a Department of Paediatrics, St Lucas Andreas Hospital, Amsterdam, PO Box 9243, 1006 AE, The Netherlands; b Department of Paediatrics, University Hospital of the Federal University of Ceara and Albert Sabin Children's Hospital, Fortaleza, Brazil; c Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, University Medical Centre, Utrecht, The Netherlands; d Department of Microbiology, Federal University of Ceara, Fortaleza, Brazil; e Department of Paediatrics, Free University Hospital, Amsterdam, The Netherlands
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Abstract |
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Introduction |
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Phenotypic methods, like antimicrobial susceptibility profiles and serotyping, are often used to distinguish bacterial isolates for epidemiological studies. However, in recent years DNA-based typing techniques have emerged as the methods of choice for typing bacterial isolates, as they are less subject to natural variation.20 Since 1993, antimicrobial resistance in pneumococcal strains isolated from patients with invasive disease from different regions of Brazil has been monitored through the Regional System for Vaccines (SIREVA), established by the Pan American Health Organization. Little is known, however, of the epidemiology of antibiotic-resistant pneumococci colonizing children in Brazil. The purpose of the present study was to investigate the clonal spread of penicillin-resistant pneumococci and to define the degree of genetic variability among strains isolated from the nasopharynx of children with community-acquired pneumonia and healthy children from day-care and immunization centres in Fortaleza, Northeastern Brazil. These strains were also compared with penicillin-susceptible isolates from the same paediatric population, and with representative multiresistant strains recovered from different parts of the world.
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Materials and methods |
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From 15 March 1998 to 15 December 1998, a single nasopharyngeal specimen was collected from children between 2 months and 5 years of age who presented at the Emergency Department of one of the three major Children's Hospitals in Fortaleza and who fulfilled the WHO criteria for community-acquired pneumonia.21 The control group consisted of children who were recruited randomly from day-care centres (DCC), and public immunization centres (IC) in the eight health districts of Fortaleza. Children were enrolled in the study only after informed consent was obtained orally from their parents.
Bacterial isolates
Nasopharyngeal specimens were collected on cotton-tipped swabs (Transwab, MW 173P, Medical Wire & Equipment Co., Wiltshire, UK) and transported in Amies medium within 4 h to the Microbiology Laboratory of the Department of Pathology of the Federal University of Ceara and processed immediately. S. pneumoniae was identified by standard laboratory procedures and stored at 70°C in Microbank vials (Pro-Lab Diagnostics, Neston, UK) until transported in dry-ice by air to the Clinical Microbiology Laboratory of the University Medical Centre Utrecht in The Netherlands, where the identity of each isolate was confirmed.22 From a total of 435 S. pneumoniae isolates, a group of 161 randomly selected isolates with reduced susceptibility to penicillin (MIC 0.12 mg/L), and 44 randomly selected penicillin-susceptible strains (MIC
; 0.06 mg/L), collected from 97 children with community-acquired pneumonia and 108 healthy controls from DCC and IC, was characterized by microbiological, serological and molecular techniques
Susceptibility testing.
MICs for penicillin, ceftriaxone, erythromycin, clindamycin, rifampicin, cotrimoxazole and chloramphenicol were determined by a microdilution method (Sensititre, AccuMed Int., Sussex, UK) and interpreted according to the National Committee for Clinical Laboratory Standards.23 S. pneumoniae ATCC 49619 was used as a control strain. Strains with an M-phenotype were resistant to erythromycin (MIC 1 mg/L) but sensitive to clindamycin (MIC
; 0.25 mg/L).24 Isolates were considered multiresistant if they were fully resistant to
3 antibiotics.4
Serotyping.
Serotyping was performed by the coagglutination method using 12 pooled antisera (Statens Seruminstitute, Copenhagen, Denmark) and protein A from Staphylococcus aureus (Cowan I strain NCTC 8530) in a disposable tray (LIP Equipment & Services Ltd, Shipley, West Yorkshire, UK) and, if necessary with the Quellung reaction using type-specific pneumococcal antisera (Statens Seruminstitute).25
Ribotyping.
Automated ribotyping was performed with the RiboPrinter Microbial Characterisation System (Qualicon Europe Ltd, Warwick, UK) according to the manufacturer's instructions. Briefly, the pneumococcal isolates were removed from storage at 70°C, subcultured overnight at 37°C in 5% CO2 on Columbia agar (Oxoid, Unipath Ltd, Basingstoke, UK) supplemented with 5% sheep blood, placed in tubes containing lysis buffer and loaded into the RiboPrinter Microbial Characterization Unit (MCU). Within the MCU, bacterial DNA was digested with EcoRI and the restriction fragments were separated by electrophoresis and transferred directly on to nylon membranes. A pattern of the restriction fragments containing rRNA genetic information was created through hybridization with a chemiluminescently-labelled DNA probe containing the rRNA operon (rrnB) from Escherichia coli. The chemiluminescence patterns were then electronically imaged and stored. For each membrane image, the software located the lane positions, reduced the background noise, scaled each lane's image intensity and used the data from the lanes containing DNA standards of known sizes to normalize the band positions.26 Similarity coefficients were calculated on the basis of the overall profile, based upon both band position and relative intensity. Output patterns were merged into a single ribogroup using an initial threshold similarity value of 0.93. Subsequently, the threshold value converged, as a function of the size of the ribogroup, until the minimal similarity value of 0.90. Reproducibility of ribogroup assignment was ascertained by running a set of control strains at regular time intervals over the study period. Thus, no inter-gel variation was detected.
A cluster was defined as a ribopattern shared by at least two S. pneumoniae isolates. Three international reference strains, including the 23F Spanish/USA clone, the 6B Spanish clone and the 19A South African clone, which were obtained from Dr Keith Klugman (Pneumococcal Diseases Research Unit, Johannesburg, South Africa), were also tested with the same method. 9,11,27 The research protocol was approved by the Ethics Committee of the Medical Council of the State of Ceara, Brazil.
Statistics
The data were analysed with SPSS-PC for Windows (version 8.0) software. Associations between categorical variables were tested by using the 2 test or Fischer's exact test, when appropriate. The t-test for independent samples and one-way ANOVA was used to compare group means. The discriminatory power of a typing method is its ability to distinguish between unrelated strains. It is determined by the number of types defined by the test method and the relative frequencies of these types. These two facets of discrimination are not generally presented as a single numerical value and therefore cannot be used for a straightforward comparison of different methods. For discriminatory power of the different typing methods the Simpson's index (SI) of diversity, based on the probability that two unrelated strains sampled from the test population will be placed into different typing groups characterized as the same type, was used.28 The degree of genetic clustering was defined as the percentage of strains displaying ribogroups that were observed twice or more.29 A P value <0.05 was considered significant.
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Results |
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Antibiogram.
The study group consisted of 44/205 (21%) penicillin-susceptible (MIC ; 0.06 mg/L), 149/205 (73%) intermediate penicillin-resistant (MIC 0.11 mg/L) and 12/205 (6%) highly penicillin-resistant (MIC
2 mg/L) isolates. The prevalence of isolates with reduced susceptibility to penicillin was similar in children with pneumonia and in healthy controls. Reduced susceptibility to cotrimoxazole (MIC
1/19 mg/L), erythromycin (MIC
0.5 mg/L), clindamycin (MIC
0.5 mg/L), chloramphenicol (MIC
8 mg/L), rifampicin (MIC
2 mg/L) and ceftriaxone (MIC
1 mg/L) was found in 52, 32, 25, 6, 3 and 2% of the isolates, respectively. Twelve of the 65 erythromycin-resistant pneumococci showed an M-phenotype. Thirty-two (16%) children carried multiresistant strains. Multiresistant strains and highly penicillin-resistant strains were more common, albeit not significantly, in children with pneumonia and children from DCC than in children from IC. Resistance to antibiotics other than penicillin was more frequent in strains with a reduced susceptibility to penicillin and only one multiresistant strain was penicillin susceptible. In addition, strains resistant to
4 antibiotics were only encountered in highly penicillin-resistant strains, suggesting co-transfer of antibiotic resistance genes among penicillin-resistant pneumococci.
Serogroups.
The most common serogroup was serogroup 6 (38%), followed by serogroup 19 (19%), 23 (12%) and 14 (10%). A higher variability of serogroups was observed among penicillin-susceptible isolates compared with isolates with reduced susceptibility to penicillin: the 44 penicillin-susceptible strains expressed 13 capsular polysaccharides, whereas the 161 strains with reduced susceptibility to penicillin expressed only 12 different serogroups (Figure). Paediatric serogroups were more often encountered in strains with reduced susceptibility to penicillin (87%) than in penicillin-susceptible strains (50%) (P < 0.0001).
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All strains proved to be typeable by automated ribotyping. Depending on the strain, 515 hybridizing fragments were observed, ranging from 1 to >20 kb. Automated ribotyping of the 205 isolates yielded 75 different genotypes: 50 ribogroups were unique, whereas 25 distinct ribogroups were shared by two or more isolates (range 223).
The SI of diversity was lower, and the degree of genetic clustering higher, in strains isolated from children with pneumonia and children from DCC compared with strains isolated from children from IC. In addition, SI was lower and the degree of genetic clustering higher in strains with reduced susceptibility to penicillin than in penicillinsusceptible strains (Table I). The 32 multiresistant strains were represented by 24 ribogroups (SI = 98%), 12 highly penicillin-resistant strains by 10 ribogroups (SI = 97%) and 12 strains with an M-phenotype included 10 ribogroups (SI = 97%), indicating a high genetic heterogeneity in these strains. Fourteen of the 25 (56%) clusters contained both isolates with reduced penicillin susceptibility and penicillin-susceptible isolates (Table II
). The discrimination index of ribotyping was higher (SI = 96.2%) than that of serotyping (SI = 79.6%). The clustering of pneumococcal strains observed by serotyping did not always correlate with the genetic relatedness observed by ribotyping and strains with an identical serogroup were distributed across different ribogroups; 12 of 25 (48%) genetic clusters contained isolates with alternative serogroups. Isolates with such alternative serogroups were more often encountered in penicillin-susceptible strains (41%) than in strains with reduced susceptibility to penicillin (7%) (P < 0.0001). The discrimination index of ribotyping was lower in the paediatric serogroups (SI = 95.2%) compared with the index in the non-paediatric serogroups (SI = 97.2%), and serogroup 14 showed the highest degree of genetic homogeneity (SI = 61%). Comparison of strains within DCC showed that strains with an identical phenotype (e.g. antibiogram and serogroup) only proved to be genetically indistinguishable in 67% of the cases. Thirty-eight (19%) isolates (including seven penicillin-susceptible strains) showed the same ribotype as the strains representative of two international epidemic clones of S. pneumoniae: ribogroup 54-S-1 (15 isolates), with a ribopattern characteristic of the 23F multiresistant Spanish/USA clone widely spread in Europe, the United States, Asia, South Africa and Latin America; and ribogroup 74-S-3 (23 isolates) with a ribopattern indistinguishable from that of the 6B multiresitant Spanish clone (Table II
). All isolates of ribogroup 54-S-1 were also resistant to cotrimoxazole, but nearly all of them were chloramphenicol susceptible and only a few were highly penicillin resistant, with occasional isolates showing resistance to macrolides. None of the isolates of ribogroup 54-S-1 isolates expressed serogroup 23: penicillin-susceptible isolates expressed serogroup 7 and isolates with reduced susceptibility to penicillin expressed serogroup 14. Most isolates of ribogroup 74-S-3 expressed serogroup 6 but three penicillin-susceptible isolates expressed serogroups 3 and 9. Most isolates of ribogroup 74-S-3 were also resistant to cotrimoxazole but hardly any showed ceftriaxone and chloramphenicol resistance, and none was highly penicillin-resistant, with occasional isolates showing resistance to macrolides.
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Discussion |
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Another interesting feature of our study was the spread among children in Fortaleza of two genotypes closely related to the international epidemic clones. Although ribotyping revealed that 15 Brazilian isolates shared the 23F Spanish/ USA clone characteristics and 23 isolates the 6B Spanish international clone's genetic features, significant phenotypic differences were found. The appearance of isolates that express serogroups not typical for the particular clone in multiresistant isolates has been described repeatedly and Barnes, Corso and Nesin and coworkers in the USA and Tomasz and colleagues in Latin-America reported capsular transformation of the international clone 23F from serogroup 23 to serogroup 14 on various occasions.33,3537
In addition, the changes in antimicrobial resistance patterns suggest that antibiotic susceptibility may have evolved in response to differing antibiotic pressures in Northeastern Brazil, as chloramphenicol is rarely used in the outpatient treatment of children in Fortaleza, in contrast to erythromycin. Erythromycin-resistant variants closely related to the 23F Spanish/USA clone have also been reported from France, the USA and South Africa.13,37,38
Two hypotheses could account for our observations: (i) the international clones have been introduced into Brazil through intercontinental spread but have undergone phenotypic changes as compared with the original Spanish clones; or (ii) strains sharing the same ribotype as the international clones have evolved toward penicillin resistance in Brazil, independently from their Spanish counterparts. Distinguishing between these hypotheses will need further investigation, using more discriminatory typing methods, of the genetic relationships between penicillin-susceptible and penicillin-resistant Brazilian isolates and comparison with the Spanish isolates, as well as determination of the genes conferring penicillin resistance in Brazil.
Finally, the data indicate the epidemic potential of certain penicillin-resistant pneumococcal ribotypes. Continuous surveillance of S. pneumoniae is urgently needed to monitor the antimicrobial resistance level and spread of penicillin-resistant clusters, and intervention studies should be planned in which the effect of reduced antibiotic pressure on the prevalence of resistant strains in the nasopharyngeal flora can be evaluated.
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Acknowledgments |
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Notes |
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References |
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2 . Shann, F. (1986). Etiology of severe pneumonia in children in developing countries. Pediatric Infectious Disease Journal 5, 24752.[ISI]
3 . Hansman, D. & Bullen, M. M. (1967). A resistant pneumococcus. Lancet ii, 2645.
4 . Klugman, K. P. (1990). Pneumococcal resistance to antibiotics. Clinical Microbiology Reviews 3, 17196.[ISI][Medline]
5 . Appelbaum, P. C. (1996). Epidemiology and in vitro susceptibility of drug-resistant Streptococcus pneumoniae. Pediatric Infectious Disease Journal 15, 9324.[ISI][Medline]
6 . Song, J. H., Lee, N. Y., Ichiyama, S., Yoshida, R., Hirakata, Y., Fu, W. et al. (1999). Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Clinical Infectious Diseases 28, 120611.[ISI][Medline]
7
.
Tomasz, A. (1995). The pneumococcus at the gates. New England Journal of Medicine 333, 5145.
8 . Soares, S., Kristinsson, K. G., Musser, J. M. & Tomasz, A. (1993). Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. Journal of Infectious Diseases 168, 15863.[ISI][Medline]
9 . Muñoz, R., Coffey, T. J., Daniels, M., Dowson, C. G., Laible, G., Casal, J. et al. (1991). Intercontinental spread of a multiresistant clone of serotype 23F Streptococcus pneumoniae. Journal of Infectious Diseases 164, 3026.
10 . Kell, C. M., Jordens, J. Z., Daniels, M., Coffey, T. J., Bates, J., Paul, J. et al. (1993). Molecular epidemology of penicillin-resistant pneumococci isolated in Nairobi, Kenya. Infection and Immunity 61, 438291.[Abstract]
11 . Smith, A. M. & Klugman, K. P. (1997). Three predominant clones identified within penicillin-resistant South African isolates of Streptococcus pneumoniae. Microbial Drug Resistance 3, 3859.[ISI][Medline]
12 . Koornhof, H. J., Wasas, A. & Klugman, K. (1992). Antimicrobial resistance in Streptococcus pneumoniae: a South African perspective. Clinical Infectious Diseases 15, 8494.[ISI][Medline]
13 . Lefèvre, J. C., Bertrand, M. A. & Faucon, G. (1995). Molecular analysis by pulsed-field gel electrophoresis of penicillin-resistant Streptococcus pneumoniae from Toulouse, France. European Journal of Clinical Microbiology and Infectious Diseases 14, 4917.[ISI][Medline]
14 . Mastro, T. D., Nomani, N. K., Ishaq, Z., Ghafoor, A., Shaukat, N. F., Esko, E. et al. (1993). Use of nasopharyngeal isolates of Streptococcus pneumoniae and Haemophilus influenzae from children in Pakistan for surveillance for antimicrobial resistance. Pediatric Infectious Disease Journal 12, 82430.[ISI][Medline]
15 . Smith, T., Lehmann, D., Montgomery, J., Gratten, M., Riley, I. D. & Alpers, M. P. (1993). Acquisition and invasiveness of different serotypes of Streptococcus pneumoniae in young children. Epidemiology and Infection 111, 2739.[ISI][Medline]
16 . Lehmann, D., Gratten, M. & Montgomery, J. (1997). Susceptibility of pneumococcal carriage isolates to penicillin provides a conservative estimate of susceptibility of invasive pneumococci. Pediatric Infectious Disease Journal 16, 297305.[ISI][Medline]
17 . Kellner, J. D., McGeer, A., Cetron, M. S., Low, D. E., Butler, J. C., Matlow, A. et al. (1998). The use of Streptococcus pneumoniae nasopharyngeal isolates from healthy children to predict features of invasive disease. Pediatric Infectious Disease Journal 17, 27986.[ISI][Medline]
18 . Ghaffar, F., Friedland, I. R. & McCracken, G. H. (1999). Dynamics of nasopharyngeal colonization by Streptococcus pneumoniae. Pediatric Infectious Disease Journal 18, 63846.[ISI][Medline]
19 . Tomasz, A. (1999). The challenge of multiresistant Streptococcus pneumoniae; international initiatives in day-care centers and the use of molecular epidemiologic techniques. Clinical Microbiology and Infection 5, Suppl. 4, S648.[Medline]
20 . Tenover, F. C., Arbeit, R. D. & Goering, R. V. (1997). How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Molecular Typing Working Group of the Society for Healthcare Epidemiology of America. Infection Control and Hospital Epidemiology 18, 42639.[ISI][Medline]
21 . World Health Organization. (1990). Acute respiratory infections in children: case management in small hospitals in developing countries. WHO, Geneva.
22 . World Health Organization. (1994). Manual for the national surveillance of antimicrobial resistance of Streptococcus pneumoniae and Haemophilus influenzae: epidemiological and microbiological methods. WHO, Geneva.
23 . National Committee for Clinical Laboratory Standards. (1999). Performance standards for antimicrobial susceptibility testing: 9th informational supplement. NCCLS, Villanova, PA.
24 . Sutcliffe, J., Tait-Kamradt, A. & Wondrack L. (1996). Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrobial Agents and Chemotherapy 40, 181724.[Abstract]
25 . Sørensen, U. B. (1993). Typing of pneumococci by using 12 pooled antisera. Journal of Clinical Microbiology 31, 2097100.[Abstract]
26 . Pfaller, M. A., Wendt, C., Hollis, R. J., Wenzel, R. P., Fritschel, S. J., Neubauer, J. J. et al. (1996). Comparative evaluation of an automated ribotyping system versus pulsed-field gel electrophoresis for epidemiological typing of clinical isolates of Escherichia coli and Pseudomonas aeruginosa from patients with recurrent Gram-negative bacteraemia. Diagnostic Microbiology and Infectious Disease 25, 18.[ISI][Medline]
27 . Muñoz, R., Musser, J. M., Crain, M., Briles, D. E., Marton, A., Parkinson, A. J. et al. (1992). Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterisation by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis. Clinical Infectious Diseases 15, 1128.[ISI][Medline]
28 . Hunter, P. R. & Gaston, M. A. (1988). Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. Journal of Clinical Microbiology 26, 24656.[ISI][Medline]
29 . Hermans, P. W., Sluijter, M., Dejsirilert, S., Lemmens, N., Elzenaar, K., van Veen, A. et al. (1997). Molecular epidemiology of drug-resistant pneumococci: toward an international approach. Microbial Drug Resistance 3, 24351.[ISI][Medline]
30 . Brandileone, M. C., Di Fabio, J. L., Vieira, V. S., Zanella, R. C., Casagrande, S. T., Pignatari, A. C. et al. (1998). Geographic distribution of penicillin resistance of Streptococcus pneumoniae in Brazil: genetic relatedness. Microbial Drug Resistance 4, 20917.[ISI][Medline]
31 . Hermans, P. W., Sluijter, M., Hoogenboezem, T., Heersma, H., van Belkum, A. & de Groot, R. (1995). Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains. Journal of Clinical Microbiology 33, 160612.[Abstract]
32
.
Brisse, S., Verduin, C. M., Milatovic, D., Fluit, A., Verhoef, J., Laevens, S. et al. (2000). Distinguishing species of the Burkholderia cepacia complex and Burkholderia gladioli by automated ribotyping. Journal of Clinical Microbiology 38, 187684.
33 . Tomasz, A., Corso, A., Severina, E. P., Echániz-Aviles, G., Brandileone, M. C., Camou, T. et al. (1998). Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries: an overview. PAHO/Rockefeller University Workshop. Pan American Health Organization. Microbial Drug Resistance 4, 195207.[ISI][Medline]
34 . Kelly, T., Dillard, J. P. & Yother, J. (1994). Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae. Infection and Immunity 62, 18139.[Abstract]
35 . Nesin, M., Ramirez, M. & Tomasz, A. (1998). Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo. Journal of Infectious Diseases 177, 70713.[ISI][Medline]
36 . Corso, A., Severina, E. P., Petruk, V. F., Mauriz, Y. R. & Tomasz, A. (1998). Molecular characterization of penicillin resistant Streptococcus pneumoniae isolates causing respiratory disease in the United States. Microbial Drug Resistance 4, 32537.[ISI][Medline]
37 . Barnes, D. M., Whittier, S., Gilligan, P. H., Soares, S., Tomasz, A. & Henderson, F. W. (1995). Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo. Journal of Infectious Diseases 171, 8906.[ISI][Medline]
38 . Klugman, K. P., Coffey, T. J., Smith, A., Wasas, A., Meyers, M. & Spratt, B. G. (1994). Cluster of an erythromycin-resistant variant of the Spanish multiply resistant 23F clone of Streptococcus pneumoniae in South Africa. European Journal of Clinical Microbiology and Infectious Diseases 13, 1714.[ISI][Medline]
Received 6 March 2000; returned 8 June 2000; revised 12 July 2000; accepted 2 August 2000