False-Positive Mycobacterium tuberculosis Detection: Ways to Prevent Cross- Contamination / 결핵및호흡기질환

The gold standard method for diagnosis of tuberculosis is the isolation of Mycobacterium tuberculosis through culture, but there is a probability of cross-contamination in simultaneous cultures of samples causing false-positives. This can result in delayed treatment of the underlying disease and drug side effects. In this paper, we reviewed studies on falsepositive cultures of M. tuberculosis . Rate of occurrence, effective factors, and extent of false-positives were analyzed. Ways to identify and reduce the false-positives and management of them are critical for all laboratories. In most cases, falsepositive is occurring in cases with only one positive culture but negative direct smear. The three most crucial factors in this regard are inappropriate technician function, contamination of reagents, and aerosol production. Thus, to reduce false-positives, good laboratory practice, as well as use of whole-genome sequencing or genotyping of all positive culture samples with a robust, extra pure method and rapid response, are essential for minimizing the rate of false-positives. Indeed, molecular approaches and epidemiological surveillance can provide a valuable tool besides culture to identify possible false positives.

Cytotoxic effect of rosemary extract on gastric adenocarcinoma [AGS] and esophageal squamous cell carcinoma [KYSE30] cell lines

Aim: The present study was conducted to survey the potential cytotoxic influence of freeze-dried aqueous extract of its fruits on gastrointestinal cell lines, namely AGS [human gastric carcinoma] and KYSE30 [human esophageal squamous cell carcinoma]

Background: Rosemary [Rosmarinus officinalis] is a wild medicinal plant shown to have anticancer activity. Carnosic and rosmarinic acids are compounds, obtained from it through several extraction methods

Methods: The aqueous extract of the fruits of R.officinalis was freeze-dried, and KYSE30 and AGS cancer cell lines were treated with crude extract. Cytotoxic effect of the extracts on the cell lines was examined using 3-[4, 5-Dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide [MTT] and neutral red assay. Apoptotic cells were detected with ethidium bromide/acridine orange [EB/AO]. Cell-cycle distributions were evaluated by flow cytometry

Results: IC50 values were 4.1, 1.8 and 1.3 mg/mL for AGS cell lines after 24, 48 and 72 hours by MTT assay, respectively, and 4.4, 2.1 and 1.1 mg/mL by neutral red assay, respectively. IC50 values for KYSE30 cell lines were 600, 180 and 150 mg/mL after 24, 48 and 72 hours by MTT assay, and 860, 270 and 230 mg/mL by neutral red. EB/AO staining increased in apoptotic cells. After 24 h of treatment at different concentrations, significant increases and decreases in population were shown at G2/M and G1 phases, respectively

Conclusion: The aqueous extract of the fruits of R.officinalis was freeze-dried, and KYSE30 and AGS cancer cell lines were treated with crude extract. Cytotoxic effect of the extracts on the cell lines was examined using 3-[4, 5-Dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide [MTT] and neutral red assay. Apoptotic cells were detected with ethidium bromide/acridine orange [EB/AO]. Cell-cycle distributions were evaluated by flow cytometry

Pulmonary Tuberculosis Diagnosis: Where We Are? / 결핵및호흡기질환

In recent years, in spite of medical advancement, tuberculosis (TB) remains a worldwide health problem. Although many laboratory methods have been developed to expedite the diagnosis of TB, delays in diagnosis remain a major problem in the clinical practice. Because of the slow growth rate of the causative agent Mycobacterium tuberculosis, isolation, identification, and drug susceptibility testing of this organism and other clinically important mycobacteria can take several weeks or longer. During the past several years, many methods have been developed for direct detection, species identification, and drug susceptibility testing of TB. A good understanding of the effectiveness and practical limitations of these methods is important to improve diagnosis. This review summarizes the currently-used advances in nonmolecular and molecular diagnostics.

Multidrug-resistant tuberculosis in north-west of Iran and republic of Azerbaijan: a major public health concern for Iranian people

Republic of Azerbaijan is considered as an area with high prevalence of multidrug resistant tuberculosis. Uncontrolled travelling of Azerbaijanis people to Iran is the issue that needs to be considered as an important issue. This study was conducted on 32 patients with tuberculosis from Baku-Nakhchivan and 48 patients from Iran during 2012 to 2014. Colonies of Mycobacterium tuberculosis were examined after isolating them from patients using proportional method on Lowenstein-Jensen media regarding resistance encounter with Rifampin, Isoniazid and Ethambutol. Among M. tuberculosis isolates belonging to 32 foreign patients; 69%, 72% and 56% of them were resistant to Rifampin, Isoniazid and Ethambutol, respectively [multidrug resistance tuberculosis: MDR-TB: 62.5%]. From 48 isolates of Iranian patients; 8%, 4% and 4% were resistant to Rifampin, Isoniazid and Ethambutol, respectively [MDR-TB: 2.1%]. Resistant strains are common in people from Baku-Nakhchivan. To prevent the transmission of these strains to Iranians, strategies such as; establishing a medical campus in border lines of both countries for clinical examinations and conducting screening tests regarding tuberculosis infection in applicants for entering Iran must be taken in to account

Incidence of giardia lamblia subspecies by PCR-RFLP in stool specimens of hospitalized children at Urmia Mutahhari hospital, West Azerbaijan province, Iran

Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children. This study was performed to determine subspecies of G.lamblia by the PCRRFLP method, targeting the glutamate dehydrogenase[gdh]locus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area. Overall,720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloroform/ isoamyl alcohol method. The single step PCR-RFLP assay was used to differentiate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used. Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples [93.3%] have the genotype BIII and 2 samples [6.7%] belong to the subgroup BIV. PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone's genes. Based on the results, an animal origin of infection cycle is suggested

Plasmodium vivax dhfr Mutations among Isolates from Malarious Areas of Iran

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.

Molecular diversity of mitochondrial DNA in Iranian Azeri ethnicities vis-a-vis other Azeris in Asia

In order to investigate the molecular diversity of mtDNA in Azeri population, 133 Azeri subjects inhabiting different regions of Azerbaijan [Iran] were selected. Blood samples were taken from these subjects for mtDNA extraction. The extracted mtDNA samples were then studied by the PCR-RFLP method. Fourteen haplogroups were characterized from which 82% were identified as European specific haplogroups. The H haplogroup was the most frequent and 79 haplotypes were specified. In this study, the Iranian Azeri population was found to be a heterogenic population where all the specific haplogroups of Asians, Europeans and Africans were present in the studied population. Comparing the haplogroups of the present investigation with other populations indicated a very close similarity with other Iranian populations, but was different from haplogroups of other Asian populations who also speak the Azeri language

[Study role of age in contact dependent transmission of Mycobacterium tuberculosis in northwest of Iran by IS6110-RFLP method]

The present study investigated the role in transmission of M. tuberculosis strains isolated from tuberculosis patients residing in Northwest [East and West Azarbaijan] of Iran. We performed restriction fragment length polymorphism analysis on IS6110 of M. tuberculosis isolated from Northwest Iran. Total of 165 isolates of M. tuberculosis were analyzed by RFLP method. The 5 copies and more IS6110 isolates comprised 30.52% of the total isolates. They formed 16 clustered groups consisting of 2 to 10 cases each. 69.48% of patients had a unique RFLP patterns. Cases from male patients were more clustered than female patients but statistically was not significant [P>0.05]. In this study patients with 56 and older age were strongly associated with clustering [59.6%], which were significantly more than younger patients [P<0.05]. In the present study we found old age as a major risk factor in contact dependent transmission of TB compared to disease recurrence. Unemployment and poor living condition were also among the risk factors in transmission of tuberculosis

Mannose binding lectin gene haplotype in Iranian patients with hepatitis C infection

Persistent infection with hepatitis C virus [HCV] leads to liver cirrhosis [LC] and often to liverm cancer. Mannose binding lectin [MBL] is a C-type serum lectin, which plays an important role in innate immunity by activating the classical complement pathway. Variants of the MBL have been shown to be associated with low serum concentrations of the protein and to predispose to bacterial, fungal and viral infections. This study was undertaken to investigate the association between polymorphisms of MBL gene and hepatitis C virus infection. We determined genotypes of two promoters and three exon 1 SNPs in mbl2 by SSP-PSR and grouped these genotypes according to related amount of functional MBL production in 100 patients infected with hepatitis C virus and 100 healthy blood donors in Iranian population. MBL gene mutations were determined by means of polymerase chain reaction and restriction fragment length polymorphism analyses. genotypes XA/O or O/O were significantly more frequent among patients infected with hepatitis C virus, where YA/YA genotype was more common among donors. Frequency of alleles X, Y, H and L did not have a significant difference between the two groups as well as alleles HYA, LYA nor LXA. MBL may be one of the factors that influence the course of HCV infection. Additional study on subjects at a high risk for infection with hepatitis C may clarify the role of carriage for the variant allele of mbl2 in a life-long risk of infection

Association of mannose binding lectin polymorphism with hepatitis C infection in northwest of Iran

Persistent infection with hepatitis C virus leads to liver cirrhosis and often to liver cancer. Mannose binding lectin is a C-type serum lectin, which plays an important role in innate immunity by activating the classical complement pathway. Variants of the mannose binding lectin have been shown to be associated with low serum concentrations of the protein and to predispose the subjects to bacterial, fungal and viral infections. This study was undertaken to investigate the association between hepatitis C virus infection and polymorphisms of mannose binding lectin gene. We assessed the single nucleotide polymorphism of mannose binding lectin in exon 1, at codon 52, codon 54 and codon 57 in 100 patients infected with hepatitis C virus and 100 controls in Iranian population. Mannose binding lectin gene mutations were determined by means of polymerase chain reaction and restriction fragment length polymorphism analyses. The occurrence of the codon 54 mutation was significantly higher in patients [OR 3.53, CI 95%: 1.94-6.44, p<0.005]. No significant difference in the frequency of codon 52 and 57 mutations was observed between patient and control groups. Mannose binding lectin may be one of the factors that influence the course of HCV infection. Our results suggest that heterozygous carriage of the variant allele of codon 54 of mannose binding lectin is associated with hepatitis C virus infection in our cases. This may not be true about codons 52 and 57 mutations