Unravelling the diagnostic methodologies for SARS-CoV-2; the Indispensable need for developing point-of-care testing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused COVID-19 pandemic that continues to be a global menace and since its emergence in the late 2019, SARS-CoV-2 has been vigorously spreading throughout the globe putting the whole world into a multidimensional calamity. The suitable diagnosis strategies are on the front line of the battle against preventing the spread of infections. Since the clinical manifestation of COVID-19 is shared between various diseases, detection of the unique impacts of the pathogen on the host along with the diagnosis of the virus itself should be addressed. Employing the most suitable approaches to specifically, sensitively and effectively recognize the infected cases may be a real game changer in controlling the outbreak and the crisis management. In that matter, point-of-care assays (POC) appears to be the potential option, due to sensitivity, specificity, affordable, and availability. Here we brief the most recent findings about the virus, its variants, and the conventional methods that have been used for its detection, along with the POC strategies that have been applied to the virus diagnosis and the developing technologies which can accelerate the diagnosis procedure yet maintain its efficiency.

Development and evaluation of a colorimetric LAMP-based biosensor for rapid detection of a nosocomial infection agent, Citrobacter freundii.

Simple and fast diagnosis of Citrobacter freundii which is an important cause of nosocomial infection in human is crucial to achieve early treatment. We have developed and evaluated an optical LAMP-based biosensor for the visual detection of C. freundii for the first time. The efficiency of the assay was investigated and compared to PCR method. The selectivity and specificity of the biosensor were analyzed using Morganella morganii, Enterobacter aerogenes, Pseudomonas aeruginosa, Yersinia enterocolitica, Shigella sonnei, Serratia marcescens, Burkholderia cepacia and Klebsiella pneumoniae and a mixed-culture medium. Endpoint analysis using hydroxy naphthol blue was applied, and the color change to sky blue and no color change from violet indicated positive and negative results, respectively. The absorption at 650 nm was measured 0.39 for the positive sample, while the mean absorption of the test samples, including water, was 0.23. The specificity of the method was equal to that of PCR. However, the sensitivity was determined as 12.24 fg/µL of the genomic content of C. freundii, higher than PCR assay. The developed LAMP-based method provided a rapid and accurate technique for molecular diagnostics of C. freundii, making it a suitable technique for point-of-care diagnostics in cases of urgent situations.

Does dexamethasone inhibit glucose oxidase: an analysis in kinetics and molecular study.

OBJECTIVES: Glucose oxidase is an enzyme that is widely used in biosensors, especially kits for measuring blood sugar. Many diabetics use this type of kit to determine their blood sugar level. Aspergillus niger is the most important source of glucose oxidase for use in biosensors. Diabetes causes secondary diseases in patients for which medications are prescribed to improve them. Dexamethasone, a corticosteroid, is one of the drugs prescribed to diabetics to cure some secondary diseases. In this study, the effect of this drug on glucose oxidase was investigated from a kinetic and molecular point of view. METHODS: In this study, the kinetics of drug binding to the enzyme was measured and the type of inhibition was determined by Lineweaver-Burk plot. The Ki value of the drug was determined by drawing the secondary curve. Using fluorescence spectrophotometry and molecular docking, the binding of the drug to the enzyme was confirmed. RESULTS: The results showed that the drug inhibits the enzyme non-competitively. Determining the kinetics parameters of the drug-enzyme interaction showed that the drug acts as a potent inhibitor. Study at the molecular level by fluorescence spectrophotometer showed that the drug attachment alters the enzyme conformation to more compaction. In silico results showed that the drug is placed between two helices that are outside the active site and binds to the enzyme by three hydrogen bonds. CONCLUSIONS: The result of this study is useful because it suggests that in diabetic patients taking dexamethasone, the amount of glucose declared by the kit may not be real due to the inhibition of glucose oxidase.

GPX2 and BMP4 as Significant Molecular Alterations in The Lung Adenocarcinoma Progression: Integrated Bioinformatics Analysis.

Objective: Non-small cell lung adenocarcinoma (NSCLC) is the most common type of lung cancer, which is considered as the most lethal and prevalent cancer worldwide. Recently, molecular changes have been implicated to play a significant role in the cancer progression. Despite of numerous studies, the molecular mechanism of NSCLC pathogenesis in each sub-stage remains unclear. Studying these molecular alterations gives us a chance to design successful therapeutic plans which is aimed in this research. Materials and Methods: In this bioinformatics study, we compared the expression profile of 7 minor stages of NSCLC adenocarcinoma, including GSE41271, GSE42127, and GSE75037, to clarify the relation of molecular alterations and tumorigenesis. At first, 99 common differentially expressed genes (DEG) were obtained. Then, functional enrichment analysis and protein-protein interaction (PPI) network construction were performed to uncover the association of significant cellular and molecular changes. Finally, gene expression profile interactive analysis (GEPIA) was employed to validate the results by RNA-seq expression data. Results: Primary analysis showed that BMP4 was downregulated through the tumor progression to the stage IB and GPX2 was upregulated in the course of final tumor development to the stage IV and distant metastasis. Functional enrichment analysis indicated that BMP4 in the TGF-ß signaling pathway and GPX2 in the glutathione metabolism pathway may be the key genes for NSCLC adenocarcinoma progression. GEPIA analysis revealed a correlation between BMP4 downregulation and GPX2 upregulation and lung adenocarcinoma (LUAD) progression and lower survival chances in LUAD patients which confirm microarray data. Conclusion: Taken together, we suggested GPX2 as an oncogene by inhibiting apoptosis, promoting EMT and increasing glucose uptake in the final stages and BMP4 as a tumor suppressor via inducing apoptosis and arresting cell cycle in the early stages through lung adenocarcinoma (ADC) development to make them candidate genes to further cancer therapy investigations.

Development of one-pot single specific primer-LAMP (SSP-LAMP) for identification of Shigella genus using 16S rDNA.

Ribosomal RNA gene as a high-copy number nucleo-biomarker is extremely conserved among bacteria which limits its application to the discriminative detection approaches. We have developed a colorimetric isothermal amplification method called "single specific primer-LAMP (SSP-LAMP)" requiring only one specific primer for the amplification of the target and applied to the identification of the 16S rRNA gene in the Shigella genus. A region with high sequence homology in the genus and low homology with other bacteria was considered as the most appropriate. In that regard, a 23 bp sequence in the 16S rRNA gene of the genus was targeted based on the alignment of the gene with fifty-three closely related bacterial species, and a single specific primer along with five degenerate primers were designed. Using hydroxy-naphthol blue (HNB) as an indicator and gel electrophoresis, the proposed approach of SSP-LAMP was able to detect S. boydii, S. sonnei, S. flexneri and S. dysenteriae specifically while other species remained unidentified. The SSP-LAMP method could provide a rapid one-pot point-of-care method for molecular diagnostics of pathogens in many circumstances mainly samples with high genetic homogeneity.

The mechanism and improvements to the isothermal amplification of nucleic acids, at a glance.

A comparative review of the most common isothermal methods is provided. In the last two decades, the challenge of using isothermal amplification systems as an alternate to the most extensive and long-standing nucleic acids-amplifying method-the polymerase chain reaction-has arisen. The main advantage of isothermal amplification is no requirement for expensive laboratory equipment for thermal cycling. Considerable efforts have been made to improve the current techniques of nucleic acid amplification and the development of new approaches based on the main drawbacks of each method. The most important and challenging goal was to achieve a low-cost, straightforward system that is rapid, specific, accurate, and sensitive.

A colorimetric nano-biosensor for simultaneous detection of prevalent cancers using unamplified cell-free ribonucleic acid biomarkers.

Early detection of cancer increases the chance of effective treatment and survival rates. The aim of this study is to develop a rapid and non-invasive nano-biosensing method to screen common lethal cancers in their early stages. In that regard, two circulating microRNA (miR-21, miR-155) biomarkers, which are upregulated in plasma in prevalent cancers, were targeted by a rapid and colorimetric nano-biosensor based on non-crosslinking Au-nanoprobes without amplification requirement. Multiple cancerous cell lines, including A549, MCF7, HT-29, A2780, AGS, MKN-45, and SW-1736 and the primary fibroblast were examined with naked eyes after the hybridization assay using exogenous biomarkers. The results were also confirmed by spectroscopy analysis. The upregulated miRNAs in cancerous cell lines caused a significant blue shift in the Au-nanoprobe absorbance spectrum while the samples isolated from normal cells remained intact red. The limit of detection (LOD) of the method was determined to be less than one ng/µL of total isolated miRNA using an instrument-free visual method. The developed geno-sensing method could serve as a simple, point-of-care platform for cancer prognosis and diagnosis, leading to operative nano-theranostics.

Early-phase nano-genosensing of cell-free nucleobiomarkers in the plasma of cancerous patients.

The extracted miR-21 and miR-155 from the plasma of clinical samples were targeted by non-crosslinking hybridization of Au-nanoprobes without the need for biomarker amplification. Thirty samples, including those suspected to cancer and chemotherapy-treated samples, were analyzed. An increase in the concentration of target biomarkers caused a blue-shift in the visible spectrum of nanoprobes. Using magnesium chloride, the change in the color of nanoprobes was shown to be dependent on time besides intensity. Samples with high averages of intensity needed less time for colorimetric differentiation than those with low average intensity. Au-nanoprobe-21 was turned to purple-gray in clinical specimens of stomach, colon, breast, esophagus, sarcoma, diaphragm, prostate, bladder, and lung while Au-nanoprobe-155 appeared as light purple-gray in colon, breast, lung, diaphragm and esophagus samples. The LOD was measured as 5 ng µL-1 of targeted biomarkers. The developed nano-biosensing method could propose a point-of-care approach for cancer prognosis and diagnosis, facilitating targeted therapeutics.

Ribonucleic-acid-biomarker candidates for early-phase group detection of common cancers.

Cancer is considered as a challenging lethal agent around the world and its detection at early stages would help prevention of the high mortality. Among the widely used biomarkers in clinical diagnosis of cancer, extracellular non-coding RNAs as ribonucleic acid biomarkers serve as state-of-the-art candidates for molecular diagnosis. In that regard, microRNAs are of great priority mainly because of high variety and stability in body fluids. Accordingly, common miRNAs among most prevalent cancers could help us (pre)diagnose cancer with high accuracy in target samples. In this study, common lethal cancers to humans were investigated in case of miRNA profiles to determine the possible common correlation between miRNA up-regulation or down-regulation (as a ribonucleic acid biomarker) and developing the cancers. It was shown that among the investigated miRNAs, five typical extracellular miRNAs (miR-18a, miR-21, miR-155, miR-221, and miR-375) dysregulation are predominant in most cancer varieties comprising breast, colon, lung, prostate, pancreas, gastric, ovarian, esophagus and liver. This could serve as an appropriate target site for developing point-of-care approaches for cancer detection e.g. designing diagnostic biosensor-based microarrays or kits for both quantification and qualification of the biomarkers. Besides, the miRNA candidates could be efficiently applied to cancer therapeutic approaches.

Development and evaluation of an improved quantitative loop-mediated isothermal amplification method for rapid detection of Morganella morganii.

A point-of-care diagnostic kit was developed for detection of Morganella morganii using optimized loop-mediated isothermal amplification (LAMP) technique within less than an hour. In that regard, dimethyl sulfoxide (DMSO) was utilized together with betaine and all variables were optimized to improve the efficiency of the method. Moreover, surface presentation of antigens protein was targeted and six unique primers were designed. Endpoint turbidity analysis was performed at 550 nm to measure the tetravalent anion (pyrophosphate) released during the reaction. The specificity of the method was evaluated using nine closely related bacterial species as well as its sensitivity. It was shown that the improved LAMP assay could significantly distinguish M. morganii from other bacteria while the sensitivity was determined to be 0.2 CFU mL-1.

Interaction of Antipsychotic Drugs with Sucrase, Kinetics and Structural Study.

BACKGROUND: In patients with the Congenital Sucrase-Isomaltase Deficiency (CSID), who lack intestinal sucrase-isomaltase enzyme, a suspension of yeast sucrase is applied as a drug to compensate the enzyme deficiency. While antipsychotic drugs are used for the treatment of schizophrenia, administering multiple drugs at the same time may counteract each other. METHODS: In this study, the interaction between trifluoperazine and haloperidol as antipsychotic drugs on oral drug yeast sucrase was investigated. In this regard, the kinetic parameters of enzyme were determined in the presence or absence of the drugs. The kinetic parameters of the drugs such as Ki and IC50 were also calculated. Lineweaver - Burk plot was used to reveal the type of inhibition. RESULTS: The results showed that both drugs could reduce sucrase activity and decrease the Vmax of the enzyme by non-competitive inhibition. The IC50 and Ki values of the drugs were determined to be 0.7 and 0.068 mM and 0.45 and 0.063 mM for haloperidol and trifluoperazine, respectively. The results suggested that trifluoperazine binds to the enzyme with higher affinity than haloperidol. Fluorescence measurement was used for conformational investigations of the drugs and sucrase interaction. It was shown that the drugs bind to free enzyme and enzyme-substrate complex which are accompanied with hyperchromicity. This suggests that tryptophan residues of the enzyme transferred to hydrophobic medium after binding of the drugs to the enzyme. CONCLUSION: The finding of this research revealed that both trifluoperazine and haloperidol could inhibit sucrase in non-competitive manner. The kinetic parameters and conformational changes due to binding of trifluoperazine to the enzyme were different from that of haloperidol.

Inhibition of pseudoperoxiadse activity of human red blood cell hemoglobin by methocarbamol.

After red blood cells lysis, hemoglobin is released to blood circulation. Hemoglobin is carried in blood by binding to haptoglobin. In normal individuals, no free hemoglobin is observed in the blood, because most of the hemoglobin is in the form of haptoglobin complex. In some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. As a result, free hemoglobin appears in the blood, which is a toxic compound for these patients and may cause renal failure, hypertensive response and risk of atherogenesis. Free hemoglobin has been determined to have peroxidase activity and considered a pseudoenzyme. In this study, the effect of methocarbamol on the peroxidase activity of human hemoglobin was investigated. Our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. Both Km and Vmax decreased by increasing the drug concentration. Ki and IC50 values were determined as 6 and 10mM, respectively. Docking results demonstrated that methocarbamol did not attach to heme group directly. A hydrogen bond linked NH2 of carbamate group of methocarbamol to the carboxyl group of Asp126 side chain. Two other hydrogen bonds could be also observed between hydroxyl group of the drug and Ser102 and Ser133 residues of the pseudoenzyme.

Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei cellobiohydrolase I expressed in Pichia pastoris.

OBJECTIVES: To modify two main N-glycosylation residues of cellobiohydrolase I from Trichoderma reesei by site-directed mutagenesis for decreasing the extent of glycosylation and exploring possible effects on its properties. RESULTS: Asparagine 45 and 64 residues were mutated to alanine to make single/double mutants and expressed in P. pastoris. Decreasing N-glycosylation of the recombinant CBH I resulted in an increased affinity of the enzyme for carboxymethylcellulose and also improved the Kcat/Km while the specific activity was decreased. Also, the enzymes were stable up to 80 °C. There was no significant change of the optimum pH and temperature by decrease of glycosylation in the mutated enzymes in comparison to the wild-type at constant incubation time of assay. CONCLUSION: Post-translational glycan-modification of CBH I in P. pastoris has different impacts on the properties of the secreted enzymes. Substrate affinity and catalytic efficiency were improved significantly while the activity and high temperature stability were negatively affected.

Enhancing effect of cimetidine on peroxidase activity of human erythrocyte hemoglobin.

BACKGROUND: Hemoglobin is released to the serum after erythrocyte lyses. Haptoglobin is responsible for carrying hemoglobin into the serum. In hemolytic disease, the amount of hemoglobin which is released to the serum is high; however, the amount of haptoglobin is not enough for binding all the released hemoglobins. Free hemoglobin has peroxidase activity (a pseudoenzyme) and has been indicated to be harmful for patients. This study is focused on the effect of cimetidine on peroxidase activity of hemoglobin. METHODS: Erythrocytes were lysed to obtain hemoglobin. Peroxidase activity of hemoglobin was detected using o-dianisidine and H(2)O(2) as substrates. RESULTS: Our results showed that the drug operated as an activator for the pseudoenzyme. Cimetidine bound to the pseudoperoxidase in an un-competitive manner and decreased the Km. Half maximal effective concentration (EC(50)) of cimetidine was determined to be about 12.5 mM. Alkaline pH increased the rate of reaction. Arrhenius plot showed that the activation energies of reactions in the absence and presence of drug were about 10.5 kJ/mol and 7.65 kJ/mol, respectively. CONCLUSIONS: The results demonstrated that cimetidine activates the peroxidase activity of free hemoglobin. Hence, it is suggested that the prescription of cimetidine for the patients with hemolyses diseases may enhance the harmful effects of free hemoglobin in these patients.

Functional and structural changes of human erythrocyte catalase induced by cimetidine: proposed model of binding.

In erythrocyte, catalase plays an important role to protect cells from hydrogen peroxide toxicity. Hydrogen peroxide is a byproduct compound which is produced during metabolic pathway of cells. Cimetidine, a histamine H2 receptor antagonist, is used for gastrointestinal tract diseases and prevents the extra release of gastric acid. In this study, the effect of cimetidine on the activity of human erythrocyte catalase was investigated. Erythrocytes were broken by hypotonic solution. The supernatant was used for catalase assay and kinetics study. Lineweaver-Burk plot was performed to determine the type of inhibition. The kinetics data revealed that cimetidine inhibited the catalase activity by mixed inhibition. The IC50 (1.54 µM) and Ki (0.45 µM) values of cimetidine determined that the drug was bound to the enzyme with high affinity. Circular dichroism and fluorescence measurement showed that the binding of cimetidine to the enzyme affected the content of secondary structure of the enzyme as well as its conformational changes. Docking studies were carried out to detect the site in which the drug was bound to the enzyme. Molecular modeling and energy calculation of the binding showed that the cyanoguanidine group of the drug connected to Asp59 via two hydrogen bonds, while the imidazole group of the drug interacted with Phe64 in the enzyme by a hydrophobic interaction. In conclusion, cimetidine could bind to human erythrocyte catalase, and its interaction caused functional and conformational changes in the enzyme.

An improved non-crosslinking gold nanoprobe-NASBA based on 16S rRNA for rapid discriminative bio-sensing of major salmonellosis pathogens.

In the absence of an appropriate detection method for simple and rapid differentiation of major salmonellosis-causing agents, nano-bio-sensing could provide ideal molecular detection approaches for food-borne pathogens. In addition, the 16S ribosomal RNA-based detection techniques would enhance specificity and sensitivity due to the presence of multiple copies of 16S rRNA per cells and also the presence of hypervariable regions in the genes. In this study, we developed a novel detection technique based on 16S rRNA gold nanoprobe-nucleic acid sequence-based amplification (NASBA) in an improved non-crosslinking mode for nanodiagnosis of the most important serovars of the Salmonella genus: Salmonella enteritidis and Salmonella typhimurium. In that regard, we designed a specific set of primers along with a thiolated oligonucleotide gold nanoprobe for one of the hypervariable regions of 16S rRNA gene. The NASBA-gold nanoprobe method was improved to lower the overall assay time to about 80min and enhance the specificity and reproducibility of the detection process using various closely related non-Salmonella species as well as some Salmonella serotypes. Finally, the sensitivity of the developed method was determined to be around 5CFUs Salmonella per amplification tube. The developed nano-bio-sensing method could provide a cost-effective and promising assay especially suited for quick identification of food-borne pathogens in the event of outbreaks.

Development and evaluation of a novel nucleic acid sequence-based amplification method using one specific primer and one degenerate primer for simultaneous detection of Salmonella Enteritidis and Salmonella Typhimurium.

Salmonella Enteritidis and Salmonella Typhimurium are the most widespread causes of salmonellosis and gastrointestinal diseases worldwide. Thus, their simple and sensitive detection is significantly important in biosafety and point-of-care diagnostics. In that regard, although present nucleic acid-based attempts are mainly focused on the detection methods encompassing all Salmonella enterica members in a single reaction, serotypes other than S. Enteritidis and S. Typhimurium are clinically and epidemiologically rare to humans. Therefore, regarding high ribosomal RNA (rRNA) copy numbers in a cell, isothermal nucleic acid sequence-based amplification (NASBA) technique was employed for simple, sensitive and simultaneous detection of the bacteria. However, due to high sequence homology among 16S rRNA genes and consequently, very few specific regions, we developed a novel NASBA method called "single specific primer-NASBA or SSP-NASBA" in which the specificity of the antisense primer is sufficient to perform a specific NASBA reaction. Accordingly, we designed highly specific NASBA antisense and degenerate sense primers for a segment of 16S rRNA variable region by universal sequence alignment to simultaneously detect S. Enteritidis and S. Typhimurium. Meanwhile, the approach was successfully evaluated for various Salmonella as well as closely related non-Salmonella serovars. Specific and simultaneous detection of both bacteria was achieved with the designed primer set in a single reaction environment with a detection limit of less than 10 CFUs mL(-1). The developed NASBA assay should facilitate the overall process and provide a simple, fast, specific and sensitive approach for molecular diagnostics of pathogens under various circumstances, e.g. outbreaks.

Non-crosslinking gold nanoprobes for detection of nucleic acid sequence-based amplification products.

Lack of an appropriate detection method for isothermal RNA amplification technique, known as nucleic acid sequence-based amplification (NASBA), is considered as a major defect for its vast applications. In that regard, novel detection methods as fast, specific, and sensitive as the gold nanoprobe detection technique are highly demanded and non-crosslinking gold nanoprobes are regarded as the ideal choice. In this study, we attempted to integrate these two techniques (RNA amplification and nanoprobe detection) into a single detection-associated amplification method. In that line, essential adjustments such as amplicon dilution, disturbing reagent extraction, ion adjustment, and modification of the hybridization protocol were needed due to the ribonucleic acid nature of NASBA products and the presence of some interfering reagents in the amplification reaction environment. The adjustments successfully resulted in the gold nanoparticle-based detection of NASBA products with naked eyes in a whole operational time of less than 3.5h (including nucleic acid extraction, amplification, and detection). Furthermore, the developed assay was successfully applied to detect dnaK messenger RNA of Salmonella typhimurium. The developed colorimetric method facilitated the detection step of NASBA leading to an ideal methodology for rapid assays and serves as an ideal alternative to the highly expensive reverse transcription polymerase chain reaction (RT-PCR) approach.

The relationship between Helicobacter pylori infection and gastric adenocarcinoma in Northern Iran.

Colonization of the human stomach with Helicobacter pylori induces chronic gastritis and is associated with the development of gastric and duodenal ulcers, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Infection with an H. pylori strain containing the cytotoxin-associated (cagA) gene (a marker for a pathogenicity island) may increase the risk of atrophic gastritis and gastric cancer. The exact role of H. pylori in gastric carcinogenesis is still being investigated. Hence, we assessed whether H. pylori infection is associated with the development of gastric adenocarcinoma in northern Iran. Gastric biopsy specimens from 168 patients suffering from gastric adenocarcinoma, gastric ulcer, and non-ulcer dyspepsia were analyzed by means of the polymerase chain reaction. H. pylori was detected in the gastric mucosa of 34 (75.5%) gastric adenocarcinoma, 56 (88.8%) gastric ulcer, and 36 (60%) non-ulcer dyspepsia. In patients with gastric adenocarcinoma, the cagA was less commonly found than those in noncancer patients (4/34 vs. 58/92, p < 0.05). Our work suggests that although H. pylori infection is significantly associated with gastric adenocarcinoma in northern Iran, the cagA is not the dominant virulence in development of gastric adenocarcinoma.

Helicobacter pylori cagA status and peptic ulcer disease in Iran.

Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA -negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene is a putative H. pylori virulence factor of unknown function. The aim of this study was to determine the prevalence of the cagA gene among H. pylori isolates and its relationship with peptic ulcer disease in 128 Iranian patients. A total of 107 (83.6%) samples were positive, including 40 (95%) of the 42 patients with duodenal ulcer, 43 (86%) of the 50 patients with gastric ulcer, and 24 (66.6%) of the 36 patients with gastritis. cagA was present in 32 (80%) of 40 strains from duodenal ulcer patients, 33 (77%) of 43 strains from gastric ulcer patients, and 11 (46%) of 24 from gastritis patients. We also attempted to investigate the subtypes of 3' region of cagA gene in H. pylori strains isolated from Iranian patients and their relation to H. pylori-associated gastroduodenal diseases. The PCR product of cagA positive strains obtained with primer set CAG1/CAG2 differed in size, varying from 642 to 651 bp (subtype A) in 33 isolates to 756 bp (subtype B/D) in 13 isolates. This does not support the view that subtypes of the 3' region of cagA gene in H. pylori isolated from Iran correlate with the clinical outcomes of H. pylori, but colonization with cagA positive strains was significantly higher among duodenal ulcer than gastritis patients in Iran.