Is Antimicrobial Resistance Pattern of Enteric Fever Changing in Kathmandu Valley ?

Introduction: Enteric fever is a public health problem in developing countries including Nepal. Antimicrobial sensitivity pattern of Salmonella sp causing enteric fever is changing over time and also differs according to geographical location. Periodic surveillance of antimicrobial resistance of Salmonella sp is mandatory for management of enteric fever rationally. The objective of this study was to analyze the resistance pattern of Salmonella sp to commonly used antimicrobials. Materials and Methods: This was a retrospective study undertaken reviewing the records of blood isolates of Salmonella sp over one year period at Kanti Children’s Hospital, Nepal. Antimicrobial sensitivity testing was done following Kirby Bauer’s disk diffusion technique using Muller Hinton agar. Results: Salmonella typhi was found to be 100% resistance to ampicillin and resistance to nalidixic acid and ciprofloxacin was also high. The resistance to chloramphenicol and ofloxacin was low. No resistance was found to amoxicillin, ceftriaxone, cefotaxime and amikacin. However, among the sensitive isolates of Salmonella typhi intermediate sensitivity to ceftriaxone and ceftazidime was reported to be high. Among the isolates of S paratyphi A, the resistance to nalidixic acid was 100% followed by 75% to ciprofloxacin. No resistance was found to chloramphenicol, ceftriaxone, cefixime, amikacin and low resistance to amoxicillin and ofloxacin. Among the sensitive isolates of S paratyphi A, majority of them were only moderately sensitive to cefotaxime and ceftazidime and about 1/3 of the organisms had only intermediate sensitivity to ceftriaxone. Conclusiion: Commonly used parenteral third generation cephalosporins, first line drugs like chloramphenicol and amoxicillin and ofloxacin among fluoroquinolones were found to be effective in vitro in treating enteric fever. However, Salmonella typhi was found to be highly resistant to the most frequently used drugs like ciprofloxacin and cefixime and Salmonella paratyphi A to ciprofloxacin. A comparative chart of antimicrobial sensitivity of enteric fever in children over 10 year period from different hospitals of Kathmandu valley is also presented in the study.


Introduction
E nteric fever is one of the public health problems in developing countries 1,2 .It continues to be endemic in poor countries 3 .Annual global incidence of enteric fever is estimated to be 0.3% 4 reaching 1.0% in disease endemic areas 5 .Worldwide, about 21 millions cases occur annually and among them 1-4% ends fatally predominantly in children, 90% of which happen in Asia 6 .Enteric fever is a public health problem in Nepal as well 7,8 .It occurs in almost every part of our country from mountain areas to Terai plains 9,10 .
Enteric fever occurs commonly in monsoon seasons.Outbreaks of enteric fever also usually occur in summer 11,12 .Children are the mostly aff ected age group 3,10 .Infected persons and healthy carriers are the source of infection."Five Fs" viz.food, fi ngers, fl ies, fomites and faeces are considered to be the main media for spread of this disease.The main factors responsible for such spread are poor sanitation and inadequate facilities for safe drinking water 13 .
The main causative agent of enteric fever is Salmonella typhi (S typhi) followed by paratyphi A (S paratyphi A).Early diagnosis and treatment reduces the morbidity and mortality from this infection.However, emergence of multi drug resistant (MDR) strains to fi rst September-December, 2012/Vol 32/Issue 3 doi: http://dx.doi.org/10.3126/jnps.v32i3.6625line antibiotics viz.chloramphenicol, ampicillin and cotrimoxazole in the late eighties and early nineties posed a great challenge to manage this disease 14,15 .Fluoroquinolones became the drug of choice for managing these cases after the emergence of MDR strains 16 .Unfortunately, these organisms developed resistance to fl uoroquionolones also within a decade of their use 7,15 .Third generation cephalosporins were then recommended to treat these resistant cases. 15But, again there has been a great concern due to report of emerging resistance of these organisms to third generation cephalosporin 17,18 .
It appears that the antimicrobial sensitivity pattern changes over time and diff ers from institution to institution in the same period of time.This could be due to the phenomenon of antimicrobial cycling 19 that may be taking place spontaneously.For instance, Salmonella sp have become sensitive to ampicillin/amoxicillin, cotrimoxazole and chloramphenicol once again 15,20 .Therefore, there is a great need of constant surveillance and antimicrobial sensitivity testing from diff erent geographical areas to keep update with emerging patterns of drug sensitivity in enteric fever 2 .The fi ndings from such studies help apply evidence based rational prescription practices because prudent and potent antimicrobial use not only prevents the emergence and spread of resistant strains of microorganisms but also benefi t the patients and a nation as whole 21 .Therefore, this study was conducted to analyze the antimicrobial sensitivity pattern of blood isolates of Salmonella sp in children at Kanti Children's Hospital.

Materials and Methods
This was a retrospective study conducted at Kanti Children's Hospital, Kathmandu, Nepal over one year period from April 2011 to March 2012.Records of all blood samples collected for cultures from children aged 1 day to 14 years attending this hospital out-patient department and admitted in the hospital were analyzed for this study.This hospital is a referral centre for children throughout Nepal; however children from outside the Kathmandu valley usually come after antimicrobial treatment for infections.Three ml of blood was collected by aseptic venipuncture and mixed in 30 ml of broth for culture.If the collected blood volume was not adequate (less than 3 ml) the volume of the broth was made 90% of the amount of blood collected.Blood samples collected were subjected to bacteriological culture and incubated at 37 0 C over night followed by sub-culture on MacConkey agar.Incubation was continued for growth negative cultures for 72 hours sub-culturing at 48 and 72 hours.Growth negative cultures even after this period were regarded as negative.
The non lactose fermenting colonies in MacConkey agar resembling Salmonella sp were further subjected to serotyping.Blood isolates other than Salmonella sp were not included in the study.Blood isolates of Salmonella sp were subjected to antibacterial susceptibility testing following Kirby Bauer's disk diff usion technique using Muller Hinton agar.The antimicrobial impregnated discs were placed on the surface of the agar plate and incubated at 37 0 C for 18 hours.Diameter of the zone of inhibition was measured for individual antimicrobial and interpreted as sensitive, intermediate and resistant on the basis of zone size as per manufacturer's instruction.Antimicroibioal discs used in this study were amikacin, amoxicillin, ampicillin, cefi xime, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol, ciprofl oxacin, cotrimoxazole, cloxacillin, nalidixic acid, norfl oxacin, ofl oxacin and tobramycin.However, only 6-7 antimicrobial discs were tested for each culture positive sample.If the culture positive sample was found resistance to all 6 or 7 antimicrobials it was tested for other antimicrobials.Selection of the antimicrobial discs to be used was based on the availability of the discs in the hospital laboratory.
The variables investigated were age and sex of the child, Salmonella species and their antimicrobial sensitivity pattern as data only on these variables were available in the records.Data analysis was done using SPSS software package version 16.
Permission to undertake this study was taken from the institutional review committee of the hospital.

Results
Total number of blood samples collected for culture during one year period was 10883, among them only 60 samples yielded Salmonella sp.Growth of Salmonella sp was not observed in children less than one year of age.Of the 60 positive samples, samples from male patients outnumbered females (60.0%vs 40.0%).The most aff ected age group was 6-10 year followed by age group of 1-5 years (Table 2).S typhi constituted more than double of the samples (72.0%vs 28.0%).Positive samples were found to be more in spring (March, April, May), summer (June, July, August) and early autumn months (September) with highest peak in June (Fig. 1).
Salmonella typhi was found to be 100.0%resistant to ampicillin and resistance to nalidixic acid and ciprofl oxacin was also high (59.4% and 46.4% respectively).No resistant strain was shown to amoxicillin, ceftriaxone, cefotaxime and amikacin.Resistance to chloramphenicol and ofl oxacin was low.However, only moderate sensitivity of Salmonella typhi to ceftriaxone and ceftazidime was worth noting here.(Table 3).

Is Antimicrobial Resistance Pattern of Enteric Fever Changing in Kathmandu Valley?
No resistance of S paratyphi A was shown to chloramphenicol, ceftriaxone, cefi xime and amikacin.The resistance to amoxicillin and ofl oxacin was low.The resistant rate of nalidixic acid was 100.0% and that of ciprofl oxacin was also high (75.0%).One fi fth of the S paratyphi A was resistant to ceftazidime and 1/3 of it to cotrimoxazole.However, it is worth noting that majority of isolated S paratyphi A were only moderately sensitive to cefotaxime and ceftazidime and about 1/3 of the organisms also had only intermediate sensitivity to ceftriaxone.(Table 4).

Discussion
This study highlights the sensitivity pattern of Salmonella sp to diff erent antimicrobials in children.
It study shows that the occurrence of enteric fever is perennial with more number in spring, summer and early autumn and a peak in the month of June.This type of seasonal distribution of enteric fever is the typical description of occurrence of enteric fever as mentioned in the textbooks.Similar observations were seen in other series as well 22,23,24,25 .The seasonal variation of occurrence of enteric fever is considered due to more chances of contamination of foods and water by faeces of infected persons and healthy carriers in the rainy season. 26Moreover, fl ies are also more abundant in this season.The "fi ve Fs" (food, fi ngers, fl ies, fomites and faeces) are considered to be the main media for spread of disease and they play more vital roles in transmission of disease in the monsoon season.
The male predominance observed in this study is in agreement with fi ndings by other authors 22,23,24,27 .This observation might be due to behavior of male children being out-door for diff erent plays.
The most commonly aff ected age group (6-10 year) reported in this series is similar to the fi ndings observed by Sharma AK 22 and Singh et al 25 .This age group is more vulnerable to exposure to contaminated foods and water and has not still developed the immunity against the disease.Ten to fourteen years was the most frequently aff ected age group in studies by Bhattarai et al 11 and Joshi et al 24 and under seven years was in studies by Prajapati et al 23 and Ansari et al 27 .A large scale study in fi ve Asian countries showed the incidence of typhoid fever to be equal in both pre-school (2-5 year old) and school aged children (5-15 year old) 28 .This discrepancy in the age group could be due to relatively small sample size and most of the studies being hospital based.
The antimicrobial resistance pattern of Salmonella sp observed by diff erent studies in children in Nepal is shown in table 5 for comparison.
Chloramphenicol, ampicillin and cotrimoxazole were regarded as fi rst line drugs in the treatment of enteric fever.The development of resistance to all these 3 drugs is called as multi drug resistant (MDR) strains.The emergence of resistance to these fi rst line antimicrobials, especially chloramphenicol in the late eighties and early nineties posed a great challenge to manage this disease 14,15 .However, Salmonella sp has been found sensitive to these fi rst line drugs once again 8,15,22,23,29 .For instance, one study reported that the resistance of S typhi to ampicillin, cotrimoxazole and chloramphenicol decreased from 80.0%, 80.0% and 50.0% in 1995 to 37.5%, 37.5% and 12.5% in 2003 respectively.On the other hand, the resistance to ciprofl oxacin and nalidixic acid rose from 0.0% and 87.2% in 1999 to 12.5% and 93.8% respectively in 2003.However, resistance to ceftriaxone remained 0.0% during that period 15 .
The present study also found chloramphenicol very eff ective against Salmonella sp in vitro.But, ampicillin resistance was 100.0%.In the contrary, another series reported ampicillin to be 100.0%sensitive to Salmonella sp 29 .These observations could be due to antimicrobial cycling that is taking place in the prescription practices spontaneously.Furthermore, physicians might have become more cautious to use chloramphenicol because of its reported side eff ect of bone marrow suppression.In the contrary, some of the studies have reported resistance of S typhi to chloramphenicol to be still high ranging from 27.0 to 57.10% 13, 17, 30.Fluoroquinolones, especially ciprofl oxacin and ofl oxacin, became the drug of choice for managing MDR cases after the emergence of MDR strains 16 .Unfortunately, these organisms also developed resistance to fl uoroquionolones within a short period of their use. 7,15For instance, the resistance of S typhi to ciprofl oxacin increased from 0.0% in 1998 to 12.5% in 2003.Manchanda et al 29 in 2004 reported the resistance rate of ciprofl oxacin as high as 21.6%.The trend of resistance developed by Salmonella sp over time as reported by diff erent studies in children in Nepal is shown in table 5 for comparison.It is seen from the table that the resistance to ciprofl oxacin increased from 0.0% in 2002 to 28.6% in 2010.The present study found resistance to ciprofl oxacin even higher (54.4%).However, this diff erence in resistance pattern should be interpreted with much caution as the methods used in these studies were diff erent (the studies conducted in 2002 and 2010 were prospective and present study being retrospective).
The Nepalese studies mentioned in Table 5 were conducted in diff erent hospitals by diff erent persons where as the Indian study 15 was conducted by the same person in the same place over time.The discrepancies observed in these diff erent studies might be due to the fact that the resistance pattern of enteric fever varies with geographical locations 28,29 .However, it is evident that the resistance pattern to ciprofl oxacin is in the increasing trend.As shown in table 5, only optimistic fl uoroquinolone seems ofl oxacin which has had constantly low level of resistance pattern over time.In the contrary, Manchanda et al 29 reported the resistance to ofl oxacin to be as high as 19.6%.Nalidixic acid resistance against Salmonella sp is considered as a Rai GK et al marker of increased minimum inhibitory concentration (MIC) of fl uoroquonolones 17 .Therefore, Jamil et al 17 recommends treating nalidixic acid resistant strains of Salmonella sp for prolonged period (14 days) with higher doses of fl uoroquonolones to avoid treatment failure.Third generation cephalosporins were recommended to treat fl uoroquinolone resistant cases of enteric fever 15 .But, reports of emerging resistance of Salmonella sp to third generation cephalosporins challenged the concerned personnel once again 17,18,30 .The commonly used third generation cephalosporins viz.cefi xime, cefotaxime and ceftriaxone for the treatment of enteric fever have been found resistant by some studies in Nepal as well, although the resistance reported is low 23,25 .The present study also observed cefotaxime and ceftriaxone eff ective in vitro treatment of Salmonella sp.However, the concern is with reported high percentages of intermediate sensitivity to ceftriaxone (45.2% for S typhi and 56.2% for paratyphi A).The high resistance rate of cefi xime (19.0%) found in this series contradicts the observations made by other studies (1.0% by Jamil et al 17 and 9.6% by Singh et al 25 ).This high reported resistance of Salmonella sp to cefi xime in the present study could be due to rampant irrational use of cefi xime in the communities.
Aminoglycosides are not recommended drugs for the treatment of enteric fever.Hundred percent sensitivity of Salmonella sp to amikacin observed in the present study is in agreement with the fi nding of study by Neopane et al. 8 However, the sensitivity reported by Bhatia et al 2 is only 84%.
The increasing resistance of Salmonella sp to diff erent antimicrobials has major implications on the management of enteric fever.It results in increase of cost of treatment as inexpensive drugs need to be replaced by the expensive newer drugs 28 .The oral antimicrobials have to be replaced by parenteral ones needing hospital admission of the patients that will further increase the cost of treatment and also increase the disease burden.Moreover, such expensive antibiotics may not be available and aff ordable to many poor people living in the communities.Thus, the emergence of drug resistance especially to common, fi rst line antibiotics and fl uoroquionolones has made very diffi cult and expensive for health services to manage the disease 31 .Furthermore, this increasing resistant pattern may lead to inadequate treatment of enteric fever resulting in more complications including persistent carriage of the organisms in the gall bladder known as post-treatment complication 26 .These chronic carriers will become the source of further dissemination of organisms causing enteric fever in the community.
The number of culture positive cases over one year period found in this study was only 60, which is considered very low.It is said that S. Typhi and S. Paratyphi A are not always culturable even in good microbiological laboratories.Furthermore, S. Typhi is ordinarily cultured from 5-10 ml of blood in 30-50 ml of broth 32 .It is diffi cult to draw such amount of blood in children, compromising diagnosis in them.However, the number of culture positive cases in 2005 and 2008 was 317 and 235 respectively in the same place and in the same period although the total number of blood samples collected for culture was almost the same in those three years.One possible reason for such decline in the number of culture positive cases could be due to eff ectiveness of the recently initiated pilot vaccination program in schools of neighbouring Lalitpur and Bhaktapur districts against Salmonella typhi 33 as the children from these districts also seek health services to the study place and students from Kathmandu district attend the schools in these districts as well.Another reason could be due to improvement in sanitary practices over the years.There is a need for further exploration to identify the exact cause of observed decline.It has been argued that mass vaccination program against typhoid along with provision of safe water supplies and eff ective sanitation in the endemic countries may be eff ective in controlling enteric fever. 13,28It has been recommended enrolling pre-school children too in vaccination programs because of their increased susceptibility to substantial clinical illness 30 as pre-school children (2-5 year old) are found equally aff ected by Salmonella sp as school aged children (5-15 year old) 28 Further studies on the estimation on the impact of disease with larger sample size and for identifying age groups at highest risk are helpful to optimize vaccination strategies in our setting 34 .
This study is considered a part of local microbiological surveillance and monitoring.It is hoped that it has added recent information about changing trends of antibiotic sensitivity pattern of Salmonella sp.It is also assumed that this study will help us choose empirical antibiotics more rationally and develop local antimicrobial policies for the treatment of enteric fever.However, limited numbers of antimicrobial discs (6-7 discs per blood isolate) used for sensitivity test are the major limitation of this study.
In conclusion, the number of salmonella sp isolates was very low.Commonly used parenteral third generation cephalosporins, fi rst line drugs chloramphenicol and amoxicillin and ofl oxacin among fl uoroquinolones seem eff ective in vitro in treating enteric fever.However, most frequently used drugs like ciprofl oxacin and cefi xime appear highly resistant.It is recommended to undertake further study to identify the causes for low yield.
Is Antimicrobial Resistance Pattern of Enteric Fever Changing in Kathmandu Valley?

Table 1 :
Distribution of blood samples for culture by age and sex (n=10883)

Table 2 :
Culture positive samples of Salmonella sp by age and sex (n=60)

Table 5 :
Comparative chart of antibiotic resistance pattern of Salmonella sp in children by diff erent studies conducted in Kathmandu valley (%).

date of study No of isolates Antibiotics tested and antibiotic resistance (%) Amoxycillin Ampicillin Chloramphenicol Cefi xime Cefotaxime Ceftriaxone Ciprofl oxacin Cotrimoxazole Nalidixic acid Ofl oxacin
25nsitivity to azithromycin in children was done only in the study by Joshi et al23, who observed the resistance to be 8.0%.Likewise, sensitivity to norfl oxacin and gentamycin was done only in the studies by Bhattarai et al 10 (14.3%) and Ansari et al25(0.0)respectively; sensitivity to amikacin and ceftazidime was done only in the present study. *