In oral cavity infection the oral micro flora may be subsequently replaced by potentially pathogenic microorganisms, such as pathogenic Gram positive cocci, Gram negative bacilli & Candida spp. There are some histological changes in oral mucosa and salivary glands such as oral mucositis, reduced phagocytic activity of salivary granulocytes and reduced amount of salivary glands facilitate their growth [1]. Opportunistic infections are common in individuals with poor health (an immunocompromised host) and are caused by several different microorganisms, among them representative microorganisms include Staphylococcus aureus, Pseudomonas aeruginosa, and Candida species [2,3]. The colonisation of bacterial and fungal infections in cancer patients varies from institution to institution and depends on use of prophylactic medication and hospital environment. The Bacterial infections are more common during the early phases of a neutropenia whereas, fungal infections occur during the latter part of cancer treatment patients with prolonged period of neutropenia [4]. Most of these organisms become drug-resistant, which has resulted in difficulties in curing the related infectious diseases. This is of utmost importance as most postoperative infections in head and neck have an endogenous source, mainly from the oropharynx [5]. Thus, the study was done to observe the change in oropharyngeal flora in head and neck cancer patients under treatment and to correlate their prevalence according with Chemotherapy cycles and Radiochemotherapy.
Materials and Methods
The present prospective case control study was carried out in the Department of Microbiology at Dr. SN Medical College, Jodhpur, from January 2015 to July 2015 after ethical clearance by local ethical committee. Study group comprised of total 110 patients of head and neck tumours of age 20-80 years, 55 patients on Chemotherapy (CT) and 55 on Radiochemotherapy (RCT). The number of patients was decided according to prevalence of hospital admission of head and neck cancer patient for cancer therapy. Fifty normal healthy attendants of patients were taken as control group whose age and sex were matched with study groups and were apparently healthy without cancer and any systemic diseases. Detailed history was taken about Patient name, age, gender, residence, complain, duration of complain, site and stage of cancer, type of cancer therapy (Chemotherapy, Radiotherapy, or both), duration of therapy, cycle of Chemotherapy, HIV status, history of diabetes, steroid or other drug use, any associated illness, TLC and DLC etc.
Exclusion criteria: The patients with other risk factors for change in oral flora such as diabetes/HIV, recent usage of corticosteroids/antibiotics/immunosuppressive drugs and patients using intra oral prostheses were excluded from the study.
Sample collection & processing: Saliva sample was collected from control and study group after taking consent by oral rinse with phosphate buffer saline for 1 minute in a sterile plastic container. Blood agar, MacConkey agar and SDA with gentamycin were used as primary culture media. The Blood agar, MacConkey agar plates were incubated at 37°C at for 24 hours and SDA plates on 25°C for 7 days. The identification of bacterial isolates was done by standard microbiological methods [6]. The creamy white, smooth, pasty colonies on SDA agar were further subjected to HiCrome Candida Differential Agar (HiMedia, M-1297A) [7], Germ Tube test, Carbohydrate Fermentation Test, and Corn Meal Tween 80 agar streak culture to identify various species of Candida.
Statistical Analysis
Statistical analysis were performed using the software SPSS Incs v.13, Chicago, IL, USA. The frequency of detection of the microorganisms was computed for each subject. Significant differences between patients were tested using the Chi-square test or Fisher’s-exact test. Differences of p<0.05 was considered statistically significant.
Result
Total patient were 110 in which male were 80 (72.8%). The Mean age of patients was 51-60 years (32.7%). Total 62% of the patients were rural and 52.7% from IPD.
The most common cancer was larynx (25.5%) followed by pharynx (20%) in head and neck region in present study group and most common histopathology of head and neck cancer was moderately differentiated squamous cell carcinoma (47.2%).
Out of 110 patients, 71% had single organism while remaining patients have poly-microbial aetiology. Among 149 culture isolates from 110 Patient, GNB was found in 70 patients i.e., 63.6%, Candida spp. in 50%, Staphylococcus aureus in 8% and Normal commensal of oral cavity in 13.6% patients in study group. This was higher than control group and this difference was statistically significant in relation to all isolates individually [Table/Fig-1].
Distribution of micro organism in study group and control group in relation with patient no. {CT= Chemotherapy; RCT= Radiochemotherapy; GPC= Gram positive cocci; GNB= Gram negative bacilli; N.C. =Normal commensal; p-value <0.05 is significant, chi-square test was used to calculate p-values}
Organism | Study Group | Control |
---|
CT | RCT | Total patient (110) |
---|
GNB | 36 | 34 | 70 (63.6%) | 1 (2%) |
GPC-Staphylococcus aureus | 3 | 6 | 9 (8.1%) | 0 |
Candida species | 20 | 35 | 55 (50%) | 10 (20%) |
N.C. (Normal Commensal) | 10 | 5 | 15 (13.6%) | 39 (78%) |
Total isolates | 69 | 80 | 149 | 50 |
Organism | p-value |
CT vs. RCT | Study vs. control |
GNB | 0.842 | <0.0001 |
GPC-Staphylococcus aureus | 0.489 | 0.002 |
Candida species | 0.007 | 0.0004 |
N.C. (Normal Commensal) | 0.266 | <0.0001 |
GNB is almost equally isolated from both group whereas, Candida spp. were more common during Radiochemotherapy (63.6%) but results were non-significant expect for Candida spp. which was significantly high in RCT group of patients. Among GNB Pseudomonas (27.7%, 32.3%) and Klebsiella (25, 29.4%) were most frequently isolated during CT, RCT respectively. Statistical Analysis showed that isolation of Acinetobacter, Citrobacter, Pseudomonas, Klebsiella were significantly high in study group as compared to control group, (Study vs. Control group, p-value <0.0001). However, the results were non-significant when compared CT vs. RCT groups. Candida spp. was more commonly isolated from patient on RCT (63.6%) than CT (36.3%) as compared to control group (20%) among which C. tropicalis was more prevalent species [Table/Fig-2]. In later Chemotherapy cycles (CT4, CT5, CT6), 64% and in early CT cycles (CT1, CT2, CT3) 36% microorganism were isolated. Normal commensal were common in early cycles of Chemotherapy. In early CT cycles percent of GNB and Candida spp. were 44.4%,30% whereas in the later cycles it was 55.6%, 70% respectively; this shows that GNB & Candida spp. were more commonly isolated in later chemotherapy cycles but results were non-significant with CT cycles [Table/Fig-3].
Distribution of micro organism in study group according to treatment status of cancer. {CT1 to CT6= Chemotherapy Cycle No. 1 to 6; RCT= Radiochemotherapy; GPC= Gram Positive Cocci; GNB= Gram Negative Bacilli}
Organism | CT1 | CT2 | CT3 | CT4 | CT5 | CT6 | Total isolates during CT | Total isolates during RCT | p-value |
---|
Total GPC (9) | |
Staphylococcus aureus (9) | 0 | 0 | 1 | 1 | 1 | 0 | 3 | 6 | 0.489 |
Total GNB (70) |
Acinetobacter (10) | 0 | 0 | 1 | 1 | 2 | 1 | 5 | 5 | 0.0 |
Citrobacter(14) | 1 | 0 | 1 | 1 | 2 | 1 | 6 | 8 | 0.779 |
Esch.coli (2) | 0 | 0 | 0 | 1 | 0 | 1 | 2 | 0 | 0.496 |
Enterobacter (4) | 0 | 0 | 2 | 1 | 1 | 0 | 4 | 0 | 0.11 |
Klebsiella (19) | 1 | 1 | 2 | 1 | 2 | 2 | 9 | 10 | 0.800 |
Pseudomonas (21) | 1 | 1 | 2 | 2 | 1 | 3 | 10 | 11 | 0.808 |
Total GNB (70) | 3 | 2 | 8 | 7 | 8 | 8 | 36 (51.4%) | 34 (48.5%) | 0.865 |
Total Candida sp. (55) |
C. albicans(6) | 0 | 0 | 0 | 1 | 1 | 0 | 2 | 4 | 0.678 |
C. dublin-iensis(5) | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 4 | 0.363 |
C. glabrata(6) | 0 | 0 | 0 | 0 | 2 | 0 | 2 | 4 | 0.678 |
C. guiller-mondii(3) | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 0.558 |
C. krusii(4) | 0 | 1 | 0 | 0 | 0 | 1 | 2 | 2 | 0.00 |
C. parap- sillosis(5) | 0 | 0 | 1 | 1 | 1 | 0 | 3 | 2 | 0.647 |
C. pseudo-tropicalis(5) | 0 | 0 | 1 | 1 | 0 | 1 | 3 | 2 | 0.647 |
C. tropicalis (21) | 1 | 1 | 1 | 1 | 1 | 1 | 6 | 15 | 0.050 |
Total Candida (55) | 1 | 2 | 3 | 4 | 6 | 4 | 20 (36.3%) | 35 (63.6%) | 0.007 |
Grand Total (134) | 4 | 4 | 12 | 12 | 15 | 12 | 59 (44%) | 75 (56%) | 0.066 |
Normal commensal |
Normal Commensal (15) | 5 | 1 | 4 | 0 | 0 | 0 | 10 | 5 | 0.143 |
(The value in the bracket denotes the total no. of isolates.
Distribution of Organism in Study Group According To Cycles of Chemotherapy; {CT1 to CT6= Chemotherapy Cycle No. 1 to 6; CT = Chemotherapy; RCT= Radiochemotherapy; GPC= Gram Positive Cocci; GNB= Gram Negative Bacilli}.
Organism | Early CT cycles(CT1, CT2, CT3) | Later CT cycles(CT4, CT5, CT6) | Totalisolate(CT) | Totalisolate(RCT) |
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Total GPC (9) |
Staphylococcus aureus | 1 | 2 | 3 | 6 |
Early vs. Late CT p-value = 1.000 |
Total GNB (70) |
Acinetobacter | 1 | 4 | 5 | 5 |
Citrobacter | 2 | 4 | 6 | 8 |
E.coli | 0 | 2 | 2 | 0 |
Enterobacter | 2 | 2 | 4 | 0 |
Klebsiella | 4 | 5 | 9 | 10 |
Pseudomonas | 4 | 6 | 10 | 11 |
Total | 13(36.1%) | 23(63.8%) | 36 | 34 |
Early vs. Late CT p-value = 0.345 |
Total Candida spp.(55) |
C. albicans | 0 | 2 | 2 | 4 |
C. dubliniensis | 0 | 1 | 1 | 4 |
C. glabrata | 0 | 2 | 2 | 4 |
C. guilliermondii | 0 | 1 | 1 | 2 |
C. krusii | 1 | 1 | 2 | 2 |
C. parapsillosis | 1 | 2 | 3 | 2 |
C. pseudo tropicalis | 1 | 2 | 3 | 2 |
C. tropicalis | 3 | 3 | 6 | 15 |
Total | 6(30%) | 14(70%) | 20 | 35 |
Early vs. Late CT p-value = 0.565 |
Grand Total(134) | 23(39%) | 36(61%) | 59(44%) | 75(56%) |
Early vs. Late CT p-value = 0.385 |
Discussion
This prospective case control study based on evaluation of microorganism of oral cavity isolated as outcomes of various cancer treatment procedures (Chemotherapy, Radiochemotherapy) for head and neck cancer patients. In study group, majority patients were in the age group of 51-60 years (32.7%) and majority of study subjects were male patients (72.8%). This is in accordance to study done by Kamath MP et al., where majority of the subjects were male (75.6%) falling in age group of 51-60 years [8]. Similar results were also seen in a study conducted by Panghal M et al., in which male cases dominated [9]. Majority of patients were of carcinoma of larynx (25.5%) followed by pharynx (20%) in head and neck region, this finding is similar to study done by Rad M et al., Dambroso DA et al., [10,11]. Oropharynx cancer was more common in a study done by Kamath MP et al., and tongue cancer in a study by Panghal M et al., [8,9].
The study done by Kamath MP et al., also showed that normal commensal was significantly reduced in cancer patients compared to controls due to radiation therapy [8]. These cancer patients colonised with a wide variety of Gram positive, Gram negative, aerobic, anaerobic and mycotic pathogens. In present study, GNB isolated in 63.6% patients which is accordance to study done by Panghal M et al., [9] but this finding is in contrast to study done by Yamashita K et al., who found Staphylococcus aureus and Candida spp. commonly isolated [12].
Another salient feature of the study was the colonization of Candida spp. in study group (50%) as compared to control group (20%). Yogitha PPV et al., showed 38% were culture positive for Candida spp. in study group and 18% in control group [13]. This could be due to reduced immunity of the patients due to chemotherapy. Bakki SR et al., also found a high prevelance of Candida colonisation in the study group (38%) as compared to control group (22%) [14]. Prevalance of colonization of Candida spp. were more in the RCT group (63.6%) than CT alone (36.3%), The epidemiology of oral candidiasis shows a wide variation (from 17 to 52.5%) in different studies [15-19].
Among Candida spp., C. tropicalis was most common isolate i.e., 38% which correlates with studies done by Yogitha PPV et al., and Bakki SR et al., [13,14] and contrast with the study done by Safdar A et al., Shaheen et al, and Suryawanshi H et al, reported C. albicans 67.3%, 78%, 78.57% respectively to be the predominant species isolated from patients undergoing RCT or CT [20-22]. The present study showed that changes in the oral and/or systemic environment due to cancer therapy can result in growth of many bacterial and fungal species in oral cavity that can lead to clinical bacterial/fungal infection.
Limitation
The sample size was small as the duration of the study was short (only 6 months) and change in the anaerobic oral micro flora was not studied as there is no anaerobic setup in the institute.
Conclusion
According to this study, colonisation of Gram Negative Bacilli & Candida spp. has linear correlation with immunosuppression and this is directly related to number of Chemotherapy cycles. Hence, it would be advisable that in patients with head and neck malignancy prophylactic medication for these two organisms should be incorporated for treatment algorithm.
(The value in the bracket denotes the total no. of isolates.