April Indian 2006 Journal of Medical Microbiology, (2006) 24 (2):101-6

April Indian 2006 Journal of Medical Microbiology, (2006) 24 (2):101-6 Original Article 101 TREATMENT OF ENTERIC FEVER IN CHILDREN ON THE BASIS OF CURRENT TRENDS OF ANTIMICROBIAL SUSCEPTIBILITY OF SALMONELLA ENTERICA SEROVAR TYPHI AND PARATYPHI A *V Manchanda, P Bhalla, M Sethi, VK Sharma Abstract Purpose: Recent reports indicate decreased susceptibility of S. typhi to fluoroquinolones, especially ciprofloxacin. Chloramphenicol has been suggested as first line therapy of enteric fever in many studies. This is a prospective study that describes the trends of antimicrobial susceptibility of S. typhi and S. paratyphi A causing bacteraemia in children and reports therapeutic failure to ciprofloxacin and evaluates the possible use of chloramphenicol, ampicillin, ciprofloxacin and third generation cephalosporins as first line therapy in the treatment of enteric fever in children. Methods: The present study was conducted from April 2004 to March 2005 in a superspeciality children hospital at New Delhi. A total of 56 S. typhi and five S. paratyphi A isolates were obtained among the 673 blood cultures performed. Antimicrobial testing was done using disk diffusion technique (NCCLS method) for 13 antimicrobials and MICs were calculated for ampicillin, ciprofloxacin, chloramphenicol and cefotaxime. Analysis of data was done using WHONET software. Results: All 56 isolates of S. typhi were sensitive to amoxycillin+clavulanate, gentamicin, cefixime, cefotaxime and ceftazidime. Multidrug resistance (MDR, resistance to three drugs) was seen in 22 cases (39%) and resistance to five drugs was seen in 12 cases (21%). Only two isolates were resistant to chloramphenicol (3%). MIC 90 for ampicillin, chloramphenicol, ciprofloxacin and cefotaxime were 1.0 µg/ml, 4.0 µg/ml, 64 µg/ml and 0.125 µg/ml respectively. All S. paratyphi A isolates were sensitive to ampicillin and chloramphenicol and resistant to nalidixic acid.mic distribution data for chloramphenicol revealed elevated MIC but still in susceptible range. Conclusions: There is an urgent need for further clinical studies to evaluate response to chloramphenicol in such cases. Antimicrobial susceptibility data and MIC distribution favour use of ampicillin as a drug of choice for the treatment of enteric fever. Third generation cephalosporins are also useful but their use should be restricted for complicated cases. Key words: Enteric fever, Salmonella, chloramphenicol, fluoroquinolones Multidrug resistant (MDR) strains (resistant to has already been reported. 16 However, reemergence of chloramphenicol, ampicillin and cotrimoxazole) of Salmonella chloramphenicol susceptible strains have also been reported enterica serovar Typhi (S. typhi) are increasingly being during recent years. 17,18 reported from India and worldwide. 1-4 Third-generation cephalosporins and fluoroquinolones have been found A prospective study was planned to study the extended effective in treatment of these cases. 5,6 However, isolates of patterns of antimicrobial susceptibilities of S. typhi and S. S. typhi with reduced susceptibility to fluoroquinolones (as paratyphi A isolated from the blood cultures at our hospital. indicated in the laboratory by resistance to nalidixic acid) have Additionally, clinical therapeutic responses were observed for now appeared in the Indian subcontinent and other regions. 7- few of these cases to ciprofloxacin, ampicillin and cefixime. 12 These nalidixic acid resistant but ciprofloxacin sensitive Also, the possible use of chloramphenicol, ampicillin, strains have increased minimum inhibitory concentrations ciprofloxacin and third generation cephalosporins as first line (MICs) for ciprofloxacin, although they are still within the therapy in the treatment of enteric fever in children was current NCCLS range for susceptibility (0.12-0.5 µg/ml). 13 evaluated. Therapeutic failures to ciprofloxacin have been reported in these cases of typhoid fever. 13-15 Furthermore, an isolate of S. Materials and Methods typhi from Bangladesh with high-level resistance to ceftriaxone The study was conducted at Chacha Nehru Superspeciality Children Hospital, Delhi from May 2004 to April 2005. Blood samples of 673 children with suspected *Corresponding author (email: <manchandavikas@hotmail.com>) enteric fever were subjected to blood culture. Salmonella Department of Clinical Microbiology (VM), Department of Pediatrics (MS), Department of Microbiology (PB, VKS); Maulana Azad enterica serovar Typhi was isolated in 56 cases (8.3%) and Medical College and Associated Chacha Nehru Children Hospital, Salmonella enterica serovar Paratyphi A in five (0.7%) cases. Geeta Colony, New Delhi - 110 031, India. Antimicrobial susceptibility patterns were determined using Received : 31-05-05 following commercial antimicrobial disks (HiMedia, India): Accepted : 07-10-05 chloramphenicol (30 µg), nalidixic acid (30 µg), ampicillin (10

102 Indian Journal of Medical Microbiology vol. 24, No. 2 µg), azithromycin (15 µg), cotrimoxazole (1.25/23.75 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), tetracycline (30 µg), cefixime (5 µg), cefotaxime (30 µg), ceftazidime (30 µg) amoxycillin + clavulanic acid (20/10 µg) and gentamicin (10 µg). Antimicrobial susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) methodology. 19 Minimum inhibitory concentrations (MICs) for ciprofloxacin and cefotaxime were determined using E - test (AB Biodisk, Solana, Sweden). Broth microdilution testing was performed to determine MICs for chloramphenicol and ampicillin in accordance with the National Committee for Clinical Laboratory Standards (NCCLS). 20 The reagent powders were dissolved in Mueller-Hinton broth and distributed to the wells of microdilution trays. Each tray was inoculated with 5 10 4 CFU per well to yield a final volume of 0.1 ml per well. Final dilutions ranged from 256 µg/ml - 0.25 µg/ml. The trays were incubated overnight at 35 C. Quality control was monitored by using Escherichia coli ATCC 25922. The recorded MIC of each antibiotic was the lowest concentration that completely inhibited visible growth of the test strain. The MIC at which 50% of the isolates tested were inhibited (MIC50) and MIC90 were calculated in accordance with the current NCCLS methodology and using WHONET software ver 5.1. Results All 56 isolates of S. typhi were found sensitive to amoxycillin + clavulanate combination, gentamicin, cefixime, cefotaxime and ceftazidime. Details of antimicrobial susceptibility patterns are shown in Table 1. Seven S. typhi isolates were sensitive to all drugs tested. Two isolates showed resistance to chloramphenicol. Multi-drug resistance (MDR, resistant to 3 drugs) was observed in 22 cases (39%) and resistance to 5 drugs was seen in 12 cases (21%). All five S. paratyphi A isolates were sensitive to ampicillin and chloramphenicol and resistant to nalidixic acid. Fourteen different types of antimicrobial profiles were observed for S. typhi. Nineteen isolates were resistant only to nalidixic acid (N), three each were additionally resistant to azithromycin (NZ) and tetracycline (NT). Eight isolates were found resistant to nalidixic acid, cotrimoxazole (Co), ofloxacin (O), ciprofloxacin (P) and tetracycline (T) (NCoOPT). Detailed antimicrobial resistance profiles are summarized in Table 2. Five S. paratyphi A isolates had three different resistance profiles. Three isolates were resistant to nalidixic acid and azithromycin (NZ) and one isolate each was resistant to nalidixic acid alone (N) and to nalidixic acid and tetracycline (NT). Seven (13%) strains were sensitive to all drugs. Minimum inhibitory concentrations (MICs) range and their distribution among the isolates are shown in Table 1 and Figures 1a-1d. Nalidixic acid resistant S. typhi (NARST) isolates were obtained in 48 cases and nalidixic acid sensitive isolates were found among eight cases of suspected typhoid fever. Among NARST, only 12 isolates met the criteria of current NCCLS break points for ciprofloxacin resistance and only two isolates had MIC < 0.12 µg/ml. The remaining 42 isolates had increased MICs between 0.12 - <1 µg/ml (Fig. 2a and 2b). Scatter plot (Fig. 2a) compares nalidixic acid zone diameters and ciprofloxacin MICs. Large numbers of isolates were found in the area of nalidixic acid resistance with slightly increased MICs for ciprofloxacin (0.12-1 µg/ml), but still below the current break point (<1 µg/ml). Another scatter plot (Fig. 2b] compares nalidixic acid zone diameters with that of ciprofloxacin zone diameters. In this figure, majority of the isolates fall in nalidixic acid resistant but ciprofloxacin sensitive area. Among S. paratyphi A isolates, MIC for Table 1: Antimicrobial susceptibility patterns of S. typhi Antimicrobials R I S MIC range MIC 50 MIC 90 Geometric mean n (%) n (%) n (%) (µg/ml) (µg/ml) (µg/ml) MIC (µg/ml) Chloramphenicol 2 (3.6) 1 (1.8) 53 (94.6) 64-0.5 1 2 1.08 Nalidixic acid 48 (85.7) 0 8 (14.3) ND ND ND ND Ampicillin 0 1 (1.8) 55 (98.2) 128-0.125 0.125 1 0.237 Azithromycin 0 14 (25) 42 (75) ND ND ND ND Cotrimoxazole 17 (30.4) 0 39 (69.6) ND ND ND ND Ofloxacin 11 (19.6) 0 45 (80.4) ND ND ND ND Ciprofloxacin 12 (21.4) 2 (3.6) 42 (75) >32-0.012 0.25 64 0.386 Tetracycline 19 (33.9) 5 (8.9) 32 (57.1) ND ND ND ND Cefixime 0 0 56 (100) ND ND ND ND Cefotaxime 0 0 56 (100) 0.125-0.023 0.064 1.25 0.073 Ceftazidime 0 0 56 (100) ND ND ND ND Amoxycillin + Clavulanate 0 0 56 (100) ND ND ND ND Gentamicin 0 0 56 (100) ND ND ND ND S- Sensitive; I- Intermediate; R-Resistant; MIC - Minimum inhibitory concentration; ND -Not determined

April 2006 Manchanda et al Treatment of Enteric Fever in Children 103 Figure 1a: MIC distribution of ciprofloxacin in S. typhi isolates. R, Resistant; I, Intermediate resistant; S, Sensitive Figure 1b: MIC distribution of chloramphenicol in S. typhi isolates. Lines in plot area denote boundaries for resistance and susceptibility cut off points. R, Resistant; I, Intermediate resistant; S, Sensitive. Figure 1c: MIC distribution of ampicillin in S. typh i isolates. Lines in plot area denote boundaries for resistance and susceptibility cut off points. R, Resistant; I, Intermediate resistant; S, Sensitive. Table 2: Antimicrobial resistance profiles for Salmonella typhi Profile N (%) T N NT NZ NCoT NZT NZP CNP NCoPT NZCoT CNCoT NCoOPT CNAZCo NZCoOPT 19 (34) 3 (5) 3 (5) 4 (7) 2 (4) 8 (14) 3 (5) C- Chloramphenicol, N- Nalidixic acid, A- Ampicillin, Z- Azithromycin, Co- Cotrimoxazole, O- Ofloxacin, P- Ciprofloxacin, T- Tetracycline Figure 1d: MIC distribution representative of third generation cephalosporins (cefotaxime in this figure) in S. typhi isolates. Lines in plot area denote boundaries for resistance and susceptibility cut off points. R, Resistant; I, Intermediate resistant; S, Sensitive. ciprofloxacin ranged between 0.125-0.75 µg/ml and for chloramphenicol ranged between 0.5-8 µg/ml. MIC distribution for cefotaxime was between 0.064-0.094 µg/ml. All five isolates had MIC of 1.0 µg/ml for ampicillin. Oral ciprofloxacin (10 mg/kg/day) was prescribed to the first five patients infected with nalidixic acid resistant but ciprofloxacin sensitive S. typhi (MIC > 0.64 µg/ml but < 1 µg/ ml) and were reviewed after five days. Of these five patients, only one responded to the therapy. In the remaining four patients, higher doses of ciprofloxacin (20 mg/kg/day) were advised and they were reviewed after another three days. Only two of them responded by this time. Oral cefixime (16 mg/kg/ day) was advised in the other two and an excellent response was observed. During the rest of the study none of the culture positive patients of typhoid fever were advised ciprofloxacin. They were advised either oral amoxycillin (100 mg/kg/day) or cefixime (20 mg/kg/day) to which, they responded well. The

104 Indian Journal of Medical Microbiology vol. 24, No. 2 Figure 2a: Scatterplots for nalidixic acid (disk diffusion) versus ciprofloxacin (MICs) for Salmonella enterica serovar Typhi. Lines in plot area denote boundaries for resistance and susceptibility cut off points. Numbers in plot area denotes number of isolates. R, Resistant; I, Intermediate resistant; S, Sensitive. Figure 2b: Scatterplots for nalidixic acid (disk diffusion) versus ciprofloxacin (disk diffusion) for Salmonella enterica serovar Typhi. Lines in plot area denote boundaries for resistance and susceptibility cut off points. Numbers in plot area denotes number of isolates. R, Resistant; I, Intermediate resistant; S, Sensitive. follow up was uneventful. Discussion In the preantibiotic era, typhoid fever case fatality rates approached 20%. Treatment with effective antimicrobial agents - ampicillin, chloramphenicol, cotrimoxazole and later ciprofloxacin - has reduced the case fatality rate to less than 1%. 21 The resistance pattern for S. typhi varies with geographical locations. Many studies have suggested that patients in Indian subcontinent or with the history of travel to the Indian subcontinent should receive ciprofloxacin as first line therapy. 8,22 However, S. typhi isolates resistant to ciprofloxacin and ceftriaxone (MIC, 64 mg/l) have been reported. 13,16,23-27 S. typhi strains with reduced susceptibility to fluoroquinolones have become a major problem in Asia and other parts of the world. 12 Although they were reported to be susceptible to fluoroquinolones, by disk testing with the use of recommended break points, these organisms were resistant to nalidixic acid and the MIC of fluoroquinolones for these strains was 10 times higher than that for fully susceptible strains. This reduction in susceptibility results in a poor clinical response to treatment. There is unpredictable response to treatment with ciprofloxacin in patients infected with these kinds of S. typhi strains. Selective pressure on the bacterial population by uncontrolled use of quinolones has likely led to emergence of resistance to this group of antimicrobials. This has been attributed to point mutation in quinolone resistance determining region (QRDR) of the topoisomerase gene gyr A, characteristically occurring at position 83 of the DNA gyrase enzyme (changing serine to phenylalanine) and position 87 (changing aspartate to tyrosine or glycine). 9 However, other mechanisms of resistance such as decreased permeability and active efflux of the antimicrobial agent may be involved. In other Enterobacteriaceae, higher levels of quinolone resistance have been associated with additional mutations in the gyr A gene, mutations in other topoisomerase genes, or alterations in fluoroquinolone uptake. 13 No such mutations have been reported yet in S. typhi, although there are sporadic reports of completely fluoroquinolone resistant isolates. Because clinical response to fluoroquinolones in patients infected with nalidixic acid resistant strains is greatly inferior to the response in those infected with nalidixic acid-susceptible strains, the break points for the classification of S.typhi strains according to their susceptibility to fluoroquinolones should be reviewed. A practical approach would be to classify strains that are resistant to nalidixic acid but susceptible to fluoroquinolones according to current disk testing criteria as resistant to quinolones or non susceptible to fluoroquinolones as suggested by other authors as well. 12 All strains that have intermediate susceptibility or resistance to fluoroquinolones on disk testing (as defined by NCCLS guidelines) should be considered fluoroquinolone resistant. There are reports that show that treatment of patients infected with similar isolates is at potential risk of therapeutic failure. 13 This study showed that resistance to nalidixic acid is generally associated with increased MICs of ciprofloxacin. Hence, nalidixic acid susceptibility testing must be included with ciprofloxacin susceptibility testing in routine microbiological laboratory. Treatment with ciprofloxacin must be avoided in nalidixic acid resistant isolates of S. typhi although higher doses may be helpful in a few cases. Most of the isolates in this study were found susceptible to chloramphenicol and ampicillin. Percentage resistance, intermediate resistance and sensitive (%RIS) data in Table 1 show that ampicillin and chloramphenicol should be

April 2006 Manchanda et al Treatment of Enteric Fever in Children 105 considered for the treatment of typhoid fever in children in Delhi, however, MIC distribution data projects different scenario. As discussed above, infection with isolates with raised MICs but still in susceptible range for ciprofloxacin (Fig. 1a) has a potential risk of therapeutic failure. MIC distribution of chloramphenicol closely resembles that of ciprofloxacin in raised MICs but still in susceptible range (Fig. 1b). Hence, theoretically, treatment with chloramphenicol also has a potential risk of therapeutic failure. More studies are needed to evaluate reuse of chloramphenicol as the first choice for treatment of typhoid fever. Minimum inhibitory concentrations of ampicillin and cefixime on the other hand were well below the susceptible limits and hence may be used safely in the treatment of typhoid fever. 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