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Eitan N. Berezin MD, Eduardo S. Carvalho MD, Silvana Casagrande MSc, Maria Cristina Brandileone MSc, Igor M. Mimica MD, Calil. K. Farhat MD
Departament of Pediatrics and Microbiology
Santa Casa de São Paulo (ENB, IMM), Instituto de Infectologia Emilio Ribas, Universidade
Federal de São Paulo (ESC, CKF), Instituto Adolfo Lutz (SC, MCB) - São Paulo, Brasil
Acknowledgements
We thank Robert Austrian, MD ( World Health Organisation Collaborating Center for Reference and Research on Pneumococci, Department of Research Medicine, University of Pennsylvania School of Medicine, Philadelphia ) for serotyping our pneumococcal strains.
Streptococcus pneumoniae
infections are an important cause of morbidity and mortality in developing countries.
Although penicillin is the antibiotic of choice for the treatment of most infections,
strains with resistance to this and other antibiotics are increasing in many parts of the
world.. The present study was conducted in S. Paulo, Brazil in two teaching hospitals
(Santa Casa de São Paulo and Instituto de Infectologia Emilio Ribas) to investigate the
prevalence of resistance to penicillin (PEN), trimethoprim- sulfamethoxazole (TMP-SMX),
and chloramphenicol (CL) in isolates from blood, cerebro- spinal fluid (CSF ) and pleural
effusion (PE) among children with invasive infections. The study also sought to identify
the most prevalent pneumococcal serotypes among children with invasive infections. From
July 1989 to July 1993 a total of 101 pneumococcal isolates were obtained, 60 from CSF ,
35 from PE and 6 from blood, from patients less than 12 years old. These strains were
routinely investigated for penicillin resistance with the disk diffusion test using a
1µg. oxacillin (OX) disk. Disk diffusion testing using the NCCLS procedure for TMP/SMX.,
and CL were also performed for every isolate. The minimal inhibitory concentration (MIC)
for PEN were determined for each isolate using the broth dilution assay. Streptococcus
pneumoniae isolates were defined as PEN susceptible if MIC were <
0.1µg/ml, and PNS-P when MIC > 0.1µg/ml. PNS-P were further subdivided into
intermediate ( 0.1µg/ml < MIC < 1.0 µg/ml) and highly resistant
strain (MIC >2.0 µg/ml).
Results- - The prevalence of PNS-P in our study was 9,8
%. Only one strain was defined as highly resistant . Of the 10 PNS-P isolates, 9 were
obtained from the 58 children under two years of age while only one was obtained from the
43 children over 2 years. Sixty three (62.7%) strains were non susceptible to TMP/SMX (53
resistant and 10 intermediate) and two were non susceptible to CL. A total of 86 strains
were serotyped . Among these, 72 (82,7%) were serotypes, contained or antigenically
related, in the 23 valent pneumococcal polysaccharide vaccine. There where 15 fatal
pneumococcal invasive infections (FI), 14 caused by meningitis and 1 due to bacteremia,
giving an overall case fatality rate of 14.8%. We were not able to correlate fatal outcome
with infections by PNS-P strains.
As infecções por Streptococcus pneumoniae são importante causa de mortalidade e morbidade em pediatria. Apesar da penicilina ser o antibiótico de escolha para o tratamento destas infecções, cepas resistentes à penicilina vem sendo descritas em todo o mundo. O presente estudo foi realizado na Santa Casa de S. Paulo e no Instituto de Infectologia Emilio Ribas para avaliar a prevalência da resistência bacteriana a penicilina (PEN), trimethropim-sulfamethoxazole(TMP-SMX), e chloranfenicol (CL) em isolados de crianças abaixo de 12 anos com infecções invasivas pelo pneumococo. O estudo também objetivou avaliar os sorotipos de pneumococo mais prevalentes nestas crianças. Entre Julho de 1989 e Julho de 1993 um total de 101 isolados de pneumococo foram obtidas, 60 de liquido cefaloraquideo, 35 de liquido pleural e 6 de sangue. Estes isolados foram investigados em relação a resistência a penicilina através do teste de sensibilidade de disco utilizando o disco de oxacilina de 1
µg (PNS-P = halo< 20mm) . Teste de sensibilidade de disco a TMP-SMX e CL foram também realizados de todos os isolados. A concentração inibitória mínima (CIM) para penicilina foi determinada de cada isolado utilizando o método de diluição em tubo. O isolado era considerado PNS-P quando a CIM era superior a 0.1µg/ml e subdividida em intermediaria (0.1µg/ml/ml <CIM <1,0µg/ml) e resistência plena (CIM >2,0µg/ml ).Streptococcus pneumoniae infections are an important cause of morbidity and mortality in developing countries. Although penicillin is the antibiotic of choice for the treatment of most infections, strains with resistance to this and other antibiotics are increasing in many parts of the world. 1, 2. Data on prevalence of penicillin non susceptible pneumococci (PNS-P) in South America are scant. In Brazil a prevalence of 17.5% was previously reported but no detailed information, regarding its importance in pediatrics was provided 3 . The present study was conducted in S. Paulo, Brazil in two teaching hospitals (Santa Casa de São Paulo and Instituto de Infectologia Emilio Ribas) to investigate the prevalence of resistance to penicillin (PEN), trimethoprim- sulfamethoxazole (TMP-SMX), chloramphenicol (CL) and ceftriaxone (CT) in isolates from blood, cerebro- spinal fluid (CSF ) and pleural effusion (PE) among children with invasive infections. The study also sought to identify the most prevalent pneumococcal serotypes among children with invasive infections.
The Santa Casa is a reference hospital in the central area of S. Paulo with 150 pediatric beds. An average 300 patients are admitted every month. The Instituto de Infectologia Emilio Ribas is a hospital for infectious diseases with 50 pediatric beds and is a reference center for meningeal infections in São Paulo. From July 1989 to July 1993 all isolates of Streptococcus pneumoniae obtained from CSF , blood or PE from patients less than 12 years old were routinely investigated for penicillin resistance. Isolates were identified as Streptococcus pneumoniae on the basis of alpha- hemolysis on blood agar and susceptibility to ethylhydrocuprein (optochin) . All strains were screened for PEN resistance with the disk diffusion test using a 1µg. oxacillin (OX) disk, according to the procedures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (4) . Inhibition zone under 20 mm was considered resistant. Disk diffusion testing using the NCCLS procedure for TMP/SMX 2.5/23.75 µg., and CL 30 µg were performed for every isolate. Inhibition zones over 19 mm to TMP/SMX and over 21mm to CL were defined as susceptible. For TMP/SMX inhibition zones between 16 and 18mm were defined as intermediate susceptibility. The minimal inhibitory concentration (MIC) for PEN were determined for each isolate using the broth dilution assay recommended by the NCCLS, using cathion adjusted Mueller-Hinton broth with 3% lysed horse blood 4. Streptococcus pneumoniae isolates were defined as PEN susceptible if MIC were < 0.1 µg/ml, and PNS-P when MIC >0.1 µg/ml. PNS-P were further subdivided into intermediate ( 0.1 µg/ml<MIC<1.0 µg/ml) and highly resistant strain (MIC>2.0 µg/ml). PNS-P isolates were evaluated for CT susceptibility using the E test manufactured by AB BIODISK, Sweden and defined as resistant to CT if MIC>2.0 µg/ml. Serotyping was performed in the WHO pneumococcal reference laboratory in Pennsylvania on the basis of capsular swelling with type-specific anti-serum (Quellung reaction) .
Patients - A total of 101 pneumococcal isolates were obtained between July, 1989 and July, 1993, 60 from CSF , 35 from PE and 6 from blood. The diagnosis and age distribution are showed in TABLE 1.
Resistant strains- The prevalence of PNS-P in our study was 9,8 %. One strain was highly resistant (MIC = 4.0 µg/ml ) and nine were intermediate resistant strains ( 0.1 µg/ml<MIC<1.0 µg/ml ). Of the 10 PNS-P isolates, 9 were obtained from the 58 children under two years of age while only one was obtained from the 43 children over 2 years. Sixty three (62.7%) strains were non susceptible to TMP/SMX (53 resistant and 10 intermediate) and two were non susceptible to CL. One PNS-P was resistant to CT (MIC= 2.0 µg/ml). The other nine strains tested to CT were susceptible (0.064 µg/ml < MIC < 1.0 µg/ml ).
Serotypes - A total of 86 strains were serotyped . Among these, 72 (82,7%) were serotypes, contained or antigenically related, in the 23 valent pneumococcal polysaccharide vaccine. TABLE 2 shows the most prevalent serotypes obtained from children younger than two years old, from patients infected by PNS-P strains and from patients whith a fatal outcome.
Outcome - There where 15 fatal pneumococcal invasive infections (FI), 14 caused by meningitis and 1 due to bacteremia, giving an overall case fatality rate of 14.8%. Of the 31 children under two years of age with meningitis, 12 (38.7%) died, while 2 (6.6 %) of the 30 children over two years of age with the same diagnosis died . We were not able to correlate fatal outcome with infections by PNS-P strains. Two of our patients with meningitis due to a PNS-P strain had a favorable outcome. Two children with meningitis due to a PNS-P strain had a poor outcome with severe neurological damage. One was infected by a CT resistant and PEN intermediate resistant strain (MIC= 2.0 µg/ml for CT and 0.125 µg/ml for PEN ). This child was initially managed with CT (100mg/Kg/day) and dexamethazone (0,6mg/Kg/day) on admission. The culture remained positive after 48 hours and the therapeutic schedule was changed to a combination of CT and vancomicin. The other child was infected by a fully PN resistant strain ( MIC=4.0 µg/ml).
The children admitted due to pneumonitis had a favorable outcome and were treated with Ampicillin (AP) or an OX and CL combination.
Strains of PNS-P have spread rapidly worldwide, however, the patterns of resistance differ from one world region to another 2. In the present study, we have shown a prevalence of PNS-P strains of 9.8% among invasive pneumococci isolates in São Paulo. Almost all the PNS-P strains exhibited intermediate resistant with the exception of one strain that was highly resistant (MIC =4.0 µg/ml) . Most of the non-susceptible strains were recovered from children younger than two years of age. A recent survey carried out in Brazil, including children and adults, found that 17,4% of strains recovered from blood, respiratory tract secretions, ocular secretions and others, between 1988 to 1992, were resistant to penicillin 3. Another survey, wich took place in Uruguay , South-America, reported a resistance prevalence of 6% 5 . Both surveys in South America demonstrated a prevalence of PR-P lower than 20%, and a highly resistant pneumococcal strain prevalence lower than 5%.
The increase of the prevalence of PNS-P strains may be associated to reduction of susceptibility to other beta-lactamic and non beta-lactamic drugs. Two strains were non-susceptible to CL and 62.4% were non susceptible to TMP/SMX. High prevalence of TMP/SMX resistance has also been observed in Asia, Europe and USA and may reflect the widespread use of this drug 2,6 . This finding is a matter of concern because this drug is recommended in outpatient management of respiratory infections in developing countries .
Patients with pneumonitis due to PNS-P were successfully treated with ampicillin or the association of oxacillin and chloramphenicol. Our data suggested that penicillin G or ampicillin treatments are probably effective for pneumococcal pneumonitis caused by strains for which the MIC of penicillin ranged from 0,12 to 1 µg/ml as has been previously reported 7.
Mortality by meningitis in younger children was high (37.8%). In Brazil most of the centers use the AP-CL combination for initial meningitis therapeutics in patients younger than five years of age. This approach has been substituted in developed countries by third generation cephalosporine due to the increasing bacterial resistance, and the poor bactericidal activity of chloramphenicol against pneumococci 8 . However, there is an increasing number of reports in wich failure of cephalosporine treatment occurs 9. Combination therapy using third generation cephalosporine and vancomicin has been proposed for the treatment of meningitis caused by PNS-P 9. Two of our patients with meningitis were effectively treated with AP or association of AP and CL. One patient was infected by a CT resistant strain (MIC= 2.0) and responded inadequately to this therapy and one was infected by a fully PEN resistant strain. The increasing prevalence of resistance to PEN and other antibiotics detected in this study may reach further implications for antibiotic therapy in the future.
In Brazil mortality due to pneumococcal invasive infection is still high. Only the development of effective conjugate pneumococcal vaccines including serotypes most prevalent in our country will raise hope of reducing mortality and morbidity due to those infections. Serotypes 1, 5, 14, 6A and 6B were the most prevalent in our study. Previous reports from South American countries have pointed similar distribution 10,11. Two formulations of pneumococcal polysaccharide conjugate vaccine have been proposed to prevent infections in children under 2 years of age. Formulation A includes serotypes 4, 6B, 9V, 18C, 19F and 23F and formulation B includes 1, 5, 6B, 14, 18C, 19F and 23F 13. The latter formulation is the most adequate for developing countries, and would prevent, according to our study, 59.6% of the total pneumococcal infections, 69,3% of infections in children younger than 2 years of age and 73.3% of deaths caused by pneumococci.
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