Laboratory Physicians

M2G1G2 white blood cell flag by three-part automated hematology analyzer: A hint to dengue infection in appropriate clinical context Maitrayee Roy, Akshay Bali Journal of Laboratory Physicians 2019 11(2):103-106 BACKGROUND: Automated hematology analyzers often generate many flags which can provide important clues to the underlying hematological abnormality. Although pathologists are generally well versed in recognizing the importance of flags indicating potential leukemic blasts, their utility in hinting toward infectious etiology, especially during epidemics, is less well known. We analyzed any abnormal flags generated by a three-part automated hematology analyzer in serologically confirmed cases of dengue. MATERIALS AND METHODS: The study included 28 patients diagnosed with serologically confirmed dengue infection. The venous samples were run on ABX Miros-60 three-part hematology analyzer. The complete blood count data and any abnormal flags were noted and correlated with peripheral blood film findings in all patients. RESULTS: An abnormal white blood cell (WBC) flag was noted in all 28 patients, including two in whom all other hematological parameters were within normal limits. In 26 (93%) patients, M2G1G2 WBC flag was noted while the samples of the remaining two patients generated M2G1 and L1M2G1G2 WBC flags, respectively. CONCLUSION: An abnormal WBC flag, when correlated in appropriate clinical context, especially during a dengue outbreak, can aid in targeting the cohort of patients who will require immediate referral for serological confirmation.

Detection of carbapenemase production in Enterobacteriaceae and Pseudomonas species by carbapenemase Nordmann–Poirel test Morubagal R Rao, Pooja Chandrashaker, Rashmi P Mahale, Sowmya G Shivappa, Ranjitha S Gowda, Vidyavathi B Chitharagi Journal of Laboratory Physicians 2019 11(2):107-110 PURPOSE: Multidrug-resistant organisms causing community-acquired and hospital-acquired infections are increasing at a dangerous rate. Carbapenemase-producing Enterobacteriaceae and Pseudomonas species are an important source of concern since these organisms are not only resistant to beta-lactam antibiotics but also show cross-resistance to other groups of antibiotics. In the present study, rapid detection of these carbapenemase-producing Enterobacteriaceae and Pseudomonas species by carbapenemase Nordmann–Poirel (Carba NP) test was evaluated by comparing with modified Hodge test (MHT). MATERIALS AND METHODS: Imipenem-resistant Enterobacteriaceae and Pseudomonas species isolated from various samples such as pus, blood, sputum, urine, and endotracheal aspirates were processed for carbapenemase detection by MHT and Carba NP test. Kappa analysis was done to evaluate the percentage agreement between the two tests. RESULTS: Seventy imipenem-resistant Enterobacteriaceae and Pseudomonas isolates were analyzed in the present study for carbapenemase production. 63.41% of Enterobacteriaceae and 34.48% of Pseudomonas species were carbapenemase producers considering both the methods. By MHT, 36 (51.42%) isolates and, by Carba NP test, 35 (50%) isolates were positive for carbapenemase production out of the 70 isolates. CONCLUSION: Carba NP test when compared to MHT is a simple, rapid, cost-effective biochemical test which can be used in all laboratories in the identification of life-threatening carbapenemase-producing Gram-negative bacteria.

Epidemiology of carbapenem-resistant Enterobacteriaceae in a Tertiary Care Center in the Kingdom of Bahrain Nermin Kamal Saeed, Safaa Alkhawaja, Nashawa Fawzy Abd El Moez Azam, Khalil Alaradi, Mohammed Al-Biltagi Journal of Laboratory Physicians 2019 11(2):111-117 PURPOSE: The purpose of the study is to estimate the rate of infection with carbapenem-resistant Enterobacteriaceae (CRE) in the main governmental tertiary care hospital in Bahrain. MATERIALS AND METHODS: All clinical samples with positive growth of CRE over 6-year period (January 2012–December 2017) were collected from the microbiology laboratory data. RESULTS: The CRE incidence was high in the first half of study period (2012–2014) and then decreased between 2015 and 2017, after implementation of intensified CRE control measure bundle. About 49.4% of CRE-positive samples were isolated from the elderly age group (above 65 years old), most of them were admitted in the intensive care unit (ICU). The most common isolated organisms were Klebsiella pneumoniae (87.0%), followed by Escherichia coli (7.9%). Isolates from deep tracheal aspirate and midstream urine specimens were the most common source of CRE isolates (27.3%) and (26.3%), respectively. Bacteremia was documented in 21.2% of cases. CRE isolates in the study showed high rates of resistance to aminoglycosides (72.2% resistant to amikacin and 67.3% to gentamicin). Alternatively, most isolates retained their susceptibility to colistin and tigecycline with sensitivity of 83.9% and 85.7%, respectively. Combined resistance to both colistin and tigecycline was observed in 0.06% of total isolates. CONCLUSION: Elderly population and ICU admission were important risk factors for CRE acquisition. Most of CRE isolates were sensitive to both colistin and tigecycline, which make them the best combination for empiric frontline therapy for suspected serious CRE infection in our facility. Implementing CRE-bundled infection control measures significantly reduced the incidence of CRE infection in our hospital.

A substitute to xylene in deparaffinization and clearing prior to coverslipping in histopathology Nasar Yousuf Alwahaibi, Sirin Hamed Aldughaishi Journal of Laboratory Physicians 2019 11(2):118-122 INTRODUCTION: Deparaffinization and clearing prior to coverslipping are important steps in all staining methods in histopathology. Xylene is the most commonly used agent worldwide. However, xylene is toxic. We evaluated safer alternative dewaxing and clearing agents prior to coverslipping in a histopathology laboratory. MATERIALS AND METHODS: Thirteen different fresh surgical tissues were cut into two halves. One half processed using xylene and the other half processed using UltraClear™. Five groups were designed. For each Group of A, B, C, and D, 100 slides were cut from xylene-processed blocks. For Group E, 100 slides were cut from UltraClear™-processed blocks. Group A is the standard method. Group B evaluates UltraClear™ as a dewaxing agent only. Group C evaluates UltraClear™ as a clearing agent prior to coverslipping only. Group D evaluates UltraClear™ as both dewaxing and clearing agents prior to coverslipping. Group E evaluates UltraClear™ as both dewaxing and clearing agents prior to coverslipping. Six parameters were evaluated: nuclear staining, cytoplasmic staining, cell morphology, clarity of staining, uniformity of staining, and cost. RESULTS: Groups B, C, and D showed 79% (P = 0.054), 83% (P = 0.221), and 80% (P = 0.079) adequacy when compared with Group A (89%), respectively. However, Group E showed only 76% (P = 0.016) adequacy. UltraClear™ is more expensive than xylene. CONCLUSION: UltraClear™ is a promising dewaxing agent. It is also a good clearing agent for use prior to coverslipping in histopathology laboratory. Cost-benefit balance between safety of laboratory workers, good quality staining, and cost-effective strategy needs to be further studied.

Prevalence and characterization of beta-lactamase-producing Escherichia coli isolates from a tertiary care hospital in India Aishwarya Govindaswamy, Vijeta Bajpai, Surbhi Khurana, Anjana Aravinda, Priyam Batra, Rajesh Malhotra, Purva Mathur Journal of Laboratory Physicians 2019 11(2):123-127 BACKGROUND: The purpose of the study was to determine the prevalence and characterize the resistance profiles of Escherichia coli isolated from various clinical specimens by various phenotypic and genotypic methods. MATERIALS AND METHODS: A total of 196 consecutive, nonduplicate strains of clinically significant E. coli isolated from various clinical specimens were included in the study. Identification and antimicrobial susceptibility testing was performed by using Vitek-2 system (Biomerieux, France). Phenotypic detection of extended spectrum beta-lactamase (ESBLs), Amp-C-β lactamase (Amp C), and carbapenemase production was done by various combination of disc diffusion methods, minimum inhibitory concentration determination by E-test, followed by polymerase-chain-reaction for the detection of β-lactamase-encoding genes. RESULTS: Overall prevalence of ESBLs, Amp C, and carbapenemase production was found to be 88.3%, 42.2%, and 65.1% by the phenotypic detection methods. Our study also revealed high resistance rates against other antibiotics such as cefepime (89%), cefotaxime (95.4%), ceftazidime (85.4%), ceftriaxone (91.8%), cefpodoxime (92.7%), aztreonam (56.3%), piperacillin/tazobactam (89.2%), and ticarcillin/clavulanic acid (76.3%). The most prevalent ESBL gene was blaTEM(67.30%), and least prevalent ESBL gene was blaVEB(2.61%). In case of Amp C, blaFOXgene (21.9%) was predominant. Among the genes encoding for carbapenemases, the most common gene was blaNDM(61.7%) followed by blaVIM(30.8%), blaKPC(10.6%), blaOXA-48 (5.3%), and blaIMP(2.1%). CONCLUSION: Our findings suggest a high rate of ESBLs, Amp C, and carbapenemase production among the E. coli isolates. A combination of both phenotypic and genotypic methods would be ideal for better characterization of resistance patterns among the E. coli isolates.

Antibiotic resistance profile and co-production of extended spectrum beta lactamases and AmpC in Acinetobacter spp. in a level 1 trauma center from India Priyam Batra, Surbhi Khurana, Aishwarya Govindaswamy, Anjana Aravinda, Vijeta Bajpai, Muruganantham Ayyanar, Purva Mathur, Rajesh Malhotra Journal of Laboratory Physicians 2019 11(2):128-132 INTRODUCTION: Acinetobacter baumannii has now emerged as a significant nosocomial pathogen in health-care setting ESP in intensive care units. Rapidly growing resistance among clinical isolates suggests a need to detect resistance mechanisms in this organism. The present study was designed to compare the various phenotypic tests available with the gold standard of genotype. METHODOLOGY: The present study was conducted to include all isolates of Acinetobacter spp. isolated over 3 years. Their resistance to various antibiotics was determined and extended spectrum beta-lactamases (ESBL) and AmpC production in the isolates showing resistance to ceftazidime/ceftriaxone/cefotaxime (CAZ/CTR/CTX) was determined. ESBL and AmpC production was confirmed using polymerase chain reaction (PCR). RESULTS: A total of 154 strains were isolated, and all the strains were tested for ESBL and AmpC detection. Of the strains tested, 15 (9.7%), 17 (11%), 24 (15.6%), 27 (17.5%), 54 (35%), 67 (43.5%), and 72 (46.7%) strains showed ESBL production using CTX/CTX-clavulanate double-disc synergy test (DDST), CTX/CTX-clavulanate E-test, CAZ/CAZ-clavulanate DDST, CAZ/CAZ-clavulanate E-test, Piperacillin/Piperacillin-tazobactam (TZ) DDST, CTR/CTR-Sulbactum DDST, and Piperacillin/Piperacillin-TZ E-test, respectively. 20 (12.9%) and 19 (12.3%) of strains were positive for AmpC production using AmpC disc test and Boronic acid inhibition test, respectively. Genotype analysis using PCR for TEM, SHV, CTXM, PER, and VEB genes was done and 69 (51.5%) strains were positive for TEM gene. DISCUSSION: ESBL detection in Acinetobacter spp. is difficult as standard guidelines for the same are not available unlike in enterobacteriaceae, and there are no zone diameter breakpoints for aztreonam and cefpodoxime. In comparison, piperacillin/piperacillin-TZ E-test had the best sensitivity and specificity for ESBL detection. CONCLUSION: Standard guidelines for ESBL detection in nil fermeners like Acinetobacter spp. must be laid down for ease of detection. Use of piperacillin/piperacillin-tazobactam E-test could be used as one of the standard methods.

Cytogenetics' impact on the prognosis of acute myeloid leukemia Monika Gupta, Manoranjan Mahapatra, Renu Saxena Journal of Laboratory Physicians 2019 11(2):133-137 INTRODUCTION: Acute myeloid leukemia (AML) is a group of disorders characterized by a spectrum of clinical, morphological, immunophenotypic, and associated chromosomal abnormalities. The identification of cytogenetic abnormalities at diagnosis is important for the evaluation of the response to therapy and the identification of an early reemergence of disease. MATERIALS AND METHODS: Newly diagnosed cases of AML were included in the study. Diagnosis of AML was based on morphology on bone marrow (BM) aspirates, cytochemistry, and flow cytometric immunophenotyping. Chromosomal analysis was performed on BM by short-term unstimulated cultures using standard cytogenetic technique. RESULTS: There were 25 males and 13 females with age group between 15 and 64 years. Cytogenetic analysis of these cases showed normal karyotype in 10 (26.3%) cases and abnormal karyotype in 28 (73.6%) cases. Cytogenetic finding in AML was divided into three groups: favorable risk, intermediate risk, and unfavorable risk. Patients in the standard risk group responded well to the chemotherapy while patients with intermediate and unfavorable karyotype had relapsed. CONCLUSION: We recommend that cytogenetics should be performed routinely in all cases of AML. A correlation must be done with various biochemical and hematological parameters, immunophenotyping, and BM morphology. Molecular studies must be integrated with cytogenetic studies for risk stratification at diagnosis to improve therapeutic strategies.

Detection of VIM and NDM-1 metallo-beta-lactamase genes in carbapenem-resistant Pseudomonas aeruginosa clinical strains in Bahrain Ronni Mol Joji, Nouf Al-Rashed, Nermin Kamal Saeed, Khalid Mubarak Bindayna Journal of Laboratory Physicians 2019 11(2):138-143 INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa has emerged as a life-threatening infectious agent worldwide. Carbapenemase genes are reported to be some of the most common mechanisms for carbapenem resistance in P. aeruginosa. No reports are available from the Kingdom of Bahrain about carbapenem resistance and the underlying cause. In this study, we determined to study the presence of the metallo-beta-lactamase (M b L) genes of VIM family and NDM-1 in carbapenem-resistant P. aeruginosa strains. METHODOLOGY: Fifty carbapenem-resistant P. aeruginosa isolates were obtained from three main hospitals of Bahrain. They were subjected to antimicrobial susceptibility testing by disc diffusion test. Subsequently, MβL was detected by imipenem-ethylene diamine tetraacetic acid (EDTA) combined disc test and conventional polymerase chain reaction. RESULTS: Among 50 P. aeruginosa strains, 40 (80%) were imipenem resistant. Among the 40 imipenem-resistant strains, 35 (87.5%) strains were positive for the imipenem-EDTA combined disc test, and 21 (52%) were carrying MβL genes. Nineteen (47.5%) strains were positive for the VIM gene; one (2.5%) strain was carrying the NDM-1 gene, while one strain was carrying both the VIM and NDM-1 genes. None of the imipenem sensitive strains carried the VIM or NDM-1 gene. CONCLUSION: This is the first study to report the presence of the VIM family gene and NDM-1 genes in imipenem-resistant P. aeruginosa isolates in the Kingdom of Bahrain. The study also confirms the multiple drug resistance by the MβL strains, attention should therefore from now on, be focused on prevention of further spread of such isolates by firm infection control measures, and to reduce its threat to public health.

Microbiological profile and antibiotic susceptibility pattern of Gram-positive isolates at a tertiary care hospital Dhruv Mamtora, Sanjith Saseedharan, Pallavi Bhalekar, Surekha Katakdhond Journal of Laboratory Physicians 2019 11(2):144-148 OBJECTIVES: Gram-positive infections such as those by Staphylococcus aureus have contributed to the disease burden by increasing the morbidity and mortality rates in India. This study aims to determine the prevalence and the antibiotic susceptibility pattern of Gram-positive pathogens at a tertiary care hospital, Mumbai, Maharashtra, India. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out from January, 2015 to December, 2017, at a tertiary care hospital in Mumbai, India. The clinical isolates were cultured, and identification was done using Vitek 2 culture system. The antibiotic susceptibility testing was done as per the Clinical Laboratory Standard Institute guidelines. RESULTS: Out of 2132 (29%) Gram-positive isolates, S. aureus (49%) was the most common encountered pathogen, followed by Enterococcus spp. (24.5%) and coagulase-negative Staphylococcus (16%). Majority of the S. aureus were observed in patients with skin and soft-tissue infections (61.2%) followed by those suffering from respiratory (41%) and bloodstream infections (35%). Among the infections caused by S. aureus, the prevalence of methicillin resistance was 30%. While the MRSA isolates showed lower sensitivity toward co-trimoxazole (39%), clindamycin (30%), erythromycin (23%), and ciprofloxacin (10%), they showed higher susceptibility to linezolid (98%), vancomycin (98%), and teicoplanin (98%). All the isolates were found to be sensitive to daptomycin and tigecycline. While vancomycin-resistant enterococci (VRE) formed 7.5%, the linezolid-resistant enterococcus species was as high as 4.1%. CONCLUSION: The study showed a high prevalence of MRSA and VRE, thereby emphasizing the increasing antimicrobial resistance pattern of the Gram-positive pathogens. Therefore, there is an urgent need for novel antimicrobial stewardship to restrict the ongoing resistance rate among the isolates.

Subclinical inflammation markers in hyperemesis gravidarum and ketonuria: A case–control study Ersin Cintesun, Serra Akar, Ayhan Gul, Feyza Nur Incesu Çintesun, Gözde Sahin, Huriye Ezveci, Fikret Akyürek, Çetin Çelik Journal of Laboratory Physicians 2019 11(2):149-153 INTRODUCTION: Subclinical inflammation markers play a significant role in hyperemesis gravidarum (HEG). Simple hematological markers such as mean platelet volume (MPV), platelet distribution width (PDW), neutrophil-to-lymphocyte ratio (NLR), red cell distribution width (RDW), plateletcrit (PCT), and platelet-to-lymphocyte ratio (PLR) have been shown to reflect inflammatory burden and disease activity in several disorders. Ketonuria is a parameter used in the diagnosis of severe HEG, but its correlation with disease severity remains controversial. The relationship of subclinical inflammation markers with degree of ketonuria has not been examined previously. In this study, we aimed to determine the diagnostic value of these subclinical inflammation markers and the relationship between these markers and grade of ketonuria in patients with HEG. MATERIALS AND METHODS: A total of 94 pregnant women with a diagnosis of HEG and 100 gestational age-matched healthy pregnant women were enrolled in this retrospective study. MPV, PDW, NLR, PLR, PCT, and ketonuria were calculated and analyzed from complete blood cell counts and total urine analyses. RESULTS: Lymphocyte count was significantly higher in the control group (P < 0,001); NLR and PLR values were significantly higher in the HEG group (P < 0,001). Among inflammation markers, RDW increased significantly (P = 0,008) with an increase in ketonuria in patients with HEG. A statistically significant correlation was found between white blood cell (WBC) and NLR, PLR, PCT. A moderate uphill relationship was observed between NLR and WBC and a weak uphill linear relationship was observed between WBC and PLR and between WBC and PCT CONCLUSIONS: PLR and NLR can be considered effective markers to aid in the diagnosis of HEG. No marker was found to correlate with ketonuria grade except RDW, although the relationship of the severity of ketonuria with severity of disease is controversial. RDW increases as the degree of ketonuria increases.

Alexandros Sfakianakis
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