|Year : 2019 | Volume
| Issue : 4 | Page : 514-520
Utility of dried blood spots in detecting helicobacter pylori infection
Abhinendra Kumar, Sharayu Mhatre, Rajesh Dikshit
Centre for Cancer Epidemiology, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Centre; Homi Bhabha National Institute (HBNI), Training School Complex, Anushaktinagar, Mumbai, Maharashtra, India
|Date of Submission||16-Nov-2019|
|Date of Acceptance||16-Apr-2020|
|Date of Web Publication||18-May-2020|
Dr. Rajesh Dikshit
Centre for Cancer Epidemiology, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Centre, Sector 22, Utsav Chowk, Kharghar, Navi Mumbai - 410 210, Maharashtra
Source of Support: None, Conflict of Interest: None
Purpose: Identifying infectious pathogens by collecting intravenous blood (IVB) is a well-established procedure, however, the collection of IVB in field epidemiological study is challenging. The dried blood spot (DBS) as an alternative to IVB has been introduced, although, there is a limited study to demonstrate the utility of DBS stored at various storage conditions and transported at different periods. This is an observational study, which evaluates the effectiveness of DBS in field epidemiological studies to identify infectious pathogens. Materials and Methods: A total of 264 paired DBS samples prepared from IVB, stored at 4°C, −20°C after period 24, 48 and 72 h. Serologically, enzyme-linked immunosorbent assay [ELISA] IgG antibody detected against Helicobacter pylori infection from DBS and compared with IVB. Results: Quantitatively, IgG antibody reactivity showed >87% correlation between IVB and DBS samples stored at 4°C or −20°C within 48 h of transport duration. DBS stored at 4°C shows, equal sensitivity 87.5% and specificity 95% before 48 h of transport duration, while at −20°C storage similar sensitivity 87.5% observed but slightly less specificity 86.36% observed as compared to 24 h of transport duration. One-way analysis of variance showed, nonsignificant difference at both (−20°C and 4°C) the stored condition with P value (P > 0.851) and (P > 0.477). Kappa values showed good inter-rater reliability between DBS and IVB in a range (0.77–0.81). Conclusion: No significant difference was observed in detecting H. pylori when ELISA was conducted using IVB or DBS stored at 4°C and transported even after 48 h. This confirms that DBS collected even in compromised conditions in the field can be used for detecting infection.
Keywords: Dried blood spot, epidemiology, gastric cancer, Helicobacter pylori, serology
|How to cite this article:|
Kumar A, Mhatre S, Dikshit R. Utility of dried blood spots in detecting helicobacter pylori infection. Indian J Med Microbiol 2019;37:514-20
|How to cite this URL:|
Kumar A, Mhatre S, Dikshit R. Utility of dried blood spots in detecting helicobacter pylori infection. Indian J Med Microbiol [serial online] 2019 [cited 2020 Sep 30];37:514-20. Available from: http://www.ijmm.org/text.asp?2019/37/4/514/284514
| ~ Introduction|| |
The idea of sampling biological fluids was given by Ivar Bang nearly a century ago. The concept that capillary whole blood, obtained by heel or finger prick and blotted on to a filter paper (Guthrie card), could be used to screen for metabolic disease in large populations of neonates. In preparation of Dried Blood Spot (DBS), few microliter bloods is required as compared to venous blood for testing of analytes and are generally nonhazardous and can be easily transported from remote area to laboratory site. However, DBS have not been used for regular serological testing as it is not been proven to be useful for all types of analytes. DBS has been used for many decades in the detection of infectious disease pathogens,,,,,,, despite of this there are no guidelines reporting the impact of transport and storage condition of DBS on the stability of antibodies on filter paper.
DBS is widely used in epidemiological studies, diagnosis and monitoring the virus infection including HIV, Cytomegalovirus, hepatitis B virus, Epstein–Barr virus, Rubella and dengue virus.,,, Few studies analysed the correlation coefficient (r = 0.92) and observed no significant changes in DBS viral load even after storage at room temperature for 12 weeks while high sensitivity and specificity (>98%), observed for DBS hepatitis C virus serology. The previous published studies shows the etiology of Helicobacter pylori infection for the development of gastrointestinal disease.H. pylori is a microaerophilic fastidious human pathogen. The bacterium has been implicated in acid peptic diseases of the stomach and duodenum and neoplasm of the stomach.,,H. pylori have been detected in individuals of all ages throughout the world and its prevalence ranges between 20% and 80%.
It is challenging to collect, transport and storage of intra-venous blood (IVB) in field settings due to limited infrastructure. The DBS as an alternative to IVB has been introduced, the objective of this study is to demonstrate the utility of DBS stored at various storage conditions and transported at different periods to identify the infectious pathogens (H. pylori) and compared the results with paired IVB used as gold standard. To evaluate the effectiveness of DBS cards, each paired DBS sample compared 6 times and results were correlated with IVB.
| ~ Materials and Methods|| |
Subject enrollment and blood collection
An observational study has been conducted between January 2018 and May 2018 at Centre for Cancer Epidemiology, Tata Memorial Centre, Mumbai (Maharashtra). The study participants with age group between 18 and 69 years have been enrolled in this study after informed consent obtained from subjects. With 0.8 power, 90% correlation at standard condition and an estimated 70% correlation at stored condition, 88 paired sample sizes were obtained. This study was approved by the Institutional Ethical Committee of the organisation. A volume of 3 ml–4 ml venous blood has been collected by trained staff in ethylenediaminetetraacetic acid tubes and immediately transported to the laboratory for further processing.
Dried blood spot preparation and storage
The Whatman 903 (GE Health Care, US) blood collection card is labeled with sample ID, date of blood collection, *transport duration and storage temperature. To maintain the homogenous nature of blood spots, 50 μl of full blood was immediately applied to 5 spots on DBS filter paper cards (Whatman 903 Protein Saver Blood Collection Cards; Schleicher and Schuell). Remaining IVB centrifuged at 3000 rpm/4°C for 15 min, plasma (gold standard) separated and stored at −80°C. Blood spots dried at room temperature for 2–3 h with the help of the Whatman card rack [Supplementary 1] without applying any direct heat, blower, dryer, etc., While in the drying process, the surface of DBS have not to touch with any other surface to prevent it from any type of contamination. DBS cards packed in airtight ziplock bags [Supplementary 2] with 1–2 g desiccant sachets and stored at respective temperatures (4°C and −20°C). Desiccant sachets act as an adsorbent to remove any moisture from blood spots to prevent any type of contamination/fungal infection. Packed DBS cards stored at 4°C and −20°C within 24 h, 48 h and 72 h of transport duration.
Terminologies used for stored dried blood spot samples
- DBS1: DBS sample stored at 4°C within 24 h of transport duration
- DBS2: DBS sample stored at −20°C within 24 h of transport duration
- DBS3: DBS sample stored at 4°C within 48 h of transport duration
- DBS4: DBS sample stored at −20°C within 48 h of transport duration
- DBS5 DBS sample stored at 4°C within 72 h of transport duration
- DBS6: DBS sample stored at −20°C within 72 h of transport duration.
Validity and processing of dried blood spot samples
In the Valid DBS card, blood-filled completely in each circle of blood collection card and spread homogeneously, whereas invalid samples are scratched, half-filled, overlapped, merged with other circles and not dried completely. Only valid DBS samples used for serology of H. pylori infection. Excise single 6 mm diameter blood spot from DBS card with the help of 6 mm steel punch plier, add 500 μl assay diluent buffer (provided in the kit) and shake at 200 rpm for 2–3 h at room temperature, the elute directly used as a sample for assay [Supplementary 3].
Assay protocol and data analysis
Commercial available enzyme-linked immunosorbent assay ELISA kit (IBL International H. pylori IgG ELISA, RE56381) used for detection of IgG antibody against H. pylori infection. As per kit standards, the cut off valve for the seropositivity of H. pylori infection was >12 U/ml. Standard curve have been prepared from available standards in kit with their tested IgG antibody concentrations (A = 1U/ml, B = 10 U/ml, C = 25 U/ml and D = 150 U/ml) and quantifying sample result according to standard curve. The scattered plot was prepared to observe the correlation coefficient [Figure 1] at all transport duration and stored thermal conditions. Prepared the Bland-Altman (BA) plot [Figure 2] of the difference between DBS and IVB to evaluate bias in the mean concentration of antibody. t-test [Table 1] and one-way Analysis of Variance (ANOVA) [Table 2] test conducted to compare mean all DBS samples stored at 4°C and −20°C with IVB. All quantitative data analysed using statistical software Excel, STATA 15.0 Details (StataCorp LLC, Texas, USA and Graph-Pad. P value (P ≤ 0.05) at 95% confidence interval (CI) considered a statistically significant difference.
|Figure 1: Scattered plot for comparison of correlation between plasma (intravenous blood) and dried blood spot stored at 4°C (dried blood spot 1, dried blood spot 3, dried blood spot 5) and −20°C (dried blood spot 2, dried blood spot 4, dried blood spot 6)|
Click here to view
|Figure 2: Bland-Altman Graph, a plot of difference of IgG antibody concentration against Helicobacter pylori infection between plasma and dried blood spot, plotted against average of plasma (intravenous blood) and dried blood spot stored at 4°C (dried blood spot 1, dried blood spot 3, dried blood spot 5) and −20°C (dried blood spot 2, dried blood spot 4, dried blood spot 6)|
Click here to view
|Table 1: t-test, immunoglobulin G antibody mean concentration comparison between intravenous blood and each group of dried blood spot stored at 4°C and -20°C|
Click here to view
|Table 2: One-way analysis of variance test, immunoglobulin G antibody mean concentration comparison between intravenous blood and paired dried blood spot samples stored at 4° and 20°C|
Click here to view
| ~ Results|| |
DBSs prepared from IVB and stored at the different thermal conditions at varying time periods. DBS samples were stored for 5 months before the experiment was conducted. Serological experiments were performed in our laboratory at 22°C temperature with humidity range 75%–85%. The sandwich ELISA assay performed as per kit protocol and data were analysed quantitatively through the standard curve.
IgG Seropositivity against Helicobacter pylori infection
A total of six groups were created from DBS1 to DBS6 with 44 DBS samples in each group, overall 264 DBS replicates tested for the detection of IgG antibody titer against H. pylori infection and compared with IVB. The blood sample >12 U/ml IgG antibody titer consider as positive for H. pylori infection. Of the 44 IVB samples, 24 (54%) samples were detected IgG positive and 20 (46%) samples were IgG negative against H. pylori infection. All 264 DBS samples were tested for IgG seropositivity in each group. 50% (22/44) samples were detected IgG seropositivity for group DBS1, 52% (23/44) samples for DBS2, 50% (22/44) for DBS3, 50% (22/44) for DBS4, 65% (29/44) for DBS5 and 50% (22/44) for DBS6. On average, 50%–52% DBS samples detected seropositive for H. pylori infection as compared to IVB (54%) irrespective of transport duration and storage condition, but group DBS5 shows the highest seropositivity (65%), and it might be possible due to chance.
Dried blood spot sensitivity and specificity
Although our method is qualitative and quantitative, the specific aim of this essay is to identify H. pylori infection through qualitative assay but quantitative data analysed through statistical methods. To evaluate the performance of our assay, we used DBS replicate samples derived from IVB. DBS sensitivity and specificity for Whatman 903 blood collection card were defined for IgG-H. pylori ELISA only. IVB IgG was used as a reference to determine the positive and negative status of antibodies, as well as to define false positive (FP) and false negative (FN) for each DBS analyzed, whereas sensitivity and specificity of DBS calculated by 2 × 2 table below [Table 3].
|Table 3: 2 x 2 table to calculate sensitivity and specificity of diagnostic test|
Click here to view
Similar sensitivity (87.5%) observed for all DBS groups except DBS5 (100%), there is no FN sample observed for DBS5. While there is slight variation observed for specificity in DBS2 (90%) and DBS5 (75%), rest of the DBS groups have similar highest specificity (95%) [Table 4].
|Table 4: Sensitivity and specificity of dried blood spot compared to intravenous blood in immunoglobulin G Helicobacter pylori ELISA|
Click here to view
The proportion of positive and negative infection also calculated with positive predictive values (PPV) and negative predictive values (NPV), and the strength of agreement between DBS and IVB was depicted by kappa statistics. At 4°C storage, excellent PPV reported (95.45%) for DBS at 48 h of transport duration, but slightly decreased in PPV (82.75%) at transport duration within 72 h. While at −20°C storage, good PPV value reported (91.30%) within 24 h of transport duration, but slightly increased in PPV value for DBS4, DBS6 (95.45%), and there is no specific difference observed at 48 and 72 h of transport duration. A large percentage of PPV and NPV values indicates that the small FP and FN detected through this testing procedure are true and correct. Kappa statistics are used to measure inter-rater reliability for a qualitative test. It is a more robust measure agreement between two raters. It is expressed as K = Po−Pe/1−Pe, where Po is a relative observed agreement among raters and Pe is the hypothetical probability of chance agreement. Overall good agreement (K = 0.77–0.81) observed between all the DBS groups (DBS1–DBS6) and IVB, respectively, indicating very strong correlation beyond chance [Table 5].
|Table 5: Positive predictive value and negative predictive value of dried blood spot compared to intravenous blood in immunoglobulin G Helicobacter pylori ELISA|
Click here to view
Evaluate correlation coefficient between dried blood spot and intravenous blood
Correlation is a statistical technique, that shows how strongly pairs of variables are related, and it is denoted as the correlation coefficient®. Correlation coefficient computed as the ratio of covariance between variables to the product of their standard deviation and its value ranges from −1 to +1. Our results indicate that overall quantitative DBS and intravenous results are well comparable. Overall the correlation ® between DBS and IVB were high, ranging from (R = 0.83 to 0.89) for all DBS groups (DBS1–DBS6). At 4°C storage of DBS, it showed small variation in correlation between DBS/IVB (R = 0.87 for DBS1, 0.88 for DBS3 and 0.86 for DBS5). These results reflect, quantitatively there is a moderate difference observed in IgG antibody titers between DBS/IVB irrespective of transport duration. While at −20°C storage, correlation decreases continuously at transport duration of 24, 48 and 72 h (R = 0.89 for DBS2, 0.88 for DBS4 and 0.83 for DBS6) [Figure 1]. The result showed, IgG antibody stability decrease with an increase in transport duration, but there is no such big difference observed when DBS transported before 48 h and −20°C storage.
Evaluate comparison between two groups: t-test
Quantitatively, the group mean of IVB (36.68 ± 45.89) at 95% CI (22.72–50.63) compared with mean of DBS1 (30.29 ± 39.10) at 95% CI (18.40–42.18), statistically non-significant difference observed with P value (P > 0.063), while significant difference observed for DBS3 (29.48 ± 37.15) at 95% CI (18.19–40.78) and DBS5 (26.74 ± 31.35) at 95% CI (17.20–36.27) with P value (P > 0.034) and (P > 0.009), respectively. Good result obtained for DBS groups stored at −20°C (DBS2, DBS4 and DBS6) as compared to 4°C, statistically non-significant difference observed for all three groups with P value (P > 0.620), (P > 0.136) and (P > 0.245), respectively. No difference observed in IgG concentration between DBS (DBS2, DBS4, DBS6) stored at −20°C and IVB. We can detect infection even if DBS transported within 72 h at ambient temperature and stored at −20°C.
Evaluate comparison between multiple groups: Analysis of variance
One-way ANOVA test has been conducted to compare IgG antibody concentration between IVB and DBS groups stored at 4°C (n = 132) and −20°C (n = 132) irrespective of transport duration. Statistically there is non-significant difference observed at both the stored temperature with their P values (P > 0.477) and (P > 0.26) and corresponding F-value (F = 0.83) and (F = 0.26). Result shows more similarity observed in IgG concentration between DBS/IVB at 4°C as compared to − 20°C storage, because F value 0.83 indicate that the within groups variance is 0.17 (1−0.83 = 0.17) times the size of the between group variance, whereas at −20°C storage it shows within group variance is 0.74 (1 − 0.26 = 0.74) times the size of the between group variance.
Evaluation of Bland-Altman plot
Altman plot is an alternative analysis, based on the quantification of the agreement between two quantitative measurements by studying the mean difference and constructing limits of agreement. It is a simple way to evaluate bias between mean differences and to estimate an agreement interval, within which 95% of the differences of the second method, compared to the first one, fall. The BA plot method only defines the intervals of agreements, it does not say whether those limits are acceptable or not. Acceptable limits must be defined a priori, based on clinical necessity, biological considerations, or other goals.
Our result plotted the difference of IgG concentration between IVB and all DBS groups (DBS1–DBS6) separately. BA plot shows, more differences observed between DBS and IVB at 4°C storage as compared to −20°C storage.(6.384), (7.193) and (9.939) mean differences observed for DBS1, DBS3 and DBS5 while (1.536), (4.793) and (4.488) observed for DBS2, DBS4 and DBS6. Based on the BA plot results, DBS should be transported within 24 h and stored at −20°C rather than beyond 24 h. At 4°C storage, it is clear that with the increase in transport duration, the mean difference also increases between DBS and IVB.
| ~ Discussion|| |
This study aimed to evaluate, whether the DBS is an alternative to IVB in the field setting, as DBS is an attractive tool for field studies as only finger prick is required for its collection and it can be easily transported from remote area to laboratory. However, its use in field studies is limited as detailed guidelines for storage and transportation are not standardised. Previous studies have demonstrated usefulness of DBS in performing HIV and other infections.,, It has been argued that different downstream application requires different storage and transport conditions. For example, DNA can be extracted effectively from DBS stored at −20°C if transported before 24 h. While different storage and transport conditions are required for analysis of biochemical parameters and infectious agents. In the present study, we discuss about conditions required for storage and transport of DBS to investigate H. pylori infection.
We demonstrated that for testing pathogens there is no effect of transport duration even if samples are transported after 48 h after proper drying. These findings are important for large scale epidemiological studies when carried out in remote areas with poor access to immediate transport and samples cannot be reached within 24 h. The study also recommends that once samples are transported to study center, the best way to store them is at −20°C before further testing is done on the samples. Many other studies have evaluated the usefulness of DBS in disease surveillance and testing for pathogens including HIV, Hepatitis B, H pylori; however, none of them have evaluated for transport duration and different storage conditions.
Our results indicate that overall DBS and IVB result are well comparable; however, we have observed slight deviation in correlation coefficient. ANOVA result shows, there is no significant difference observed at both the storage temperature, while quantitatively less difference observed in IgG concentration at − 20°C rather than at 4°C. With qualitative ELISA, infectious agents can be detected at both the storage temperature due to observed higher sensitivity and specificity; however quantitatively, low IgG antibody titer detected at 4°C as compared to −20°C. With these results, we can justify that DBS can be transport within 48 h and must be stored at −20°C or −80°C to restore the stability of antibodies titers on DBS.
| ~ Conclusion|| |
DBS offer an excellent method for blood collection to measure infectious pathogens. As stomach cancer is common cancer sites, DBS can be used to understand burden of H. pylori infection in India and to conduct randomised trials to eradicate H pylori infection and reduce mortality from stomach cancer.
Authors are thankful to the Director, Tata Memorial Centre, Mumbai for providing the necessary facilities to carry out the research on DBS.
Financial support and sponsorship
This study was financially supported by Tata Memorial Centre, Mumbai.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Schmidt V. Ivar Christian bang (1869-1918), founder of modern clinical microchemistry. Clin Chem 1986;32:213-5.
Guthrie R, Susi A. Simple phenylalanine method for detecting phenylketonuria in large populations of infants. Pediatrics 1963;32:338-43.
Fischer A, Lejczak C, Lambert C, Servais J, Makombe N, Rusine J, et al
. Simple DNA extraction method for dried blood spots and comparison of two PCR assays for diagnosis of vertical human immunodeficiency virus type 1 transmission in Rwanda simple DNA extraction method for dried blood spots and comparison of two PCR assays fo. J Clin Microbiol 2004;42:1-6.
Mercader S, Featherstone D, Bellini WJ. Comparison of available methods to elute serum from dried blood spot samples for measles serology. J Virol Methods 2006;137:140-9.
Barbi M, Binda S, Caroppo S. Diagnosis of congenital CMV infection via dried blood spots. Rev Med Virol 2006;16:385-92.
Luo W, Yang H, Rathbun K, Pau CP, Ou CY. Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay. J Clin Microbiol 2005;43:1851-7.
Fachiroh J, Prasetyanti PR, Paramita DK, Prasetyawati AT, Anggrahini DW, Haryana SM, et al
. Dried-blood sampling for epstein-barr virus immunoglobulin G (IgG) and IgA serology in nasopharyngeal carcinoma screening. J Clin Microbiol 2008;46:1374-80.
Iroh Tam PY, Hernandez-Alvarado N, Schleiss MR, Hassan-Hanga F, Onuchukwu C, Umoru D, et al
. Molecular detection of Streptococcus pneumoniae
on dried blood spots from febrile Nigerian children compared to culture. PLoS One 2016;11:e0152253.
Waterboer T, Dondog B, Michael KM, Michel A, Schmitt M, Vaccarella S, et al
. Dried blood spot samples for seroepidemiology of infections with human papillomaviruses, Helicobacter pylori
, Hepatitis C Virus, and JC Virus. Cancer Epidemiol Biomarkers Prev 2012;21:287-93.
Snijdewind IJ, van Kampen JJ, Fraaij PL, van der Ende ME, Osterhaus AD, Gruters RA. Current and future applications of dried blood spots in viral disease management. Antiviral Res 2012;93:309-21.
Helfand RF, Keyserling HL, Williams I, Murray A, Mei J, Moscatiello C, et al
. Comparative detection of measles and rubella IgM and IgG derived from filter paper blood and serum samples. J Med Virol 2001;65:751-7.
Condorelli F, Scalia G, Stivala A, Gallo R, Marino A, Battaglini CM, et al
. Detection of immunoglobulin G to measles virus, rubella virus, and mumps virus in serum samples and in microquantities of whole blood dried on filter paper. J Virol Methods 1994;49:25-36.
Stene-Johansen K, Yaqoob N, Overbo J, Aberra H, Desalegn H, Berhe N, et al
. Dry blood spots a reliable method for measurement of hepatitis b viral load in resource-limited settings. PLoS One 2016;11:e0166201.
Croom HA, Richards KM, Best SJ, Francis BH, Johnson EI, Dax EM, et al
. Commercial enzyme immunoassay adapted for the detection of antibodies to hepatitis C virus in dried blood spots. J Clin Virol 2006;36:68-71.
Judd A, Parry J, Hickman M, McDonald T, Jordan L, Lewis K, et al
. Evaluation of a modified commercial assay in detecting antibody to hepatitis C virus in oral fluids and dried blood spots. J Med Virol 2003;71:49-55.
Perez-Perez GI, Rothenbacher D, Brenner H. Epidemiology of Helicobacter pylori
infection. Helicobacter 2004;9 Suppl 1:1-6.
Correa P, Houghton J. Carcinogenesis of Helicobacter pylori
. Gastroenterology 2007;133:659-72.
Bayerdörffer E, Neubauer A, Rudolph B, Thiede C, Lehn N, Eidt S, et al
. Regression of primary gastric lymphoma of mucosa-associated lymphoid tissue type after cure of Helicobacter pylori
infection. MALT Lymphoma Study Group. Lancet 1995;345:1591-4.
Zetterström R. The Nobel Prize in 2005 for the discovery of Helicobacter pylori
: Implications for child health. Acta Paediatr 2006;95:3-5.
Malaty HM. Epidemiology of Helicobacter pylori
infection. Best Pract Res Clin Gastroenterol 2007;21:205-14.
Mei JV, Alexander JR, Adam BW, Hannon WH. Use of filter paper for the collection and analysis of human whole blood specimens. J Nutr 2001;131:1631S-6S.
Kumar A, Mhatre S, Godbole S, Jha P, Dikshit R. Optimization of extraction of genomic DNA from archived dried blood spot (DBS): Potential application in epidemiological research bio banking. Gates Open Res 2018;2:57.
Katherine E. Schlusser, Christopher Pilcher, Esper G. Kallas, Breno R. et al
. Eshleman, Oliver Laeyendecker: Comparison of cross sectional HIV incidence assay results from dried blood spots and plasma. PLoS ONE 2017;12:1-10.
Giavarina D. Understanding Bland Altman analysis. Biochem Medica. 2015;25:141-51.
Smit PW, Elliott I, Peeling RW, Mabey D, Newton PN. Review article: An overview of the clinical use of filter paper in the diagnosis of tropical diseases. Am J Trop Med Hyg 2014;90:195-210.
Parker SP, Cubitt WD. The use of the dried blood spot sample in epidemiological studies. J Clin Pathol 1999;52:633-9.
Rawla P, Barsouk A. Epidemiology of gastric cancer: Global trends, risk factors and prevention. Prz Gastroenterol 2019;14:26-38.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]