Jundishapur Journal of Microbiology
Volume:10 Issue: 4, Apr 2017

Molecular typing techniques are reliable tools for epidemiological study of tuberculosis because of their power in detecting recent transmission and differentiating reinfection and relapses.
The present study investigated epidemiological diversity among Mycobacterium tuberculosis strains circulating in three Khorasan provinces, Iran, using 12-loci MIRU-VNTR and spoligotyping.
This study was performed on 140 M. tuberculosis strains selected from the sputum of new cases of pulmonary tuberculosis patients in three Khorasan provinces, Iran. 12 loci MIRU-VNTR and Spoligotyping were performed on all isolates.
By MIRU-VNTR analysis, 76 distinct patterns comprising 19 clusters and 57 unique patterns were identified. Based on the results, MIRU10, MIRU26, and ETRF were highly discriminative, ETRD was poorly discriminative and other loci were designated as moderately discriminative. Spoligotyping of isolates revealed 51 distinct patterns: 26 patterns containing 33 strains (23.6%) corresponding to orphan strains and 14 patterns containing 107 strains (76.4%) corresponding to shared-types in the SITVIT2 database. Totally, 103 isolates (73.6%) were classified into 14 clusters containing 2 - 56 isolates; the remaining 37 isolates (26.4%) were unique patterns. By combining two techniques, 94 distinct patterns (15 clusters) contained 61 isolates (43.6%), and 79 unique patterns were identified. The discriminatory power (HGDI) of combination of two techniques was 0.962, which was higher than that of each technique alone. Based on the trees designed by Bionumerics software, we differentiated isolates with similar genetic patterns and grouped them together. Two great clusters were Haarlem and CAS lineage. All strains with combined drug resistance related to Beijing strains. Also, all mono drug-resistant strains related to Haarlem family; other strains were susceptible to the first-line anti-tuberculosis drugs. Also, homoplasy was observed in a number of patterns.
In MIRU-VNTR typing method, according to the genotype of each area, the loci with high discriminatory power (such as miru10, miru26, and ETRF in Iran) are recommended to be used and the loci with poor discrimination (such as ETRD in Iran) are not.

Legionnaires’ disease (LD) is a common form of severe pneumonia, caused by Legionella spp. Legionella pneumophila is an important agent of severe pneumonia including 15 serogroups, which are all human pathogens. However, L. pneumophila serogroup 1 is the most prevalent agent of LD. Fatality rates among elderly and immunocompromised patients are high and may occur as a result of infection with this pathogen.
The aim of this study was to detect the LD agent in clinical samples of patients with respiratory symptoms by culture, urinary antigen and polymerase chain reaction (PCR) of the 16SrRNA gene.
In this study, a total of 200 specimens (including 100 urine and 100 respiratory samples), which were collected from hospitalized patients with respiratory symptoms were examined. The respiratory specimens were inoculated to the buffered charcoal-yeast extract and modified Wodowsky and Yee agar media for isolation of the Legionella spp. The 16S rRNA gene in the respiratory specimens was amplified by the PCR method and the urinary antigen of L. pneumophila serogroup 1 was detected by EIA (enzyme immunoassay) test using the Coris Legionella V-test kit.
From a total of 200 specimens from patients with respiratory symptoms, 5% of urine specimens and 3% of respiratory specimens were positive for L. pneumophila using the EIA test and PCR of the 16SrRNA gene, respectively. The results of the culture of the respiratory samples showed that 1% of them were positive for Legionella spp.
In this study, the LD agent was detected by the rapid EIA test. In addition, the sensitivity of the urinary antigen test using the Coris Legionella V-test kit for detection of L. pneumophila in respiratory specimens was more than those of the PCR and culture methods.

There should be a public environmental reservoir for Helicobacter pylori in the developing countries, such as Iran, due to their high infection rate of over 70%. Epidemiological findings revealed that water could be a possible source of H. pylori transmission. However, high prevalence of H. pylori in drinking water in Kermanshah, West of Iran, was detected in the authors’ previously published study. The current study aims at designing a more accurate and rapid procedure to investigate the prevalence of Helicobacter species and cagA gene in drinking water samples in Kermanshah, from October to December 2012.
In the current study, 60 tap water samples were obtained and specific polymerase chain reaction (PCR) targeted cagA and 16s rRNA was performed. A loop-mediated isothermal amplification (LAMP) targeted ureC gene was developed to accurately detect H. pylori in water samples.
The prevalence of ureC by PCR, ureC by LAMP and 16s rRNA by PCR were 26.67%, 38%, and 61.67%, respectively. Among 24 samples (40%), 1 of the 2 tests was positive. The prevalence of cagA gene among ureC positive, 16s rRNA positive and all samples were 18.75%, 13.51%, and 10%, respectively.
Helicobacter pylori contamination in drinking water was considerably higher using LAMP compared with PCR. It is noteworthy that some H. pylori positive samples were also positive for Caga.

Salmonella is one of the major agents of food-borne diseases that cause severe illness in humans. The conventional detection methods of these bacteria are time-consuming with low sensitivity and specificity, which limit their applications. Therefore, developing more rapid and accurate methods is urgently required in food safety programs.
In this study for the first time, polymerase chain reaction-Temporal temperature gradient gel electrophoresis (TTGE) was optimized for the identification of Salmonella enterica subspecies enterica serovars in processed food samples using a single-copy sequence.
DNA was isolated from pure cultures of Salmonella and non-Salmonella strains. The single copy target sequence was selected and amplified by employing the polymerase chain reaction (PCR) with specific primers, designed in this study, and their specificity and sensitivity were determined. The TTGE parameters, especially the temperature gradient and the time of reaction, were optimized and then this method was applied to investigate spiked food samples.
The PCR detection sensitivity was recorded as 12 × 103 CFU/mL in artificially-contaminated food samples. The best resolution was observed at a temperature gradient from 62.5 to 67.5°C, with a ramp rate of 1°C/hour and electrophoresis for 5 hours at a constant voltage of 130 V. The TTGE patterns obtained from the artificially-contaminated samples were similar with the respected standard bacteria strains. The optimized TTGE protocol resulted in the separation of the same length PCR products into different band positions on the polyacrylamide gel.
By optimization of PCR-TTGE and determination of distinct band pattern for standard bacteria, this method can be used as a fast screening test to investigate the presence of S. enterica, subspecies enterica, directly in food samples.

Infection by certain types of human papilloma virus (HPV) is known as a causal and essential factor for cervical cancer, the second most common malignancy in women around the world.
The aim of this study was to determine the frequency and types of HPV among women with normal and abnormal cytology in Southern Khorasan, eastern Iran.
This was a cross-sectional study with 253 randomized Pap smear samples from women who were referred to gynecologist clinics. Human papillomavirus-DNA testing (a nested PCR with primers MY09/ MY11 and GP5 GP6 ) was performed on Pap smear samples. The first round PCR product was subjected for sequencing to determine the HPV types. Phylogenic analysis with Mega 6 was carried out to determine the relationship between HPV types.
The mean age of patients were 34.47 ± 5.38 years; 85.77% with normal cytology, and the rest were with an abnormality; atypical cells of undetermined significance (ASCUS) and Low-grade squamous intraepithelial lesions (LISL). Human papilloma virus-DNA was detected in 18.57% of population (15.66% of normal and 36.11% of abnormal group) with the most prevalent HPV types 6 and 11. The HPV type 84 was identified in a case.
The result of this study revealed a partially high prevalence of HPV in women with normal cytology which are high risk for transmission in population. It is suggested that HPV testing should be carried out along with Pap test in screening programs to enhance early detection of neoplasia and intended infections.

Cryptosporidium is a protozoan parasite that effects rodents, dogs, calves, humans, and cats. Infection with this parasite is known as cryptosporidiosis. Cryptosporidium spp. may induce clinical or subclinical signs in infected hosts. In the life cycle of this parasite infected dogs freely living in urban and rural areas of Khuzestan province are the definitive hosts that should be considered as a real problem in public health for humans.
This study aimed at determining the frequency of cryptosporidiosis in dogs in southwest of Iran.
Overall, 350 fresh fecal samples were collected from domestic dogs living in 43 villages, from June 2012 to September 2013. All samples were investigated by Sheather’s concentration method and fecal smears were stained with modified Ziehl-Neelsen followed by light microscope examination, and polymerase chain reaction (PCR).
The results revealed that frequency of Cryptosporidium infection was 8% and 12.3%, using direct smear and molecular method, respectively.
The present findings indicated that domestic dog feces from southwest of Iran may contain zoonotic parasites such as Cryptosporidium spp. and may be a potential risk for humans and other animals, especially when they contaminate the environment. The role of dogs as source of human infection should be investigated by further studies.

About 90% of cutaneous leishmaniasis (CL) cases are reported from 7 countries including Iran. In this study, the cutaneous leishmaniasis species causing CL in Gonabad, Bardaskan, Kashmar cities in central Khorasan (Iran) were identified by kDNA-PCR.
During the study, 93 suspected patients with CL, who were referred to the dermatology research center in these cities, were evaluated based on age, clinical forms, and place of residence. Direct microscopy was employed for parasitological diagnosis and PCR from skin ulcers performed using specific kDNA primers. Data were analyzed using the SPSS software.
Among 93 individuals with skin ulcers suspected to CL, the results of 81 direct smears were positive. PCR bands were observed in 84 examined samples, of which 68 (81%) samples were identified for Leishmania tropica and 16 (19%) samples for L. major. 12 patients were from Bardaskan with 10 cases of L. tropica, 23 patients from Gonabad with 20 cases of L. tropica, and 49 patients from Kashmar with 38 cases of L. tropica. Most of the lesions were located on hands (37%), the most clinical feature was papule (75%), and most of patients were 21 - 30 years old.
Previous epidemiologic studies have indicated that anthroponotic cutaneous leishmaniasis is the only dominant CL in the center and south of Khorasan. However, this study introduced new foci of zoonotic cutaneous leishmaniasis in these areas. Kashmar city was introduced as a new focus of zoonotic cutaneous leishmaniasis.

Voriconazole is a triazole antifungal agent with considerable inter and intra-individual variability in plasma concentrations. Therapeutic drug monitoring of voriconazole plays an important role in optimizing the efficacy and safety of the drug in patients.
This study aimed at evaluating the performance of a simple agar well diffusion bioassay and comparing its utility with high-performance liquid chromatography (HPLC).
The clinical isolate of voriconazole hyper susceptible Candida kefyr was used for a simple agar well diffusion bioassay method. Acetonitrile precipitations followed by reverse-phase HPLC on C18 column, with ultra violet detection, were used for HPLC. Cross validation was done by evaluating the accuracy and precision of both methods in a large cohort of 180 samples from 60 voriconazole-treated patients.
The validated bioassay method and HPLC, as a reference method, were found to be accurate and precise. A good correlation was found between the 2 methods with similar analytical range (0.25 – 16 µg/mL). The result of linear regression analysis revealed bioassay = 0.961 (HPLC) .148; R2 = 0.965; correlation coefficient = 0.982; n = 180. Voriconazole serum concentration of patients ranged from 0.25 µg/mL to 5.41 µg/mL when using HPLC, and from 0.25 µg/mL to 5.71 µg/mL when using the bioassay method.
When laboratories are not equipped with HPLC, bioassay may be a reliable technique with sufficient accuracy and precision for monitoring voriconazole plasma concentration.

Human papilloma virus (HPV) is the most common sexually transmitted viral infection worldwide. On the basis of HPV clinical associations, this disease is divided to 2 types; high risk and low risk. high risk HPV (HR HPV) is the causative agent of cervical intraepithelial Neoplasia (CIN) and cervical cancer. low risk HPV (LR HPV) is the causative agent of diseases, such as anogenital warts and oral papillomas. Polymerase chain reaction (PCR) is one of the molecular diagnostic methods for detection of HPV DNA that has many advantages.
This study aimed at exploring the frequency of HPV genotypes using polymerase chain reaction reverse dot blot (PCR RDB) method on cervix and wart samples obtained from patients in Isfahan, Iran.
The population included 111 females and 7 males. These samples were collected during 2011 and 2013. DNA was extracted from these samples and PCR was performed using reverse dot blot (RDB) method to detect HPV types.
With this molecular diagnostic method, different HPV types were detected in 45 samples out of 118. In 18 samples, co-infection with different HPV types was detected. Data analysis exhibited a significant correlation between age and HR HPV infection (P value 0.05). In this study, among all HPV types, HPV 16 was detected more frequently than the other HPV types.
The PCR-RDB method has the ability to diagnose HPV infection by screening Pap smear samples and to determine the risk factor of detected HPV genotypes. This study showed that HPV16 was 1 of the most common agents in Iran, similar to other countries.