Jundishapur Journal of Microbiology
Year:2017 | Volume:10 | Issue:3|Papers Count:11

Background: Tuberculosis remains a major global threat. Two billion of the world’ s population is latently infected with Mycobacterium tuberculosis and is at the risk of progression to active disease. Bacillus Calmette-Guerin (BCG), as the only licensed vaccine, has prophylaxis strategy, which protects children from disseminated form of tuberculosis. Therefore, postexposure vaccine strategy, which targets individuals with latent tuberculosis infection, is an important strategy to control this disease globally. Objectives: In the present study, we designed a novel postexposure multi-epitope DNA construct based on 3 latency-associated antigens of Rv2029c, Rv2031c, andRv2627candmicrotubule-associated protein light chain 3 (LC3) as a hallmark protein of the autophagy system. Methods: A mouse construct was designed based on predictedMHCclass I-and class II-restricted T-cell epitopes that fused together tandemly. MHC class I-and class II-restricted epitopes were linked by AAY and GPGPG motifs, respectively. LC3 directly fused to the MHCclass II-restricted epitopes at the C-terminus of the peptide. The varieties of expressed construct features were analyzed by bioinformatics tools. Finally, construct codons were optimized and mRNA structure of optimized construct was analyzed. Results: MHC class I-and class II-predicted epitopes showed a high potential to binding to human HLAs alleles, with global broadspectrum population coverage. The construct had no allergenicity, and the analysis indicated a desirable antigenicity of the construct. The construct had several posttranslational modifications, no signal peptide, and cytoplasmic localization with high score. Also, mRNA analysis showed low
G which demonstrated high stability and efficient translation. Conclusions: The results revealed that the novel multi-epitope DNA construct could be an effective candidate in tuberculosis vaccine development, and it is qualified to investigate its potential to induce CD4 and CD8 T-cell immune response in the experimental animal model.

Background: Membrane vesicles are non-viable structures released by pathogenic bacteria. They contain numerous antigenic materials from the bacterial outer membrane, making them attractive targets for use as vaccine antigens. The membrane vesicles are related to the virulence because of their capacity to concentrate immunomodulatory molecules and toxins. Objective: In the present study, we examined the membrane vesicles of Mycobacterium tuberculosis as adjuvant and vaccine candidate. Methods: Mycobacterium tuberculosis standard strain CRBIP7. 11 was cultured at 37° C in Loewenstein Johnson (LJ) media for 3-4 weeks. To confirm the species, standard microbiological and biochemical tests were performed. After preparation of membrane vesicles, the amount of protein in membrane vesicles was measured by SDS-PAGE and Nanodrop. To analyze the integrity and morphology of extracellular vesicles, transmission electron microscopy was used. The lipopolysaccharide was determined using the Limulus Amebocyte Lysate (LAL) kit. Results: The total mass of vesicular fraction was 4. 8 mg. SDS-PAGE showed protein bands in the approximate regions of 35, 40, 70, and 90 kDa. The amount of membrane vesicles total protein was 1. 26 and 1. 29 mg/mL. Transmission electron microscopy analysis of pellets revealed that the extracted vesicles are 50-200 nm in size. Also, LaL test showed negative results (values were less than 300 IU). Conclusions: The results of the present study give important evidence that actively released mycobacterial vesicles are delivery instruments for immunologically active molecules involved in mycobacterial virulence.

Background: Multiple detection temperature (MuDT) technique is an advanced method for the analysis of multiple Ct (cycle threshold) values in a single channel. Objectives: The advantage of this method has been shown only in DNA samples, restricting its diagnostic applicability. This technique was evaluated in this study for its efficacy in the analysis of target RNA. Methods: Allplex GI-virus assay was developed to detect pathogens causing viral gastroenteritis, one of the major diseases caused by RNAviruses. This one-step multiplex real-time polymerase chain reaction (PCR) basedontheMuDTtechnique permits simultaneous amplification and detection of target nucleic acids of norovirus GI, norovirus GII, rotavirus A, adenovirus F, astrovirus, and sapovirus genogroups. The assay was tested for analytical sensitivity, cross-reactivity, repeatability, and applicability to clinical samples. Results: The analytical performance was validated for each target. The assay demonstrated high analytical sensitivity and no crossreactivity, and the repeatability tests showed excellent performance with high accuracy. Analytical performance validation indicated high positive agreement and negative agreement for this method. In the analyses comparing Allplex GI-virus Assay and commercial Seeplex Diarrhea-V ACE Detection using clinical specimens, the positive and negative agreements between the test results were found to be 94. 9% and 98. 8%, respectively. Statistical analysis showed that there was no difference in the performance between the two products. Conclusions: The Allplex GI-virus Assay can rapidly detect six viruses in a single tube without the complementary DNA synthesis step, and this assay was shown to represent an improved molecular diagnostic tool for the simultaneous detection of several RNA viruses. Therefore, our results suggest that theMuDTtechniquemayrepresent anewmolecular diagnostic method for the detection of RNA viruses.

Background: With theimprovementof quantitative molecular hepatitisCvirus (HCV)RNAassays, the usefulness of these assays has been indicated for management of HCV infection. Recently, various real time assays with different methodology and performance characteristics have been introduced. Objectives: This study aimed at designing, developing, and evaluating an in-house 1 step TaqMan real time-polymerase chain reaction (RT-PCR) assay for detection and quantification of HCV-RNA. Methods: The primers and probe were selected from a highly conserved region of the HCV genome, which allowed the detection of 4 common HCV genotypes in Iran. Using 4 quantification standards from 101 IU/ L to 104 IU/ L and clinical specimens, the current study determined analytical sensitivity, linear range, precision, analytical and clinical specificity, and validity of the assay. Data was analyzed by the SPSS statistical software (version 16). Results: The sensitivity of the assay with 95% probability, determined by probit analysis, was 15 IU/ L. The assay showed a linear range of 101 IU/ L to 104 IU/ L (R2 = 0. 989). The coefficient of variation for intra and inter assay precision of the assay, based on threshold cycle value, ranged from 0. 24 to 0. 4, and from 1. 94 to 3. 19, respectively. The analytical and clinical specificity was 100%. No bias in relation to concentration between the results of 29 HCV RNA positive clinical specimens, simultaneously tested by artus HCV LC RT-PCR reagents and in-house reagents, was observed in the method comparison. Conclusions: The in-house 1 step TaqMan Real Time RT-PCR assay showed acceptable performance characteristics. Our study presents the robustness and cost-effectiveness of the method for detection and quantification of HCV RNA.

Background: Human Papillomavirus (HPV) plays a necessary etiological role in cervical cancer, it is logical to use HPV as a marker for the early detection of cervical cancer and precancerous. Prevalence of HPV infection and HPV genotypes vary among different regions. Objectives: The aim of the present study was to investigate the frequency of HPVand its genotype distribution in cervical specimens of the patients who had attended to the pathology laboratories of Kermanshah, Iran. Methods: Cervical swaps, genital, and skin biopsy were obtained from each participant. DNA of HPV specimens were extracted using a genomics extraction kit. PCR amplification and genotyping were employed to investigate the presence of High-risk (HR) and Low-risk (LR) genotypes and also the types of HPV. Results: Human Papillomavirus was found in 62. 1% of the samples obtained from the patients. Among the HR and LR types, HPV-6 was the major genotype 64. 8%. Type 16 and 18 were the most frequent HR-HPV types (presented in 26% of the patient), followed by type 50s (24. 7%), 45, 30s, and 11 (1. 8% from each). Multiple HPV infections were presented in 40. 7% of the positive genotyping specimens. Conclusions: In comparison with previous surveys done in other parts of Iran, we noticed higher frequency of HPV among the collected isolates and also the higher prevalence of HPV types 6, 16, 18, and 50s in this region of Iran. It seems that HPV vaccination as well as an organized prevention program can be safe and efficient to reduce HPV types.

Background: Timely diagnosis of influenza virus is important because this virus can cause severe illness. The 2009 pdmH1N1 influenza virus spread rapidly throughout the world as the first infectious pandemic of the 21st century. Objectives: The aim of this study was the investigation of clinical and epidemiological figure of influenza virus A/H1N1, A/H3N2, and Influenza B infection among patients with respiratory syndrome in Golestan province, Southeast of Caspian see, Iran. Methods: This prospective, cross sectional study took place since November 2010 through March 2014. Demographic and clinical data were collected. Nasopharyngeal (NP) swabs were taken from patients with respiratory syndrome in virus transport medium and were extracted with High Pure Viral RNA Extraction Kit. Real time PCR were performed according to the CDC recommended protocol. Results: A total of 790 suspected cases were assessed; pandemic A H1N1, A/H3N2, and influenza B virus were confirmed in 25 cases (3. 2%), 21 cases (2. 7%), and 22 (2. 8%), respectively. The greatest number of confirmed cases occurred in the age group of 25 to 34 years. There was no significant association between positive cases and age, sex, residency, and clinical symptoms. Conclusions: The prevalence of pandemic Influenza viruses in recent years has caused financial losses as well as mortalities around the world. This shows the importance of the rapid diagnosis of common serotypes in our society. Using real-time RT-PCR is recommended for the early diagnosis and the rapid identification of the individuals infected with pandemic influenza virus.

Background: Loss of hepatitis B surface antigen (HBsAg) in chronic hepatitis B (CHB) infection is a favorable outcome. Type I interferon (IFNI) has an essential role to fight virus infections when they bind to the IFN- / receptor (IFNAR). Free-circulating IFNARs, known as IFNAR2, perform as carrier proteins to keep the ligands from proteolysis as well as antagonists for ligand binding. Objectives: In this study, we evaluated the HBsAg titer and IFNAR2 in serum baseline of a subcohort of Iranian HBeAg-negative patients with chronic hepatitis B. Methods: Sixty-four patients who spontaneously cleared HBsAg and 100 chronic hepatitis B patients enrolled in this study for assessment of the serum levels of HBsAg and IFNAR2. Results: Serum levels of HBsAg and IFNAR2 were both powerfully associated with loss of HBsAg. The baseline HBsAg titer was significantly lower (333. 72 1300 IU/mL vs 3811 6779 IU/mL, P = 0. 00) and the IFNAR2 serum level was significantly higher (1. 64 0. 6 vs 0. 87 0. 5 ng/mL, P = 0. 00) in those who cleared HBsAg compared to the CHB patients. Conclusions: These findings indicated the association of the HBsAg titer and serum IFNAR2 in HBsAg clearance in hepatitis B virusinfected patients. In consequence, immune mechanisms related to IFN- / signaling might be responsible in CHB outcome.

Background: Human cationic antibacterial peptide hCAP18/LL-37 is the only known cathelicidin, which is part of the innate immune system of humans. The antimicrobial activity of LL-37 against multi-drug-resistant Acinetobacter baumannii has been reported recently, however, whether LL-37 and its analogues (LL/CAP18 and FF/CAP18) have antimicrobial activity against pan-drug-resistant Acinetobacter baumannii (PDRAB) is still unknown. Objectives: This study aims to clarify the antimicrobial activity of LL-37 and its analogues against PDRAB. Methods: In this study, we evaluated LL-37 and its truncated analogs inhibitory effect on the growth of PDRAB by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) analysis, we also examined the effect of LL-37 and its analogs on bacterial biofilm formation by using a quantitative crystal violet assay. Results: LL-37 and its truncated analogs were effective in inhibiting the growth of PDRAB and had a rapid killing ability. After exposure to the peptides, the development of the PDRAB biofilm was inhibited. Strong inhibitory and dispersion effects of FF/CAP18 on the biofilm were confirmed by fluorescence microscopy and field emission scanning electron microscopy. Conclusions: This results show that FF/CAP18 is a potential antimicrobial agent for treating pan-drug– resistant bacterial infections.

Background: HIV/AIDS is a serious global health problem with an adverse impact on human health and his socioeconomic status in different countries. Objectives: The aim of the present study was to estimate the prevalence of HIV infection in Mashhad city. Methods: This cross sectional study was performed since May to September 2009 in Mashhad, Iran. A total of 1, 678 individuals ranged 1-90 years of age were selected randomly from different geographical regions of the city, proportionate to sex and age distribution of the population according to 2006 census. Enzyme-linked immunosorbent assay (ELISA) was used to screen anti-HIV antibodies and the positive samples were confirmed byWestern Blot (WB) assay. In anti-HIV positive cases, antibodies to human Tlymphotropic virus type 1 (HTLV-1) and hepatitis C virus (HCV) as well as the surface antigen of hepatitis B virus (HBV) were evaluated by the ELISA Kits. Results: Atotal of 1, 651serumsamples were analyzed for anti-HIV antibodies. Atotalnumberof 751 of participants were males (45. 5%) and 900 were females (54. 5%). The mean age was 27. 9 19. 0 and 30. 0 18. 0 years, respectively. Anti-HIV seropositivity was detected in 12 cases (0. 73%, 95% CI: 0. 38-1. 27 percent). No samples were further confirmed by WB technique, thus the overall prevalence of HIV infection was 0 (95% CI: 0. 00-0. 22 %). No case with co-infection of HBV, HCV, or HTLV-1 was observed in individuals who showed seroactivity for HIV antibodies. Conclusions: This first population-based survey showed no evidence of HIV infection in the general population of Mashhad. It seems that implemented health policies and strategies have contributed to this low prevalence and this shall be continued.

Background: Despite the implementation of the national control program, tuberculosis is one of the greatest health problems in Iran. The prevalence of different strains in specific ethnic populations suggests that Mycobacterium tuberculosis transmission has been limited and restricted to close contact. Objectives: In the present review, we describe the epidemiology of tuberculosis in Iran. Methods: In this review article, databases including Scopus, PubMed, and Google scholar were used to search for the epidemiology of tuberculosis in Iran. Results: Since 1996, tuberculosis incidence has been decreased as the national tuberculosis control program was established in Iran. However, due to the emergence of drug-resistant strains, recurrence of the disease, and association of tuberculosis with HIV pandemic, tuberculosis is becoming a health problem in Iran like many other parts of the world. Moreover, several other factors such as poverty, homelessness, inadequate access to health services, and lack of infrastructure in public health play an important role in worsening the situation. The distribution of the disease is not similar in all parts of Iran and it is higher in the western and eastern parts than the central areas. Conclusions: Review of the studies revealed that levels of multidrug-resistant tuberculosis among new tuberculosis patients are increasing in Iran and the vaccination system needs to be reformed. Because of the increasing number of patients coming from neighboring countries with multidrug-resistant tuberculosis, this area needs more stewardship and control in order to prevent the outbreak of multidrug-resistant tuberculosis. Finally, by improving the healthcare system, it is trusted that a more noteworthy number of these patients will be cured in Iran.

Background: Staphylococcus aureus is an important opportunistic pathogen and can cause a wide range of infections. The ability of this pathogen to successfully persist within the hospital and the community is largely due to its remarkable ability to acquire resistance against various antimicrobial agents. Objectives: The aim of this study was to determine the carriage of antibacterial resistance genes and virulence markers of S. aureus isolates from hospitalized patients in intensive care units in Tehran, the capital of Iran. Methods: In this cross-sectional study that was conducted during an 11-month period since March 2015 to January 2016, a total of 105 S. aureus strains were investigated. MRSA screening was performed by phenotypic and genotypic methods. The Kirby-Bauer disk diffusion method was used to assess the sensitivity of S. aureus strains. The strains were typed based on the polymorphisms in SCCmec types. The presence of resistance (ermA, ermB, ermC, mupA, msrA, msrB, tetM, ant (4´ )-Ia, aac (6´ )-Ie/aph (2˝ ), aph (3´ )-IIIa) and toxin (etb, eta, pvl, tst) encoding genes were investigated by the polymerase chain reaction (PCR) technique. Results: In this study, 105 isolates of S. aureus were obtained from 299 various clinical specimens. Ninety five (90. 5%) strains were confirmed as methicillin-resistant S. aureus. The lowest levels of resistance were related to quinupristin-dalfopristin (16. 8%) while the highest levels of resistance were related to penicillin (94. 7%). Multi-drug resistance was observed in 91. 5% of the isolates. Type IV was the most prevalent SCCmec type (57. 9%), followed by type III (22. 1), type V (12. 6%), I (5. 3%), and II (2. 1%). Overall, 25 isolates (26. 3%) harbored PVL-encoding genes, andall of thembelonged toSCCmec type IV. Thepresence of resistance genes ant(4´ )-Ia, aac(6´ )-Ie/aph(2˝ ), aph(3´ )-IIIa, ermA, ermB, ermC, msrA, msrB, and tetM was detected in 94. 7%, 81. 1%, 31. 6%, 31. 6%, 15. 9%, 18. 9%, 47. 3%, 21. 1%, 56. 8%. The frequency of the etb, eta, and tst genes were 1. 1%, 4. 2%, and 32. 6%, respectively. Conclusions: The results illustrated the diversity of antibacterial resistance and virulence gene profiles among different SCCmec types of S. aureus. The increased prevalence of methicillin-resistant S. aureus isolates containing different toxin and antibiotic resistance genes is a serious threat for the hospitalized patients in the intensive care units.