Human parvovirus B19 infection in malignant and benign tissue specimens of different head and neck anatomical subsites.

BACKGROUND: The role of human parvovirus B19 (B19V) infection in malignant and benign lesions such as head and neck squamous cell carcinomas (HNSCCs) and oral mucocele lesions has not been established. Herein, we examined, for the first time, the presence of B19V in HNSCCs from Iranian subjects. METHODS: One hundred and eight HNSCC specimens were analyzed for the presence of B19V using nested polymerase chain reaction (nPCR) and TaqMan quantitative PCR assays. Immunohistochemistry procedures were performed to evaluate the expression of B19V VP1/VP2 proteins, p16INK4a, and NF-κB in tumor tissues and their adjacent non-tumor tissues. In addition, 40 oral mucocele, 30 oral buccal mucosa swabs, and 30 nasopharyngeal swabs obtained from healthy adults were analyzed as controls. RESULTS: B19V DNA was detected in 36.1% of HNSCCs. Further, 23.3% of HNSCC specimens showed immunoreactivity against B19V VP1/VP2 proteins. There was a significant difference in the frequency of B19V DNA-positive cases between the patient and control groups (p < 0.0001). Moreover, comparing tumoral tissues and their adjacent non-tumor tissues in terms of immunoreactivity against B19V structural proteins, a significant association was found between tumor tissues and B19V infection (p < 0.0001). Finally, investigating the simultaneous presence of B19V and high-risk human papillomaviruses (HPV) DNA, we found a significant association between these two viral infections in HNSCCs (p = 0.031). CONCLUSIONS: To sum up, B19V was frequently present in HNSCC tissues of Iranian patients but mostly absent in the adjacent non-tumor tissues as well as oral mucocele lesions, buccal, and nasopharyngeal swabs of healthy subjects. HPV possibly contributes to B19V persistence in HNSCC tissues. Additional research is required to investigate potential etiological or cofactor roles of B19V in the development of HNSCCs.

Type distribution of human papillomaviruses in ThinPrep cytology samples and HPV16/18 E6 gene variations in FFPE cervical cancer specimens in Fars province, Iran.

BACKGROUND: There exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran. METHODS: A total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees. RESULTS: Overall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively. CONCLUSION: The present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening.

Simultaneous detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacteria coexisting in patients with pulmonary tuberculosis by single-tube multiplex PCR.

Background and Objectives: In clinical diagnostics, molecular methods are used to detect Mycobacterium tuberculosis bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube. Materials and Methods: 100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the rpoB gene, encoding the beta-subunit of RNA polymerase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis. Results: Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuberculosis drugs. Conclusion: A single-tube mPCR assay based on the rpoB gene provides a rapid and reliable means of detecting and differentiating MTB and NTM in sputum specimens.

Human herpesvirus 6A and 6B and polyomavirus JC and BK infections in renal cell carcinoma and their relationship with p53, p16INK4a, Ki-67, and nuclear factor-kappa B expression.

There are a limited number of studies regarding the involvement of viruses in the development and pathogenesis of renal cell carcinoma (RCC). In this study, we aimed to discover whether human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) and human polyomavirus JC (JCV) and BK (BKV) are associated with RCC and the expression of p53, p16INK4a, Ki-67, and nuclear factor-κB (NF-κB) in patients with RCC. A total of 122 histologically confirmed RCC tissue specimens and 96 specimens of their corresponding peritumoral tissues were included in this prospective study. Nested PCR was performed to amplify viral DNA sequences. Restriction endonuclease analysis was carried out to discriminate between HHV-6A and HHV-6B. p53, p16INK4a, Ki-67, and NF-κB immunostaining data of the studied tissue specimens were available from our previous study. Statistical analysis was performed to demonstrate the potential associations. HHV-6B and JCV were detected in 10.7% and 13.9% of patients with RCC, respectively. We did not detect HHV-6A and BKV in any of RCC tissue specimens. Moreover, no association was found between either of these viruses and RCC. Our study revealed a significant association between HHV-6B and p53 overexpression. No other associations were found between cellular biomarkers p53, p16INK4a, Ki-67, and NF-κB and the studied viruses. The data of this study, though very limited, disprove the involvement of HHV-6A, HHV-6B, BKV, and JCV in the initiation or progression of RCC.

The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases.

Background: The nonstructural protein (NS1) of human parvovirus B19 (hPVB19) is considered to be a double-edged sword in its pathogenesis. NS1 protein promotes cell death by apoptosis in erythroid-lineage cells and is also implicated in triggering and the progression of various inflammation and autoimmune disorders. Objectives: We investigated the possible role of hPVB19 NS1 in the modulation of proinflammatory cytokines in nonpermissive HEK-293T cells. Methods: A plasmid containing the fully sequenced NS1 gene (pCMV6-AC-GFP-NS1) was transfected into HEK-293T cells. Transfection efficiency was assessed by fluorescent microscopy over time. Mock (pCMV6-AC-GFP) transfected cells were used as controls. The percentage of apoptotic cells was measured by flow cytometry at 24, 48, and 72 h posttransfection. Interleukin 6 (IL-6) mRNA, as a pleiotropic cytokine, was measured by real-time PCR. Furthermore, cellular supernatants were collected to determine the type and quantity of cytokines produced by mock- and NS1-transfected cells using flow cytometry. Results: Fold change in the expression level of IL-6 mRNA in transfected cells after 72 hr of incubation was found to be 3.01 when compared with mock-transfected cells; however, cell apoptosis did not happen over time. Also, the concentration of cytokines such as IL-2, IL-6, IL-9, IL-17A, IL-21, IL-22, interferon (IFN)-γ, and tumor necrosis factor α (TNF-α) increased in NS1-transfected cells. Conclusions: Overall, our results indicated that proinflammatory cytokine levels had increased following the expression of hPVB19 NS1 in HEK-293T cells, consistent with a role for NS1 expression facilitating the upregulation of inflammatory reactions. Therefore, hPVB19 NS1 function may play a role in the progression of some chronic and inflammatory diseases.

Epstein-Barr virus infection is associated with the nuclear factor-kappa B p65 signaling pathway in renal cell carcinoma.

BACKGROUND: There have been few studies regarding viral involvement in the pathogenesis of renal cell carcinoma (RCC). The aim of this study was to examine the possible association of Epstein-Barr virus (EBV) infection with clinicopathological features and cellular biomarkers including p53, p16INK4a, Ki-67 and nuclear factor-kappa B (NF-κB) in RCC tumors. METHODS: In this prospective study, 122 histologically confirmed Formalin-fixed Paraffin-embedded RCC tissue specimens along with 96 specimens of their corresponding peritumoral tissues and 23 samples of blunt renal injuries were subjected to nested polymerase chain reaction (nPCR) in order to amplify EBV DNA sequences. The expression of p53, p16INK4a, Ki-67 and NF-κB was investigated by immunohistochemistry (IHC) assay. Statistical analysis was employed to demonstrate the possible associations. RESULTS: Infection with EBV was found to be significantly associated with RCC. Our results indicate that p65 NF-κB signaling pathway is probably involved in EBV-mediated RCC pathogenesis. Moreover, we found p53, Ki-67 and cytoplasmic NF-κB expression to be associated with tumor nuclear grade in RCC patients. The expression of p53 and Ki-67 was associated with primary tumor category as well. In addition, p53 overexpression was significantly more frequent among nonconventional RCC tumors than the conventional histologic type. CONCLUSIONS: Infection with EBV is likely to play an important role in the development of RCC through the constitutive and permanent activation of NF-κB p65 signaling pathway. However, more experiments and supporting data are required to reach a decisive conclusion.

Immobilization of horseradish peroxidase on lysine-functionalized gum Arabic-coated Fe3O4 nanoparticles for cholesterol determination.

BACKGROUND: Horseradish Peroxidase (HRP) is ranked as one of the most important industrial enzymes that is extensively used in industry. Cholesterol is routinely detected indirectly by cholesterol oxidase in the presence of O2, liberating H2O2 as a by-product. The H2O2 content is determined through the HRP activity in the presence of a redox dye, producing a red colored quinoneimine which can be measured quantitatively. Herein, we have designed a magnetic nanoparticle for reusing and easily separating HRP as the most expensive compartment for the low-cost cholesterol assay. METHODS: The gum Arabic coated magnetic nanoparticles were functionalized with L-lysine linker for maintaining protein flexibility on nanoparticle. Enzyme-loaded nanoparticles were characterized by TEM, FTIR, DLS, VSM and XRD analysis. RESULTS: The immobilization efficiency was ∼65% and the immobilized HRP retained 60% of its activity after 8 times reuse. The optimum pH and thermal stability shifted from 7.0 to 8.0 and 60 to 70 °C after immobilization, respectively. Storage stability of HRP was improved by 10%, at 4 °C for 60 days. Immobilized HRP showed more catalytic activity in presence of Fe2+, Ca2+ and Na+. The designed system has cholesterol detection linearity range from 0.2 to 5.0 mM and detection limit of 0.08 mM and acceptable correlation coefficient of 0.9973 and 0.9982 on sample serum using both chromogens. CONCLUSION: The HRP-loaded magnetic nanoparticles are capable of being used as a cost-effective system for cholesterol determination in laboratory due to its reusability and stability benefits.

Emergence of Highly Multidrug-Resistant Bacteria Isolated from Patients with Infections Admitted to Public Hospitals in Southwest Iran.

BACKGROUND: The emergence of multidrug-resistant (MDR) microorganisms causing infections is increasing worldwide and becoming more serious in developing countries. Among those, Acinetobacter species are becoming prominent. OBJECTIVES: The aim of this study was to determine the rate of antimicrobial resistance of the bacteria causing infections, Acinetobacter species in particular, in local public hospitals in Firuzabad, Fars province, Iran. METHODS: This cross-sectional study was performed on different clinical specimens collected from patients who were suspected of infections hospitalized from March 2016 to March 2019 in local hospitals of Firuzabad, Fars province, Iran. The bacterial isolates were identified following standard microbiological methods. Clinical and Laboratory Standards Institute guidelines were used to identify the antibiotic susceptibility of these isolates. RESULTS: Overall, 1778 bacterial etiologies were isolated from 1533 patients diagnosed with infection. Of these, 1401 (78.8%) were Gram-negative and the remaining were Gram-positive bacteria. Escherichia coli (37.1%), Klebsiella spp. (13.9%), and Acinetobacter species (10.4%) were the most common isolated bacteria. Antibiotic sensitivity testing in this study showed a high resistance rate of Acinetobacter species to all antibiotics tested except Colistin. During the study period, the rate of infection with highly multidrug-resistant Acinetobacter species increased from 7.2% to 13.3%. CONCLUSIONS: This study highlights the emergence of MDR bacterial agents such as Acinetobacter species as a new threat in our region. However, a decrease in the rate of infection with Pseudomonas aeruginosa was noticeable.