Jundishapur Journal of Microbiology
Volume:12 Issue: 8, Aug 2019

Mycobacterium tuberculosis genotyping is an essential step for understanding the epidemiology of tuberculosis. Mycobacterial-interspersed-repetitive-units (MIRU)-variable-number-of-tandem-repeats (VNTR) typing is an important method for this purpose. The study aimed to determine the reproducibility of 15-loci MIRU-VNTR method. DNA extraction and 15-loci MIRU-VNTR were carried out for genotyping of 60 M. tuberculosis isolates collected from different clinical samples of 27 M. tuberculosis patients, each with two to four clinical isolates of M. tuberculosis. The similarity of M. tuberculosis isolates of the same patient was investigated by MIRU-VNTRplus. The patterns of MIRU-VNTR were identical in 43 M. tuberculosis isolates collected from 20 tuberculosis patients, giving the repeatability of 71.6% for the test. In 12 isolates of M. tuberculosis belonging to five patients, the variable-number tandem repeat (VNTR) in only one locus was different. In three M. tuberculosis isolates of one patient, two different genotypes were identified. This study confirms the high reproducibility of 15-loci MIRU-VNTR, except for limited cases.

Staphylococcus aureus is a frequent cause of hospital and community-associated infections on a global scale. This pathogen is responsible for causing an extensive range of diseases and sometimes create biofilms for their survival. Biofilm formation, leads to difficulty in treatment with antibiotics and antibiotic resistance which is another rising concern in health centers. Some S. aureus virulence factors encode on the core genome, such as alpha-toxin and phenol-soluble modulins (PSMs), which are produced by nearly all strains. There is no information about the expression of PSM A gene in clinical isolates of biofilm-producing S. aureus in Iran. This study was performed on clinical samples to determine the prevalence and expression of PSM A gene in biofilm-producing S. aureus clinical isolates. The clinical samples werecollected and examinedfor S. aureusby microbiological and biochemical tests. Then, thebiofilm formation in S. aureus isolates was detected by microtiter plate. Finally, the expression of PSM A was determined using SYBR Green real-time PCR. From a total of 60 isolates of S. aureus, 47 strains (78.3%) had ability of biofilm formation and the others were negative for biofilm formation. Real-time PCR testing showed that 100% of the strains were positive for biofilms and PSM A genes. The results of phenotypic and genotypic tests of biofilm were closely related to each other and the expression of PSM A gene was 80%. It was found that 100% of strains were biofilm producing and PSM A gene was present in 78.3% (47 strains) of them. The prevalence of biofilm production in S. aureus strains isolated from clinical samples was high, so it is highly important to monitor the prevalence of these organisms in hospitals and community as well as their antimicrobial resistance.

In the era of drug resistant organisms, there exists a huge need to overcome this issue. Drug repositioning is the action of repurposing current drugs against alternative targets. Herein weas sessed the antileishmanial activity of ethinyl estradiolandtolmetinonthe promastigotes and amastigotes of Leishmania infantum, using methyl thiazolyl tetrazolium (MTT) and atomic force microscopy (AFM) methods. Following previous in silico evaluation of 3358 FDA-approved compounds, ethinyl estradiol and tolmetin drugs were selected. Promastigote assay was done in RPMI 1640 culture in 96-well plates (105/100L). Also, axenic amastigotes cultured in acidic RPMI 1640 were seeded in 96-well plates (5  104 parasites). Raw 264.7 macrophages were seeded at a density of 3  106 cells/well. Then, the macrophages were infected with metacyclic promastigotes of L. infantum. Also, drug solvent and amphotericin B were considered as control wells. Drug challenge was performed in triplicate with different concentrations of ethinyl estradiol (250, 125, 62.5, 31.25, 15.62 and 7.81 g/mL) and tolmetin (200, 100, 50, 25, 12.5 and 6.25 g/mL) and cell viability was assessed by MTT assay. AFM imaging was done to observe morphological changes. The main finding of our investigation was the substantial effect of ethinyl estradiol on promastigotes, and amastigotes of L. infantum during 24, 48 and 72 h, according toMTT results and cellular morphology observations. Thus, the IC50 of this drug on promastigote, axenic amastigotes and macrophage-dwelling amastigotes during 72 h was 23, 45 and 32 g/mL, respectively. However, tolmetin was not effective against L. infantum parasites. Cellular alteration, was observed by AFM technique. This study showed that ethinyl estradiol had good anti-leishmanial activity and it is recommended to test this drug under in vivo condition. With the aid of drug repurposing we could substitute the old drugs with novel compounds for a specific disease.

Leishmaniasis is a protozoan disease caused by Leishmania genus and its most common form is cutaneous leishmaniasis. The number of reported cases of cutaneous leishmaniasis in Khuzestan, southwest Iran, continues to increase. Therefore, early, accurate diagnosis is crucial for successful therapy. The Nested PCR is a molecular method with high sensitivity and specificity in detecting leishmaniasis, but thismethod requires advanced equipment and skilled labor. Therefore, developing a simple yet accurate technique to diagnose leishmaniasis is essential. This study was designed to evaluate the possibility of replacing Nested PCR with loop-mediated isothermal amplification (LAMP) method for diagnosis of cutaneous leishmaniasis. We obtained 75 clinical samples from cutaneous leishmaniasis patients whose infection had already been confirmed by microscopic examination. The LAMP assay with pre-added malachite green was performed using a set of four primers targeting conserved sequences of the18S ribosomal RNA gene. The nested PCR method was performed using specific kinetoplast minicircle DNA primers. Cultured promastigotes of Leishmania major (MHOM /IR/75/ER), L. tropica (MHOM/IR/02/Mash10), and the virulent RH strain of Toxoplasma gondii were used as controls. Our results showed that LAMP was positive for 100% of microscopically positive samples, which was similar to Nested PCR. The detection of L. major was improved at 104 parasites/ml using the LAMP method. Our findings suggest that considering the simplicity, specificity, and sensitivity of LAMP, it would be a potentially useful method in the diagnosis of leishmaniasis and detection of its various strains/species. This cost and time-effective method can be used as a suitable alternative for surveillance of leishmaniasis, as well as in epidemiological studies.

Cryptosporidium is one of the most important causes of gastroenteritis in humans. This study aimed to investigate the distribution of Cryptosporidium species in children with acute diarrheal disease in Zahedan, Iran. Stool specimens collected from 764 children aged < 10 years with diarrheal disease admitted to Aliasghar Pediatric Hospital and the Reference Laboratory in Zahedan were assessed for the presence of Cryptosporidium oocysts using the conventional microscopic examination and acid-fast staining. To characterize the oocysts at the molecular level, the 18S rRNA was nested-PCR amplified and sequenced. PCR products were also digested using Vsp1 restriction enzyme for genotype differentiation. Of the 764 stool specimens, 7 (0.91%) were positive for Cryptosporidium oocysts using the microscopic examination. Restriction endonuclease digestion pattern successfully revealed the presence of C. parvum in two microscopic positive samples that were confirmed by DNA sequencing. C. parvum is responsible for cryptosporidiosis in children under 10 years of age in the study region. Although C. parvum may be an important pathogen associated with diarrhea, it is the cause of only a small proportion of diarrheal episodes.

Vaccination is known as one of the effective methods for the prevention of infectious diseases. Birth season as an environmental factor can play an important role in the immune response to vaccines.

This study aimed to determine the association between hepatitis B virus (HBV) surface antibody (HBsAb), the expression of TLR2, 3 and 4 genes in response to the hepatitis B vaccine, and the birth season of children aged 3 - 5 years.

In this study, 72 children aged 3 - 5 years born in winter and summer (36 in each group) were enrolled for blood sample collection. Then, the HBsAb titer and TLR2, 3, and 4 were determined by the ELISA method and real-time PCR, respectively.

The results indicated that the expression of TLR2, 3, and 4 genes was higher in the winter-born group than in the summer-born group. However, this difference was not statistically significant (TLR2 P value = 0.5; TLR3 P value = 0.06; TLR4 P value = 0.16). The overall efficacy of HBsAb was 72.2%. The average level of antibody was greater in the summer-born group than in the winter-born group but this difference was not statistically significant (P value = 0.3).

Although TLR2, 3, and 4 expression levels were higher in the winter-born group than in the summer-born group and the antibody titer was higher in summer than in winter, these differences were not statistically significant. However, more research is needed to prove these relationships.