Abstract
The study aimed at identifying the profile of gut colonization of patients with acute leukemia who underwent induction chemotherapy and its association with induction events and outcome. Baseline bacterial stool culture with resistance pattern of isolates were recorded. Multi-drug resistance was defined as resistance to at least two antibiotic classes including beta lactam and fluoroquinolones. During induction chemotherapy, blood and clinically indicated cultures were taken during febrile neutropenic episodes. Association studies were done between gut colonization and induction events/outcome. Among 109 patients enrolled, 71 (65.13%) patients undergoing induction chemotherapy were colonized with bacteria, with nearly 50% of colonizers harboring multi-drug resistant bacteria. Organisms isolated from stool pre-induction were mostly gram negative (98%), with Escherichia coli and Klebsiella pneumoniae being the commonest. 65.13% patients developed febrile neutropenia. Overall multi-drug resistant positivity during febrile neutropenia was 70.14%. Concordance of 8.45% was observed between isolates from stool and organisms isolated from cultures during febrile neutropenia. There were significant proportion of gut colonized gram-negative multi-drug resistance bacteria among patients with acute leukemia. There was a low concordance rate between baseline stool isolates and subsequent cultures during the induction. There was no significant association between gut colonization and induction events/outcomes studied.
Keywords: Surveillance stool culture, Acute leukemia, Febrile neutropenia, Leukemia induction, Microbiologically documented infection
Introduction
Febrile neutropenia is a life-threatening complication of chemotherapy. It results in morbidity, healthcare expenditure, reduced efficacy due to chemotherapy delays. The mortality rates are around 18% in Gram-negative and 5% in Gram-positive bacteremia [1]. Bacterial translocation from gut is a common mechanism of sepsis in cancer patients owing to immunosuppression, mucositis and antibiotic use favoring colonization with multi-drug resistant pathogens [2].
Infection with multi-drug resistant bacteria is common in India. In absence of well-designed studies regarding association between gut colonization and sepsis in leukemia, majority of such events go undiagnosed. Some cancer centers have screening programs for isolation and differential treatment of colonized patients. However robust evidence supporting this practice is lacking.
Our primary objective was to study the profile and resistance pattern of bacteria colonizing gut of patients with acute leukemia who underwent induction chemotherapy. Secondary objectives included study of association between gut colonization and microbiologically documented infection during febrile neutropenic episodes and induction events/outcomes.
Materials and Methods
Newly diagnosed cases of acute leukemia registered in department of Medical Oncology and eligible for induction chemotherapy were included in a prospective cohort study. The sample size was estimated with expected proportion of patients with antibiotic resistance colonizing bacteria at 30% at 95% confidence interval and 5% relative precision. Bacterial stool culture was performed prior to induction and isolates were tested for antibiotic resistance. Blood and fluid cultures with resistance pattern were taken during episodes of febrile neutropenia during the induction protocol as and when indicated. Association studies were done between gut colonization and induction events like development of febrile neutropenia, number, and days of antibiotics, need for intensive care, development of multi-organ dysfunction and induction mortality. Concordance rate between stool isolates and organisms isolated during febrile neutropenia was calculated. Patients were followed till recovery of counts from induction chemotherapy.
All stool samples were collected and transported in Cary Blair medium. They were then plated on Meropenem (1 mcg/ml) containing MacConkey, without drug MacConkey, deoxycholate citrate agar and xylose lysine deoxycholate agar with intermittent heating. Since as a protocol we were also looking for any possible diarrhoeal disease bacterial pathogens, they were subsequently inoculated into Selenite F broth and further sub-cultured after 16 h of incubation. Following overnight incubation, plates were examined. Colonies that came up were observed and processed. Biochemical tests were read, and slide agglutination tests were performed for relevant pathogens. Antibiotic susceptibility testing for isolates was performed as per Clinical and Laboratory Standards Institute guidelines using Kirby-Bauer disc diffusion method. For control, a known isolate of meropenem resistant Pseudomonas aeruginosa was used for the drug containing medium.[3]. Multi-drug resistance was defined as resistance to at least two antibiotic classes including beta lactam and fluoroquinolones.
Statistical analyses were performed with IBM SPSS 19.0 version. Association between categorical variables were performed by Chi-square test. P value of less than 0.05 was considered significant. Institute ethics clearance was obtained.
Results
Baseline Characteristics
A total of 109 patients (73 acute lymphoblastic leukemia-ALL and 36 acute myeloid leukemia-AML) at baseline were included. Fever was present in 43.8% patients with ALL and 47.2% patients with AML at presentation. Infection was more common in AML (27.7%) compared to ALL (8.2%) at presentation. The most common site of infection and organism were blood stream infection and Klebsiella pneumonia respectively. There were no multidrug resistant organisms isolated in blood pre-induction (Table 1).
Table 1.
Baseline characteristics of patients
| Baseline characteristics of patients | ALL (n = 73) | AML (n = 36) | Total (n = 109) | ||
|---|---|---|---|---|---|
| Pediatric (n = 50) | Adult (n = 23) | Pediatric (n = 10) | Adult (n = 26) | ||
| Age (mean in years) | 8.62 (SD 5.21) | 34.87 (SD 0.00) | 11.00 (SD 4.83) | 35.14 (SD 10.93) | |
| Sex (M/F) | 31/19 | 14/9 | 6/4 | 8/18 | 59/50 |
| Duration of symptoms (median in months) | 0.7 | 1 | 0.7 | 1 | |
| Baseline fever (Y/N) | 24/26 | 8/14a | 5/5 | 12/14 | 49/59 |
| Pre-induction microbiologically documented infection | 6 (12%) | 0 | 4 (40%) | 6 (23.07%) | 16 (14.67%) |
| Induction chemotherapy protocol | MCP 841 (n = 42), BFM 95 (n = 14), GMALL (n = 17) | 3 + 7 induction (n = 30), All-trans-retinoic acid + daunorubicin (n = 6) | |||
Y Yes, N No
aOne patient, data on baseline fever not available
Profile and Resistance Pattern of Gut Colonization
Gut colonization was seen in 65% of patients, with Escherichia coli (73.33%) and Klebsiella pneumoniae (17.33%) as the most common organisms. There were 50% isolates which were multi-drug resistant. High carbapenem resistance was seen in isolates of E. coli (40.54%), K. pneumoniae (62.50%) and Enterobacter (50%; Table 2).
Table 2.
Profile of organisms and resistance pattern of common isolates from stool
| Type of isolates | Isolates | Antibiotics | |||||
|---|---|---|---|---|---|---|---|
| AG | CS | FQ | CP | ||||
| Cf | Mg | Cx | |||||
| MDR (n = 37) | E. coli (n = 24) |
22 91.66% |
22 91.66% |
6 25% |
24 100% |
23 95.83% |
15 62.50% |
| K. pneumoniae (n = 8) |
7 87.50% |
8 100% |
1 12.50% |
8 100% |
8 100% |
5 62.50% |
|
| Enterobacter spp. (n = 2) |
2 100% |
1 50% |
1 50% |
2 100% |
2 100% |
1 50% |
|
| Non MDR (n = 38) | E. coli (n = 31) |
1 3.22% |
19 61.29% |
1 32.25% |
23 74.19% |
18 58.06% |
0 |
| K. pneumoniae (n = 5) |
1 20% |
1 20% |
0 |
2 40% |
2 40% |
0 | |
| Enterobacter spp. (n = 1) | 0 | 0 | 0 |
1 100% |
0 | 0 | |
AG aminoglycoside, CS cephalosporine, Cf ceftazidime, Mg magnex, Cx ceftriaxone, FQ fluoroquinolones, CP carbapenem, MDR multi-drug resistant, E Escherichia, K Klebsiella
Profile of Febrile Neutropenia in Patients Undergoing Induction Chemotherapy
Patients underwent standard induction chemotherapy protocol for leukemia. Febrile neutropenia was seen in 71 (65%) patients who had 118 episodes of febrile neutropenia. Clinically documented infection was seen in 84% patients and microbiologically documented infection in 55%. Lungs were the most common site of clinically documented infection (66%). Blood stream infection was most common source of microbiologically documented infection (79.48%). Multi-drug resistant blood stream infection was seen in 69% patients. Most common organism isolated from blood was K. pneumoniae. Carbapenem resistance for multi-drug resistant E. coli, Enterobacter spp. and Pseudomonas spp. was 75% each and for K. pneumoniae was 88.88% (Table 3) Escherichia coli and Pseudomonas spp. were the most commonEscherichia coli and Pseudomonas spp. were the most common organisms from non blood sources like exudate with high degree of antibiotic resistance including carbapenem.
Table 3.
Profile and resistance pattern of organisms causing blood stream infection during febrile neutropenia
| Type of isolates | Antibiotics | |||||||
|---|---|---|---|---|---|---|---|---|
| AG | CS | FQ | CP | APB | ||||
| Cf | Mg | Cx | ||||||
| MDR isolates: (n = 30) | ||||||||
| E. coli (n = 4) | 3 | 4 | 2 | 4 | 4 | 3 | 1 | |
| K. pneumoniae (n = 9) | 9 | 9 | 4 | 9 | 8 | 8 | 4 | |
| Enterobacter spp. (n = 4) | 4 | 4 | 4 | 4 | 3 | 3 | 2 | |
| Pseudomonas spp. (n = 8) | 8 | 8 | 3 | NT | 8 | 6 | NT | |
| Acinetobacter baumannii (n = 5) | 4 | 5 | 2 | NT | 5 | 5 | 2 | |
| Non MDR isolates: (n = 6) | ||||||||
| K. pneumoniae (n = 2) | 0 | 1 | 0 | 2 | 1 | 0 | 0 | |
| Enterobacter spp. (n = 2) | 2 | 2 | NT | 2 | 2 | 1 | NT | |
| Pseudomonas spp. (n = 2) | 1 | 1 | 0 | 0 | 0 | 0 | 0 | |
AG aminoglycoside, CS cephalosporine, Cf ceftazidime, Mg magnex, Cx ceftriaxone, FQ fluoroquinolones, CP carbapenem, MDR multi-drug resistant, E Escherichia, K Klebsiella
Concordance Between Gut Colonization and Microbiologically Documented Infection During Febrile Neutropenia
Eight patients had the same organism isolated from pre-induction stool culture and cultures during febrile neutropenia. Of these, six patients had the same resistance pattern. Concordance rates between baseline stool and clinically directed cultures at febrile neutropenia was 8.45%.
Induction Events, Outcome and Association with Gut Colonization
The complete remission rates (CR) for adult and pediatric acute lymphoblastic leukemia was 70%. The CR rates for pediatric and adult AML were lower at 20% and 38.46% respectively. Induction mortality was seen in 16.51% of the study population. There was no association between gut colonization and development of febrile neutropenia (P = 0.49). There was also no association between gut colonization and total number/days of intravenous antibiotic use, need for intensive care and development of multi-organ dysfunction. Pre-induction gut colonization was not associated with increased induction mortality (P = 0.34) (Table 4).
Table 4.
Association between baseline colonization and induction events/outcomes
| Induction events/outcome | Baseline stool culture | Chi square test | |
| Growth | Normal flora suppressed | P value | |
| Development of FN | |||
| Yes | 48 | 23 | 0.49 |
| No | 23 | 15 | |
| Total IV antibiotic use | |||
| > 3 | 41 | 16 | 0.73 |
| < / = 3 | 30 | 22 | |
| Duration of IV antibiotic | |||
| > 7 days | 42 | 23 | 0.35 |
| < / = 7 days | 29 | 15 | |
| Transfer to ICU | |||
| Yes | 32 | 15 | 0.45 |
| No | 39 | 23 | |
| Development of MODS | |||
| Yes | 10 | 9 | 0.65 |
| No | 61 | 29 | |
| Requirement of ventilation | |||
| Yes | 7 | 8 | 0.74 |
| No | 64 | 30 | |
| Induction mortality | |||
| Yes | 12 | 6 | 0.35 |
| No | 59 | 32 | |
Discussion
Majority of enrolled patients were ALL. Fever at presentation was lower in our study compared to others [4] possibly due to higher over the counter use of oral antibiotics in India.
Nearly 65% patients had gut colonized with bacteria. Rate of colonization was higher in our study compared to others [5, 6]. Other studies have looked only at one or two organisms whereas we analyzed all isolates that came up in culture. Multi-drug resistant bacteria were seen in 50% of the colonized patients. Hess et al. [6] looked at colonization by gram negative organisms and showed resistance rates of 37% which was lower compared to ours. They studied the resistance pattern to three antibiotics whereas we have tested for an extensive panel of antibiotics. The broader spectrum of organisms isolated combined with large panel of antibiotics tested makes our study more comprehensive.
Febrile neutropenia was lower in our study than reported by Sasmita Biswal (89.16%) and higher than reported by Pratupjai et al., (47.8%) [7, 8]. Incidence of clinically documented infection was higher than described in similar studies from Pakistan [9, 10]. Incidence of microbiologically documented infection was similar to other studies [9]. Most common organisms isolated were K. pneumoniae, Pseudomonas spp. (16.41%). Although incidence of gram-negative infection was higher than similar studies, it is consistent with the fact that gram-negative organisms are commonest cause of febrile neutropenia [10, 11]. Carbapenem resistance is higher in our study compared to other centers [12]. Complete remission rates and induction mortality for ALL was comparable to data from other centers in India [13, 14]. The acute leukemia induction mortality of 16.51% was comparable with other studies.[15–18].
Concordance rate of 8.45% was seen between stool isolates and organisms from clinically indicated cultures. Liss et al. [19] reported a concordance rate of 6.6% for extended spectrum beta-lactamase resistant enterococci and 2% for vancomycin resistance enterococci. Zaas et al. [20] found concordance of 13.4% between gut colonization and blood stream infection with vancomycin resistance enterococci.
There was no association between pre-induction gut colonization with induction outcomes studied including induction mortality rate, despite 50% colonizers harbored multi-drug resistant organism.
This study, to the best of our knowledge is the first study from India that studied the entire spectrum of gut bacteria in patients with acute leukemia and looked at the association between gut colonization and isolates during febrile neutropenia from both blood and non-blood isolates.
Limitations of the Study
This study included a heterogenous population of adult and pediatric patients with two different types of leukemia viz, acute lymphoblastic leukemia and acute myeloid leukemia, is exploratory and not powered for subset analysis. A polymerase chain reaction study to characterize the bacteria to establish the definitive concordance was not done in our study. The study was not powered to look for association between the gut colonizers and induction outcomes which needs to be confirmed in a larger population.
Conclusion
Almost 2/3rd of acute leukemia patients are colonized by bacteria with 50% colonizers harboring multi-drug resistant bacteria. Gram negative organisms are the most common cause of febrile neutropenia with high rates of multi-drug carbapenem resistance. The concordance rate between the colonizing bacteria and subsequent blood infection was low. There was no association between gut colonizers and adverse induction outcome. Further studies need to be conducted to understand the role of gut bacteria in febrile neutropenia.
Acknowledgements
I would like to thank JIPMER for providing the intramural grant to conduct the study. I would like to thank the staff and residents of departments of Medical oncology and Microbiology
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
Footnotes
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Contributor Information
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