|Year : 2017 | Volume
| Issue : 6 | Page : 1505-1510
|Study of possible association of chronic typhoid infection in patients with chronic leukemia
Nilesh Kumar1, Pankaj Kannauje1, Yashwant Kashyap2, Kailash Kumar1, Ranjan Bhattanagar1, Ravindu Tiwari1
1 Department of General Medicine, IMS, BHU, Varanasi, Uttar Pradesh, India
2 Department of Medical Oncology, TMH, Mumbai, Maharashtra, India
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|Date of Web Publication||11-Jan-2018|
| Abstract|| |
Objective: The study of the possible association of chronic typhoid infection in patients with chronic leukemia. Materials and Methods: A total of twenty patients with chronic myeloid leukemia (CML) either newly diagnosed or follow-up patients with or without chemotherapy in the chronic phase of the disease and ten patients with chronic lymphocytic leukemia had comprised of the study. Bone marrow (BM) samples of 20 controls had been taken from the Orthopedic department during surgery. BM was aspirated from iliac crest under aseptic precautions and BM biopsy was done, samples were stored at 4°C. BM samples were subjected for polymerase chain reaction (PCR) analysis for Salmonella typhi. Blood and stool sample from the patients were also collected for Salmonella culture, serological assays. BM samples from control had been taken during orthopedic surgery. Results: S. typhi infection, the comparison between CML patients and healthy controls had been made based on the PCR results. The result is significant with 40% positivity for S. typhi PCR in CML patients when compared to 20% positivity in healthy controls with P = 0.028 which is statistically significant. In patients with chronic lymphocytic leukemia, 10% of BM samples showed positivity for S. typhi by PCR targeting flagellin gene-specific nucleotide sequences and 10% of controls showed positivity for S. typhi and was statistically insignificant (P = 1.00). Conclusion: The early incidence of the disease in tropical country like India favors the hypothesis of chronic inflammation in the early process of leukemogenesis. The result of the study also questions whether attenuated bacteria can be used safely for vaccination and delivering therapeutic agents. Moreover, in a tropical country like India where S. typhi infection is endemic, the infection with the same can be taken as a marker of chronic inflammation and its role in the etiopathogenesis of CML remains to be understood.
Keywords: Leukemia, polymerase chain reaction, typhoid
|How to cite this article:|
Kumar N, Kannauje P, Kashyap Y, Kumar K, Bhattanagar R, Tiwari R. Study of possible association of chronic typhoid infection in patients with chronic leukemia. Ann Trop Med Public Health 2017;10:1505-10
|How to cite this URL:|
Kumar N, Kannauje P, Kashyap Y, Kumar K, Bhattanagar R, Tiwari R. Study of possible association of chronic typhoid infection in patients with chronic leukemia. Ann Trop Med Public Health [serial online] 2017 [cited 2018 Aug 18];10:1505-10. Available from: http://www.atmph.org/text.asp?2017/10/6/1505/222658
| Introduction|| |
The molecular pathogenesis of chronic myeloid leukemia (CML) is well-known but what is the mechanism for gene translocation is still unclear. Various factor such as radiation, impaired immunity, inflammatory bowel disease, obesity, exposure to the pesticide, and benzene had been linked with CML.,
As we know that BCR-ABL fusion transcripts are detectable at low frequency in the blood of many healthy individuals so only presence of the BCR-ABL translocation in a hematopoietic cell is not only sufficient to cause leukemia, because even today it is hidden fact why Ph-positive leukemia develops in a minority of these persons , which further show the importance of dysfunction of immune control in individuals developing full-blown disease. Since CML is the disease of elderly people, the role of immune alteration and chronic infection in its genesis can be considered. The chronic lymphocytic leukemia (CLL) cells are Naive B lymphocytes cell that express CD5, CD19, and CD23, with weak or no expression of surface immunoglobulin (Ig), CD20, CD79b, and FMC7, Ongoing current research suggests that these tumor cells are derived from memory B cells, and there is definite heterogeneity in CLL in form of cellular morphology, phenotype, biology, molecular genetics, and prognosis.,,
Unlike other leukemia, occupational exposure are not related to an increased incidence of CLL., However, a recent study has observed that CLL patients have a higher incidence of having had an infection before the diagnosis as compared to normal population suggesting a role for infection as an etiology CLL. However, no such study was performed from our institution, and there is lack of study which shows there is any association between chronic typhoid infection and leukemia. Therefore, this study aimed to observe and explore possible association of chronic typhoid infection and chronic leukemia particularly CML and CLL.
| Materials and Methods|| |
The present study entitled “Study of the possible association of Chronic Typhoid infection in Patients with Chronic Leukemia” was carried out in Department of Medicine, and Department of Microbiology, Sir Sunderlal Hospital, Banaras Hindu University, Varanasi. A total of 20 cases of CML in chronic phase and 10 cases of CLL comprised of the study. Twenty controls had taken from orthopedic department admitted for orthopedic related surgeries. The study period was from January 2014 to May 2015.
To evaluate the evidence regarding the association of bacterial infection (Salmonella typhi [S. typhi]) with the development of chronic leukemia's (CML and CLL) observational study, patients attending Hematology outpatient department with CML and CLL. Twenty patients with CML either newly diagnosed or follow-up patients with or without chemotherapy in the chronic phase of the disease and ten patients with chronic lymphocytic leukemia had comprised of the study. Bone marrow (BM) samples of 20 controls had been taken from the orthopedic department during surgery. Any patient having hepatitis B virus surface antigen, hepatitis C virus, HIV positivity or having any history of radiation therapy or exposure to chemical carcinogens were excluded from study.
BM was aspirated from iliac crest under aseptic precautions and BM biopsy was done, samples were stored at 4°C. BM samples were subjected for polymerase chain reaction (PCR) analysis for S. typhi. Blood and stool sample from the patients were also collected for Salmonella culture, serological assays. BM samples from control had been taken during orthopedic surgery.
Blood sample was inoculated directly onto blood agar, McConkey agar (MA), deoxycholate citrate agar (DCA) and Selenite F broth (for enrichment). Inoculated media was incubated overnight at 37°C aerobically. Subcultures were made from Selenite F Broth on solid plates (DCA and MA). The bacterial growth was identified using standard recommended techniques.
For detection of S. typhi infection, antibody titer against somatic (TO) and flagellar (TH) were determined by the Widal test using a colored antigen kit (Span Diagnostics, Surat, India). A titer ≥1:160 are considered to be positive result.
Identification of Salmonella typhi antigen in bone marrow samples through polymerase chain reaction
It had been successful in detecting S. typhi in specimen by PCR targeting flagellin (fliC) gene sequences. Initially, the bacterial DNA is extracted from the BM aspirate following the standard phenol chloroform method following which the same will be subjected to PCR using primer specific to fliC gene of S. typhi.
Preparation of reagents/chemicals
- TE Buffer (Tris- ethylenediaminetetraacetic acid [EDTA]) – 2 ml of 1M tris HCl (pH 7.5) and 0.8 ml of 0.25 M EDTA (pH 8.0) were added and volume was raised up to 200 ml with distilled water. It was autoclaved and stored at 4°C. Working concentration = ×1
- Ten percent sodium dodecyl sulfate (SDS) – Dissolved 10 g of SDS in 100 ml of Distilled water. This stock solution is stable for 6 months at room temperature
- Proteinase-K – 5 mg Proteinase K was dissolved in 1 ml of sterile distilled water. This solution is stored in small aliquots at 4°C in refrigerator
- 10% C-TAB – 10 ml of 1M TrisHCl, 28 ml of 5M NaCl, 4 ml of 0.5M EDTA and 10 g of CTAB (cetryltrimetythyl ammonium bromide) was added in 100 ml of sterile distilled water
- Phenol: Chloroform: Isoamyl alcohol (25:24:1) – 2 ml of isoamyl alcohol and 48 ml of chloroform and 50 ml of Phenol solution was mixed to prepare the solution when required
- Chloroform: Isoamyl alcohol (24:1) – 2 ml of isoamyl alcohol and 48 ml of chloroform was mixed to prepare solution when required. It helps in deprotenization
- Iso-propanol – It selectively precipitates DNA
- 70% Ethanol – 30 ml of Distilled water was added to absolute ethanol. It is used to wash the DNA pellets
- TE buffer (pH = 8.0) – 2 ml of 1M trisHCl (pH 7.5) and 0.8 ml of 0.25 M EDTA (pH 8.0) were added and volume was raised up to 200 ml with distilled water. It was autoclaved and stored at 40°C adjust pH to 8.0. Working concentration = ×1.
Polymerase chain reaction for primary cycle
Procedure for polymerase chain reaction mix preparation
At the start of PCR, the DNA from which a segment is to be amplified, and excess of two primer molecules, the four deoxyribose triphosphates and the DNA polymerase are mixed together in the reaction in the fallowing manner– First, the master mixture for the PCR was made in an Eppendorf and template was added at last after distributing the master mixer, i.e., the reaction volume (25 μl) in each PCR tubes. All the tubes are kept in thermo cycler and the corresponding program is used for PCR.
Master mix preparation for primary cycle
×10 buffer with MgCl2 2.5 μL
dNTP mix 2.0 μL
Primer F1 1.0 μL
Primer R1 1.0 μL
Taq polymerase 0.3 μL
Milique Water 13.2 μL
Templete 5.0 μL
Total Volume = 25 μL × n Numbers Of Samples + negative control and positive control
Master mix was prepared and 20 μl was aliquot in numbered PCR tubes and to them added 5 μL of Templates.
The primers used are:
- Flagellin (fliC) gene
- Primary amplification
- F1-5'-ACT GCT AAA ACC ACT ACT-3'
- R1-5'-TTA ACG CAG TAA AGA CAG-3'
- 495 bp
Nested Polymerase chain reaction
Nested PCR involves two sets of primers, used in two successive runs of PCR, the second set intended to amplify a secondary target within the first run product. It increases the specificity and eliminates unwanted products in PCR.
Master mix preparation for nested cycle
10X buffer with MgCl2 2.5 μL
dNTP mix 2.0 μL
Primer F2 1.0 μL
Primer R2 1.0 μL
Taq polymerase 0.3 μL
Milique Water 17.2 μL
Template 1.0 μL (Primary PCR product)
Total volume = 25 μL × n numbers of samples + negative control and positive control
Master Mix was prepared and 24 μL was aliquoted in numbered PCR tubes and to them added 1 μL of Primary PCR Products.
The primers used for nested polymerase chain reaction are:
- Flagellin (fliC) gene
- Nested amplification
- F2-5'-AGA TGG TAC TGG CGT TGC TC-3'
- R2-5– TGG AGA CTT CGG TCG CGT AG-3'
- 364 bp
Procedure for agrose gel electrophoresis
- Mixed 750 mg (1.5%) of agarose in 50 ml of 1X TBE (Trisbiric acid EDTA) buffer
- Boiled this till it becomes transparent liquid
- Let this cooled to 45°C–50°C
- Mixed 5 μl of ethedium bromide for florescence in this cooled liquid, this acts as intercalating agent that binds to major groove of DNA
- Pour the liquid in already cleaned comb and tray for solidification.
- Kept it in Electrophoresis unit for DNA loading
- On paraffin paper 2 μl of bromophenol blue dye is mixed with 10 μl of PCR product
- Transfer the mixture in specified wells
- First given high Voltage 200+ for 10 min. when the DNA is out of the well, then 75 Volts is given to precede the bands to desired distance
- Kept the gel in alpha imager to see the result on computer.
After running the gel the fragments can be seen with a UV light. The reason we can see the fragments with UV light is due to the Ethidium bromide dye that has been added to the gel. This dye will adhere to the DNA in the gel and when the gel is finished it can be viewed under a UV light and can be photographed.
Mean, standard deviations had been calculated for quantitative variables. For qualitative and categorical variables, Chi-square and Z-test had been applied to test the significant difference between two proportions. P < 0.05 considered as statistically significant. Sensitivity, specificity, positive productive value, negative productive value, and accuracy of serological tests had been calculated at different cutoff points in respect of PCR for S. typhi.
| Results|| |
Out of 20 cases of CML, 12 cases were female (60%) and 8 cases (40%) were male, most of the patients were between the age group of 40 and 60 years (60%), most common presentation of the patient was generalized weakness (90%), 11 cases (55%) have hemoglobin <10 during presentation, 11 cases (55%) have total leukocyte count between ten thousand to one lakh, 13 cases (65%) are newly diagnosed cases, The mean age of the Patients studied is 46 years which is quite low when compared to western population where the incidence of CML is after 60 years of age, more proportion of female patients (50% vs. 25%), more proportion of patients belonging to lower socioeconomic state (50% vs. 40%) showed positive result for S. typhi in our study, although none of the result is statistically significant and probably because of small number of cases.
Since detection and sequencing of fliC gene-specific nucleotide sequences of S. typhi and nested PCR is superior to culture and serology in detecting the presence of S. typhi in study samples and PCR is considered the gold standard in diagnosing S. typhi infection, the comparison between CML patients and healthy controls had been made based on the PCR results. The result is significant with 40% positivity for S. typhi PCR in CML patients when compared to 20% positivity in healthy controls with P = 0.028 which is statistically significant [Table 1], [Table 2], [Table 3].
|Table 1: Comparison of nested polymerase chain reaction for Salmonella typhi in bone marrow sample of patients with chronic myeloid leukemia and controls|
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|Table 2: Comparison of Widal test in patients with chronic myeloid leukemia and healthy controls|
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|Table 3: Comparison of Salmonella typhi blood culture in patients with chronic myeloid leukemia and healthy controls|
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The following table compares the sensitivity, specificity, positive predictive and negative predictive values of serological assay (Widal test) and blood culture with respect to PCR results in this study. The comparative study made between Widal test and blood culture for S. typhi vis-a-vis with nested PCR as standard test, showing Widal as more sensitive and blood culture as more specific in detection of S. typhi infection [Table 4].
|Table 4: Comparative evaluation of tests (Widal and blood culture) used in detection of Salmonella typhi taking Bone marrow in chronic myeloid leukemia patients as the standard|
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Out of 10 cases of CLL, 6 cases were female (60%) and 4 (40%) were male. Most of the patients were between the age group of 40 and 60 years (60%). Most common presentation of the patient was generalized weakness (80%). Only three cases (30%) have hemoglobin <10 during presentation. Six cases (60%) have total leukocyte count between 10,000 and 100,000. The mean age of the patients studied is 55 years which is low when compared to western population where the incidence of CML is after 60 years of age.
In patients with CLL, 10% of BM samples showed positivity for S. typhi by PCR targeting fliC gene specific nucleotide sequences and 10% of controls showed positivity for S. typhi and was statistically insignificant (P = 1.00). Blood culture was negative in test as well as control group whereas, Widal positivity in both group comparable [Table 5], [Table 6], [Table 7].
|Table 5: Nested polymerase chain reaction for Salmonella typhi in bone marrow of patients with chronic lymphocytic leukemia and controls|
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|Table 6: Widal test in patients with chronic lymphocytic leukemia and healthy controls|
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|Table 7: Salmonella typhi blood culture in patients with chronic lymphocytic leukemia and healthy controls|
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| Discussion and Conclusion|| |
Although scientific knowledge in viral oncology has exploded in the 20th century, the role of bacteria as mediators of oncogenesis has been less well-elucidated. Understanding bacterial carcinogenesis has become increasingly important as a possible means of cancer prevention. As cancer continues its climb as the leading cause of death in developed nations, understanding the long-term effects of bacteria has become increasingly important as a possible means of cancer prevention. Chronically inflamed tissue may lead to cancer development which may be either infectious or noninfectious inflammation. I have tried to show relationship of bacteria in causation of oncogenesis.
Much effort has been applied to understanding the bacterial mechanisms that might influence oncogenesis. Posited mechanisms include deleterious alterations in physiological host processes such as inflammation, antigen-driven lymphoproliferation, and induction of hormones that increase epithelial cell proliferation. Bacteria may also promote cancer through direct effects on cell transformation or through the production of toxic, carcinogenic metabolites.
Areas of tissue injury and inflammation trigger regenerative cell division from tissue and marrow-derived stem cells. Increased cell division may lead to point mutations, deletions, or translocations as damaged DNA escapes the repair system; the aberrant DNA is then propagated by subsequent cell division. This can result in disordered cell differentiation and ultimately, oncogenesis. Hence, chronic inflammation instigates a cycle of cell damage, repair, and compensatory proliferation that promotes the development of cancer cells.,
Chronic typhoid carrier state was shown to be the only independent risk factor for the development of gallbladder carcinoma. Finally, studies demonstrated that typhoid carriage was not only associated with an increased risk of gallbladder cancer but also with malignancy in the pancreas, lung, and colorectum.
The challenge of finding and understanding the true associations between bacterial infections and human cancers is indeed great, but it also promises great rewards. Unlike viral infections, bacterial infections are typically curable, and the prospect of antibiotic treatments to prevent, alleviate, or cure cancers is obviously alluring. When the pathways toward malignancy are initiated and when they become irreversible, though, are not fully understood. Vaccination against etiologic pathogens to prevent infection and thus eliminate the risk of cancer is yet another hopeful prospect for researchers.
Pinpointing specific bacterial causes of cancer however has been challenging. The colon alone, for example, harbors >500 species of bacteria.
Recent research has uncovered a great deal of information regarding the bacterial mechanisms used to cause, colonize, or cure cancer; however, many questions remain. For example, Do the bacteria in question initiate, promote, or merely show affinity for the neoplasm? Does cancer weaken the host which facilitates acquiring the infection? Can the highly site specific colonization of certain bacteria for a tumor be clinically useful in diagnosis or treatment?
Could attenuated bacteria be used in vaccines to safely and effectively deliver therapeutic agents? The continued exploration of these questions will bring research ever closer to the prevention, early diagnosis, and truly effective treatment of this scourge of humanity.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of General Medicine, IMS, BHU, Varanasi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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