Evaluation of Mycobacterium kansasii extracellular vesicles role in BALB/c mice immune modulatory.

Background: Mycobacterium kansasii as a nontuberculosis mycobacteria, naturally release extracellular vesicles (EVs) with widespread utilities. The aim of the present study was the extraction and biological evaluation of M. kansasii EV and its role in BALB/c mice immune modulatory by considering EVs medical usage specificities. Method: Density gradient ultracentrifugation method was used to EVs extraction from standard species of M. kansasii. Biologic validation of EVs has been performed by physicochemical experiments. Immunization has been done by subcutaneous injection to BALB/c mice, then spleen cell isolation and lymphocyte transformation test and eventually ELISA cytokine assays were made for interleukin-10 (IL-10) and interferon-gamma (IFN-γ).IBM SPSS version 22 software (SPSS. Inc., Chicago, IL, USA) was used for the data calculation. The evaluation of variables was conducted using one sample t-test. Results: Physicochemical experiment results contribute that extracted EVs have intransitive capability to use in immunization schedule. Finally, ELISA test results showed that EVs induced IL-10 production, but have no effect on IFN-γ. Conclusions: In this current study, EVs were prepared in high-quality composition. The results of cytokine assay revealed that the extracted EVs have anti-inflammatory property. Accordingly, this macromolecule can be used as immune modulatory agents to prevent severe immune reactions, especially in lungs disorders.

A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician's skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as "Patho-tb", for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen's kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 103 cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.

The impact of genetic variation in IL28B, IFNL4 and HLA genes on treatment responses against chronic hepatitis C virus infection.

Single nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B), interferon lambda 4 (IFNL4) and the human leukocyte antigen (HLA) gene are associated with treatment responses in patients with chronic hepatitis C (CHC) virus infection treated with pegylated interferon-α and ribavirin (pegIFN-α/RBV). We compared the role of IL28B SNPs (rs12979860, rs12980275, and rs8099917), IFNL4 ss469415590 and HLA rs4273729 with treatment outcomes in patients with CHC virus. A total of 520 Iranian patients with CHC infection were enrolled. SNPs in IL28B, IFNL4 ss469415590 and HLA rs4273729 were genotyped by PCR-restriction fragment length polymorphism, TaqMan® Real-Time PCR and direct sequence. Out of 520 CHC treatment-naive patients, 42.9% were infected with HCV-1a, 15.4% with HCV-1b, 9.8% with HCV-2, and 31.9% with HCV-3a. Rapid virologic response (RVR), complete early virologic response (cEVR), and sustained virologic response (SVR) were 53.3%, 73.8%, and 66.7%, respectively. Multivariate logistic regression analysis showed that IL28B rs12980275 and IFNL4 ss469415590 in all HCV genotypes were associated with RVR. In addition, IL28B rs12979860 and RVR in all HCV genotypes and IL28B rs12980275, IFNL4 ss469415590, and HLA rs4273729 in HCV subtypes 1a, 1b, and 3a correlated with cEVR. In patient's achieving-SVR, IL28B rs12980275, and RVR in all HCV genotypes and IL28B rs12979860, IFNL4 ss469415590, and HLA rs4273729 in HCV subtypes 1a, 1b, and 3a were the powerful predictor factors. As the first report of its kind published in Iran, we indicated that beside IL28B SNPs and HLA rs4273729, IFNL4 ss469415590 was a powerful predictor factor for RVR, cEVR and SVR. Genotyping these SNPs may be a helpful priority in the treatment of patients with HCV infection, especially in countries where access to triple or double therapy with a viral protease inhibitor is limited.

A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis.

This study aimed to evaluate the frequency of resistance to first- and second-line drugs using phenotypic and genotypic methods and its correlation with resistance-linked mutations in Mycobacterium tuberculosis (M. tb) isolated in Iran. Three different methods, including the indirect proportion method(PM), direct and indirect nitrate reductase assay(NRA), and direct sequencing were used to assess drug resistance. In this study, sensitivity, specificity, agreement, costs, and turnaround time of these methods were compared in 395 smear positive isolates. Compared to the PM, the NRA and the direct sequencing methods demonstrated higher specificity, sensitivity, and agreement for detection of all anti-tuberculosis drugs. The NRA had a short turnaround time and was more cost-effective than the other methods. Mutations in codon 531 in rpoB, 315 in katG, 18 in rpsL, and 306 in embB were associated with high-level resistance to the first-line drugs, and mutations in codon 94 in gyrA, and A1401G in rrs were correlated with resistance to the second-line drugs. We found that the NRA is a highly sensitive, specific, inexpensive, and rapid test with strong potential to be a useful and interesting alternative tool, particularly in low-income countries. In addition, these molecular data will be helpful for developing new molecular methods for detecting first- and second-line drug-resistant M. tb.

Distribution and Diversity of hmw1A Among Invasive Nontypeable Haemophilus influenzae Isolates in Iran.

BACKGROUND: The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. Almost 38-80% of NTHi clinical isolates produce proteins that belong to the High Molecular Weight (HMW) family of adhesins, which are believed to facilitate colonization. METHODS: In the present study, the prevalence of hmwA, which encodes the HMW adhesin, was determined for a collection of 32 NTHi isolates. Restriction Fragment Length Polymorphism (RFLP) was performed to advance our understanding of hmwA binding sequence diversity. RESULTS: The results demonstrated that hmwA was detected in 61% of NTHi isolates. According to RFLP, isolates were divided into three groups. CONCLUSION: Based on these observations, it is hypothesized that some strains of nontypeable Haemophilus influenzae infect some specific areas more than other parts.

High resolution melting curve assay for detecting rs12979860 IL28B polymorphisms involved in response of Iranian patients to chronic hepatitis C treatment.

BACKGROUND: A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-α/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-α/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. MATERIALS AND METHODS: The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. RESULTS: The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10% (10/100), 35% (35/100), and 6% (6/100) and in genotype 3a were 13% (13/100), 31% (31/100), and 5% (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18%, 34% and 4%, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio [ORs], 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. CONCLUSIONS: The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-α/RBV treatment. The present results may help identify subjects for whom the therapy might be successful.

Portable, battery-operated, low-cost, bright field and fluorescence microscope.

This study describes the design and evaluation of a portable bright-field and fluorescence microscope that can be manufactured for $240 USD. The microscope uses a battery-operated LED-based flashlight as the light source and achieves a resolution of 0.8 microm at 1000x magnification in fluorescence mode. We tested the diagnostic capability of this new instrument to identify infections caused by the human pathogen, Mycobacterium tuberculosis. Sixty-four direct, decontaminated, and serially diluted smears were prepared from sputa obtained from 19 patients suspected to have M. tuberculosis infection. Slides were stained with auramine orange and evaluated as being positive or negative for M. tuberculosis with both the new portable fluorescence microscope and a laboratory grade fluorescence microscope. Concordant results were obtained in 98.4% of cases. This highly portable, low cost, fluorescence microscope may be a useful diagnostic tool to expand the availability of M. tuberculosis testing at the point-of-care in low resource settings.

Immunogenicity of a triple diphtheria-tetanus-whole cell pertussis vaccine in Iranian preschool children.

BACKGROUND: Immunization against diphtheria, tetanus and pertussis has been applied in Iran since 1950. WHO suggests periodical evaluation of effectiveness of the triple diphtheria-tetanus-whole cell pertussis (DTwP) vaccine, worldwide. OBJECTIVES: To determine the immunogenicity of locally manufactured DTwP vaccine administered to preschool children in a number of health centers of Tehran in 2006. METHODS: In this prospective study, 350 children aged 4-6 years were injected with DTwP vaccine manufactured by Razi Institute of Iran. Blood samples were collected before and 2-4 weeks after the vaccination. The immunogenicity of the vaccine was assayed by measurement of specific antibodies using enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: Of the 337 children who were vaccinated, 99.4% and 100% had protective anti-diphtheria and anti-tetanus antibody titers, respectively. The vaccine response and seroconversion for pertussis was achieved in 70.3% of the subjects. The geometric mean titers (GMT) of the antibodies produced against diphtheria, tetanus and pertussis by DTwP vaccine were 7.76, 9.37 IU/ml and 30.20 EU/ml after booster vaccine dose, respectively. CONCLUSIONS: Comparison of the results obtained from this study with those of previous studies performed in other countries reveals that immunogenicity of diphtheria and tetanus components is similar to other vaccines, but the immunogenicity of pertussis vaccine was less efficient. The lower immunogenicity of DTwP against pertussis may be related to the bacterial strain used or the formulation protocol adopted for the vaccine preparation.