The present prospective cohort analysis was conducted on the patients undergoing treatment in the radiotherapy unit of Regional Cancer Institute, Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana, during the period of January 2007 to October 2009. A total of 186 patients with squamous cell carcinoma of oral cavity were analyzed in the study. The present study was approved by Human Ethical Committee of the University (M.D. University, Rohtak) and written consent was also taken from the patients.
The patients were divided into three groups depending on their treatment protocol; each category was having 62 cases. First group patients were radiotherapy treated (RT) only (total dose of radiotherapy ranging from 51 to 60 grays in dose of 200cGY/day, 5 days a week), second group patients were given chemotherapy treated (CT) only (3 or 4 Courses of carboplatin, 5-FU, docetaxel/methotrexate/cisplatin given after 21 days gap) and third category patients were given radio chemotherapy simultaneously (RCT) (Between irradiation, chemotherapy courses of paclitaxel, carboplatin and 5-FU given).
About 108 CFU/ml of bacteria and 105 CFU/ml of fungi cells were considered as pathogenic for the study. The main predisposing factors that can cause oral cavity and blood stream infection in the three studied groups were following:
Bloodstream infection (BSI): BSIs were defined, as isolation of a recognized pathogen (aerobic bacteria and fungi) from one or more blood cultures (BCs) that were unrelated to an infection at another site with, or without fever or hypotension[29, 30].
Oral infection: Oral infection was defined as, isolation of recognized pathogens (aerobic bacteria and fungi) from one or more oral swab.
Episodes of BSI: Since any given patients could have a BSI more than once, so we use the term episode of BSI for each separate event[29, 30].
Episode of bactermia: The isolation of one bactermia (unimicrobial) or more (polymicrobial) microorganisms in the same blood culture or in a separate blood culture obtained within 24–48 hours[29, 30].
Neutropenia: Neutropenia was defined as, an ANC of less than 500 neutrophil/μL that may increase susceptibility to infection [29. 30].
Fever: Oral temperature of ≥ 38.5°C or more within a 24-hours period after initiation of therapy[29, 30].
Anemia: A pathologic deficiency for oxygen-carrying hemoglobin in the red blood cells. In case of males Hb. < 14 g/dl and in case of females Hb. < 12 g/dl were considered as anemic cases.
Community acquired infection: Any infection acquired before, or within 48 hours of admission to hospital and which was not related to any hospital procedure[29, 30].
Nosocomial Infection: Nosocomial infection was defined as, at least one blood culture positive for significant pathogens in patients before, or within 48 hours of admission to hospital[29, 30].
Catheter related infection: Infection was considered catheter related when at least one of following conditions were, meet (1) Isolation of same pathogens from catheter tip and blood. (2) Isolation of pathogens from a blood culture obtained from the catheter, but not from another blood obtained from peripheral vein at the same time[29, 30].
Oral mucositis: WHO describe oral mucositis into 4 categories, like: grade 0- no change; grade 1 soreness/erythema; grade 2 erythema, ulcers, can eat solids; grade 3 ulcers, requires liquid diet only; grade 4 alimentation not possible.
Patients were excluded from the study if they had clinical or microbiological evidence of bloodstream infection of unknown origin. Patients who developed, fever within 24 hours after administration of chemotherapy and fever subsided within next 24 hours after completion of chemotherapy were also excluded from study. Common skin isolates, including Coryneforms and Bacillus species excluded from analysis. Coagulase negative Staphylococci (CoNS) were only considered as causative pathogens if two or more blood samples drawn on separate occasions showed the growth of the pathogen.
Clinical and laboratory data
The data on patient’s age, sex, underlying cancer, clinical stage of cancer, medications (antibiotics, cytotoxic drugs), fever, and exposure to radiotherapy or chemotherapy were recorded over the preceding 30 days and an invasive procedure performed over the proceeding 10 days. For every febrile episode of oral infection and blood cavity infection, the data on: date of onset, date of admission, sources of infection, presence of venous catheters and period of their insertion, result of complete blood count, severity and duration of neutropenia were collected.
Oral cavity specimen handling
Before antibiotics were started, Oral swab were taken by gently rubbing a sterile cotton swab over the labial mucosa, tongue and cancerous lesion. The swabs were incubated in sheep blood agar, saboured dextrose agar, macconkey agar, nutrient agar, and other selective media for primary isolation of the pathogens. These plates were than aerobically incubated for 24–48 hours at 37°C temperature for bacterial pathogens isolation and for 24–72 hours at 30°C in B.O.D. incubator for fungal species isolation.
Blood Specimen handling
Before antibiotics were started, blood samples (5 ml each) for cultures were obtained from each patient who developed, fever within 21 days following radiotherapy, chemotherapy and radio chemotherapy. One samples isolated from central venous catheter (if present) and other from peripheral vein. Blood cultures were drawn with a sterile system after a sterile pad was placed below the catheter hub and the hub was disinfected with 10% povidone–iodine. Blood samples were than transferred in culture bottles of brain heart infusion broth. Bottles were incubated at 37°C for 7 days. Simultaneously bottles showing positive growth index from blood culture were gram stained and sub cultured on sheep blood agar, saboured dextrose agar, macconkey agar and nutrient agar, simmon citrate agar and cetrimide agar plates. These plates were than aerobically incubated for 24–48 hours at 37°C temperature for bacterial pathogens isolation and for 24–72 hours at 30°C in B.O.D. incubator for fungal species isolation.
The bacterial pathogens were identified after appearance of growth on sub cultured, plates of blood and oral swab by standard microbiological and biochemical procedures. These biochemical tests include: Carbohydrates fermentation tests, urease tests, oxidase test, haemolysis of blood, catalase test, motility tests and growth pathogens on specific media etc. A preliminary examination of fungal colony on SDA was done through gram stained, smear, formation of germ tube, study of micro morphology, morphology on KOH stained smear, assimilation of carbon and nitrogen[33–36].
All isolated pathogens were compared with MTCC standard strains like S. aureus with MTCC 96 strain, S. epidermidis MTCC 435 strain, P. vulgaris MTCC 426 strain, P. mirabilis MTCC 425 strain, E. coli MTCC 443 strain, K. pneumonia MTCC 109 strain, P. aeruginosa MTCC 741 strain, C. albicans 3017 strain and A. fumigatus 2550 strain.
Absolute neutrophils count
The absolute neutrophils count (ANC) was done by multiplying the total WBC count by percentage of neutrophils (segmented + band)[37
On the basis of ANC the patients were divided into two categories:
Neutropenia: When ANC was less than 500 (severe risk of infection).
Non neutropenia: When ANC was more than 500 (moderate risk of infection).
Basic statistical methods
Means values were reported ± standard deviation (SD). Continuous variables mean values were compared by ‘t’ tests. For independent samples difference in proportion of two groups were compared by chi – square test (with Yates correction) or Fisher’s exact test, when appropriate. All test of significance were two tailed. Alpha was set at 0.05. For the logistic regression odd ratio with 95% confidence interval (CI95) were calculated. Univariate analysis of dichotomous and ordinal variables was performed by using the procedures for matched data seta in the EpiInfo computer Programme (Epi 6.03: centre for disease control and prevention, USA). Conventional statistical methods were used to calculate means and standard deviation with the help of Microsoft excel 2007.