Staphylococcus aureus meningitis. A review of 104 nationwide, consecutive cases.

METHODS: Based on a nationwide registration, the clinical and bacteriologic data from 61 postoperative and 43 hematogenous cases of Staphylococcus aureus meningitis in Denmark from 1986 through 1989 were reviewed. RESULTS: Postoperative meningitis was a foreign body infection in 89% of the cases and had a lower mortality (18% [11/61]) compared with hematogenous meningitis (56% [24/43]). Hematogenous S aureus meningitis seems to be part of an overwhelming, disseminated infection as indicated by the following: 81% of the patients had bacteremia, 21% had endocarditis, and 12% had osteomyelitis. Most patients were older, often with underlying diseases, community-acquired infections, and a clinical picture of severe meningitis. The major findings were mental status changes and a high rate (34%) of focal neurological changes. The initial leukocyte count in the cerebrospinal fluid sample was low, and the bacteria were seen in Gram's stain smears in 40% of cases only. The prognosis was related to the age of the patients and the initial antibiotic treatment. Patients treated with penicillinase-stable penicillins in combination with fusidic acid may have a better prognosis. Three (12%) of 25 surviving patients had severe sequelae. CONCLUSIONS: Hematogenous S aureus meningitis is a severe disease with a high mortality related to age, presence of shock, and infection with strains of phage type 95.

Clinical features of Staphylococcus aureus endocarditis: a 10-year experience in Denmark.

BACKGROUND: Both morbidity and mortality resulting from Staphylococcus aureus endocarditis are known to be high, and the incidence of this disease seems to increase. The Statens Serum Institut, Copenhagen, Denmark, made it possible for us to analyze the clinical features of S aureus endocarditis in a nation-wide population of non-drug addicts. METHODS: Almost all Danish cases of bacteremia due to S aureus are reported to the Staphylococcus laboratory, Statens Serum Institut. The medical records were reviewed in cases reported from 1982 to 1991 in which the diagnosis of endocarditis was reported or suspected. RESULTS: A total of 260 patients, 145 males and 115 females, fulfilled the diagnostic criteria. The median age was 67.5 years. In 83 patients, the diagnosis of endocarditis was not suspected clinically. The overall mortality rate among those patients whose disease was diagnosed clinically was 46%. Among the subset of patients who received medical therapy only and appropriate antistaphylococcal treatment, mortality was significantly associated with late congestive heart failure, age, and involvement of the central nervous system. CONCLUSIONS: A raised awareness of the paucity of clinical findings and a more frequent use of echocardiography as a screening method seem essential to improve the prognosis of patients with S aureus endocarditis. Involvement of the central nervous system constitutes a relative indication of early valve replacement.

Neurologic manifestations in Staphylococcus aureus endocarditis: a review of 260 bacteremic cases in nondrug addicts.

PURPOSE: To investigate the neurologic manifestations of infective endocarditis caused by Staphylococcus aureus in a population of nondrug addicts with special emphasis on the clinical presentation, epidemiology, and mortality. PATIENTS AND METHODS: During the period from 1982 to 1991 a total of 8,514 cases of bacteremia with S aureus were reported to the Staphylococcus Laboratory, Copenhagen, Denmark. The medical records of cases of suspected infective endocarditis were retrospectively reviewed and classified according to the new diagnostic criteria for endocarditis proposed by Durack. RESULTS: A total of 260 cases from 63 hospitals fulfilled the diagnostic criteria. Overall, 91 patients (35%) experienced neurologic manifestations. Sixty-one presented with neurologic symptoms, whereas 30 patients developed neurologic complications at various intervals (median: 10 days) after the debut of the disease. The most frequent neurologic manifestation was unilateral hemiparesis, which occurred in 41 patients (45%). Forty-two percent of the females had neurologic manifestations compared to only 30% of the males (P = 0.06). Cases with native mitral valve infection had a significantly higher frequency of neurologic manifestations compared with all other valvular involvement (44% versus 29%, P = 0.02) but the frequency of neurologic complications was only nonsignificantly higher in those patients with native mitral valve infection than in those patients with native aortic valve infection (44% versus 31%, P = 0.10). Only two of the patients with tricuspid valve infection and none of those with congenital heart disorder experienced neurologic manifestations. A neurologic manifestation occurred in 22 (35%) of the 63 episodes in which vegetations were detected on the echocardiograms, compared with 17 (26%) of the 65 episodes without vegetations (P = 0.38). The mortality was 74% in patients with major neurologic manifestations and 56% in patients without neurologic manifestations (P = 0.008). In patients with neurologic complications the mortality was significantly higher among those treated with antibiotics alone as compared with those treated surgically (65 of 81, 80% versus 2 of 10, 20%; P = 0.0003). CONCLUSIONS: In a population of nondrug addicts with infective endocarditis caused by S aureus the following main conclusions can be drawn: neurologic manifestations occur with a higher frequency in patients with native mitral valve infection. The presence of vegetations on echocardiograms is not a risk factor for developing neurologic complications but this conclusion is based on the results of transthoracic echocardiograms performed in only one half of the patients. The majority of the neurologic manifestations occur on presentation or shortly thereafter and the risk of recurrent embolism is low. Mortality is increased in patients with neurologic manifestations. A neurologic event per se may constitute an indication for surgical treatment.

A new Danish Listeria monocytogenes phage typing system.

A phage typing system was developed for Listeria monocytogenes. Phages were released from clinical and from food strains of L. monocytogenes by mitomycin C induction. The system consists of two subsystems, one of which is used to type L. monocytogenes serotype 1 strains, containing 12 phages, and a second which is used to type serotype 4 strains, containing 14 other phages. For the serotype 1 subsystem, the reproducibility was > or = 90%, the typability 92%, and the discriminatory power, as judged by the discriminatory index, 80%. The corresponding figures for the serotype 4 subsystem were: reproducibility > or = 94%, typability 87%, and discriminatory index 87%. The performance of the whole typing system is sufficient for it to be used for screening purposes.

Genome fingerprinting as a typing method used on polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Phenotypical changes occur in the surface of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients. It is difficult with the classical typing methods, such as serotyping, phage typing and pyocin typing, to decide if a patient has been colonized with a new strain or whether it is the same strain which has reappeared, for instance after chemotherapy in the lungs. This investigation was carried out to evaluate genome fingerprinting as a typing method and to see how it correlated with classical methods and with DNA probe typing. Forty Pseudomonas aeruginosa isolates, 34 polyagglutinable and six monoagglutinable, from 14 cystic fibrosis patients were analysed using genome fingerprinting. The bacterial chromosomes were digested with the restriction endonucleases Dra 1 and Xbal, and separated by field inversion gel electrophoresis. The results were compared with those of a previous work (Ojeniyi et al. 1990) concerning typing with a DNA probe, serotyping using both polyclonal and monoclonal sera, phage typing, pyocin typing and reverse phage typing. The results of genome fingerprinting and DNA probe typing showed the best correlation, followed by pyocin typing. The correlation between the results of genome typing and the other typing methods was low. The discriminatory effect of genome fingerprinting was higher than that of DNA probe typing, and genome fingerprinting was found to be the best single method for epidemiological investigations of polyagglutinable isolates from cystic fibrosis patients.

Prevalence and persistence of polyagglutinable Pseudomonas aeruginosa in isolates from cystic fibrosis patients.

During the 17 years from 1972 to 1988 1010 Pseudomonas aeruginosa isolates from sputum samples of 183 cystic fibrosis patients attending the Danish Cystic Fibrosis Centre at Rigshospitalet were serogrouped and phage typed. The patients were chronically infected with Pseudomonas aeruginosa (range of duration of infection: 1-17 years). They attended the Centre monthly and since 1976 all had been hospitalized for 14 days every three months. The number of patients with Pseudomonas aeruginosa infection increased from 29 to 123 during the period 1972 to 1988 and the number of isolates typed ranged from 14 to 197 a year. About half of the patients had one to five monoagglutinable isolates before they had polyagglutinable isolates. The prevalence of polyagglutinable isolates each year increased during the observation period (range: 24% to 88%). In all, three quarters of the patients had polyagglutinable isolates in their last typed sample. During the 17-year period 81% of the patients harboured polyagglutinable isolates. 77% of these patients, however, were for periods colonized with monoagglutinable isolates (0-3, 0-6, 0-1, 0-2 (including 0-2, 0-5, 0-2/5 due to cross-reactions between 0-2 and 0-5) and 0-9 were most prevalent) either alone or concurrent with polyagglutinable isolates, whereas 5% harboured polyagglutinable isolates exclusively during the whole observed period of infection. Polyagglutinable isolates with a short phage pattern were more frequent than those with a long phage pattern during the entire period from 1972 to 1988, and the prevalence increased during the period. Twenty-eight percent of the patients had a persistent phage pattern for an average of seven years in spite of change of serogroup. Twenty-seven percent of the patients had a persistent monoagglutinable serogroup and phage pattern for an average of eight years. The increasing prevalence and persistence of polyagglutinable strains correlate with the improved survival of the patients and thereby with the increased duration of the infection. The reason for this is discussed.

Contaminating coagulase-negative staphylococci isolated in a lysis-centrifugation (Isolator) blood culture system. Application of different epidemiological markers for deduction of mode of contamination.

The lysis-centrifugation blood culture system, Isolator, is a promising system with respect to detection of many significant microorganisms, e.g. Staphylococcus aureus and Enterobacteriaceae, as compared with conventional systems. A drawback of the Isolator system is a disturbingly high rate of clinically insignificant, supposedly contaminating coagulase-negative staphylococci, which leads to considerable waste of time and materials in the laboratory. Several sources of these isolates have been proposed (viz. the patient, the ward environment, the laboratory handling, and the plate media). The aim of this study was to pinpoint the origin of these clinically doubtful coagulase-negative staphylococci, using different epidemiological markers, such as species identification, antibiotic susceptibility patterns, phage-types, and plasmid profiles. Plasmid profile analysis proved to be more discriminating than the other techniques and made it possible to conclude that the laboratory handling of the Isolator system was a major source of coagulase-negative staphylococci in this system.

Erythromycin resistance in Danish Staphylococcus aureus hospital strains with emphasis on erythromycin consumption.

Erythromycin resistance was analyzed in 280,415 Danish Staphylococcus aureus strains isolated from hospitalized patients and phage-typed in the period 1967 to 1987. Multiresistant, erythromycin-resistant strains decreased during the whole period, while strains resistant only to penicillin and erythromycin increased after 1972. This increase coincided with an increase in erythromycin consumption in Denmark from 0.4 DDD/1000 persons/day in 1978 to 1.9, in 1987. Erythromycin consumption in nine large Danish hospitals was significantly associated with the level of erythromycin-resistant S. aureus at the hospitals.

erm genes in erythromycin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

Erythromycin-resistant S. aureus from general practice and a dermatology ward with a very high consumption of erythromycin was studied for ermA and ermC content by Southern blot analysis. The prevalence of these erm genes was also studied in coagulase-negative staphylococci from the same dermatology ward and in a collection of 15 S. aureus and 18 coagulase-negative staphylococci found in the same specimen from 15 different patients. ermA was only found as a chromosomal insert and ermC only on small plasmids. In erythromycin-resistant S. aureus from general practice ermC was responsible for 84% of erythromycin resistance, while 16% of the strains contained ermA. In 17 of 18 S. aureus strains from the dermatology ward a 2.5 kb plasmid contained ermC. Among 58 erythromycin-resistant coagulase-negative staphylococci only 2 (3%) had ermA, whereas 44 strains (76%) had ermC. ermA or ermC genes were common in S. epidermidis (36/38). However, in 20 isolates of erythromycin-resistant coagulase-negative staphylococci of other species, 10 had neither ermA nor ermC. ermC was the most common erm gene in both coagulase-negative staphylococci and S. aureus. In 11 of 15 patients with erythromycin-resistant S. aureus the co-isolated erythromycin-resistant coagulase-negative staphylococcus had another resistance mechanism, either another gene or a different phenotypic expression of the same gene. Resistance to pristinamycin, a streptogramin antibiotic only used in animals, was not found in S. aureus but was found in 14% of erythromycin-resistant coagulase-negative staphylococci.

Evaluation of methods for the detection of nasal carriage of Staphylococcus aureus.

In the present study we investigate the optimal methodology for determination of the nasal carriage rate of Staphylococcus aureus. Tests were performed on 91 healthy laboratory staff. The reproducibility of different sampling, transportation, storage and culture methods was examined. We compared sterile dry cotton wool swabs with sterile dry cotton wool swabs impregnated with charcoal and 5% blood agar plates with mannitol salt agar plates after different incubation periods. Finally, we investigated the detection rate for S. aureus following direct plating compared to storage in Stuart's transport medium for 7 days. There were no differences in isolation rates from the right or left nostril using either cotton or charcoal swabs. Charcoal swabs gave an increased isolation rate as compared to cotton swabs, and incubation in broth enrichment medium containing 6.5% NaCl also increased the isolation rate. Storage in Stuart's transport medium for 7 days gave an increase in isolation rate as compared to direct plating on blood agar. With mannitol salt agar plates the increase in isolation rate when incubation was performed for from 2 to 4, 2 to 7, and 4 to 7 days was 5.9%, 16.7%, and 11.5%, respectively. For the detection of S. aureus nasal carriers we find the use of charcoal swabs and Stuart's transport medium combined with cultivation on mannitol salt agar for 7 days to be the optimal method.

Identification of coagulase-negative staphylococci and typing of Staphylococcus epidermidis by a 4 h micromethod.

A new 4 h micromethod (Minibact-S) for identification of coagulase-negative staphylococci (CNS) important in human medicine, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and Staphylococcus saprophyticus, has been investigated. The reproducibility for species identification was 100% and the reproducibility for the single reactions was 95%. When compared to a modified Kloos & Schleifer conventional identification method, discrepant identification was obtained with the Minibact-S in only 8 of 382 clinical and reference strains. The system has been used for typing and biotyping of 201 S. epidermidis strains, and gave a subdivision in 69 different types; 4 related types accounted for 91 (45%) of the isolates, whereas 44 types were represented by only 1 isolate. The discriminatory index was 0.82. In a comparison with other typing methods performed on 81 isolates from 15 patients Minibact-S gave about the same number of types as antibiograms, but in 20% of the patients extra types were obtained with Minibact-S alone compared to antibiogram alone and vice versa. The Minibact-S is of some value in the routine clinical microbiology laboratory as a first screening method for identification of coagulase-negative staphylococci and typing of S. epidermidis in combination with the antibiogram.

A comparison of the efficiency in serotyping of Pseudomonas aeruginosa from cystic fibrosis patients using monoclonal and polyclonal antibodies.

A well-known problem in serotyping Pseudomonas aeruginosa strains from cystic fibrosis patients using polyclonal sera is that more than half of the strains cannot be assigned to a single O-serogroup because of the high occurrence of polyagglutinable strains and nontypable strains. In the present study, the efficiency of a set of O-specific monoclonal antibodies in the simple slide agglutination test was compared with polyclonal sera for the serotyping of 243 isolates of P. aeruginosa from cystic fibrosis patients. Using the monoclonal antibodies, 213 (88%) strains were found to be typable and only 30 (12%) strains were nontypable. In contrast, when the polyclonal sera from Statens Seruminstitut were used, only 53 (22%) strains were typable. Similar results were obtained when polyclonal sera manufactured by Difco were used where only 61 (25%) strains were typable. We also investigated the consistency of each set of antibodies in typing of the same isolates on different days and found that the polyclonal sera showed higher reproducibility. The Statens Seruminstitut sera were found to have an 86% reproducibility while the Difco sera scored 81%; however, the percentage of strains that are nontypable remains below 25% despite the highly reproducible results obtained. The monoclonal antibodies were found to score a 75% reproducibility when the serotyping of the same strains was done both in the laboratories of Copenhagen and of Guelph. However, it should be noted that although the reproducibility was somewhat lower with the monoclonal antibodies, these highly specific antibodies are still clearly superior when compared with polyclonal sera used because more than 80% of the strains from cystic fibrosis can now be assigned to a single O-serogroup.

Staphylococcus lugdunensis: an important cause of endocarditis. A case report.

Staphylococcus lugdunensis endocarditis was diagnosed in a 55-year-old woman maintained on chronic haemodialysis. S. lugdunensis was isolated from blood and a Gore-Tex graft fistula at admission, and vegetations with Gram-positive cocci were found on the mitral cusps at autopsy.

Biotyping, phage typing, and O-serotyping of clinical isolates of Enterobacter cloacae.

The purpose of this study was to make an independent evaluation of the methods of bio-, phage-, and O-serotyping which had been used only in the laboratory of origin, and to assess the extent of possible cross-infection of Enterobacter cloacae in a Danish university hospital. The material consisted of 237 clinical isolates of E. cloacae from the clinical microbiology laboratory at Hvidovre Hospital. The typability of bio-, phage-, and serotyping was 100%, 83%, and 85%, respectively. Reproducibility of serotyping was 90% and of phage typing 96% if two major differences were allowed to differentiate between patterns. O-serotyping had the highest discriminatory power and combination of all typing methods further increased discrimination. Outbreaks of E. cloacae were not evident in clinical departments, but cross-infections from one department to another could not be completely ruled out. We concluded that the combination of bio-, phage- and O-serotyping is sufficiently discriminating and will be satisfactory in the majority of clinical situations.

Coagulase-negative staphylococci in a newborn with a cardiac vascular prosthesis. Case report.

A case of septicemia with coagulase-negative staphylococci in a newborn with a vascular graft is discussed. Nineteen different isolates of coagulase-negative staphylococci were isolated from four different blood cultures. Eleven of the isolates belonged to one strain, which showed slight variation; the remaining seven isolates belonged to six different strains. The patient was treated for a prolonged period with antibiotics and seemed to respond. The diagnosis and proper treatment are discussed.

In vitro activity of dicloxacillin against methicillin-susceptible and methicillin-resistant Staphylococcus-aureus.

A total of 54 strains of Staphylococcus aureus, 37 methicillin-susceptible (MSSA) and 17 methicillin-resistant (MRSA), were investigated for their susceptibility to dicloxacillin as compared to methicillin and oxacillin by agar plate dilution at two different temperatures of incubation (30 degrees C and 37 degrees C). Against MSSA strains we found a slight but significant increase (0-2 dilution steps) in minimal inhibitor concentrations (MICs) for all three antibiotics with decrease in incubation temperature. Against MRSA strains methicillin and oxacillin showed a 3-6 fold increase in median MIC with decrease incubation temperature. For dicloxacillin, in contrast, there were no significant differences in median MICs (i.e. 0.4 mg/l) against MSSA strains at 30 degrees C or MRSA strains at either incubation temperature. Population-analysis of the MRSA strains revealed, however, that a highly dicloxacillin-resistant subpopulation appeared with a frequency of 10(-6) to 10(-7). Such heterogenous resistance of MRSA strains to dicloxacillin probably prohibits the use of dicloxacillin against serious infections caused by these pathogens.

Resistance to dicloxacillin, methicillin and oxacillin in methicillin-susceptible and methicillin-resistant Staphylococcus aureus detected by dilution and diffusion methods.

A total of 54 Staphylococcus aureus strains of varying methicillin resistance were investigated for their resistance to methicillin, oxacillin and dicloxacillin by different diffusion tests. Inhibition zones were measured around locally prepared paperdiscs with 10 micrograms methicillin, 5 and 10 micrograms oxacillin, 5 and 10 micrograms dicloxacillin, PDM paperdiscs with 10 micrograms methicillin or oxacillin and Neo-sensitabs tablets with methicillin or oxacillin. All diffusion tests were performed both with Mueller-Hinton agar and Danish Blood agar as well as at 37 degrees C and 30 degrees C and read after overnight incubation. Differences in zone diameter under different conditions were found to be independent of the susceptibility level of the strains. Seventeen of the strains were detected as methicillin-resistant (MRSA) by two methods including high inoculum and prolonged incubation at 30 degrees C. The minimum inhibitory concentration (MIC) of the 54 strains was determined by a plate dilution method at 30 degrees C and 37 degrees C. A 10 micrograms locally prepared methicillin disc detected all MRSA strains with no false reactions either at 37 degrees C or 30 degrees C on Mueller-Hinton agar. Investigations with oxacillin discs had to be performed at 30 degrees C or with a 5 microgram disc in order to detect correctly. PDM paperdiscs gave reactions identical to the corresponding locally prepared discs. Methicillin Neo-sensitabs detected all MRSA strains but also included a few susceptible strains among the resistant ones. Addition of blood increased the number of not-detected MRSA strains. All 17 MRSA strains were susceptible to dicloxacillin by the dilution method, and the disc diffusion test showed similar results. Dicloxacillin discs therefore did not detect the presence of MRSA strains. The implications of replacement of the methicillin/oxacillin disc by a dicloxacillin disc are discussed.

Evaluation of staphylococci isolated from a blood culture system (Colorbact).

During a one-year period, all blood cultures positive for staphylococci from two of the smaller Danish counties with non-university hospitals only were evaluated. The isolates were speciated and coagulase-negative staphylococci (CoNS) were biotyped. Furthermore, antibiogram, phage typing and lectin typing were performed for all isolates. Clinical information was obtained by telephone, and before any bacteriological identification was performed, a preliminary judgment was made as to whether the positive blood culture was of clinical significance. A total of 3,500 blood cultures were evaluated and 426 (12.2%) were positive. One hundred and sixty blood cultures from 137 patients contained staphylococci; 36 of these patients had a pure culture of Staphylococcus aureus. One hundred and twenty-four CoNS were found and identified as S. epidermidis (81), S. hominis (19), S. haemolyticus (8), S. simulans (1), and Micrococcus species (3), and another 12 staphylococcal isolates which could not be identified to species level. A total of 35 patients had mixed cultures, including 6 with S. aureus as one of the isolates and 15 with mixed CoNS. Clinical relevance was estimated in 90% of cases where the later bacterial identification showed S. aureus, whereas clinical relevance was absent in the majority of cases including CoNS. Methicillin and gentamicin resistance was absent among S. aureus, but frequent among CoNS, especially S. epidermidis, where 40% were resistant to methicillin and 30% to gentamicin.

Phage-typing of coagulase-negative staphylococci. Factors influencing typability.

Factors influencing the phage-typability of coagulase-negative Micrococcaceae have been studied in 2,778 clinical isolates comprising A) 209 consecutive isolates from one laboratory, B) 2,107 clinical strains submitted for phage-typing for epidemiological reasons, and C) 462 strains representing all isolates of presumed clinical significance found in two laboratories during one month. The reproducibility was acceptable at duplicate repeated typing of the same strains as well as by typing epidemiologically-related pairs of strains from the same patient. Strains of Staphylococcus haemolyticus were seldom typable, whereas strains of S. epidermidis and S. hominis had a higher typability. Methicillin-resistant strains and other multiple-resistant strains were rarely typable (11-13%). The typability was higher among susceptible strains (36%) and strains resistant to penicillin only (43-50%). The typability of strains of the same species and antibiotic-resistance pattern differed between hospitals compared and decreased markedly over the years for multiple-resistant S. epidermidis isolates.

Bacterial colonization and healing of venous leg ulcers.

UNLABELLED: The aim of the study was to evaluate a possible influence of selected bacterial species on healing of venous leg ulcers. Fifty-nine patients with venous leg ulcers were followed via frequent semiquantitative culture of bacteria from the ulcer surface and determination of the ulcer area over a period of 180 days. Occurrences of cellulitis were treated with systemic antibiotics. There was a significant difference in relative areas on days 90 and 180 when ulcers with growth of Pseudomonas aeruginosa were compared to those without (p = 0.0080 and 0.0133, respectively). Ulcers with P. aeruginosa were characterized to a great extent by enlargement in contrast to those without. Ulcers with growth of Staphylococcus aureus or haemolytic streptococci healed significantly more slowly than those without when relative areas were compared on day 180 (p = 0.0079 and 0.0492, respectively). Complete healing within the observation period of 180 days was observed in 10.5% of patients with P. aeruginosa and 35% of those without (p = 0.0631), in 21.6% of patients with S. aureus and 62.5% of those without (p = 0.0278), and in 10.5% of patients with haemolytic streptococci and 35% of those without (p = 0.0631). The initial areas of ulcers colonized with P. aeruginosa or S. aureus were significantly larger than those without, but no significant correlation between initial areas and ulcer healing was revealed. CONCLUSION: Our results suggest that P. aeruginosa in venous leg ulcers can induce ulcer enlargement and/or cause a healing delay. The results also suggest a healing delay caused by S. aureus and haemolytic streptococci. However, conclusions have to be treated with caution since P. aeruginosa was found in combination with haemolytic streptococci in 15.3% of the patients.