fragmentation of chromatin DNA to oligonucleosomal lengths. These changes were obvious in 48 h. In contrast, 1000 nM TPA for five days did not induce apoptosis in the PET-cell. DNA fragmentation was induced in both cell lines by A23187 (0.25 microM) and etoposide (7 microM). Novobiocin (600 and 900 microM) induced DNA fragmentation in S-cells, but higher concentrations inhibited fragmentation. Novobiocin is believed to induce DNA fragmentation by a direct action on DNA. In the case of PET-cells, novobiocin did not induce DNA fragmentation at any concentration, and prior treatment of PET-cells with novobiocin (300-1200 microM for 30 min) inhibited DNA fragmentation induced by A23187. Novobiocin inhibited cell growth equally in S-cell and PET-cells. It is concluded that the promyelocytes have the capacity to undergo apoptosis in response to agents which activate protein kinase C, and that the PET-cell has a mutation which disables both protein-kinase C-induced and novobiocin-induced DNA fragmentation, leaving intact the ability of novobiocin to protect DNA from calcium-entry-initiated fragmentation. The elucidation of the lesion responsible for the PET phenotype is likely to increase our understanding of this important pathway for regulating cellular proliferation and how it bears on leukemogenesis and chemotherapy.

CpG-oligodeoxynucleotide-resistant variant of WEHI 231 cells.

Bacterial DNA and synthetic single-stranded oligonucleotides having unmethylated CpG motifs (CpG-ODN) powerfully stimulate cellular immune responses by an unknown mechanism. There is evidence that internalization of the nucleotide is required for activity. Both CpG-ODN and engagement of CD40 protects WEHI-231 murine B lymphoma cells from apoptosis induced by antibody to surface IgM, and both agents induce interleukin-6 (IL-6) production by these cells. We now report the isolation of CpG-ODN-resistant subclones (designated CR) from WEHI 231 cells, as well as subclones that are sensitive to CpG-ODN (designated CS). CR clones completely fail to respond to CpG-ODN but they retain the capacity to respond normally to engagement of CD40. CR cells incorporate CpG-ODN into small, acidified perinuclear vesicles in the same way as do the parent WEHI 231 cells. The CR, CS, and WEHI 231 cells all had identical cytogenetics. The described CR clones have a stable and specific defect in the mechanism responsible for the intracellular recognition and response to CpG-ODN, suggesting that they harbor a mutation that disables the CpG-ODN detection mechanism. These clones may be useful to determine at a molecular level which proteins and cell components are required for immune cells to detect and respond to CpG-ODN.

Isolation of RNA from cells in culture using Catrimox-14 cationic surfactant.

Traditional RNA isolation methods use chaotropic agents and anionic detergents to lyse cells and solubilize nucleic acids. In contrast, the cationic surfactant, Catrimox-14, lyses cells and simultaneously precipitates RNA, thereby protecting it from RNases. We describe and compare four methods for extracting RNA from cultured cells that differ in the technique used to extract the RNA from the precipitate. The first uses a high-salt solution (guanidinium isothiocyanate). In the second, the RNA is extracted with a polar solvent (formamide). The third involves conversion of the RNA to the sodium salt by treatment of the precipitate in situ with sodium acetate in ethanol. The fourth uses 2 M lithium chloride to convert the RNA in the pellet to the lithium salt in situ. We applied these methods to human leukemia cells growing in culture and each method resulted in excellent yields of RNA (typically 23 micrograms/million K562 cells, 13 micrograms/million HL-60 cells) over a wide range of cell concentrations (1 x 10(5) - 3 x 10(7)/ml) and of good to excellent quality as judged by agarose electrophoresis and UV absorbance data (OD260/280 1.90-2.05). The advantages and limitations of each method are discussed.

Depletion of platelet amine storage granules by the antithrombotic agent, suloctidil.

Suloctidil, a compound with antithrombotic properties in animal models, causes depletion of human platelet serotonin stores during 1-5 hr incubation with platelet-rich plasma in vitro. This effect is not attended by leakage of cytoplasmic nucleotides or by alterations in cyclic AMP metabolism, malondialdehyde production or energy balance. 5HT uptake was also inhibited but uptake of adenine was not. 5HT released by suloctidil appeared in the supernatant as oxidation products, though the amine also accumulated when re-uptake was blocked by imipramine.

Malondialdehyde production by platelets during secondary aggregation.

A method is described for increasing the sensitivity of the thiobarbiturate assay for malondialdehyde by concentrating the coloured reaction product. The basal level of malondialdehyde-like material in plasma was found to be about 0.03 micrometer. Platelets synthesized malondialdehyde when stimulated by collagen or thrombin and also during the second phase of aggregation induced by ADP or adrenaline.

Protein kinase C is not necessary for transforming growth factor beta-induced growth-arrest in leukemia cell lines.

Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC beta, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D3 induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC beta, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC beta, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D3 is caused by upregulation of PKC beta isozyme gene expression. The influence on growth of combinations of five cytokines (TNF alpha, TGF beta 1, gamma-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D3 on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPM1 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D3 followed by TGF beta 1. The expression by each cell line of mRNA encoding PKC alpha, beta, and delta, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D3, was determined. Cell lines sensitive to TGF beta 1 each expressed PKC delta endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D3. U937 cells underexpressed PKC alpha, and HL-60 PET cells underexpressed PKC beta. These data suggested that PKC delta could be responsible for mediating growth-arrest by TGF beta 1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D3 and TGF beta 1. Surprisingly, the PKC inhibitors did not block the growth-arrest induced by 1,25-dihydroxyvitamin D3 and TGF beta 1. This experiment strongly suggests that neither growth-arrest induced by TGF beta 1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D3 is mediated by a PKC isozyme which is inhibitable by the bisindolymaleimides.

Evaluation of automated large-scale screening tests for syphilis.

Two methods of performing serological screening tests for syphilis are compared. One consisted of the Venereal Diseases Reference Laboratory (VDRL) slide test, the cardiolipin Wassermann reaction (CWR), and the Reiter protein complement fixation test (RPCFT) performed manually; the other was a fully automated system using two Technicon AutoAnalyzers (AAII), one for the automated reagin test (ART) and the other for automated complement fixation tests. The absorbed fluorescent treponemal antibody test (FTA-ABS) was used as a final arbiter in all cases found to be seropositive by either method. A pooled antigen consisting of a mixture of cardiolipin and Reiter protein was used for the automated complement fixation test, thus increasing the scope and capacity of the system. The AutoAnalyzer was shown to be capable of performing 400 cardiolipin and Reiter complement fixation tests and 700 automated reagin tests in an 8-hour day. Modification of the complement fixation test method to take advantage of the highly sensitive colorimeter resulted in a significant increase in sensitivity and a corresponding saving in reagents. Of the 7843 sera tested, 258 gave a positive result in one or more of the screening tests. The automated test detected many more Reiter positive sera (127) than the manual test (83). Conversely, fewer CWR positive sera were detected by the automated test (60) than by the manual test (82). There was little difference between the number of positive sera detected by the ART (73) and the VDRL slide test (71). In 19 instances the automated tests detected positive sera which registered as completely negative in the manual tests, and four seropositive cases which the automated tests had failed to detect were detected by the manual tests, and four seropositive cases which the automated tests had failed to detect were detected by the manual tests. It was concluded that a combination of the ART and automated Reiter protein complement fixation test (ARPCFT) would be ideal for use in a large-scale screening programme for the detection of syphilis.

Improved media for the culture of Neisseria gonorrhoeae.

The ANM medium of Hafiz and McEntegart (1976) was found to be deficient in ability to support the growth of various strains of N. gonorrhoeae. Strains that did grow required a large starting inoculum which invariably suffered a substantial drop in the number of viable organisms during the first 2 h. Investigation of the various deficiencies led to the development of liquid (G77L) and solid (G77S)media which were shown to have several advantages over existing media for the cultivation of N. gonorrhoeae. These media, which are simple to prepare, do not contain blood or serum and yet gave excellent growth. The inclusion of antibiotics, glucose and phenol red allowed the selective isolation and partial identification of N. gonorrhoeae from routine specimens. These media also gave good results in fermentation tests for the identification of neisseriae.

Prostacyclin biosynthesis in vascular endothelium is not inhibited by cyclic AMP. Studies with 3-isobutyl-1-methylxanthine and forskolin.

We have previously reported (Proc. Natl. Acad. Sci. 79, 495-499, 1982) that the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), stimulates cyclic AMP accumulation and inhibits prostacyclin (PGI2) production in primary monolayer cultures of human umbilical vein endothelium. The present study was carried out to determine whether these effects are causally related. Incubation of endothelial monolayers with the diterpene, forskolin, increased the intracellular concentration of cyclic AMP by 10-fold. Despite this marked increase in cyclic AMP, neither baseline production of PGI2 nor release in response to stimulation by thrombin or the divalent cation ionophore, A23187, was affected. Both forskolin and isoproterenol were found to potentiate the effect of IBMX on cyclic AMP accumulation without causing further inhibition of PGI2 biosynthesis. Inhibition of cyclic nucleotide phosphodiesterase activity with 2,6-bis-(diethanolamino)-4-piperidinopyrimido-[5,4-d]pyrimidine increased cyclic AMP levels to the same extent as IBMX; however, this agent had no effect on PGI2 biosynthesis. These findings demonstrate that increases in the intracellular concentration of cyclic AMP have no short-term effects on PGI2 biosynthesis in vascular endothelium and suggest that inhibition of PGI2 production by IBMX is the result of some other, cyclic AMP-independent action of the drug.

Bacteriology of sickle cell leg ulcers.

The bacteria isolated on aerobic and anaerobic culture were compared in 80 unilateral ulcers in patients with homozygous sickle cell (SS) disease, 62 superficial skin lesions, and in 30 diabetic ulcers. In SS disease, the bacterial flora was predominantly aerobic and polymicrobial with Staphylococcus aureus, Pseudomonas aeruginosa and beta-haemolytic streptococci being the major isolates. Repeat sampling of 26 ulcers over a period of 23 weeks indicated the persistence of these three organisms, either singly or in combination in 21 ulcers. Although a variety of Enterobacteriaceae were recovered no single genus predominated and these organisms did not normally persist on follow-up. Simultaneous swabs from bilateral ulcers revealed similar if not identical flora in most cases, indicating good predictive value of a single swab in patients with multiple ulcers. Corynebacterium diphtheriae was recovered from eight ulcers and four of these strains were toxigenic. By contrast, the superficial skin lesions grew mainly S. aureus and beta 6-haemolytic streptococci, and the diabetic ulcers yielded a mixed growth of streptococci, Enterobacteriaceae and anaerobes. The recovery of known skin pathogens from most sickle cell leg ulcers, the persistence of these organisms, and the presence of associated lymphadenopathy, indicates that infection may be a significant factor in the pathology of these lesions.

Topical antibiotics in chronic sickle cell leg ulcers.

A randomized, controlled, trial of a topical antibiotic preparation (neomycin, bacitracin, and polymyxin B) was performed in 30 patients with homozygous sickle cell (SS) disease and chronic leg ulceration. Over a period of 8 weeks, the reduction in ulcer size was significantly (P less than 0.05) greater in the treatment group than in the control group. The results suggest that these topical antibiotics may contribute to the therapy of chronic leg ulceration associated with sickle cell disease.

CpG-oligodeoxynucleotide supports growth of IL-6-dependent 7TD1 murine hybridoma cells.

The mouse-mouse hybridoma plasma cell line 7TD1 requires exogenous IL-6 for growth, and has been used to characterize and assay IL-6. Oligodeoxynucleotides containing CpG (CpG-ODN) and bacterial DNA are immunostimulatory, preventing B-cell apoptosis and inducing cytokine synthesis. We report that the phosphorothioate CpG-ODN 5'-ATAATCGACGTTCAAGCAAG-3' (half maximal effect at 0.3 microg/ml) supports the growth of 7TD1 cells in the absence of added IL-6. A non-CpG control ODN was without effect. No IL-6 production by 7TD1 cells incubated with CpG-ODN was detected by sensitive immunoassay. CpG-ODN-supported growth was not influenced by IL-6-neutralizing antibody, but was completely abolished by chloroquine and quinacrine, agents which inhibit CpG-ODN responses in other cells. Our results show that CpG-ODN can substitute for IL-6 in this cell line. This cell line may be useful for assaying CpG-ODN activity and for screening congeners of chloroquine and quinacrine for their ability to inhibit CpG effects.

Bacteraemia at the University Hospital of the West Indies--a report of 222 cases.

A total of 222 cases of septicaemia was recorded at the University Hospital of the West Indies between June 1982 and June 1983. This gave an overall incidence of 16.1 per 1000 admissions. The 233 bacterial strains isolated comprised 100 Gram-positive and 133 Gram-negative organisms with Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus being the most frequent. Highest rates of septicaemia were recorded in patients less than 1 year and over 50 years of age. Septicaemia caused by Gram-positive organisms was predominantly a disease of children whereas that caused by Gram-negative organisms arose more often in neonates and in patients over 50 years of age. A predisposing factor was noted in 104 patients of whom 42 had neoplastic disease. The most frequently identified initial sites of infection were the respiratory tract, the gastro-intestinal tract and the meninges. Most blood stream infections were community-acquired, three quarters of all septicaemic patients being admitted to the departments of medicine or paediatrics. There were 11 cases of polymicrobial septicaemia caused predominantly by Gram-negative organisms in patients with underlying disease. Appropriate antimicrobial drugs were administered to 57% of septicaemic patients whereas 17% received superfluous antimicrobial therapy. In those patients who received inappropriate antimicrobial therapy there was a marked increase in mortality. Forty of 61 deaths were attributed to septicaemia. Mortality from septicaemia caused by Gram-negative organisms was 21% compared with 13% for that caused by Gram-positive organisms. The organisms associated with the highest case fatality rates were Escherichia coli, 53%; Enterobacter sp., 27%; and beta-haemolytic streptococci 24%. There were no deaths from septicaemia caused by Haemophilus influenzae, Salmonella sp. or Serratia sp. The highest mortality rates were associated with neoplastic disease, diabetes, polymicrobial septicaemia, urinary tract infections and old age.

Flavobacterium odoratum ventriculitis treated with intraventricular cefotaxime.

A 6-week-old infant admitted to the University Hospital of the West Indies with hydrocephalus later developed ventriculitis. A heavy growth of Flavobacterium odoratum susceptible to gentamicin and cefotaxime was recovered from the ventricular fluid. Since intraventricular therapy was envisaged, a Pudenz reservoir was installed and ventricular fluid aspirated every 24 h to monitor treatment. Initial therapy consisted of intravenous cefotaxime, 50 mg/kg q.i.d. for 4 days. No significant reduction in the number of organisms in the ventricular fluid was achieved with this regimen. Intravenous therapy was therefore discontinued. On day 5 intraventricular therapy began with 5 mg cefotaxime 24 h for 6 days, followed by 1 mg/24 h for 4 days. Daily monitoring of intraventricular fluid indicated a high degree of antibacterial activity with rapid elimination of bacteria. Ventricular fluid remained sterile 10 days after therapy stopped. The Pudenz reservoir was removed, a ventriculoperitoneal shunt installed, and the patient discharged from hospital 4 days later without noticeable sequelae.