Integration and evaluation of cutaneous leishmaniasis laboratory diagnosis in the primary health care laboratory network.

Background: The lack of an integrated national system prevents the Islamic Republic of Iran from registering and reporting all cases of cutaneous leishmaniasis. Aim: To establish a laboratory network for the improvement of diagnosis and surveillance of cutaneous leishmaniasis in endemic areas of the Islamic Republic of Iran using parasitological and molecular methods. Methods: This descriptive, cross-sectional, pilot study examined 49 laboratories in the 2 endemic areas for cutaneous leishmaniasis in the Islamic Republic of Iran. Samples were taken for identification of the dominant Leishmania species from individuals with cutaneous leishmaniasis referred to the laboratories and had not travelled to other endemic regions. Statistical analysis was conducted using SPSS version 25.0. Using the primary healthcare laboratory network, we established a 3-level surveillance system. We compared misdiagnosis, new cases, clinical relapses, treatment resistance, and treatment failure before and after establishment of the network. Results: Network implementation reduced relapse of cutaneous leishmaniasis. After the laboratory training, the average misdiagnosis rate decreased from 49.3% to 4.2% for positive microscopic slides and from 31.6% to 12% for negative slides. Correct diagnosis was significantly higher in the study areas after the intervention. Conclusion: Implementation of a cutaneous leishmaniasis laboratory network can enhance diagnosis, unify diagnostic methods and improve patient care.

Corrigendum to 'How Iran responded to expanding need for laboratory services for COVID-19?'

[This corrects the article DOI: 10.1016/j.hlpt.2021.100506.].

Insight into blood pressure targets for universal coverage of hypertension services in Iran: the 2017 ACC/AHA versus JNC 8 hypertension guidelines.

BACKGROUND: We compared the prevalence, awareness, treatment, and control of hypertension in Iran based on two hypertension guidelines; the 2017 ACC/AHA -with an aggressive blood pressure target of 130/80 mmHg- and the commonly used JNC8 guideline cut-off of 140/90 mmHg. We shed light on the implications of the 2017 ACC/AHA for population subgroups and high-risk individuals who were eligible for non-pharmacologic and pharmacologic therapies. METHODS: Data was obtained from the Iran national STEPS 2016 study. Participants included 27,738 adults aged ≥25 years as a representative sample of Iranians. Regression models of survey design were used to examine the determinants of prevalence, awareness, treatment, and control of hypertension. RESULTS: The prevalence of hypertension based on JNC8 was 29.9% (95% CI: 29.2-30.6), which soared to 53.7% (52.9-54.4) based on the 2017 ACC/AHA. The percentage of awareness, treatment, and control were 59.2% (58.0-60.3), 80.2% (78.9-81.4), and 39.1% (37.4-40.7) based on JNC8, which dropped to 37.1% (36.2-38.0), 71.3% (69.9-72.7), and 19.6% (18.3-21.0), respectively, by applying the 2017 ACC/AHA. Based on the new guideline, adults aged 25-34 years had the largest increase in prevalence (from 7.3 to 30.7%). They also had the lowest awareness and treatment rate, contrary to the highest control rate (36.5%) between age groups. Compared with JNC8, based on the 2017 ACC/AHA, 24, 15, 17, and 11% more individuals with dyslipidaemia, high triglycerides, diabetes, and cardiovascular disease events, respectively, fell into the hypertensive category. Yet, based on the 2017 ACC/AHA, 68.2% of individuals falling into the hypertensive category were eligible for receiving pharmacologic therapy (versus 95.7% in JNC8). LDL cholesterol< 130 mg/dL, sufficient physical activity (Metabolic Equivalents≥600/week), and Body Mass Index were found to change blood pressure by - 3.56(- 4.38, - 2.74), - 2.04(- 2.58, - 1.50), and 0.48(0.42, 0.53) mmHg, respectively. CONCLUSIONS: Switching from JNC8 to 2017 ACC/AHA sharply increased the prevalence and drastically decreased the awareness, treatment, and control in Iran. Based on the 2017 ACC/AHA, more young adults and those with chronic comorbidities fell into the hypertensive category; these individuals might benefit from earlier interventions such as lifestyle modifications. The low control rate among individuals receiving treatment warrants a critical review of hypertension services.

Is salt intake reduction a universal intervention for both normotensive and hypertensive people: a case from Iran STEPS survey 2016.

PURPOSE: There is a direct association between salt intake and blood pressure (BP), one of the main risk factors for CVDs. However, yet there has been a debate that how strong is this association in people with and without hypertension. This study was conducted to evaluate the magnitude of the association between salt intake and BP in hypertensive and normotensive population among a nationally representative population. METHODS: The study was conducted on a nationally representative sample of 18,635 Iranian adults aged 25 years and older who participated in the STEPS survey 2016 and provided urine sample. Salt intake was estimated through spot urine sample and Tanaka equation. Multiple linear regression model in survey data analysis was used to assess the independent effect of salt intake on BP. RESULTS: After adjusting for covariates, there was a significant association between salt intake and SBP in hypertensive (p < 0.001) and normotensive people (p < 0.001). In hypertensive people, with 1 g of increase in salt intake, the SBP and DBP increased 0.37 mmHg and 0.07 mmHg, respectively. Whereas in normotensive people, with 1 g of increase in salt intake, the SBP and DBP increased 0.26 mmHg and 0.05 mmHg, respectively. Moreover, there was a significant trend toward an increase of SBP across salt intake quartiles in both hypertensive (p < 0.001) and normotensive people (p = 0.002), though the slope was steeper in hypertensive than in normotensive people. CONCLUSIONS: The present study demonstrated that salt intake significantly increased SBP in both hypertensive and normotensive people, though the magnitude of this increase was greater in hypertensive people as compared with normotensive people.

New design, development, and optimization of an in-house quantitative TaqMan Real-time PCR assay for HIV-1 viral load measurement.

Background Viral load measurement is commonly applicable to monitor HIV infection in patients to determine the number of HIV-RNA in serum samples of individuals. The aim of the present study was to set up a highly specific, sensitive, and reproducible home-brewed Real-time PCR assay based on TaqMan chemistry to quantify HIV-1 RNA genome. Methods In this study, three sets of primer pairs and a TaqMan probe were designed for HIV subtypes conserved sequences. An internal control was included in this assay to evaluate the presence of inhibition. Standard curve and threshold cycle values were determined using in vitro transcribed RNA from int region of HIV-1. A serial dilution of RNA standards was generated by in vitro transcription, from 10 to 109 copies/ml to find the sensitivity and the limit of detection (LOD) of the assay and to evaluate its performance in a quantitative RT-PCR assay. Results The assay has a low LOD equivalent to 33.13 copies/ml of HIV-1 RNA and a linear range of detection from 10 to 109 copies/ml. The coefficient of variation (CV) for Inter and Intra-assay precision of this in-house HIV Real-time RT-PCR ranged from 0.28 to 2.49% and 0.72 to 4.47%, respectively. The analytical and clinical specificity was 100%. Conclusions The results indicate that the developed method has a suitable specificity and sensitivity and is highly reproducible and cost-benefit. Therefore, it will be useful to monitor HIV infection in plasma samples of individuals.

Cost-price estimation of clinical laboratory services based on activity-based costing: A case study from a developing country.

BACKGROUND: It is believed that laboratory tariffs in Iran don't reflect the real costs. This might expose private laboratories at financial hardship. Activity Based Costing is widely used as a cost measurement instrument to more closely approximate the true cost of operations. OBJECTIVE: This study aimed to determine the real price of different clinical tests of a selected private clinical laboratory. METHODS: This study was a cross sectional study carried out in 2015. The study setting was the private laboratories in the city of Kerman, Iran. Of 629 tests in the tariff book of the laboratory (relative value), 188 tests were conducted in the laboratory that used Activity Based Costing (ABC) methodology to estimate cost-price. Analyzing and cost-price estimating of laboratory services were performed by MY ABCM software Version 5.0. RESULTS: In 2015, the total costs were $641,645. Direct and indirect costs were 78.3% and 21.7% respectively. Laboratory consumable costs by 37% and personnel costs by 36.3% had the largest share of the costing. Also, group of hormone tests cost the most $147,741 (23.03%), and other tests group cost the least $3,611 (0.56%). Also after calculating the cost of laboratory services, a comparison was made between the calculated price and the private sector's tariffs in 2015. CONCLUSION: This study showed that there was a difference between costs and tariffs in the private laboratory. One way to overcome this problem is to increase the number of laboratory tests with regard to capacity of the laboratories.

Reproducible and Reliable Real-time PCR Assay to Measure Mature Form of miR-141.

miR-141 is one of the miRNAs that has significant expression variations in different human malignancies including prostate cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, and ovarian cancer. Furthermore, various studies have designated miR-141 as a prognostic and diagnostic biomarker in different types of cancer. Thus, accurate and precise quantification of miR-141 is very essential for clinical diagnostics. In this regard, development of a reproducible and reliable assay for miR-141 can be the first step to standardize quantification of this valuable biomarker for in vitro diagnostics assays. Using stem-loop approach, we designed a Taqman real-time PCR assay for miR-141. This method allowed us to reproducibly and reliably quantify miR-141. The specificity, sensitivity, interassay and intraassay, and the dynamic range of the method were determined. The assay had a linear dynamic range of 3E-9.6E copies/reaction and the limit of detection was determined to be between 960 and 192 copies/reaction with 95% confidence interval. In addition, the R2 rate was >0.99 and the slope of the standard curve >-3.27, indicating great amplification efficiency, which is >99%. The coefficient of variation for Ct values was <1.9% and 2.39% for intraassay and interassay, respectively. Therefore, this study can be the first step to standardize miR-141 evaluations, which consequently assist the physicians for improved prognosis, diagnosis, and treatment.

Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples.

In diagnostic research challenges, quantitative real-time PCR (QPCR) has been widely utilized in gene expression analysis because of its sensitivity, accuracy, reproducibility, and most importantly, quantitativeness. Real-time PCR base kits are wildly applicable in cancer signaling pathways, especially in cancer investigations. T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia that is more common in older children and teenagers. Deregulation of the Notch signaling pathway promotes proliferation and inhibits apoptosis of the lymphoblastic T cells. The aim of this study was to investigate the effect of Notch signaling activation on the expression of target genes using real-time QPCR and further use this method in clinical examination after validation. Two T-ALL cell lines, Jurkat and Molt-4, were used as models for activation of the Notch signaling via over-expression of the Notch1 intracellular domain. Expression analysis was performed for six downstream target genes (NCSTN, APH1, PSEN1, ADAM17, NOTCH1 and C-MYC) which play critical roles in the Notch signaling pathway. The results showed significant difference in the expression of target genes in the deregulated Notch signaling pathway. These results were also verified in 12 clinical samples bearing over-expression of the Notch signaling pathway. Identification of such downstream Notch target genes, which have not been studied inclusively, provides insights into the mechanisms of the Notch function in T cell leukemia, and may help identify novel diagnoses and therapeutic targets in acute lymphoblastic leukemia.

Isolation of Asian endemic and livestock associated clones of methicillin resistant Staphylococcus aureus from ocular samples in Northeastern Iran.

BACKGROUND AND OBJECTIVES: Methicillin Resistant Staphylococcus aureus (MRSA) strains are divided into Community Associated (CA-) and Hospital Associated (HA-) MRSA. These strains vary in antimicrobial resistance and pathogenicity. S. aureus is one of the most common microorganisms in ocular infections. This study was aimed to determine antimicrobial resistance patterns and genetic characteristics of MRSA strains isolated from ocular infections in Iran. MATERIAL AND METHODS: Out of 171 S. aureus strains isolated from various clinical samples during September-December 2011 at Mashhad Emam Reza Hospital, 3 were cultured from eye discharge samples. Antimicrobial resistance tests were performed with MIC and disk diffusion methods and also genetic evaluation was done with Staphylococcal Cassette Chromosome mec (SCCmec), Accessory Gene Regulator (agr) and Staphylococcal Protein A (spa) typing, Multi Locus Sequence Typing (MLST) and determination of toxin gene profile. RESULTS: All strains were MRSA and showed resistance to tetracycline, gentamicin and clindamycin too. Vancomycin, minocyclin and trimethoprim/sulfamethoxazole were effective on all ocular isolates. All isolates belonged to SCCmec IV type. MRSA1 belonged to ST239, CC8, Spa type t7688 and agrIII and had tst1 and hla toxin genes. MRSA2 belonged to ST239, CC8, Spa type t037 and agrI and had the hla toxin gene. Finally, MRSA3 belonged to ST291, CC398, Spa type t304, and agrI and had pvl and hla toxin genes. CONCLUSION: Phenotypic and genotypic evaluation of the isolated MRSA strains revealed that these strains belong to endemic Asian and livestock related clones that could reach from other body sites or environment to the eye of patients and developed ocular infection.

Genetic characterization of a vancomycin-resistant Staphylococcus aureus isolate from the respiratory tract of a patient in a university hospital in northeastern Iran.

Emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains has led to global concerns about treatments for staphylococcal infections. These strains are currently rare even though there is an upward trend in their reported incidence. Therefore, appropriate screening and epidemiological evaluation of VRSA strains can affect future global health care policies. Isolates of Staphylococcus aureus were obtained from various clinical samples and were then evaluated with agar screening, disk diffusion, and MIC methods to determine resistance to vancomycin and methicillin. After confirmation of the isolated VRSA strain, genetic analysis was performed by evaluating mecA and vanA gene presence, SCCmec, agr, and spa types, and toxin profiles. Multilocus sequence typing (MLST) and plasmid analysis were also performed. The VRSA strain was resistant to oxacillin (MIC of 128 µg/ml) and vancomycin (MIC of 512 µg/ml). Disk diffusion antimicrobial susceptibility tests showed resistance to oxacillin, vancomycin, levofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, clindamycin, rifampin, and tetracycline. The isolate was susceptible to minocycline and gentamicin. PCRs were positive for the mecA and vanA genes. Other genetic characteristics include SCCmec type III, agr I, spa type t037, and sequence type (ST) 1283. The plasmid profile shows five plasmids with a size of ~1.7 kb to >10 kb. The isolated VRSA strain was obtained from a critically ill hospitalized patient. Genetic analysis of this strain suggested that the strain was a methicillin-resistant S. aureus (MRSA) clone endemic in Asia that underwent some genetic changes, such as mutation in the gmk gene and acquisition of the vanA gene.

Genetic Characterization of Methicillin Resistant and Sensitive, Vancomycin Intermediate Staphylococcus aureus Strains Isolated from Different Iranian Hospitals.

Background. Global concerns have been raised due to upward trend of Vancomycin Intermediate Staphylococcus aureus (VISA) and Vancomycin Resistant Staphylococcus aureus (VRSA) reports which mean casting doubt on the absolute effectiveness of the last line of antibiotic treatment for S. aureus, vancomycin. Hence, epidemiological evaluation can improve global health care policies. Methodology. 171 Isolates of Staphylococcus aureus were collected from different types of clinical samples in selected hospitals in Isfahan, Mashhad, and Tehran, Iran. Then, they were evaluated by agar screening, disk diffusion, and MIC method to determine their resistance to vancomycin and methicillin. The isolated VISA strains were then confirmed with genetic analysis by the evaluation of mecA and vanA genes, SCCmec, agr, and spa type, and also toxin profiles. MLST was also performed. Results and Conclusion. Our data indicated that 67% of isolated S. aureus strains were resistant to methicillin. Furthermore, five isolates (2.9%) had intermediate resistance to vancomycin (VISA). In contrast to usual association of VISA with MRSA strains, we found two isolates of MSSA-VISA. Therefore, our data suggests a probable parallel growing trend of VISA towards MSSA, along with MRSA strains. However, more samples are required to confirm these primarily data. Moreover, genetic analysis of the isolated VISA strains revealed that these strains are endemic Asian clones.

Glutathione S-transferase mu gene variants and colorectal cancer development--use of sequence-specific probes for an Iranian population.

BACKGROUND: Enzymes of the glutathione S-transferase (GST) family are encoded by a set of polymorphic genes as an important part of cellular chemical defense. The aim of this study was to investigate the possible effect of GSTM1 deletion on susceptibility to developing clinical outcome of colorectal cancer in a group of CRC patients from Isfahan province, Iran, in comparison to age and gender matched control group. METHODS: DNA was extracted from blood of 140 CRC patients and 90 healthy individuals and a set of sequence specific hybridization probes was used for GSTM1 genotyping by real-time PCR in Light-Cycler instrument. Chi-squared test was used to assess the statistical significance of observed differences between the patient and control subjects of different genders and ages. To estimate the risk for overall and stratified analyses, odds ratios (OR) with 95% confidence intervals (CI) computed with logistic regression. RESULTS: No difference in GSTM1 null genotype frequency was found in CRC patients and controls stratified by gender (p value=0.14). The data were suggested a trend of increasing risk for GSTM1 null genotype in patients over 60 years old compared with controls (p value=0.05). GSTM1 null genotype carried an increase of the odds of developing CRC in patients over 60 years old (OR=2.7; 95% CI: 1.03-7.05). No significant association was found (P>0.05) between the GSTM1 null genotype with tumor site (right, left, rectum) or tumor differentiation (well, moderately). CONCLUSION: Our findings suggest that the GSTM1 null genotype may contribute to colorectal cancer development in people over 60 years old.

Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR Green I.

BACKGROUND: We intend to design and validate a low-cost assay for the detection of hepatitis C virus (HCV) RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours. METHODS: A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DNA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay. RESULTS: The minimum detection level of our assay was less than 50 IU/mL. The results on 100 plasma samples were comparable with commercial assays. CONCLUSIONS: This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications.

Mutation in pfmdr1 gene in chloroquine-resistant Plasmodium falciparum isolates, Southeast Iran.

OBJECTIVES: The main objective of the present study was to detect point mutations at positions 86, 184, 1034, 1042, and 1246 of the Plasmodium falciparum multidrug resistance gene (pfmdr1) in blood samples collected from malaria patients in Chabahar, a harbor city located in Southeast Iran. METHODS: Twenty-six blood samples from patients infected with P. falciparum, who had a chloroquine (CQ) response failure, were collected pre-treatment. Following treatment with CQ, drug susceptibility was assessed using an in vivo test. Molecular detection of single nucleotide polymorphisms (SNPs) was carried out using the LightCycler hybridization probe assay. RESULTS: The pfmdr1 N86Y mutation was found in six isolates (23.1%). Mutations at the four other positions were not observed in any isolates. CONCLUSION: The present study showed no mutation at codon positions 184, 1034, 1042, and 1246 of pfmdr1 in any of the Iranian P. falciparum isolates; thus these alleles cannot serve as markers for CQ resistance in Iran.