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or the BEAM regimen (4) or high-dose chemotherapy alone (17). A median number of 7-3 (x 10$/kg) nucleated cells (3-6-26-1) corresponding to 15.4 (x l()4/kg) CFU-GM (1 -5-217) was infused. No patient died during marrow aplasia. The median times to granulocyte (0-5 x 109/1) and platelet (50 x 109/1) recovery were 11 days (9-24) and 10 (8-59),

cyclophosphamide (4)

respectively. These results reported by Sheridan et al.

are not

very different from those

Thus, at least for patients with acute lymphoblastic leukaemia or non-Hodgkin lymphoma, the use of peripheral blood stem cells alone seems sufficient to ensure quick and stable engraftment. Haematopoietic growth factors are expensive and the combined use of G-CSF, PBSC, and bone-marrow cells to rescue haematopoiesis after massive chemotherapy may not be necessary in every situation. Bone Marrow Transplant Unit, CHR Bordeaux,

Hôpital Haut-Levêque, 33604 Pessac, France

J. REIFFERS C. FABERES G. MARIT

SIR,-We too would like to report our experience with (and make the efficacy of) G-CSF as a "recruiting agent" for blood progenitor cells (PBPC). We are investigating the peripheral some coments on

feasibility of G-CSF-mobilised PBPC after intensive chemotherapy. Ten patients with refractory lymphoma have been enrolled in a salvage protocol (mitoxantrone, methylprednisolone, carboplatin, and cytarabine followed by G-CSF 5 Ilg/kg subcutaneously for 10 days starting 24 h after cessation of cytarabine). All patients are submitted to leukapheresis to collect PBPC. A median number of eight leukaphereses was necessary to achieve a median number of 1-22 monocytes x 10/kg. Five patients have been transplanted so far; all had bone marrow harvested and cryopreserved. Transplanted patients were conditioned with busulphan and cyclophosphamide and received PBPC only. The total number of cells transfused was 11 -8-17-9 monocytes x 10/kg and CFU-GM numbers ranged between 79 and 257 x’104/kg. Our data confirm the reduction in recovery time for platelets and white cells. The mean times to achieve white cell counts of greater than 1000/pl, polymorphs above 500/il, and platelets above 50000/lll were 13, 12-5, and 12.5 days, respectively. Bone-marrow biopsy was done on days 7 and 14 after PBPC transfusion and demonstrated in all patients megakaryocytes from day 7. This effect is well known and is seen with PBPO,2 but it seems to be amplified, especially for platelets, if G-CSF is used. We would like to emphasise that in our experience the use of the PBPC collected after chemotherapy and G-CSF was sufficient to obtain the same results as Sheridan et al did, making the use of G-CSF after transplantation almost pointless. In our patients too the thrombocytopenia was short-lasting. A median of 2 units of single donor platelets (range 0-3) was necessary to maintain plateletcounts above 20 OOO/il. However, in our patients there was an unexpected and considerable reduction in platelet count 30 days after PBPC reinfusion. This late thrombocytopenia lasted only few days and transfusional support was not needed. This pattern may be hidden by the protocol of Sheridan et al. It probably reflects the expansion, in peripheral blood, of megakaryocytic progenitors cells at a different stage of commitment. How and why G-CSF induces a megakaryocytic commitment is not clear. This cytokine generates a large number of PBPC, most of them committed; however, sufficient are true stem cells to permit the use of PBPC alone after myeloablative chemotherapy.

Supported in part by grant from AIRC.

Istituto di Semeiotica Medica, Catholic University,

00168 Rome, Italy

SIMONA SICA PRASSEDE SALUTARI LUCIANA TEOFILI GIACOMO MENICHELLA GIUSEPPE LEONE

1. Gianni AM, Bregni M, et al: Rapid and complete hemopoietic reconstitution following combined transplantation of autologous blood and bone marrow cells.A changing role for high dose chemotherapy? Hematol Oncol 1989; 7: 139-48. 2.Corbling M, Martin H. Autologous transplant of hemaphereses derived hemopoietic stem cells: a new concept in the treatment of patients with malignant lymphroemopoietic disorders. Plasma Ther Transfu Technol1988;9: 119-32.

Selective decontamination of the digestive tract and methicillin-resistant

Staphylococcus aureus SIR,-Patients needing intensive care are at high risk of nosocomial infections, especially pneumonia. A prophylactic strategy called selective decontamination of the digestive tract (SDD) has been commonly used in many intensive care units (ICUs) in Europe. The aim of SDD is the selective abolition of carriage of aerobic disease-causing microorganisms (ie, Staphylococcus aureus, gram-negative bacilli, and yeasts). During the past eight years this technique has been evaluated in several centres.1 These studies showed a significant reduction of carriage by SDD and a protective effect for the risk of respiratory tract infection, although the survival benefit was less clear .1,2 Results of a French study3 are in line with a meta-analysis.2 Although SDD has not led to a clinically relevant emergence of resistance, this issue is still of concern and controversial. In April, 1989, we started SDD in seriously ill and mechanically ventilated patients admitted to a multidisciplinary ICU at University Hospital, Aachen. All patients who were expected to need endotracheal intubation for more than four days received SDD. The SDD regimen, a suspension of 50 mg polymyxin B and 80 mg gentamicin, was given daily via the oropharynx and nose, and simultaneously via a nasogastric tube at 6 hour intervals; an initial course of a systemic antibiotic for prophylaxis was not routinely included.4 SDD led to a pronounced decrease of colonising gram-negative bacilli, and to a slight reduction in the duration of postoperative ventilation. However, neither the pneumonia rate nor mortality was substantially reduced. Before the introduction of SDD, methicillin-resistant S aureus (MRSA) strains were very uncommon in our ICU. In May, 1990, an MRSA was isolated from the tracheal secretion and subsequently from the blood of a patient. The MRSA was resistant to all clinically relevant antibiotics, apart from co-trimoxazole and vancomycin. During the next 16 months an outbreak due to the same organism developed. Identification of the epidemic strain was based on the same antibiotic resistance pattern as well as on phage typing. Nearly all patients who remained longer than two weeks in ICU became colonised shortly after admission, and subsequently had at least one episode of microbiologically proven MRSA septicaemia. All patients were successfully treated by prompt administration of a glycopeptide. Surveillance cultures of ICU staff yielded the epidemic MRSA strain from the nose in two. They were excluded from work until they were no longer MRSA-positive. Systemic co-trimoxazole or rifampicin failed to eradicate MRSA in several patients. Moreover, traditional infection control measures including the isolation of colonised and infected patients, the enforcement of hand washing, and, finally, the closure of the unit in January, 1991, failed to eradicate MRSA. Control of the spread of MRSA was hampered in part by the fact that some patients were already carriers of MRSA on admission to the unit. In September, 1991, all seven long-stay ICU patients were MRSA-positive, and systemic MRSA infection developed in four of them. SDD was discontinued as a final measure. MRSA infection immediately began to decline. In the next few weeks the organism was isolated only sporadically from patients’ specimens, and infections due to the organism stopped. A possible explanation for this observation may be that SDD is not active against MRSA exhibiting gentamicin resistance; the result is selective pressure in the ICU associated with carriage and subsequent endogenous infections by MRSA following the control of gram-negative bacilli by SDD. SDD was not thought to be the primary cause of the MRSA outbreak (with often life-threatening infections) but rather had contributed to its spread. Although there is no definite proof that the abolition of SDD was responsible for the virtual disappearance of MRSA from the ICU, our observation could support this hypothesis. This report emphasises the high failure rate seen with traditional measures in the control of outbreaks with MRSA.5,6 Some investigators believe in more aggressive approaches, including the use of topical mupirocin in combination with rifampicin and minocycline.7 Darouiche and co-workers’ study’ confirms our observation that medical personnel

1412

had no obvious role in the transmission of MRSA. Therefore, as with infections due to gram-negative bacilli and yeasts, an endogenous source of the organism (ie, nasopharyngeal and gastrointestinal carriage preceding infection) may be pivotal in the development of hospital-acquired MRSA infections. General hygienic measures, including nasal application of mupirocin and isolation, mainly intended to prevent exogenous infections, do not control primary and secondary endogenous infection. Finally, we would like to emphasise that SDD should not be applied without careful microbiological monitoring (ie, surveillance cultures twice a week) and that its indiscriminate use may cause difficulties in certain

settings. We thank Dr

Lenz, University of Bonn, for phage typing.

Institute of Medical Microbiology, and Clinic of Anaesthesiology, Technical University (RWTH), 5100 Aachen, Germany, and Department of Medical Microbiology, University of

Liverpool,

UK

ACHIM KAUFHOLD WALTER BEHRENDT THOMAS KRAUSS HENDRICK VAN SAENE

Fig 2-lmmunofluorescent staining of schizont in red cell.

(Left) FITC fluorescence of schizont following Incubation with a-TRAP polyclonal antibody Parasite pigment can be seen in centre of schizont. Red cells are negative ( x 1400) (Right) Control bright field corresponding to (a), showing haemozoin pigment of schizont ( x 1400). .

man has proved problematical. We are investigating thrombospondin-related anonymous protein (TRAP), a polypeptide encoded by P falciparum that shares a short aminoacid consensus sequence with a diverse group of proteins.l These include two malarial sporozoite proteins, thrombospondinz (a mammalian adhesive glycoprotein) and properdin3 (a component of the alternative complement pathway). The function of this motif in

in

1. Van Saene HKF, Stoutenbeek CP, Hart CA. Selective decontamination of the digestive tract (SDD) in intensive care patients a critical evaluation of the clinical, bacteriological and epidemiological benefits. J Hosp Infect 1991; 18: 261-77. 2. Vandenbroucke-Grauls CMJE, Vandenbroucke JP. Effect of selective decontamination of the digestive tract on respiratory tract infections and mortality in the intensive care unit. Lancet 1991; 338: 859-62 3. Gastinne H, Wolff M, Delatour F, Faurisson F, Chevret S A controlled trial in intensive care units of selective decontamination of the digestive tract with nonabsorbable antibiotics. N Engl J Med 1992; 326: 594-99. 4. Unertl K, Ruckdeschel G, Selbmann HK, et al. Prevention of colonization and respiratory infections m long-term ventilated patients by local antimicrobial prophylaxis. Intensive Care Med 1987; 13: 106-13 5. Preheim LC, Rimland D, Bittner MJ. Methicillin-resistant Staphylococcus aureus in Veterans Administration Medical Centers. Infect Control 1987; 8: 191-94. 6. Vandenbroucke-Grauls CM, Frénay HME, van Klingeren B, Savelkoul TF, Verhoef J. Controls of epidemic methicillm-resistant Staphylococcus aureus in a Dutch university hospital. Eur J Clin Microbiol Infect Dis 1991, 10: 6-11. 7. Darouiche R, Wnght C, Hamill R, Koza M, Lewis D, Markowski J. Eradication of colonization by methicillin-resistant Staphylococcus aureus by using oral minocycline-rifampin and topical mupirocin. Antimicrob Agents Chemother 1991; 35: 1612-15

Expression of thrombospondin-related anonymous protein in Plasmodium falciparum sporozoites SIR,-Plasrnodium falciparum malaria is responsible for the deaths of 1-2 million individuals annually and the widespread multidrug resistance of the parasite is narrowing available options for treatment. During infection of the human host the parasite completes a complex lifecycle which includes several morphological changes. Although analysis of the induction of protective immunity by irradiated sporozoites has highlighted the importance of T-cell responses to the liver-stage parasite, identification of parasite antigens that are recognised by cytotoxic T lymphocytes (CTLs)

vivo is unknown, but its conservation in such different molecules suggests that it is significant. We have raised polyclonal and monoclonal antibodies against recombinant TRAP and used them to localise TRAP at the resolution of light microscopy by indirect immunofluorescence. Erythrocytes infected with laboratory cultured P falciparum were air-dried onto slides and fixed in 90% acetone/10% methanol. Sporozoites were obtained by dissection of the salivary glands of infected mosquitoes and fixed on slides in the same way. Antibodies were applied to these samples and incubated at room temperature for 30 min before washing and the addition of secondary antibodies labelled with a fluorescent marker. Controls (circumsporozoite protein [CSP] monoclonal antibody, stage-specific antibodies, non-immune mouse serum, no primary antibody, no secondary antibody) were included in each experiment. We have demonstrated the expression of TRAP both in sporozoites (fig 1) and in the erythrocytic stage (fig 2) of P falciparum. The presence of TRAP in the sporozoite may help to explain the remarkable polymorphism of TRAP (52 of 53 identified nucleotide changes are non-synonymous4), a pattern similar to the polymorphic regions of CSP that appear to have been subject to significant selection pressure, possibly by cytotoxic lymphocytes.5 Selection pressure of this type would be more pronounced at the liver stage of infection where a single sporozoite enters the liver cell, so that mutants that escape CTL recognition have a considerable advantage. In Pyoelii infection of mice a sporozoite antigen termed SSP2 elicits protective CTL responses.6 SSP2 and TRAP share sequence similarity that extends behond the consensus motif,7 and we have failed to find any other P falciparum genes (excluding that for CSP) that share this motif (unpublished). We propose that TRAP is the P falciparum homologue of SSP2. Although CTL to CSP have been detected in human immunisation studies,8 precursor frequencies were low, possibly because the small dose of parasites that infect the liver induces only a weak CTL response. An antigen such as TRAP, which is expressed during both the liver and blood stages of infection, might induce a more potent and protective CTL response. Our results suggest that studies of CTL response to TRAP in man may identify an immune response to P falciparum of protective value. MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK

Fig 1-lmmunofluorescent staining of a P falciparum sporozoite. FITC fluorescence showing patchy staining of sporozoite following incubation with a-TRAP polyclonal antibody ( 3400)

GILL COWAN SANJEEV KRISHNA

Institute of Parasitology, University of Rome,

ANDREA CRISANTI

Rome, Italy MRC Molecular

Haematology Unit

KATHRYN ROBSON

Selective decontamination of the digestive tract and methicillin-resistant Staphylococcus aureus.

1411 or the BEAM regimen (4) or high-dose chemotherapy alone (17). A median number of 7-3 (x 10$/kg) nucleated cells (3-6-26-1) corresponding to 15.4...
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