PCR to CRISPR: Role of Nucleic Acid Tests (NAT) in detection of COVID-19.

COVID-19 infection has emerged as an unparalleled pandemic with morbidity and mortality tolls challenging diagnostic approaches and therapeutic interventions, and raising serious questions for healthcare policy-makers. From the diagnostic perspective, Reverse transcriptase polymerase chain reaction remains the gold standard. However, issues associated with gene primer variation in different countries, low analytical sensitivity, cross-reactivity with certain human coronaviruses have raised serious concerns within the scientific community. Alongside longer turnaround times, requirements of sophisticated equipment and trained technicians are the other challenges for conventional reverse transcriptase polymerase chain reaction testing. The recent biotechnological boom has now allowed newer nucleic acid testing options for diagnosing severe acute respiratory syndrome Coronovairus 2 (SARS-CoV2) with much better diagnostic efficiency, reduced turnaround times and possible benefit for use as a point-of-care test. Isothermal techniques with simple equipment requirements along with uniform temperature for analysis have emerged to be more sensitive and specific with turnaround times as low as 10-15 minutes. Similarly, Cluster Regularly Interspaced Short Palindromic Repeats have also been seen to play a very decisive role in COVID-19 diagnostics with much superior diagnostic efficiency and feasibility as a point-of-care test and its possible use for sequencing. The current narrative review was planned to consolidate data for all possible nucleic acid testing options under research/clinical use, and to provide a comparative assessment from the perspective of both the clinician and the laboratory.

Comparative Fluoride Release and Antimicrobial Analysis of Commercial and Experimental Bioactive Glass/Nano-Oxide-Based Dentifrices.

OBJECTIVES: The objectives were to measure fluoride release and assess the antimicrobial behavior of fluoride-doped nano bioactive glass (F-nBG) and nano zinc oxide (ZnO)-enriched novel dentifrices. MATERIALS AND METHODS: Experimental dentifrices were synthesized by incorporating ZnO nanoparticles and F-nBG (1.5 wt% and 4 wt%) as active ingredients. The fluoride release behavior of suspensions and elutes of samples were analyzed by ion selective electrode. Antimicrobial activity and minimum bactericidal concentration against Streptococcus mutans and Lactobacillus casei were evaluated. Microbial stability against contamination was also assessed by a challenge test. RESULTS: The fluoride release behavior of experimental dentifrices was higher than that of commercial dentifrices and was dependent on filler loading. The fluoride release was more from suspensions than elutes. Zones of inhibition (ZOIs) and minimum bactericidal concentration values for novel dentifrices showed direct proportionality with filler loading, and effectiveness was exhibited against both strains. Experimental dentifrices exhibited effective antibacterial potential, which could possibly be due to release of sufficient fluoride and zinc ions in aqueous media from F-nBG and ZnO present in their formulations. CONCLUSION: Combination of F-nBG and ZnO may provide a multi-benefit approach for simultaneously treating early white spot lesions, reducing bacterial growth, and providing core plaque control.

Efficacy of Antimicrobials Against Multidrug-Resistant Acinetobacter baumannii from Patients in a Tertiary Care Hospital.

Introduction: In just two decades Acinetobacter baumannii has attained considerable importance, evolving from an insignificant organism to a leading pathogen especially in intensive care unit settings globally. Treatment options are already very limited and have almost run out due to the rapid emergence of antimicrobial resistance. Evaluation of antimicrobials that are currently in use to determine their effectiveness against multidrug-resistant (MDR) strains and developing newer options is of utmost importance. We thus set out to determine the efficacy of routinely used antibiotics against MDR A. baumannii. Materials and Methods: This was a cross-sectional study conducted at the Department of Microbiology, Army Medical College, National University of Medical Sciences (Rawalpindi, Pakistan) from December 2015 to June 2016. The organisms were identified on the basis of colony morphology, gram staining, catalase, oxidase, motility test, and API (analytical profile index) 20NE. The organisms were considered to be MDR when the isolate was found to be resistant to at least one agent in more than three antimicrobial groups. Antibiotic sensitivity was determined using the modified Kirby-Bauer disc diffusion method according to Clinical Laboratory Standards Institute (CLSI) guidelines. Results: The 77 isolates were found to have good sensitivity to tigecycline (94.8%) and minocycline (80.5%). Most of the isolates were resistant to other routinely used antibiotics. Conclusion: A few antibiotics, tigecycline and minocycline, are still effective against these MDR A. baumannii. We need to remain up to date regarding the efficacy of antibiotics to effectively treat patients with these MDR bacteria.

Determination of reference intervals of thyroid markers during pregnancy in Urban area of District Rawalpindi Pakistan.

OBJECTIVE: To determine the reference values for thyroid stimulating hormone, free tetra-iodothyronine and total tri-iodothyronine for healthy pregnant women. METHODS: This cross sectional study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from January 2016 to June 2017. Pregnant women with normal, single intrauterine, uncomplicated pregnancy were recruited from the local population. Blood sample was taken to analyse thyroid stimulating hormone, free tetra-iodothyronine and total tri-iodothyronine using chemiluminescence immunoassay. For thyroid hormone levels during each trimester 5th and 95th percentiles were calculated as reference intervals. Data was analysed using SPSS 24. RESULTS: Out of 384 subjects, 188(48.95%) were in their first trimester and 196(51.04 %) females were in their second trimester. There were 109(57.97%) primigravida in the first trimester and 137(69.9%) in the second trimester. Mean age of subjects presenting in the first and second trimester was 25.37±3.78 years and 26.54±4.65 years respectively. Reference intervals for those in the first trimester for thyroid stimulating hormone was 0.05-2.8uIU/ml, for free tetra-iodothyronine14.4-22.7pmol/l and total tri-iodothyronine1.5-3.3nmol/l. For those in second trimester the corresponding values were 0.16-3.3 uIU/ml, 14.2-24.6.0 pmol/l and 1.6-3.1nmol/l. CONCLUSIONS: Laboratories should adopt trimester-specific reference intervals for thyroid function tests in pregnancy..

Spectrum of renin angiotensin aldosterone system disorders in young hypertensives.

OBJECTIVE: To analyse the spectrum of renin angiotensin aldosterone system disorders in young hypertensive patients in hospital settings. METHODS: This cross-sectional study was conducted at the Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from January to December, 2016. It comprised hypertensive subjects aged 17-40 years of either gender presenting in the outpatient department. All subjects were having blood pressure more than 140/90mmHg and were not on any anti-hypertensive medicine. Blood sample was taken from each patient to analyse arterial blood gases, plasma renin, serum aldosterone and electrolytes. Association of qualitative variables like age, systolic and diastolic blood pressure with essential hypertension and primary hyperaldosteronism was explored. RESULTS: Of the 80 patients, 72(90%) were diagnosed with essential hypertension and 8(10%) with primary hyperaldosteronism. None of the patients had Liddle syndrome, apparent mineralocorticoid excess or Gordon syndrome. Mean age of patients having essential hypertension was 30.97±7.1 years, whereas, for those with primary hyperaldosteronism it was 29.25±7.1 years. Systolic blood pressure was significantly higher (p = 0.000) among all patients. No statistically significant association was found between age, systolic and diastolic blood pressure (p<0.05). CONCLUSIONS: Primary hyperaldosteronism as compared to other renin angiotensin aldosterone system disorders was found to be the leading cause of hypertension in young population.

ICU Pathogens: A Continuous Challenge.

OBJECTIVE: To determine the frequency and antibiogram of pathogens in an intensive care unit (ICU). STUDY DESIGN: Cross-sectional, observational study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, National University of Science and Technology, Islamabad, from January 2013 to January 2014. METHODOLOGY: Clinical samples, received from patients admitted in ICU, were inoculated on various medias like blood agar, chocolate agar, MacConkey agar and urine samples on CLED. These were then incubated at 37°C for 24 hours. Isolates were identified by colony morphology, Gram reaction, catalase test, oxidase test. Species identification in case of Gram Negative Rods was done by using API 20E (BioMérieux). Antibiotic susceptibility was done by using modified KirbyBauer disc diffusion technique. Bacterial isolates were prepared and inoculated on Mueller-Hinton agar plates followed by application of various antibiotic disc (Oxoid, UK) as per manufacturer's instructions. The plates were then incubated at 37°C aerobically for 18 - 24 hours. Zone diameters were measured and interpreted as sensitive and resistant, according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Out of the 367 positive cultures, 116 (31.08%) were Acinetobacter baumanniisusceptible to minocycline and tigecycline followed by Klebsiella pneumoniae (n=71, 16%) susceptible to tigecycline and meropenem. Others were Pseudomonas aeruginosa, Escherichia coli,Coagulase Negative Staphylococcus, Staphylococcus aureus, Enterococcus spp., Streptococcus spp., Klebsiella oxytoca, Stenotrophomonas maltophilia,and Candida spp. CONCLUSION: Acinetobacter baumanniiwas the most frequently isolated pathogen. Most of the cultures yielding pathogens were from respiratory tract samples. Gram negative isolates were multidrug resistant but most were tigecycline and susceptible to meropenem.

Methicillin resistant coagulase negative staphylococcus: From colonizer to a pathogen.

The objective of our study was to determine the frequency of methicillin resistance in coagulase negative Staphylococcus (CoNS) and to determine its in-vitro antimicrobial susceptibility to various other routinely used antibiotics. It was a cross sectional study conducted at the department of Microbiology, Army Medical College, Rawalpindi, Pakistan from June 2011 to May 2012. The organisms were identified on the basis of colony morphology, Gram staining, catalase, DNAase and slide/tube coagulase tests. The organisms were considered to be methicillin resistant when the diameter of zone of inhibition was less than 25mm around 30µg cefoxitin disc. Antibiotic sensitivity was determined using the Modified Kirby-Bauer disc diffusion method. From a total of 337 CoNS, 201 were methicillin resistant and were included in the study. All were resistant to Penicillin, followed by Erythromycin (93•1%), Ciprofloxacin (77%), Co-trimoxazole (74•8%), Gentamicin (68•3%), Clindamycin (51•06%), Tetracycline (44•6%), Fusidic acid (40%), Rifampicin (39•5%), Chloramphenicol (19•3%), Linezolid (2%), Minocycline (1•1%), and Vancomycin (0%). More than half of CoNS were methicillin resistant. Vancomycin is the only drug to which all of the MRCoNS were sensitive, with more than 98% of the isolates being sensitive to Linezolid and Minocycline.

Evaluation of phenotypic tests for detection of Amp C beta-lactamases in clinical isolates from a tertiary care hospital of Rawalpindi, Pakistan.

OBJECTIVE: To evaluate sensitivity and specificity of disc approximation test compared to three-dimensional extract test as a phenotypic gold standard test for detection of AmpC beta-lactamase producing Escherichia coli and Klebsiella pneumoniae. METHODS: The cross-sectional validation study was conducted from November 2014 to April 2015 at Army Medical College, Rawalpindi, Pakistan. Extended spectrum beta lactamases (ESBLs) were isolated from various clinical specimens. Screening for AmpC beta-lactamases was done by using cefoxitin disc (30µg) showing inhibition zone diameter of <18mm. Screen-positive isolates were subjected to disc approximation test (DAT) and three-dimensional extract test(3-DET).SPSS 20 was used for statistical analysis. RESULTS: A total of 120 ESBL producing Gram negative rods were included in the study. Out of these 120, 82(68.33%) were found to be AmpC beta-lactamase producing on screening with cefoxitin disc. Amongst these 82 isolates, Escherichia coli were identified in 57(69.51%) and Klebsiella pneumoniae in 25 (30.48%). Phenotypic confirmation by disc approximation test (DAT) identified 43(52.43%). AmpC beta-lactamase producing isolates, whereas gold standard 3-DET showed 38(46.34%) of AmpC beta-lactamase producing isolates. Hence, sensitivity of disc approximation test (DAT) was found to be 88%, specificity was 92%, positive predictive value was 92.68%, negative predictive value was 87.80% and diagnostic accuracy was 90.24%. CONCLUSIONS: Implementation of disc approximation test in the laboratories can help in identifying AmpC beta-lactamase harbouring organisms.

In Vitro Efficacy of Doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii by E-Test.

OBJECTIVE: To assess the in vitro efficacy of doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii using Epsilometer strips. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi and National University of Sciences and Technology, Islamabad, from May 2014 to September 2014. METHODOLOGY: A total of 60 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa collected from various clinical samples received from Military Hospital were included in the study. The specimens were inoculated onto blood, MacConkey and chocolate agars. The isolates were identified using Gram staining, motility, catalase test, oxidase test and API 20NE (Biomeriux, France). Organisms identified as Acinetobacter baumannii and Pseudomonas aeruginosa were included in the study. Bacterial suspensions equivalent to 0.5 McFarland turbidity standard of the isolates were prepared and applied on Mueller Hinton agar. Epsilometer strip was placed in the center of the plate and incubated for 18-24 hours. Minimum Inhibitory Concentration (MIC) was taken to be the point where the epsilon intersected the E-strip. MIC of all the isolates was noted. RESULTS: For Pseudomonas aeruginosa isolates, MIC(50) was 12 µg/mL and MIC(90) was 32 µg/mL. For Acinetobacter baumannii MIC(50) and MIC(90) was 32 µg/mL. CONCLUSION: Doripenem is no more effective against Pseudomonas aeruginosa and Acinetobacter baumannii in our setting.

Coagulase negative staphylococci - a fast emerging threat.

OBJECTIVE: To determine the frequency of isolation of coagulase-negative staphylococci and their resistance to methicillin over a period of time. METHODS: The descriptive cross-sectional study was carried out at Army Medical College, Rawalpindi, from June 2009 to May 2012, and comprised clinical samples mostly from patients admitted to the intensive care unit. They were inoculated onto appropriate culture media depending upon the specimen. After 24-hour incubation at 35°C, coagulase-negative staphylococci were identified on the basis of colony morphology, gram staining, a positive catalase and a negative tube coagulase test.Methicillin resistance among the isolated staphylococci was determined using a 30µg Cefoxitin disc as per the Clinical and Laboratory Standards Institute protocol. Number of coagulase-negative staphylococci for each year and their methicillin resistance rates were calculated. A comparison was made with methicillin resistant staphylococcus aureus) isolated during the same period. RESULTS: Of the total 1331 specimens studies over three years, 581(43.65%) were coagulase-negative staphylococci. The rate of coagulase-negative staphylococci and methicillin resistance was higher each year; 110(26.6%) in May 2009-Jun 2010, 134(36.5%) in 2011, and 337(61%) in 2012. Methicillin resistance rates also increased from 25(22.7%) to 46(34.3%) and then to 201(59.6%) in 2012.Maximum isolated specimens came from blood 311(53.5%), followed by pus/swabs 204(35.1%). CONCLUSIONS: The frequency of isolation of coagulase-negative staphylococci and its methicillin resistance among hospitalised patients is on the rise.

Comparative Efficacy of Ceftaroline with Linezolid against Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus.

OBJECTIVE: To compare the in vitro antimicrobial efficacy of ceftaroline with linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus. STUDY DESIGN: Quasi-experimental study. PLACE AND DURATION OF STUDY: Microbiology Department, Army Medical College, Rawalpindi, from January to December 2013. METHODOLOGY: Clinical samples from respiratory tract, blood, pus and various catheter tips routinely received in the Department of Microbiology, Army Medical College, Rawalpindi were innoculated on blood and MacConkey agar. Staphylococcus aureus was identified by colony morphology, Gram reaction, catalase test and coagulase test. Methicillin resistant Staphylococcus aureus detection was done by modified Kirby Bauer disc diffusion method using cefoxitin disc (30 µg) and the isolates were considered methicillin resistant if the zone of inhibition around cefoxitin disc was ≤ 21 mm. Bacterial suspensions of 56 Staphylococcus aureus isolates and 50 MRSA isolates were prepared, which were standardized equal to 0.5 McFarland's turbidity standard and inoculated on Mueller-Hinton agar plates followed by application of ceftaroline and linezolid disc (Oxoid, UK), according to manufacturer's instructions. The plates were then incubated at 37 °C aerobically for 18 - 24 hours. Diameters of inhibition zone were measured and interpretated as per Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Out of 106 isolates all of the 56 Staphylococcus aureus (100%) were sensitive to ceftaroline and linezolid. However, out of 50 methicillin resistant Staphylococcus aureus, 48 (96%) were sensitive to ceftaroline whereas, 49 (98%) were sensitive to linezolid. CONCLUSION: Ceftaroline is equally effective as linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus.

Comparison of urine dipstick test with conventional urine culture in diagnosis of urinary tract infection.

OBJECTIVE: To determine the sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of Urine Nitrite (NIT) and Leukocyte Esterase (LE) test compared with urine culture for diagnosis of UTI. STUDY DESIGN: Validation study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi, from January 2013 to December 2013. METHODOLOGY: Three hundred fresh uncentrifuged urine samples with suspicion of UTI, were collected and tested for LE and NIT by using (COMBI-10SL, UK) strip. Nitrite was considered as positive if there was a change in color of dipstick from colorless towards pink within 60 seconds. Leukocyte esterase was considered as positive if there was a change in color from off-white towards purple within 2 minutes. Quantitative urine culture was performed by using the strips calibrated to deliver 0.02 ul of urine on Cystine Lactose Electrolyte Deficient (CLED) medium agar. All plates were incubated at 37°C and read after 24 and 48 hours. Culture was considered as gold standard to evaluate the performance of dipstick test. RESULTS: Out of 300 samples, 136 were culture positive and 164 were culture negative. Out of 136 positive culture results, 103 were dipstick positive and 33 were negative. Sensitivity, specificity, positive predictive value and negative predictive value of both nitrite and leukocyte esterase were 75.74%, 68.90%, 66.66% and 77.40% respectively considering culture as gold standard. CONCLUSION: Dipstick test for the detection of leukocyte esterase and nitrite in urine are sensitive and specific and can be used reliably for the detection of UTI in resource limited setup.

Frequency and antibiogram of vancomycin resistant enterococcus in a tertiary care hospital.

OBJECTIVE: To determine the frequency of Vancomycin Resistant Enterococcus (VRE) in a tertiary care hospital of Rawalpindi, Pakistan. STUDY DESIGN: Observational, cross-sectional study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi, from May 2011 to May 2012. METHODOLOGY: Vancomycin resistant Enterococcus isolated from the clinical specimens including blood, pus, double lumen tip, ascitic fluid, tracheal aspirate, non-directed bronchial lavage (NBL), cerebrospinal fluid (CSF), high vaginal swab (HVS) and catheter tips were cultured on blood agar and MacConkey agar, while the urine samples were grown on cystine lactose electrolyte deficient agar. Later the antimicrobial susceptibility testing of the isolates was carried out using the modified Kirby-Bauer disc diffusion method on Mueller Hinton agar. RESULTS: A total of 190 enterococci were isolated. Of these, 22 (11.57%) were found to be resistant to vancomycin. The antimicrobial sensitivity pattern revealed maximum resistance against ampicillin (86.36%) followed by erythromycin (81.81%) and gentamicin (68.18%) while all the isolates were 100% susceptible to chloramphenicol and linezolid. CONCLUSION: The frequency of VRE was 11.57% with the highest susceptibility to linezolid and chloramphenicol.