International Journal of Medical Laboratory

85-99

Authors: Sara Taghizadeh 1 Ph.D., Farkhondeh Behjati 2, 3* Ph.D.

In recent years, viral epidemics such as the coronavirus disease (COVID- 19) have spread, and this outbreak is thought to be the result of animalto- human transmission. Hence, accurate diagnostic tests to detect COVID-19 and antiviral antibodies in infected individuals are of utmost importance. This report describes the structure, history, taxonomy, and molecular and immunological techniques for diagnosing this disease. Tests for early diagnosis of COVID-19 depend on the reverse transcription-polymerase chain reaction (RT-PCR). However, tests based on isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)-based methods are promising options. Identifying people whose activated antibodies require serological tests, including enzyme-linked immunosorbent assays (ELISA). The search for efficient, cost-effective, and accurate laboratory techniques that can be used on a large scale continues. The RT-PCR technique is a dominant technique for the detection of viral RNA. Other acidic nucleic-based assays such as isothermal amplification, microarray hybridization, amplicon-based metagenomics sequencing, and CRISPER-based techniques have been developed. Along with molecular methods, different efficient serological and immunological methods such as ELISA, rapid antigen test, lateral flow immunoassay, luminescent Immunoassay, and biosensors are also developed.

110-121

Authors: Victor Fondoh

Background and Aims: Improving the Quality Management System (QMS) of clinical laboratories and achieving accreditation are important in health care delivery. It can be achieved by implementing the World Health Organizaton Stepwise Laboratory Improvement Process Towards Accreditation (SLIPTA). The SLIPTA program was introduced to the Bamenda Regional Hospital (BRHL) in 2010. Our objectives were to identify improvements and evaluate the QMS at the BRHL. Training, mentorship, and improvement of laboratory infrastructure were considered for the program to succeed. Materials and Methods: Secondary data from the WHO SLIPTA assessment reports of the BRHL between November 2009 and March 2018 were extracted. The assessments were conducted by the WHO African Society for Laboratory Medicine (ASLM) SLIPTA certified and competent auditors, using the SLIPTA checklist. The final percentage score(s)/star(s) of the assessments was/were identified as improvements, and the evaluation was done by taking the difference between an absolute score of the Quality System Essentials (QSE) of the baseline recent follow-up assessment. Results: A total of nine SLIPTA assessments were carried out. The results indicated great improvements in the QMS from a baseline score of 18% (0-star) to 82% (3-stars) at the recent follow-up assessment. There were also significant changes in the QSE, with the final absolute scores ≥ 58% in all aspects and the greatest change registered in the management review (94%). Conclusions: We identified incredible improvements and magnificent changes in the QMS at the BRHL that were due to training, mentorship, and improvements in infrastructure resulting from the implementation of the SLIPTA program.

131-140

Authors: Michael Chukwuemeka Ugwu 1* M.Sc., Innocent Ikechi Ogbu 1 Ph.D., Nancy Amalachukwu Mbachu 2 Ph.D., Perekeme Frank Kendabie 3 SBMS., Ozoemena Christian Ike 4 M.Sc.

Background and Aims: This cross-sectional study examined the nutritional status, serum proteins, and some trace elements in human immunodeficiency viruses (HIV) drug-naive patients on highly active antiretroviral therapy. Material and Methods: Twenty-five drug-naive subjects, 25 subjects on highly active antiretroviral therapy (HAART), and 20 control subjects aged 21 to 65 years were conveniently recruited. Serum total protein, albumin, and hemoglobin were assayed spectrophotometrically. In contrast, iron, copper, zinc, and selenium were assayed using Atomic Absorption Spectroscopy. CD4 count was done by Flow Cytometry. Then, the nutritional assessment was performed using a subjective global assessment questionnaire. Results: Weight, body mass index, and mid-upper arm circumference were significantly lower (p = 0.000) in the drug-naive HIV subjects than in subjects on HAART and control. Serum total protein and copper were significantly higher (p = 0.000) in drug-naive HIV subjects compared to subjects on HAART and control. In contrast, albumin, globulin, albumin-globulin ratio, hemoglobin, iron, zinc, and selenium were significantly lower (p = 0.000) in drug-naive HIV subjects compared to HIV subjects on HAART and control subjects. The CD4 count of drug-naive HIV subjects was significantly lower (p = 0.000) compared to subjects on HAART. In addition, malnutrition was higher in the drug-naive subjects. Conclusions: Monitoring the course of HIV infection and malnutrition can be aided by incorporating measurements of nutritional status and some trace elements into routine laboratory tests.

150-155

Authors: Zahra Shakeri Fini 1 M.Sc., Payam Kheirmand Parizi 2,3 MD., Seyed-Alireza Esmaeili 4,5 Ph.D., Abbas Samadi-Khouzani 6 M.Sc., Sara Hosseini 4,5 M.Sc., Mostafa Jafarpour 1* Ph.D.

Background and Aims: Psoriasis is a chronic inflammation of the skin caused by the proliferation of inappropriately differentiated horn cells, resulting in plaque psoriasis formation. It is not often fatal, and it may lead to physical disabilities and severe mental stress in which professional and social activities could be highly affected. It is generally accepted that the human leukocyte antigen (HLA)-CW06 allele has a significant role in the onset of this disease. Materials and Methods: This study determines the association between the HLA-CW06 allele and psoriasis. The present case-control study was conducted to evaluate the relation between HLA-CW06 and psoriasis in a population in Iran. This study was performed on 30 samples of patients with psoriasis. Results: The results of polymerase chain reaction- sequence-specific primers for the detection of HLA-CW06 showed positive amplification in 7 out of 30 psoriasis patients as compared to 4 among 30 controls. Conclusion: This study showed that due to different allele frequencies associated with psoriasis in different parts of the world, it seems that other genetic and epigenetic factors may be involved in this disease.

6-16

Authors: Rajabi1, 2 Ph.D., Majid Pourentezari1,2* Ph.D., Hengameh Dortaj1, 3 Ph.D., Abbas Shahedi1,2 Ph.D., Maryam Yadegari1,2 Ph.D., Sepideh Izadi 1,2 M.Sc., Fatemeh Zakizadeh1,2 M.Sc., Zeinolabedin Sharifian Dastjerdi1, 4 Ph.D.

Recently, it has been proven that cartilage healing is difficult. The most commonly used treatments are autogenously cartilage grafting and allogeneic bone grafting, but grafts cannot fully meet treatment goals because of source, price, safety, and other concerns. Thus, a combination of biological materials and tissue engineering technology has become a recent trend in studies. Among the studies performed on tissue engineering cartilage materials are hydrogels that exhibit biological activity, postdecomposition adsorption, flexibility, and easy preparation. Cellcontaining hydrogels are often used in cartilage tissue engineering because of their biocompatibility, ease of use, and ability to adapt to different defects. Hydrogels are used to mimic extracellular matrices. Although multiple materials can configure and form hydrogels, hyaluronic acid and its derivatives are distinguished. Hyaluronic acid (HA) is an extracellular molecule with several physical and biological functions found in many tissues, including cartilage. HA is formed in several biomaterial systems and scaffolding. HA hydrogels have many interests, including increased adhesion, cell proliferation, and wound healing. In addition, they represent adequate biological acting for stimulating a microenvironment for the survival of cells. However, their disadvantages include a slow degradation rate and low mechanical properties. Here, HA-based hydrogels and their applications in cartilage tissue engineering are briefly reviewed.

26-33

Authors: Alireza Torki 1,3 Ph.D., Mahlagha Zahedi 2 MD., Nour Amirmozafari 3* Ph.D.

Background and Aims: Due to a wide variety of causes, various bacteria can acquire resistance to antibiotics. This study investigated the antibiotic susceptibility patterns of the etiologic bacterial agents of various infections in patients referred to three hospitals in Yazd city, Iran. Materials and Methods: A total of 336 clinical specimens including wound discharge, sputum, blood, bronchial fluid, pleural fluid, ascitic fluid, synovial fluid, stool, and trachea secretions were collected in three hospitals. Microbiological culturing in order to grow and identify the causative bacteria were performed. Antibiotic susceptibility determinations were done by the Kirby Bauer disk diffusion method. Result: Among all organisms isolated, Escherchia coli, Staphylococcus. aureus, Staphylococcus. suprophyticus, Acinetobacter spp., Pseudomonas spp., Klebsiella spp., Staphylococcus. epidermidis and Entrococcus spp. were the most prevalent, respectively. The Escherchia coli isolates were the most antibiotic-resistant Gram-negative bacterium. It seems that the same organisms recovered from different hospital wards show different antibiotic susceptibility patterns. Conclusion: The increased prevalence of resistant organisms in different hospitals may reflect a lack of proper antibiotic usage policy, resulting in the prolonged and indiscriminate use of antimicrobial agents.

47-54

Authors: Amita R* MD., Arya AB PGDBBT., Sathyabhama S MBBS., Debasish Gupta MD.

Background and Aims: Leukoreduction of blood components has reduced the incidence of transfusion-associated adverse events. Leucofiltration is the most effective method of leukoreduction. We encountered haemolysis in a series of leucofiltered units. This stressed our precious inventory, added to financial loss, increased our turnaround time to issue leucofiltered blood units, and placed doubts on the safety of our leucofiltered products. A systematic root cause analysis was done to identify the reason for haemolysis. Materials and Methods: A total of 69 units were leucofiltered during the study period, of which 13 units showed lysis following filtration. Results: This study warranted a review of our existing leucofiltration standard operating procedures to keep strict adherence to manufacturer instructions to ensure quality in the end product. Conclusions: Among the leucofiltered units showing hemolysis, 62% were due to higher temperature, 23% due to increased time for filtration, and 15% due to increased holding time of red cell units.

65-72

Authors: Soheila Khaksari 1 M.Sc., Ehsan Aghaei Moghadam 2 M.D., Ahoura Nozari 1,3 Ph.D., Zahra Boroughani 4 M.Sc., Saghar Ghasemi Firouzabadi 1 Ph.D., Farkhondeh Behjati 1,5* Ph.D.

Background and Aims: Congenital heart defects (CHD) are the most common type of congenital disability. Copy number variations (CNVs) have been found as one of the genetic etiology of nonsyndromic CHD, and researchers have detected several pathogenic CNVs in patients with cardiac defects. Materials and Methods: In the present study, 70 patients with familial (20 patients) and sporadic (50 patients) non-syndromic CHD were evaluated to find whether CNVs in the GATA4, NKX2-5, TBX- 5, CREL, BMP4 genes, and 22q11.2 region contribute to the pathogenesis of non-syndromic CHD. We have used the Multiplex Ligation-dependent Probe Amplification (MLPA) technique as a molecular method to identify CNVs in predefined loci. Results: Normal MLPA results were demonstrated for GATA4, NKX2-5, TBX-5, CRELD, and BMP4 genes for all sporadic and familial cases. However, we found three patients with imbalances for the 22q11.2 region. One patient with 22q11.2 deletion showed tetralogy of fallot, and the other had ventricular septal defects/ pulmonary atresia/ multiple aortopulmonary collateral arteries. A duplication of the 22q11.2 region was detected in one patient with patent ductus arteriosus. Conclusion: Identifying genomic imbalances in 6% of the nonsyndromic sporadic patients indicates that recurrent CNVs could be associated with non-syndromic CHD. It seems that it is the first CNV analysis using MLPA carried out in Iranian patients with cardiac defects. We suggest that 22q11.2 imbalances should be considered in patients with cardiac lesions to provide an accurate diagnosis and appropriate genetic counseling in affected families.

272-277

Authors: Archana Garg 1 M.D., Lakshmi Agarwal 2 M.D., Mamta Gupta 3 M.D., Manmohan Agarwal 4* M.D.

Background and Aims: Emerging antimicrobial resistance is one of the major public health threats worldwide. It can result in increased morbidity and mortality rates along with increased treatment costs and hospitalization stays. Methicillin-resistant Staphylococcus aureus (MRSA) has become a global challenge. It is a major cause of nosocomial and community-acquired infections. Its prevalence varies with country and within hospitals within a country. The current study estimates the prevalence of MRSA. Materials and Methods: A total of 162 Staphylococcus aureus strains were isolated from various clinical specimens, and antibiotic susceptibility tests and identification by cefoxitin disc (30 μg) were performed as per Clinical and Laboratory Standards Institute guidelines. Results: Among 162 Staphylococcus aureus 92 isolates were found to be MRSA. The highest rate of resistance was detected for penicillin (100), followed by erythromycin (97%), clindamycin (57%), tetracycline (13%), sulfamethoxazole-trimethoprim (SXT) (55%), ciprofloxacin (23%), and gentamicin (2%). All of the isolates were susceptible to linezolid and vancomycin. Conclusions: Treatment of multi-drug resistant MRSA is still problematic because of the limited choice of antibiotic. This analysis will assist clinicians to choose an appropriate course of action for MRSA infections.

286-294

Authors: Esmaeil Sarani 1 M.Sc., Maryam Hataminejad 2 Ph.D., Hakim Azizi 3* Ph.D.

Background and Aims: Toxocariasis is a zoonotic parasitic disease with worldwide distribution caused by the larval stage of ascaridoid nematodes of dogs (Toxocara canis) and cats (Toxocara cati). The study was accomplished to determine the sequence variation in ITS2 and COX1 genes within isolates of Toxocara canis. Materials and Methods: Two hundred Stool samples were collected randomly from dogs in public parks and streets from different regions of Zabol. Thirty samples containing eggs were isolated from the feces using Formalin ether 10% and centrifugal flotation. Genomic DNA was extracted, and COX1 and ITS2 were amplified by PCR-RFLP and sequenced. Sequence data were aligned using the BioEdit software and BLAST program and compared with published sequences in GenBank. The phylogenetic relationship between isolates of T. canis from Zabol city with other regions based on sequences obtained from COX1 and ITS2 genes and using MEGA7.0 software was investigated. Results: For all samples, amplicons of about 388 and 422 base pairs were produced by PCR for ITS2 and COX1, respectively. Drawing the phylogenic tree of ITS2 and COX1 sequences of isolates of T. canis showed that the identified genotypes are not only different from each other but also from other parts of the world. Conclusions: Our result showed that genetic variation among isolates of T. canis from Zabol is very low. For a deeper understanding of genetic diversity among populations of Toxocara, it is recommended to analyze more isolates from various geographical areas and variable genetic markers.

304-310

Authors: Seyed Ali Salari 1 M.Sc., Seyed Mohsen Miresmaeili 1* Ph.D., Mahta Mazaheri Naeini 2 Ph.D.

Background and Aims: One of the most important genes involved in Alzheimer's disease (AD) is the presenilin2 (PSEN2) gene, which is one of the main constituents of the gamma-secretase complex. Mutations in this gene promote the formation of amyloid plaques resulting in AD. The study aimed to evaluate the mutation variant in exon 6 of the PSEN2 gene in patients with Late-Onset Alzheimer's disease (LOAD). Due to the important role of the PSEN2 gene in the formation of beta-amyloid aggregates and the investigation involves an association between PSEN2 mutations and their pathogenicity in LOAD progression, we presented this exon as a more efficient alternative. Materials and Methods: The thirty patients with LOAD and 16 healthy subjects as a control group were involved in this experimental study. DNA was extracted from blood samples and purified. The desired gene fragment was propagated using polymerase chain reaction and the products were electrophoresed and the results were analyzed. Result: A novel mutation was found in PSEN2 IVS6 + 30 G → C at the intron region of exon 6 in 20 cases of patients suffering from LOAD and 12 subjects in control cohort. In this mutation a guanine base was substituted by cytosine base which this position was 30 nucleotides separated from coding region. Conclusions: The novel mutation was identified in both studied groups. These findings reveal no relationship between PSEN2 mutation and pathogenicity of LOAD disease. However, further studies are required to find the role of PSEN2 mutation in LOAD progression.

169-186

Authors: Mohammad Tollabi, Navid Ghasemzadeh , Ali Dehghani Firoozabadi *

Background and Aims: Substantial damage to the kidney tissue and diabetic nephropathy (DN) can be caused by chronic hyperglycemic conditions and exposure to a high level of blood glucose. In the current study, we explored the capability of adipose-derived mesenchymal stem cells (ADSCs) and Toll-like receptor-4-primed mesenchymal stem cells (TLR4-primed MSCs) on kidney regeneration, resolution of inflammation, and alleviation of diabetic nephropathy in DN in the rats. Materials and Methods: STZ-induced diabetic rat models were divided into 5 subgroups, including 1) DN group, 2) DN group received insulin, 3) DN group received human foreskin fibroblast (DN-HFF), 4) DN group received single pulse of 1×106 cells of MSCs and 5) DN group received single pulse of TLR4-primed MScs. Thereafter, biochemical and histological analysis was performed on DN-model groups. Biochemical analysis exhibited a blood urea nitrogen level recovery in both MSCs and TLR4-primed MSCs-treated groups. Administration of MSCs also up-regulated mRNA expression of Bcl-xL, while the expression of Bax was significantly down-regulated. Results: Histological and molecular results showed that TLR4-primed MSCs have an anti-inflammatory and anti-apoptotic effect on inflamed kidneys and effectively reduced DN indicators in the TLR4-primed MSCs group compared to the unprimed MSCs group. Conclusion: Priming with LPS improves the therapeutic effects of MSCs in the rat model of DN, consequently lessening the symptoms of DN rats. This study proposes that primed MSCs can be served as a potential therapeutic approach in diabetes mellitus and DN management.

198-208

Authors: Ali Nosrati Andevari , Soheila Moein , Durdi Qujeq * , Zoleika Zmoazezi , Karimollah Hajian-Tilaki

ackground and Aims: Atorvastatin may alter glycemic traits and lipid profiles. This study aimed to evaluate the effects of atorvastatin on biochemical variables in patients with type 2 diabetes and pre-diabetes (borderline diabetes). Materials and Methods: This study included 80 individuals divided into five groups. Patients with type 2 diabetes mellitus and pre-diabetes used atorvastatin 20 mg/day for three months. After three months, variables such as serum fasting blood glucose (FBS), cholesterol, triglyceride, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), and glycosylated hemoglobin (HbA1c) levels were measured to assess the status of diabetes and pre-diabetes condition. Linear regression was applied to determine the association between atorvastatin uses and alters of biochemical variables levels. Results: The serum FBS and HbA1c levels in patients with diabetes and pre-diabetes who use atorvastatin were significantly lower than in patients with diabetes and pre-diabetes who did not use atorvastatin (p=0.001). Serum cholesterol and LDL-C levels decreased in diabetic and pre-diabetic patients who used atorvastatin in comparison with diabetic and pre-diabetic patients who did not use atorvastatin (p=0.001). In patients with pre-diabetes, the use of atorvastatin slightly increased serum HDL-C levels. However, in patients with diabetes, the use of atorvastatin slightly decreased serum HDL-C level (p= 0.001). Diabetic and pre-diabetic patients who use atorvastatin significantly decreased serum triglyceride levels (p=0.016), while in diabetic and pre-diabetic patients, using atorvastatin slightly increased the serum insulin level (p= 0.003). Conclusions: Atorvastatin using alters fat and sugar indices in diabetic and pre-diabetic patients.

218-225

Authors: Seyed Hamidreza Mirabutalebi, Mohammadreza Dehghani, Nasrin Ghasemi , Mohammad Yahya Vahidi Mehjardi , Mojtaba Movahedinia , Seyed Mehdi Kalantar *

Background and Aims: One of the neurotransmitters in the brain is Histamine which acts as several biological mechanism regulators like inflammation, gastric acid secretion, and neuromodulation. Inactivation of Histamine occurs by histamine N-methyltransferase (HNMT) enzyme. The HNMT transfers a methyl group from S-adenosyl-L-methionine to Histamine and is the main process for the termination of neurotransmission actions of Histamine in the mammalian central nervous system. Materials and Methods: In this case, a family was referred to the genetic clinic to diagnose the cause of their disorder. The clinical form, pedigree, and questionnaire were completed for the family, and the parents gave their written consent for all tests and photographs publication. Both siblings have moderate learning and intellectual disability. Whole exome sequencing was performed and Sanger sequencing for co-segregation was used. Results: Bioinformatics analysis revealed a homozygous missense variant in HNMT (c.623T>C p.Leu208Pro) which causes non-syndromic autosomal recessive intellectual disability in this consanguineous family. Analysis of segregation confirmed this mutation. P.Leu208Pro mutation reduces the stability of the protein, which reduces the inactivation of Histamine. Conclusion: HNMT should be considered an important gene in the genetic evaluation of consanguineous families with intellectual disability.

237-247

Authors: Mohammed Mehdi Saghafi , Ahmad Tavakoli , Shahnaz Ahmadi , Seyed Hamidreza Monavari *

Background and Aims: Currently, there are antiviral chemicals used to treat viral infections accompanied by limitations such as high levels of toxicity and adverse effects in humans, the emergence of drug-resistant viral strains, low numbers, and limited diversity. Therefore, it is necessary to evaluate new photochemical to obtain new therapeutic methods. The present study was conducted to evaluate the antiviral activity of Arnebia Euchroma (A. Euchroma) extract, amniotic membrane, a mixture of A. euchroma extract and amniotic membrane and its carrier (comprised of Sesam and ostrich oil) against three different viruses, including Herpes simplex virus type 1 (HSV-1), influenza A, and rotavirus in-vitro. Materials and Methods: A methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cytotoxic effect of the above compounds on Vero, MDCK, and MA-104 cell lines. After the determination of non-toxic concentrations, Tissue culture infection dose 50 (TCID50) test was performed to determine antiviral activity. Results: Among all the above-mentioned compounds, the combination of A. euchroma extract and amniotic membrane at the highest non-toxic concentration for the cells had the highest antiviral activity against all three viruses, leading to 1 log10 TCID50 reduction in influenza A and HSV-1 titers and 0.6 log10 TCID50 reductions in rotavirus titer when compared to the virus control. Conclusions: The combination of A. euchroma and amniotic membrane can be considered a new antiviral agent to treat viral infections.