pilot study on microbial flora of water contained in shisha water pipes and their identification by MALDI-TOF MS

Objective: The water containing jar of the shisha water pipe is constantly moist and is a potential site for bacterial growth and biofilm formation. This study aims to examine the microbial flora and bacterial load of the water in the shisha water jar so as to determine possible risks to users

Materials and methods: Three shisha cafes participated voluntarily in the study. Seventeen de-identified water samples were collected from Shisha pipes being used by various customers at the participating Shisha Cafes. Samples were collected using sterile precautions. Ten micro L from the water samples were inoculated on a blood agar plate and incubated at 37[degree sign]C for 24 hours. The bacterial CFUs/mL were counted manually and organisms isolated were identified by Proteomic fingerprinting of ribosomal proteins using a Bruker Biotyper [Trade Mark] working on the principle of Matrix Assisted Laser Desorption Ionization- Time of Flight Mass Spectrometry, [MALDI-TOF MS]

Results: Seventeen samples were collected. Eleven samples showed microbial growth of 100 to 3000 CFUs/ml with a median value of 400 CFUs/ml. Six samples were sterile. Of the eleven samples, six samples had one organism each, two samples had two organisms each, one sample had three organisms and the last sample had five organisms. The 18 organisms isolated were distributed among nine species: Acidovorax temperans [3 isolates], Burkholderia vietnamiensis [3 isolates], Candida_pelliculosa [1 isolate], Delftia acidovorans [1 isolate], Enterobacter cloacae [3 isolates], Escherichia hermannii [1 isolate], Klebsiella pneumonia [2 isolates], Pseudomonas aeruginosa [1 isolate], and Raoultella ornithinolytica [1 isolate]. Two isolates could not be identified

Conclusions: The organisms isolated have been referenced in literature as pathogens or emerging pathogens capable of causing infections. Presence of significant amounts of pathogens in Shisha water suggests the strong possibility of inhalation of aerosols containing microbes by shisha smokers. The poly microbial nature of the isolates especially suggests the formation of bacterial biofilms. Interestingly almost all the isolates were gram negative bacteria, which contain lipopolysaccharide [LPS] in their cell walls. LPS is a microbial protein that stimulates innate immunity. LPS has been shown to be an etiological agent of acute and chronic airway obstruction and disease. Thus LPS from bacterial aerosols can contribute to the causation of COPD in chronic users, simultaneously exposing them to the risk of infection by newer emerging pathogens

Application of a rapid assay to detect targeted numerical and structural anomalies in patient with congenital malformations

Bacterial artificial chromosomes [BACs]-on-Beads [BoBs] is one of the novel and rapid technologies that has been a part of recent advances in genomic technologies. BACs-on- Beads technology [Trade Mark] that assists in speedy detection of copy number changes [CNVs] in targeted genomic regions from minimal amount of DNA. We compared this molecular multiplex, bead-based suspension array that is used in prenatal invasive testing, with conventional cytogenetic and G-banded karyotype techniques. We present the initial BoBs analysis data of 4 patients referred to CABRI with congenital malformations. As per the manufacture's information the targeted region covers at least 4-5 bacs for each region. The selected loci represent the relatively common chromosomal syndromes associated with deletions that can be missed by karyotype analysis. The syndromes are known with definable phenotype and deletion as the major means giving rise to the syndrome. In addition to this the BAC's for the common aneuploidies of chromosomes 13, 18, 21, X, and Y are also present. The method not only detected the known trisomy 21 but also identified a deletion on the long arm of chromosome 7 at q11.2 region that represents the Williams - Beuran Syndrome [WBS] critical region in a patient with suspected trisomy 21. BoBs is potentially a very useful first row test for aneuploidy detection because of its lower cost and rapid detection with minimal amount of DNA especially in newborns with suspected congenital malformations. The results suggest that it is a reliable technique to detect common microdeletions that get missed out by conventional chromosomal analysis

comparison of in-house hemoglobin DNA mutation analysis for beta thalassemia by ARMS PCR with a commercial line probe assay

This study was done with a view to find a correlation between two molecular tests for beta Thalassemia, our in-house developed haemoglobin DNA mutation analysis using ARMS PCR and a Commercial Line Probe Assay. De-identified samples from known beta thalassemia patients characterised by HPLC for HbA2, Peripheral smear [Target cells] and CBC [microcytosis and erythrocytosis] were used for the study. DNA was extracted using the DTAB/CTAB method. Amplified DNA from the samples was hybridised for mutations using a line probe assay. The extracted DNA was also examined for wild type genes and mutant genes using an Amplification refractory mutation system [ARMS] PCR. In this study fifteen beta Thalassemia patients were involved. The in-house ARMS PCR tested for six mutations and detected thalassemia trait in 66.7% of the samples tested for. The line probe assay tested for 22 mutations and detected thalassemia trait in 93.7% of cases examined. One case was missed by both methods and will require sequencing. The importance of stratification of testing for a cost effective strategy for Thalassemia diagnostics is discussed

DNA sequencing and molecular modelling of HLA-B 27 gene sub types in patients with acute anterior uveitis and ankylosing spondylitis

HLA-B 27 is a human leukocyte antigen [HLA] class I cell surface molecule located on the short arm of chromosome 6. It is strongly associated with Acute anterior uveitis [AAU] and Ankylosing spondylitis [AS], the disorders are not only by genetic inherited disease. Several genetic and environmental factors likely play a vital role in determining the risk of developing these disorders. In this study, we try to find out the structure and functional relationship of HLA-B 27 sub types [HLA B[asterisk]27:112, HLA B[asterisk]27:04:01, HLA B[asterisk]27:06, HLA B[asterisk]27:05:02]. The molecular modeling [3D structure] of these subtypes are constructed and ligand binding sites are predicted. The HLA-B 27 gene were amplified from the DNA isolated from the patients with AS and AAU are sequenced. The protein sequences of the HLA-B 27 subtypes obtained from the IMGT/HLA database are aligned with each other and structure of all the subtypes models were constructed by in-silico method. The binding sites of the HLA-B 27 protein and their subtypes was predicted using 3DLigandSite server. The accurate prediction of ligand binding sites on the HLA-B 27 protein surface can be very helpful for rational drug design of AAU and AS

Method developing: standardization of Hemoglobin variant detection by HPLC from dried blood spot on card

Objective: To detect Hemoglobin variants D and S in dried blood spot collected on Guthrie card

Materials and Methods: This study was conducted in Gulf Medical University Ajman on known positive samples for hemoglobin S and hemoglobin D. Known EDTA blood sample was spotted on Guthrie card, the hemolysate of a punched disc collected from blood spot was eluted and the elute examined by Tosoh G7 using HPLC method1

Results: HPLC analysis of the eluate could correctly identify the hemoglobin's subtypes with 100% concordance in detection for hemoglobin D and hemoglobin S

Conclusion: HPLC analyzed the eluate from blood spot on card can correctly detect hemoglobin D and hemoglobin S. Dried blood spot card could be used to screen newborn for presence of hemoglobin S and hemoglobin D

Line probe assay for detection of alpha thalassemia: a pilot study

This pilot study was initiated with a view to find alpha thalassemia genotypes on de-identified samples from patients diagnosed with anaemia at the Centre for Advanced Biomedical research and Innovation [CABRI] at Gulf Medical University [GMU] in June 2015. Amplified DNA from the samples was probed for mutations using a line probe assay. Results obtained are presented. The study has shown the 3.7 single gene deletion in three cases, and alpha 2 IVS1 [-5nt] mutation seen in one case suggesting these cases have alpha + thalassemia. One sample showed wild type alpha 2 Poly A missing along with the alpha 2 poly a-1 [AATAAA>AATAAG] mutation with a suggestive diagnosis of HbH disease. A SEA double gene deletion was seen in one case suggesting alpha 0-thalassemia. Further studies are being carried out to enhance the data base

Assessment of commercial specific IgE assays for detection of allergens in allergic patients

Allergy is a serious health problem throughout the world, affecting people of all ages. Allergic diseases such as asthma, rhinitis and atopic dermatitis are becoming epidemic in all countries. The cost of investigating these diseases is increasing and becoming very expensive. There are many ways to explore allergenic antibodies to assess the presence and the amount of specific IgE. These are: Skin test [Prick], Specific IgE [ELISA], RAST Sp. IgE and Elimination Challenge methods. Skin test produces pain, local or anaphylactic reaction and patient discomfort. Other procedures are expensive to the patient. So, a modified procedure, based on the same principle of previous tests, was studied in Allergy and Immunology laboratory of Ain Shams University. The procedure has suitable cost for all patients; it is very simple, accurate, cheap and does not produce any problems for patients. It depends on ELISA technique and measures the quantitative amount of the following different allergens: Food and Drug allergens such as, Milk, Eggs, Banana, Maize, Fish, Chocolate, Wheat, Nuts, Strawberry, Shrimps, Spices and Aspirin as a drug allergen. Inhalants, as House dust, Mite, Mixed Pollens, Mixed Moulds, Hay dust, Wool, Latex and Cat Hair. The results of this test for 150 allergic patients were compared with those of national specific IgE kits [ELISA], Sp.IgE [RAST], Skin test and elimination challenge test. Statistical results of sensitivity showed respectively: 88.9%, 89.6%, 91.2%, 71.4%, 93.1%. As regards specificity, the results were 93.1%, 94.7%, 95.3%, 65.5%, 91.6%, respectively. These results conclude that the test is in line with all other standard tests. It can also be noted that it is not only the cheapest and most commercial technique using the immediately available, locally prepared reagents, plates and other requirements found in any standard laboratory, but, additionally, probably it can be unique in using foods, drug and inhalants allergens at the same time. Now, it is applied successfully in Allergy and Immunology unit in Ain Shams university hospitals in Egypt. The test has also been recently introduced at the Center for Advanced Bio Medical Research and Innovation [CABRI] at the Gulf Medical University, Ajman, UAE, using a commercial system from Phadia. About two hundred different IgE levels against specific antigens are tested using the Immunocap 100. The allergen of interest, covalently linked to the Immunocap is incubated with the serum being tested. The unbound IgE is washed away and the bound specific IgE is detected using a fluorescent reader. The concentration is calculated using a calibration curve

Utilization of high density Oligo/SNP array to analyze patients with autism - a step towards high definition GENOMICS

Array comparative genome hybridization [aCGH] has been a powerful tool that allows a high resolution whole genome analysis of copy number variations and single nucleotide polymorphisms [SNPs] that can reveal submicroscopic deletions, duplications loss of homo or heterozygosity [LOH] and uniparental disomy. We present aCGH analysis data of 4 patients referred with autism that was analyzed using a high density oligo/SNP array with over 1.9 million markers for copy number variations [CNV's] and about 750,000 SNPs [Affymetrix]. After careful evaluation, we found no genomic CNVs that were previously described but noticed several regions with loss of heterzygosity [LOH] in 2 of the 4 patients analyzed. The regions of LOH range from 7Mb to over 29Mb in patient A2 and from over 3Mb to over 63Mb in patient A3. Some of the LOH noticed in these patients are seen associated with genes responsible for causing Autism. The genes noticed have been fully characterized and classified as Autism genes. Patient A2 has only one gene involved [DPP10 [2q14.1]] and patient A3 has 5 genes [MBD5 [2q23.1], SCN1A, SCN2A [2q24.3], KCTD13 [16p11.2] and PAFAH1B1 [17p13.3]]. Several regions of LOH detected in these two patients encompass -101 Mb and 201 Mb of the total genome respectively. The results suggest that it is not related to a specific disorder but involvement of at least gene in the LOH region can raise the possibility of a recessive condition. The importance of LOH and the details of the genes will be discussed

Enhancing laboratory turnaround time performance by combining the implementation of a laboratory instrumentation interfacing solution with six sigma methodology

Six Sigma is considered to be an important quality improvement tool in the industry. This paper outlines the application of six sigma methodology along with the implementation of a laboratory instrumentation interfacing solution in bringing down turnaround time [TAT] failures in the hospital laboratory sample collection, testing and reporting cycle at GMC Hospital Ajman. This was implemented since the GMC Hospital laboratory was facing numerous client complaints due to TAT failure in reporting of diagnostic test results. To address the problem of high TAT failures the following steps were taken 1. Bar coding of all samples was facilitated and all samples barcoded 2. Random access analyzers interfaced through R5232 ports with HIMS 3. TAT defined for all in-house tests 4. Barcoded samples were scanned and time points noted automatically at collection, receipt in lab, testing on bench, initial technical verification, secondary verification and printing of report. This data was monitored for adherence to the pre-analytical, analytical and post analytical phases. 5. Three cycles of the DMAIC process was used to decrease defect rate and enhance adherence to TAT. a. In the first cycle analytical Issues, b. in the second cycle pre-analytical issues and c. in the third cycle post-analytical issues were defined, measured, analyzed, intervention carried out and changes consolidated [DMAIC]. The interventions in three phases of two months each, over a six months period, sequentially brought down TAT failures 45% [pre-intervention] to 33%, 13%, 11%, 4%,3% and 1% [post-intervention] respectively. A barcode enabled laboratory equipment interfaced HIMS system combined with Six Sigma Tools was able to significantly enhance adherence to TAT, improve services to physician, resulted in better staff utilization and improve the quality and value of the report by ensuring timely reporting. To consolidate gains and for continual improvement it is now proposed to improve performance by further reduction of TAT by introducing POCT, introducing a dedicated LIMS and auto-verification of samples

DNA diagnostics: Using the polymerase chain reaction for detecting HLA B27 in patients of acute anterior uveitis

The human leukocyte antigens [HLA] are a group of cell surface molecules encoded for by the major histocompatability locus on the short arm of human chromosome six. Individuals carrying the HLA-B27 gene, have been shown to have a higher incidence of isolated acute anterior uveitis [AAU]. This study aims to examine the incidence of the HLAB27 gene in individuals presenting with AAU at GMCHRC. This pilot study was carried out for a period from May 2013 to July 2013 at GMCHRC Ajman UAE. Four patients of clinically diagnosed AAU were examined for the presence of the HLA B27 gene. Genomic DNA was amplified using primers and cycling parameters were as described by Bunce et al. using exon-2, B-locus- specific primers. The PCR products were run in a 1% agarose gel stained with ethidium bromide [0.5 microg/ml] and visualized under U.V. light. Four cases of AAU were subjects in this study. Of the four cases examined two [50%] were positive for the HLAB27 gene. The M:F ratio of the cases 3:1. All the HLAB27 positive cases were males. The average age of the positive cases was 37.5 years as compared to 53.5 years in the negative cases. There was no racial predisposition seen. Using the above set of primers, DNA from HLA B27 positive individuals gave a 150 bp band. AAU is the most common form of uveitis. Of the cases reported in literature half of all cases of AAU are HLA-B27 positive and this is similar to the results displayed in our series of four cases. Cases positive for HLAB27 in AAU require active treatment using immunomodulators for early resolution and topical cycloplegic agents and steroids are the cornerstones of treatment. Methotrexate, Salazopyrine, anti-TNF and anti-CD20 therapy may be used to prevent recurrent attacks. This study exemplifies the use of PCR and DNA diagnostics in the identification of HLAB27 gene in cases of AAU. Further studies using the Dideoxy Sequencing are proposed to be carried out

Accuracy of POCT device, Afinion[TM], for tests for diabetes and dyslipidemia

The aim was to evaluate the analytical performance of a point of care system, based on boron affinity chromatography, for recommended laboratory tests for monitoring of diabetes and lipids e.g. Hemoglobin A 1 c and lipid profile tests. The Afinion[TM] system [Afinion] is intended for use as POCT [Point Of Care Testing]. It is a fully automated boronate affinity assay. Ten samples for HbA1c, CRP and Lipids [Total Cholesterol, HDL-C, LDL-C and Triglycerides] were analyzed on the Afinion[TM] system and chemistry analyzer Cobas 6000 for comparison. Cobas 6000 is a fully automated chemistry analyzer from Roche Diagnostics. The analyzer uses NGSP traceable, immunoturbidimetry based method and lipids by standard photometric methods traceable to reference respective methods. Five samples were also tested in duplicate to check imprecision for each of the tests. The percentage bias for each of the tests was evaluated using Total Error Allowable [TEA] based on biological variation. The bias percentage was 1.643% for HbA1C, -2.764% for CRP, 1.439% for Total Cholesterol, -1.794% for HDL Cholesterol, 3.013% for LDL cholesterol, 3.473% for Triglycerides as compared to those on Cobas 6000. The strong correlations of HbA1c, CRP and Lipid profile performed on POCT with state of art automated chemistry analyzer indicate that the POCT can be used for monitoring of treatment at sites remote from the central laboratory with confidence. This can result in a faster turnaround time, prevent sample integrity issues as well as save transport costs. Accurate check of a new point of care system [Afinion TM AS 100 Analyzer, Axis-Shield PoC, AS, Oslo, N] prior to consideration for procurement was to evaluate usability of the device for a small physician-office type of laboratory

Evaluation of point of care device for hemoglobin, total WBC and differential counts at clinical laboratory, GMC Hospital Ajman, UAE

Recently, GMCH Laboratory present a point of care analyzer, HemoCue, for Hemoglobin, total WBC and differential counts to reduce turnaround time, especially for pediatric patients. The objective of this study is to validate these tests before introducing it to the patient care. There are 2 units; one for the measurement of Hemoglobin, called Hb201+ and the other for total WBC and differential counts, called HemoCue WBCDIFF. Hb201+ performs photometric measurement of hemoglobin by converting it to hemoglobinazide by special capillary action microcuvettes. In the cuvette for HemoCueWBCDIFF, red blood cells are lysed and white blood cells are stained. Cells are counted and differentiated by image analysis. Whole blood collected in EDTA from patients covering [known value] high to low hemoglobin and total WBC counts was used for precision study. 5 replicates in 5 different runs were used.%CV was calculated and compared against allowable imprecision based on total allowable error TEa for all the parameters. For correlation study, 20 previously analyzed whole blood EDTA samples from patients were analyzed for all the parameters. The values obtained were compared with the results of Sysmex 2000i, automated hematology analyzer. The%bias for the parameters was calculated and compared with allowable%bias based on TEa for each of the parameter and linear regression analysis was performed to calculate coefficient of variance [r].TEa was based on biological variation. Hemoglobin showed a maximum CV of 0.96% and bias of 0.16% and r=0.997. Total WBC count showed maximum CV of 1.27% and the differential WBC count a maximum CV of 3.6% [neutrophils and lymphocytes]. Both, accuracy and precision for the analyzer for all the parameters are acceptable as per the quality goal of TEa and comparable to fully automated hematology analyzer. The POCT is relatively inexpensive, simple to use, battery operated, thus helps to reduce turnaround time and venous sample collection, especially in pediatric patients

Evaluation of point of care cardiac and thromboembolic markers in GMC Hospital Laboratory

The objective of the present work was to perform validation study on a group of cardiac biomarkers so that they could be confidently used for the on-spot diagnosis of a range of cardiovascular events like acute coronary syndrome, heart failure and thromboembolic phenomena. A group of cardiac markers including NT-pro BNP, Troponin T, OK- MB, Myoglobin and D-Dimer were recently introduced in GMC Hospital as a point of care using Cobas h232, a handheld device from Roche Company. Precision study was carried out using commercially available control material. Five replicates were carried out across 4 different runs and performed over 5 different days. Coefficient of variation%CV was calculated both for within run and between runs and compared with Total Error Allowable [TEa] for the analytes. The accuracy study for Troponin T was determined by using 8 previously tested heparinized whole blood patient samples obtained from Rashid Laboratory, Dubai, and comparing the result with Cobas h232 available in GMC lab. Percent CV calculated for all analytes was found to be less than TEa. Bias between the achieved target and manufactured target was found to be within one- third of the TEa. The bias achieved, based on the medical decision values was found to be acceptable when compared with TEa. For D-Dimer assay, the bias between the achieved target and manufacture's target was greater than the TEa/3. However it was found within the reference allowable range of the QCcompared with the conversional method the turnaround time was extremely low in the average of 45 minutes. Due to its good analytical agreement with the laboratory methods and ease of use Cobas h232 system for POCT could be reliably used to support on site decision making regarding cardiovascular patients in acute and non-acute settings