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Introduction Pseudomonas aeruginosa is the most common opportunistic pathogen associated with a broad range of infections, including cystic fibrosis, ocular, otitis media, and burn infections. The aim of this study was to show the frequency of the pilS2 gene, and its association with P. aeruginosa plasmid pKLC102 and PAPI-1 pathogenicity island among P. aeruginosa strains. Methods The samples were collected from patients with cystic fibrosis, ocular, otitis media, and burn infections between January 2016 and November 2017. DNA was extracted using the DNA extraction kit and was used for PCR assay. PCR with 4 primer-pairs including 976 F/PAPI-1R, 4542 F/intF, SojR/4541 F, and intF/sojR was performed to identify PAPI-1. pKLC102 was detected using three other primer-pairs including cp10F/cp10R, cp44F/cp44R, and cp97F/cp97R. Results A total of 112 P. aeruginosa isolates were collected from patients with cystic fibrosis (36), burn (20), otitis media (26), and ocular (30) infections. The results of PCR showed that pilS2 gene was identified in 96 (85%) strains. PAPI-1-attB integration was detected among 38 (33.9%) isolates and the circular form of PAPI-1 detected among 17 (14%) isolates. In addition, 79 (70.5%) strains were found to be positive for pKLC102. Conclusion We found that the majority of the isolates may be susceptible to transfer this significant island and the related element pKLC102 into recipient isolates lacking the island owing to high association of the PilS2 pilus with the islands in the studied strains. It is anticipated that strains isolated from burn and eye with the highest rate of PilS2, PAPI-1, and pKLC102 association have a high level of antibiotic resistance.

Encapsulation of peptide and protein-based drugs in polymeric nanoparticles is one of the fundamental fields in controlled-release drug delivery systems. The molecular mechanisms of absorption of peptides to the polymeric nanoparticles are still unknown, and there is no precise molecular data on the encapsulation process of peptide and protein-based drugs. Herein, the self-assembly of different polymers and block copolymers with combinations of the various molecular weight of blocks and the effects of resultant polymer and copolymer nanomicelles on the stability of magainin2, an α-helical antimicrobial peptide, were investigated by means of all-atom molecular dynamics (MD) simulation. The micelle forming, morphology of micellar aggregations and changes in the first hydration shell of the micelles during micelles formation were explored as well. The results showed that the peptide binds to the polymer and copolymer micelles and never detaches during the MD simulation time. In general, all polymers and copolymers simultaneously encapsulated the peptide during micelles formation and had the ability to maintain the helical structure of the peptide, whereas the first hydration shell of the peptide remained unchanged. Among the micelles, the polyethylene glycol (PEG) micelles completely encapsulated magainin2 and, surprisingly, the NMR structure of the peptide was perfectly kept during the encapsulation process. The MD results also indicated that the aromatic and basic residues of the peptide strongly interact with polymers/copolymers and play important roles in the encapsulation mechanism. This research will provide a good opportunity in the design of polymer surfaces for drug delivery applications such as controlled-release peptide delivery systems.

Summary Introduction The measurement of adenosine deaminase (ADA) level in cerebrospinal fluid (CSF) has generated as a suitable test for tuberculous meningitis (TBM) diagnosis. The main objective in the present meta-analysis focused on analyzing the ADA test accuracy in order to diagnose TBM. Methods We searched several databases including Medline, Embase and Cochrane databases to identify studies addressing the diagnosis of TBM. The quality of included reports were assessed by RevMan5 software (via QUADS2 checklist). Accuracy measures of ADA test (sensitivity, specificity and others) pooled with random effects models. In addition, the data was elicited by using midas and metan packages in stata (version 12). Result: Twenty studies were eligible for inclusion within the meta-analysis. The pooled sensitivity and specificity for TBM diagnosis hallmarks were 89% (95% CI: 0.84–0.92) and 91% (95% CI: 0.87–0.93), respectively. The positive likelihood ratio was 9.4 (95% CI: 7–12.8), negative likelihood ratio was 0.12 (95% CI: 0.09–0.17), and diagnostic odds ratio was 77 (95% CI: 45–132). Indeed, the area under the summary receiver operating characteristic (SROC) was 0.96. Conclusion It was magnificently attained that ADA test had a relatively high accuracy for TBM diagnosis.

Background: Multidrug-resistant tuberculosis (MDR-TB) is one of the main challenges to TB control, particularly in developing countries such as Iran. Continuous TB drug resistance surveillance is required for the effective management of TB patients. Methods: The antibiotic susceptibility patterns of clinical Mycobacterium tuberculosis isolates were retrospectively analyzed from April 2012 to March 2018. Conventional and molecular methods were used for the identification and drug susceptibility testing (DST) of M. tuberculosis isolates. Results: A total of 3,012 clinical specimens were collected form TB-suspected patients. Of them, 100 (3.3%) were culture-positive and assigned as M. tuberculosis by phenotypic and molecular methods. According to DST, 17 (17%) isolates were MDR-TB. Conclusions: Improved diagnosis and treatment of MDR-TB may lead to better control of the disease in Iran.

IntroductionTuberculosis (TB) remains a leading cause of death worldwide, especially in developing countries. Early detection of resistance is extremely important to reduce the risk of death. This study was aimed to compare the diagnostic accuracy of high resolution melting (HRM) analysis in comparison with Xpert MTB/RIF as well as conventional drug susceptibility testing (DST) for the detection of rifampicin (RIF) resistance inMycobacterium tuberculosisin Iran.Materials and methodsA comparative cross-sectional study was carried out from April 2017 to September 2018. A total of 80 culture-positive clinical samples selected during the study period were analyzed for detection of RIF-resistant TB by conventional DST, Xpert MTB/RIF, and sequencing. Sensitivity and specificity of the HRM calculated according to DST as our gold standard test in this study.ResultsThe overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HRM assay were found to be 100%, 89.33%, 38.46%, and 100% respectively.ConclusionsThe analysis has demonstrated that the diagnostic accuracy of HRM tests is insufficient to replace Xpert MTB/RIF and conventional DST. HRM test may be used in combination with culture due to the advantage of the time to result. Further work to improve molecular tests would benefit from standardized reference standards and the methodology.

Among different nanomaterials, dendrimers stand out because of their inherent broad range of activity against viruses, bacteria, and cancer cells. Herein, a total of 15 molecular systems have been simulated to characterize the structural features and geometry of low generation polyamidoamine (PAMAM) dendrimers (G0-G2) after they attached to various membranes (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG) with a different ratio of cholesterol (CHL) to explain their membrane activity in cancer cells. The results indicated that the local membrane order parameters were increased in membranes containing 20 and 40 mol% cholesterol molecules. Furthermore, the PAMAM dendrimers were able to make a substantial number of holes in the membranes containing cholesterol. The results also showed that hydrogen bonds, and electrostatic interactions, govern the interactions between the PAMAM dendrimers and the membranes. Neutral bilayers could drive back the PAMAM dendrimers from the bilayer surface, whereas phosphatidylcholine (PC) bilayers mixed with phosphatidylglycerol (PG) phospholipids (as mimics of negatively charged cancer cell membranes) bond firmly to the hyperbranched polymers. Moreover, the negatively charged phospholipids crowded in regions where dendrimers interacted with membranes. This investigation provided an explanation and mechanistic insight for the activity of PAMAM dendrimers against the negatively charged cancer cells.

This study aimed to compare the diagnostic accuracy of high-resolution melting (HRM) analysis in comparison with Xpert MTB/RIF as well as conventional drug susceptibility testing (DST) for the detection of rifampicin (RIF) resistance in Mycobacterium tuberculosis in Iran. A comparative cross-sectional study was carried out from April 2017 to September 2018. A total of 80 culture-positive clinical samples selected during the study period were analysed for detection of RIF-resistant TB by conventional DST, Xpert MTB/RIF, and sequencing. Sensitivity and specificity of the HRM calculated according to DST was our reference standard test in this study. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HRM assay were found to be 100%, 89.33%, 38.46%, and 100% respectively. The analysis demonstrated that the diagnostic accuracy of HRM tests is insufficient to replace Xpert MTB/RIF and conventional DST. HRM tests have the advantage of time to result and may be used in combination with culture. Further work to improve molecular tests would benefit from standardized reference standards and the methodology.

Background Staphylococcus epidermidis is one of the common causes of bacterial keratitis and post-operation infections. One of the most important virulence factors of S. epidermidis is biofilm formation. Poly-N-acetylglucosamine (PNAG) production is critical for biofilm formation in S. epidermidis. The intercellular adhesion (ica) operon is formed by icaA, icaD, icaB, and icaC genes, which participate in the biosynthesis of PNAG. Strains of S. epidermidis from different infections show different genotypes in relation to adhesion operon genes. Therefore, regarding the fact that the pathogenic strain in each community has unique genotypes, this study investigated the relation between ica operon genotypes and various ocular infections. However, the correlation between the ica operon genes and the mecA gene was analyzed in the isolates. Methods For this study, samples of the conjunctiva, cornea, and intraocular fluid of patients with ocular infection were collected. After culture and incubation, detection of S. epidermidis was performed using biochemical and coagulase tests. The antibiotic susceptibility of the bacteria was evaluated by the disk diffusion method. After this, DNA was extracted from the bacteria and the presence of icaA, icaD, is256, and mecA genes was analyzed using polymerase chain reaction. Results All 50 coagulase-negative Staphylococcus samples isolated from different eye infections were characterized as S. epidermidis. Most of the samples (36%) were isolated from the cornea and the others were, respectively, from the conjunctiva (24%), vitreous (20%), anterior chamber (8%), eyelid (6%), and nasolacrimal duct (6%). The icaA, icaD, and is256 genes were detected with different genotypes in isolates from keratitis and endophthalmities compared with conjunctivitis. Overall, the most isolated genotype from ocular infections was icaA+. icaD+. is256+. (46%). Most of the isolates (82.60%) had mecA, icaA, and icaD genes simultaneously, which indicates a strong relationship between the adhesion genes and the antibiotic resistance gene. Conclusions The adhesion operon genes were observed with different genotypes in S. epidermidis samples isolated from various ocular infections.

Pseudomonas is the most common cause of microbial keratitis especially in people who use contact lens. The virulence of Pseudomonas aeruginosa in different eye infections is associated with different virulence factors . In this study, 54 P. aeruginosa isolates including 39 isolates from keratitis and 15 isolates from conjunctivitis were evaluated for their ability to form biofilm, production of protease, elastase, alkaline protease and their antibiotic-resistant patterns. The distribution of the exoS and exoU genes in the test strains were determined using PCR assays. Most of the eye infections (90.74%) were seen in people who used contact lenses, and in most of patients (72.22%), the infection was presented as keratitis. None of the isolates were resistant to a single antibiotic as tested. Multidrug resistance (MDR) was detected in two isolates (3.5%) which were resistant to more than one category of antibiotics. The exoU+/exoS+ isolates were in majority although in total, compared to exoS, there were more exoU in a greater number of samples. Most of the strains produce elastase but among all of ocular isolates, only 5.8% of the strains showed alkaline protease activity. Most of the ocular isolates were not capable of producing biofilm. In our study, a high prevalence of virulence factors was observed in P. aeruginosa isolates from contact lens wearer with keratitis. As the P. aeruginosa isolates from different infection origins and different geographic region may have different virulence factors, having a better perception of these differences could help to improve development of clinical instructions for the control of keratitis.

Antimicrobial peptides (AMPs) are part of the innate host defense system, and they are produced by living organisms to defend themselves against infections. Pardaxin is a cationic AMP with antimicrobial and antitumor activities that has potential to be used as a novel antibiotic or for drug delivery in cancer therapy. This peptide acts on the membrane of target cells and can lead to lysis using different mechanisms of action. Here, we conducted 4.5 μs all-atom molecular dynamics (MD) simulations to determine the critical fragments and residues of Pardaxin for early insertion into different lipid bilayers. Our results revealed that the N-terminal domain of the peptide, particularly the Phe 2 and (/or) Phe 3 residues, has a crucial role in early insertion, independent of the type of lipid bilayers.

Background: Chlamydia trachomatis is the most common cause of chronic follicular conjunctivitis. As a rapid diagnosis is important in the reducing the long-term squeal of the diseases, the objective of this study was to compare the three methods, direct immunofluorescence (DIF), staining and PCR, for detection of Chlamydia trachomatis in patients with follicular conjunctivitis. Material and methods: Overall 90 patients with conjunctival were enrolled in this study smears were prepared for DIF and Giemsa staining. PCR amplification after Extraction performed using CT1 and CT5 primers designed from Omp1 gene. Results: Of the 90 patients, 28 (31.1%) were positive by DIF and 13 (14.4%) by Giemsa staining; and 35 patients (38.8%) showed positive results in PCR. Sensitivity, specificity, predictive positive value, and negative predictive value of DIF in comparison to PCR respectively were calculated as 88.33, 100, 100 and 88.70. Sensitivity, specificity, predictive positive value and negative predictive value of DIF in comparison to PCR respectively were calculated as 61.40, 100, 100 and 71.42. Therefore, sensitivity and negative predictive value of DIF are significantly higher than Giemsa staining. Conclusion: DIF is more sensitive and more reliable than Giemsa staining for detection of Chlamydia trachomatis in the conjunctiva samples of patients with follicular conjunctivitis.

Objective/background Mycobacterium tuberculosis pyrazinamidase (PZase) is known an enzyme that is involved in degradation of pyrazinamide to ammonia and pyrazinoic acid. Pyrazinamide is an important first-line drug used in the short-course treatment of tuberculosis. Previous investigations have indicated that the pyrazinamide (PZA)-resistant M. tuberculosis strains are caused by point mutations in the PZase enzyme which is the activator of the prodrug PZA. Although the general fold of PZase was determined, the structural and functional properties of the enzyme in solution were not understood very well. In this study, the PZase enzyme was overexpressed and purified. In addition, two polyols, namely sorbitol and glycerol, were chosen to study their effects on the structure, dynamics, and stability of the enzyme. To gain a deeper insight, molecular dynamics simulation and spectroscopic methods, such as fluorescence spectroscopy and circular dichroism (CD), were used. Methods The genes were cloned in Escherichia coli BL21 (DE3), harboring the recombinant pET-28a (+) plasmid, overexpressed and purified by Ni-NTA Sepharose. The far UV–visible CD spectra were measured by a Jasco-810 spectropolarimeter. The intrinsic fluorescence spectra were measured on a Cary Varian Eclipse spectrofluorometer. For molecular dynamics (MD) simulations, we have applied GROMACS4.6.5. Results The results showed that glycerol and sorbitol increased the enzyme activity up to 130% and 110%, respectively, at 37 °C. The stability of PZase was decreased and the half-life was 20 min. Glycerol and sorbitol increased the PZase half-life to 99 min and 23 min, respectively. The far UV CD measurements of PZase indicated that the CD spectra in glycerol and sorbitol give rise to an increase in the content of α-helix and β-sheets elements. The average enzyme root mean square deviation (RMSD) in sorbitol solution was about 0.416 nm, a value that is higher than the enzyme RMSD in the pure water (0.316). In dictionary of protein secondary structure (DSSP) results, we observed that the secondary structures of the protein are partially increased as compared to the native state in water. The experimental and simulation data clearly indicated that the polyols increased the PZase stabilization in the order: glycerol > sorbitol. Conclusion It can be concluded that the native conformation of the enzyme was stabilized in the sorbitol and glycerol and tend to exclude from the PZase surface, forcing the enzyme to keep it in the compactly folded conformation. The glycerol molecules stabilized PZase by decreasing the loops flexibility and then compacting the enzyme structure. It appears that more stability of PZase in glycerol solution correlates with its amphiphilic orientation, which decreases the unfavorable interactions of hydrophobic regions.

Urea and GdmCl are widely used to denature proteins at high concentrations. Here, we used MD simulations to study the denaturation mechanisms of helical peptide in different concentrations of GdmCl and urea. It was found that the helical structure of the peptide in water simulation is disappeared after 5 ns while the helicity of the peptide is disappeared after 70 ns in 2 M urea and 25 ns in 1 M GdmCl. Surprisingly, this result shows that the helical structure in low concentration of denaturants is remained more with respect to that solvated in water. The present work strongly suggests that urea interact more preferentially to non-polar and aromatic side chains in 2 M urea; therefore, hydrophobic residues are in more favorable environment in 2 M urea. Our results also reveal that the hydrogen bonds between urea and the backbone is the dominant mechanism by which the peptide is destabilized in high concentration of urea. In 1 M and 2 M GdmCl, GdmCl molecules tend to engage in transient stacking interactions with aromatics and hydrophobic planar side chains that lead to displacement of water from the hydration surface, providing more favorable environment for them. This shows that accumulation of GdmCl around hydrophobic surfaces in 1 M and 2 M GdmCl solutions prevents proper solvation of the peptide at the beginning. In high GdmCl concentrations, water solvate the peptide better than 1 M and 2 M GdmCl. Therefore, our results strongly suggest that hydrogen bonds between water and the peptide are important factors in the destabilization of peptide in GdmCl solutions.

Background: In this study, we have experimentally investigated the effects of different osmolytes including sucrose, sorbitol, urea, and guanidinium chloride (GdmCl) on the stability and structure of the Mycobacterium tuberculosis pyrazinamidase (PZase). PZase converts pyrazinamide to its active form. Methods: In addition, in order to gain molecular insight into the interactions between osmolytes and PZase, we have conducted 1000-ns molecular dynamics simulations. Results: The results indicated that sucrose and sorbitol increase the stability and compactness of the enzyme, whereas in the presence of urea and GdmCl, PZase loses its stability and compactness. Furthermore, the activity of PZase in the presence of sucrose was more than the other solutions. The energetic analyses imply that the electrostatic and van der Waals interactions are the major factors in the osmolyte–PZase interactions. Sorbitol and sucrose, as protective osmolytes, protect the protein structure by utilizing the van der Waals interaction from denaturation. In addition, urea molecules affect the structure of the protein using the hydrogen bonds and van der Waals interactions. Conclusion: The results show that the most important factor in the denaturing effect of GdmCl is the strong interactions of positively charged guanidinium ions with the aspartate and glutamate residues.

Pyrazinamide (PZA) is an important first-line drug used for the short-course treatment of tuberculosis in combination with isoniazid and rifampin. It has been reported that mutations in pncA gene correlate well with PZA resistance depending on the geographic area. On the other hand, different genotypes of Mycobacterium tuberculosis show different affinities to acquire resistance-related mutations. To determine the relative significance of various mutations in the pncA gene in Iranian PZA-resistant M. tuberculosis isolates and to analyze the association of different genotypes of M. tuberculosis with PZA resistance, 34 PZA-resistant M. tuberculosis isolates were analyzed for their pncA mutations using direct sequencing. These isolates were genotyped by IS6110 fingerprinting and spoligotyping methods. Mutations in the pncA gene were identified in 24 of 34 of these isolates (70.58%). No mutations were found in 10 PZA-resistant isolates, which implied that alternative mechanisms of resistance existed in these strains. PZA resistance was strongly (41.2%) associated with multidrug-resistant tuberculosis. Genotyping revealed the Central Asian (CAS) and East-African Indian families as the most prevalent families between PZA-monoresistant isolates versus the Beijing and Haarlem families which were the most frequent families between PZA including multidrug-resistant isolates.

Pyrazinamide (PZA) is one the first line anti-tuberculosis drugs that require activation by the pyrazinamidase (PZase). Most PZA-resistant Mycobacterium tuberculosis strains have mutations in the pncA gene which encoding PZase that result in the reduction or loss of the enzyme activity. Herein, we have examined how various mutations, which have been found from the PZA-resistant M. t uberculosis strains in Iran, modify the PZase activity. To elucidate the possible role of these mutations, namely A143T (MUT1), L151S (MUT2), A143T/T168A/E173K (MUT3), in the bioactivity of the enzyme, the PZase and mutant genes were cloned, functionally expressed and biochemically and computationally characterized. In comparison to the PZase enzyme, the enzymatic efficiency of mutant enzymes was decreased, with MUT2 indicating the largest enzymatic efficiency reduction. Homology models of mutants were constructed based on the PZase X-ray crystal structure. Molecular modeling and substrate docking revealed that the wild-type has much stronger binding affinity to PZA than the mutants whereas MUT2 has the weakest binding affinity. In addition, the molecular dynamics simulations and the essential dynamics results illustrated that the positions of the 51st to 71st residues were more dynamics in MUT2 as compared to the other atoms in PZase, MUT1 and MUT3 which could decrease the K m and k cat values of the enzymes.

Co-solvents such as glycerol and sorbitol are small organic molecules solvated in the cellular solutions that can have profound effects on the protein structures. Here, the molecular dynamics simulations and comparative structural analysis of magainin, as a peptide model, in pure water, 2,2,2-trifluoroethanol/water, glycerol/water, and sorbitol/water are reported. Our results show that the peptide NMR structure is largely maintained its native structure in osmolytes–water mixtures. The simulation data indicates that the stabilizing effect of glycerol and sorbitol is induced by preferential accumulation of glycerol and sorbitol molecules around the nonpolar and aromatic residues. Thus, the presence of glycerol and sorbitol molecules decreases the interactions of water molecules with the hydrophobic residues of the peptide, and the alpha helical structure is stabilized.

Considering the significant increase of isoniazid (INH) resistance in Iranian Mycobacterium tuberculosis isolates in the last few years and to investigate the prevalence and diagnostic potential of the most commonly reported mutations associated with INH resistance in Iran, we analyzed parts of the katG gene and fabG1-inhA and oxyR-ahpC regulatory regions in a sample of 48 INH-resistant and 25 INH-sensitive isolates. Mutations in the katG 315, fabG1-inhA, and oxyR-ahpC regulatory regions were detected in 58.3%, 18.7%, and 39.6% of isolates, respectively. The R463L polymorphism in the katG gene and the ahpC46A were detected with high frequency in both INH-resistant and -sensitive isolates. Spoligotyping and IS6110-based restriction fragment length polymorphism patterns revealed that most of the isolates containing ahpC46A and katG 463Leu polymorphism belonged to the Central Asian (CAS) super family. The tight relationship between ahpC46A and katG 463Leu polymorphisms and the CAS super family highlights the importance of the CAS super family in INH resistance in Iran. In conclusion, mutations at katG codon 315 or the fabG1-inhA regulatory region were identified in 77.0% of the INH-resistant isolates and in none of the INH-sensitive strains, and are highly predictive of isonizid resistance in Iranian isolates.

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Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.

The Bacillus Calmette-Guerin (BCG) vaccine is administered to all newborns within the first days of life for the prevention of tuberculosis (TB) in many of the developing countries, including Iran. BCG vaccine is prepared from a special strain of Mycobacterium bovis . This vaccine is safe, but local and systemic side effects are associated with BCG vaccine. Severe adverse effects of BCG vaccination are extremely rare in immunocompetent children, but lymphadenitis, which is the most common side effect of BCG vaccination in children, is seen with high frequency in Iran. Different studies have shown that the vaccine strain and its genetic variations are important in inducing these side effects. Therefore, in this study, the aim is to analyze the genetic characterizations of vaccine strains used in Iran. Methods The samples were collected from infants showing lymphadenitis induced by BCG vaccination. After acid-fast staining and culture of samples on Lowenstein–Jensen medium, DNA was extracted from samples using Phenol–chloroform method. Using primer for 16sRNA gene, the genus of the isolates was characterized. Then using RD1, RD14 and DU1 primers and followed by the next step using RD9, RD4 Deleted, RD4 Present and RD1 Deleted primers, the species and strains of the isolates were characterized. Results All 30 samples of acid-fast bacilli that were isolated from infants with BCG complications confirmed as Mycobacterium genus using 16sRNA PCR and using RD1, RD14, DU1, RD9, RD4 Deleted, RD4 Present and RD1 Deleted primers confirmed that all isolates were M. bovis BCG strain Pasteur. Conclusion As all of the strains isolated from the patients were confirmed as M. bovis BCG strain Pasteur, the other possible factors causing BCG complications, including BCG overdose, faulty intradermal technique, and disturbance of cellular immunity, must be considered.