Emergence of plasmid-mediated mcr genes from Gram-negative bacteria at the human-animal interface.

BACKGROUND: The global emergence of plasmid-mediated colistin resistance (Col-R) conferred by mcr genes in gram-negative rods (GNRs) has jeopardized the last treatment option for multidrug-resistant bacterial infections in humans. This study aimed to assess the emergence of mcr gene-mediated Col-R in GNRs isolated from humans and animals in Pakistan. METHODS: Animal and clinical specimens collected from various sources were prospectively analysed using standard microbiological procedures. Pathogens were identified using the API 20E and API 20NE systems (bioMerieux). Minimum inhibitory concentration (MIC) against colistin was determined using the MIC detection methods, and multiplex polymerase chain reaction (PCR) was used to amplify the mcr-1 to mcr-5 genes. RESULTS: We isolated 126 (88.1%) animal and 17 (11.9%) human Col-R phenotypes, among which there was a significant association (P < 0.01) of Escherichia coli and Proteus mirabilis with animals and of Acinetobacter baumannii with humans. Animal strains exhibited statistically significant (P < 0.05) resistance to co-trimoxazole, chloramphenicol, and moxifloxacin, and the human pathogens exhibited statistically significant (P < 0.05) antibiotic resistance to cephalosporins, carbapenems, and piperacillin-tazobactam. For Col-R strains, MIC50 values were > 6 µg/mL and > 12 µg/mL for human and animal isolates, respectively. mcr genes were detected in 110 (76.9%) bacterial strains, of which 108 (98.2%) were mcr-1 and 2 (1.8%) were mcr-2. CONCLUSIONS: The detection of a considerable number of mcr-1 and mcr-2 genes in animals is worrisome, as they are now being detected in clinical pathogens. The acquisition of mcr genes by colistin-susceptible bacteria could leave us in a post-antibiotic era.

Infection Prevention and Control Measures at the Children Hospital Lahore: A My Child Matters Collaborative Project.

PURPOSE: Infection prevention among children with cancer is a major challenge at Children Hospital Lahore (CHL), a public health care facility in Pakistan with 1,000 new pediatric cancer admissions annually. The objective has been to reduce infections through collaboration between CHL and the St Jude Children's Hospital Global Infectious Disease program via a grant by the Sanofi Espoir foundation through the My Child Matters program. The aim of the current study was to describe the effect of the collaborative improvement strategy on existing infection prevention and control (IPC) standards at CHL. MATERIALS AND METHODS: Our work was a prospective before-and-after study to improve IPC standards. We compared the WHO Hand Hygiene Self-Assessment Framework and four modules of the St Jude modified Infection Control Assessment Tool (ICAT) scores over a 3-year period. Our strategy included creating a multidisciplinary team of pediatric oncologists, infectious disease physicians, nurses, a microbiologist, and a data manager; engaging in monthly online IPC mentoring sessions with St Jude Children's Hospital Global Infectious Disease program and My Child Matters mentors; performing daily inpatient health care-associated infection surveillance rounds; and performing regular hand hygiene training and compliance audits. RESULTS: Baseline needs assessment showed health care-associated infections identified by positive blood cultures as 8.7 infections per 1,000 patient-days. Deficient hand hygiene supplies, health education measures, and bed sharing of neutropenic patients were identified as major challenges. Our hand hygiene facility level, per WHO scores, increased from Inadequate to Intermediate/Consolidation by the end of the 3-year implementation (122 v 352 WHO Hand Hygiene Self-Assessment Framework scores). The sink:bed and hand sanitizer:bed ratios improved to 1:6 and 1:1, respectively. The ICAT general infection control module increased by 40% (45 v 78 ICAT scores) and hygiene compliance improved by 20%. CONCLUSION: Implementing a collaborative improvement strategy improved IPC standards in our center, which can be easily replicated in other pediatric oncology centers in lower- and middle-income countries.

COVID-19 and comorbidities: Deleterious impact on infected patients.

The pandemic situation with the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from China has endangered human lives. Coronavirus disease 2019 (COVID-19) is presented with asymptomatic, mild, or severe pneumonia-like symptoms. COVID-19 patients with diabetes, chronic obstructive pulmonary disease (COPD), cardiovascular diseases (CVD), hypertension, malignancies, HIV, and other comorbidities could develop a life-threatening situation. SARS-CoV-2 utilizes ACE-2 receptors found at the surface of the host cells to get inside the cell. Certain comorbidities are associated with a strong ACE-2 receptor expression and higher release of proprotein convertase that enhances the viral entry into the host cells. The comorbidities lead to the COVID-19 patient into a vicious infectious circle of life and are substantially associated with significant morbidity and mortality. The comorbid individuals must adopt the vigilant preventive measure and require scrupulous management. In this review, we rigorously focused on the impact of common morbidities in COVID-19 patients and recapitulated the management strategies with recent directions. We found limited resources describing the association of comorbidities in COVID-19; however, our review delineates the broader spectrum of comorbidities with COVID-19 patients.

AmpC beta-lactamases in Klebsiella pneumoniae: An emerging threat to the paediatric patients.

OBJECTIVE: To determine the burden of AmpC beta-lactamase producing Klebsiella pneumoniae and its antimicrobial profile among paediatric patients. METHODS: This cross-sectional study was conducted at the Microbiology Department of The Children's Hospital and the Institute of Child Health in Lahore, Pakistan, from May 2014 to April 2015, in which isolates of Klebsiella pneumoniae were screened by using the cefoxitin disc. Confirmation was done by inhibitor-based method using 400 micro grams of boronic acid dispensed on the cefoxitin discs. The zone sizes of cefoxitin with and without the boronic acid were compared. The antimicrobial susceptibility testing was performed using Kirby Bauer disc diffusion method. RESULTS: Positive cultures yielded 585 Klebsiella pneumoniae out of which 220(37.6%) strains were AmpC beta-lactamase-positive on the basis of cefoxitin screening and 126(21.53%) were positive on the basis of inhibitor-based confirmatory method. Most of the infected patients 73(57.9%) were neonates. All AmpC beta-lactamase-producing strains were resistant to cephalosporins. They also exhibited resistance to ciprofloxacin 109(86.5%), amikacin 98(77.8%), levofloxacin 8(77.8%), cefoperazone-sulbactam 81(64.3%), piperacillin-tazobactam 82(65.1%), meropenem, 56(44.4%) and imipenem 32(25.4%). CONCLUSIONS: Prompt identification of AmpC beta-lactamases using inhibitor-based confirmatory test can help reduce the burden of these pathogens.

Phenotypic characterization of extended-spectrum-beta-lactamase producing E. coli from healthy individuals, patients, sewage sludge, cattle, chickens and raw meat.

OBJECTIVE: The present study aimed to determine the frequency and antimicrobial profile of ESBL-producing isolates of E. coli in different environments. METHODS: This cross-sectional study was conducted at The Children's Hospital and The Institute of Child Health, Lahore from July to December 2015. The faecal specimens from healthy individuals, patients, sewage sludge, cattle, chickens and raw meat (n = 122) were processed for microbiological analysis using MacConkey agar supplemented with cefotaxime. The identification of organisms was confirmed by API 10S and antimicrobial resistance profile was recorded by Kirby-Bauer disc diffusion method. RESULTS: On the basis of screening, 77 (63.0%) specimens were found to be positive for ESBL production. The confirmation of 74 (60.0%) ESBL producing E. coli was done using double disc synergy test (DDST). The frequency of ESBL producing E. coli was found to be 17 (57.0%) in healthy individuals, 15 (53.0%) in patients, 10 (66.0%) in cattle faeces, 5 (71.0%) in sewage sludge, 14 (70.0%) in raw meat and 13 (59.0%) in chicken faeces. All of these isolates were resistant to cephalosporins and some of these were resistant to fluoroquinolones and meropenem. None of the isolates showed resistance to cefoperazone-sulbactam, imipenem, piperacillin-tazobactam and amikacin. CONCLUSION: The prevalence of ESBL-producing E. coli was recorded in all the environments, suggesting a global expansion of these enzymes.

Antibacterial activity of penicillins alone and in combination with different agents against Staphylococcus aureus.

Difficulties in the treatment of the resistant strains of Staphylococcus aureus, which is a frequent cause of nosocomial infections in paediatric patients, has prompted this research to empower the usage of various combinations of penicillin. During the study period 17,452 clinical samples were processed for culture. The positive cultures yielded 564 strains of S. aureus. Out of these, 362 (64.2%) isolates were found to be methicillin sensitive S. aureus (MSSA) and 202 (35.8%) methicillin resistant S. aureus (MRSA). The frequency of S. aureus isolates from male patients (355; 63.1%) was found to be higher than female patients (209; 36.9%) and those from indoor wards (441; 78.2%) were more than the outdoor wards (123; 21.8%). Frequency distribution of S. aureus showed to be highest among blood 342 (60.6%) and cerebrospinal fluid 100 (17.8%) samples. The sensitivity pattern of MSSA with piperacillin-tazobactam was 344 (95.0%), ampicillin-sulbactam 340 (93.9%), co-amoxiclav 332 (91.8%) and ampicillin-oxacillin 257 (71.0%). MRSA susceptibility to piperacillin-tazobactam was 143 (71.0%), ampicillin-sulbactam 114 (56.6%), co-amoxiclav 61 (30.2%) and ampicillin-cloxacillin 18 (9%). The Cochran Mantel Haenszel test showed that the effectiveness for each penicillin was associated significantly (p<0.05) with both the MSSA and MRSA. The combinations of piperacillin-tazobactam, ampicillin-sulbactam, co-amoxiclav and ampicillin-cloxacillin exhibited higher efficacy than using them alone to combat Staphylococcal infections.

Acute bacterial meningitis in children presenting to The Children's Hospital Lahore before and after pneumococcal vaccine in Pakistan National Immunization Program; A comparison.

OBJECTIVE: To describe bacteriological profile, morbidity and mortality of acute bacterial meningitis (ABM) in children and to compare these parameters before and after the introduction of Pneumococcal vaccine in Pakistan National Immunization Program. METHODS: The present descriptive study was conducted at the Department of Paediatric Medicine of The Children's Hospital Lahore from January 2012 to December 2015. A total of 503 children one month to five years of age admitted with diagnosis of meningitis were included. Complete blood count, CSF cytology, biochemistry, culture sensitivity and blood culture sensitivity were performed. RESULTS: Frequency of meningitis decreased by 50% in 2013-2015 (199 [2012] vs 304 [2013-2015). Most children in both groups were under one year of age. More neurological complications were seen in the group 2, 20% vs 17%. CSF culture positivity decreased from 12% to 6.6%. Streptococcus pneumoniae isolation decreased from 5 (2.5%) in 2012 to 4 (1.3%) in 2013-2015. Refusal to take feed (p=0.002), impaired sensorium (p=<0.001), severe malnutrition (p=0.001), prolonged duration of symptoms (p=<0.001) and incomplete vaccination status (0.005) were associated with mortality. Mortality rate decreased from 20 (10%) in 2012 to 17 (5.6%) in 2013-2015 but more children developed neurological sequelae 2.7% versus 1%. CONCLUSION: Acute bacterial meningitis mostly affected children <1 year. Frequency of Streptococcus pneumoniae and mortality of meningitis decreased significantly after PCV but more neurological complications developed in those children who were unvaccinated in 2013-2015 compared to 2012.

Metallo-beta-lactamase producing Escherichia coli and Klebsiella pneumoniae: A rising threat for hospitalized children.

OBJECTIVE: To determine the frequency of metallo-beta-lactamase (MBL) producing E.coli and Klebsiella pneumoniae, better phenotypic techniques for MBL detection and choices of treatment available for such cases. METHODS: This study was conducted in The Children's Hospital, Lahore during March, 2013 and February, 2014. A total number of 17,651 samples including blood, urine, CSF, pus and catheter tips from suspected cases of bacterial infections were processed and test organisms were identified using standardized microbiological techniques. MBL phenotypic identification was performed by Modified Hodge Test, Double Disc Synergy Test and Combined Disc Test. RESULTS: Carbapenem resistance was observed in 134/1168 (11.47%) strains which comprised of 89 (67.4%) Klebsiella pneumoniae and 45 (32.6%) E.coli. All of these carbapenem resistant isolates were found to be carbapenemase producers (CP) by MHT test. Among these CP strains, MBL was detected in 131/134 (97.8%) isolates both by CDT and DDST including 87 (66.4%) Klebsiella pneumoniae and 44 (33.6%) E.coli. Majority of these organisms were resistant to most of the antibiotics used in the study. The isolates showed good susceptibility to colistin (90.1%), chloramphenicol (60.3%) and fosfomycin(31%). CONCLUSIONS: Isolation of such a high number of MBL producers is a serious threat for hospitalized paediatric patients.

Phenotypic Detection of Metallo-Beta-Lactamases in Carbapenem Resistant Acinetobacter baumannii Isolated from Pediatric Patients in Pakistan.

Multidrug resistant A. baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections especially in neonates and immunocompromised patients because they have least capacity to fight against infections. Carbapenems are used as last resort antibiotics for treating these infections but currently resistance against carbapenems due to MBL production is on the rise. The objective of this study was to determine the frequency of antibiotic resistance in A. baumannii and also to compare the efficacy of combined disk test and double disk synergy test for detection of metallo-beta-lactamases. A total of 112 A. baumannii were identified from various clinical samples and antibiotic susceptibility profile was determined by Kirby-Bauer Disk Diffusion method. Out of 112, 66 (58.9%) isolates were resistant to both imipenem and meropenem (OXOID). These resistant isolates were tested for carbapenemase production, and 55 (83.3%) were carbapenemase producers by Modified Hodge Test. These isolates were further tested for MBL production by combined disk test and double disk synergy test. Out of 66, 49 isolates were positive by both methods, CDT and DDST, and only one isolate was detected as negative (with kappa value = 0.038). All MBL producing strains showed remarkable resistance to cephalosporins, fluoroquinolones, aminoglycosides, and piperacillin/tazobactam (OXOID). The antibiotic resistance was very high in A. baumannii which were isolated from children in Pakistan specially attending a nephrology unit.

Detection of AmpC ß-lactamase producing bacteria isolated in neonatal sepsis.

OBJECTIVE: The objective of this study was to determine the occurrence and antimicrobial profile of AmpC ß-lactamase producing bacteria. METHODS: The study was conducted at The Children's Hospital and The Institute of Child Health Lahore, Pakistan, during September 2011 to June 2012. A total number of 1,914 blood samples of suspected neonatal septicemia were processed. Isolates were identified using Gram's staining, API 20E and API 20NE tests. Gram negative isolates were screened for AmpC ß-lactamase production against ceftazidime, cefotaxime and cefoxitin resistance and confirmed by inhibitor based method. RESULTS: Total number of 54 (8.49%) Gram positive and 582 (91.5%) Gram negative bacteria were identified. Among Gram negative isolates 141 (22%) were AmpC producers and found to be 100% resistant to co-amoxiclav, cefoxitin, ceftazidime, cefotaxime, cefuroxime, cefixime, ceftriaxone, cefpodoxime, gentamicin, amikacin and aztreonam. Less resistance was observed against cefepime (30.4%), sulbactam-cefoperazone (24.8%), piperacillin-tazobactam (10.6%), ciprofloxacin (20.5%) and meropenem (2.1%). All the isolates were found sensitive to imipenem. The patients harbored AmpC ß-lactamases were on various interventions in which intravenous line was noted among (51.1%), naso-gastric tube (37.6%), ambu bag (8.5%), endotracheal tube (3.5%), ventilator (2.1%) and surgery (0.7%). CONCLUSION: Extensive use of invasive procedures and third generation cephalosporins should be restricted to avoid the emergence of AmpC beta-lactamases in neonates.

Comparison of fluorescence microscopy and Ziehl-Neelsen technique in diagnosis of tuberculosis in paediatric patients.

Early diagnosis of tuberculosis is very important for therapeutic reasons and to control the spread of infection. The purpose of this study was to compare the efficacy of fluorescence (FL) microscopy in comparison to Ziehl-Neelsen (ZN) staining. A total of 103 samples were collected from paediatric tuberculosis (TB) suspects and processed using Petroff's method. The smears were subjected to ZN and FL staining for the detection of acid-fast bacilli (AFB). Positive smears were graded according to scale of International Union Against Tuberculosis and Lung Disease and World Health Organisation (IUATLD/WHO). Out of 103 pulmonary and extra-pulmonary samples, 7(6.8%) were positive for AFB on the ZN method, while the positivity increased to 9(8.7%) on the FL method. Two positive samples were missed on ZN staining which were found to be positive with FL microscopy; thus overall positivity increased by 2/9 (22.22%) by FL microscopy over the conventional ZN method. The difference in case detection was found to be statistically significant (p<0.00). FL technique has a better diagnostic value and is less time-consuming compared to ZN in diagnosing tuberculosis in paediatric patients.

Multidrug resistant AmpC ß-lactamase producing Escherichia coli isolated from a paediatric hospital.

UNLABELLED: Objective : The objective of the study was to observe the antimicrobial resistance of AmpC ß-lactamase producing E. coli. METHODS: Six hundred and seventy E. coli were isolated from 20,257 various pathological samples collected from The Children's Hospital and Institute of Child Health, Lahore, Pakistan. The isolates showed resistance to ceftazidime which were further examined for AmpC ß-lactamase activity by Disc Potentiation method. RESULTS: There were 670 isolates of E. coli out of which 85 (12.6%) were AmpC ß-lactamase producers. Risk factors like intravenous line (76.5%), endotracheal tube (22.4%), surgery (12.9%) and urinary catheters (7.1%) were found to be associated with infection caused by AmpC ß-lactamase producing E. coli. Antimicrobial resistance pattern revealed that AmpC producing E. coli were highly resistant to co-amoxiclav, ceftazidime, cefotaxime, cefuroxime, cefixime, ceftriaxone and cefoxitin (100% each). Least resistance was observed against sulbactam-cefoperazone (14.1%), cefepime (7.1%), piperacillin-tazobactam (5.9%) and none of the isolates were resistant to imipenem and meropenem. CONCLUSION: The minimum use of invasive devices and strict antibiotic policies can reduce the spread of AmpC ß-lactamase producing E. coli.

Phenotypic characterization of ESBL producing Enterobacter cloacae among children.

OBJECTIVE: The emergence of ESBL producing Enterobacter cloacae in clinical isolates is posing a serious threat for treating nosocomial infections. The aim of the study was to determine the frequency of extended spectrum ß-lactamase (ESBL) producing Enterobacter cloacae and to compare the phenotypic methods used for the characterization of ESBL producing strains. METHODOLOGY: This cross sectional observational study was conducted during April 2011 to March 2012 at Microbiology department of The Children's Hospital and Institute of Child Health, Lahore. A total number of 20,257 various clinical samples were analyzed during the study period. Enterobacter cloacae were identified using API 20E system and ESBL detection was carried out using double-disk synergy test (DDST) and CLSI confirmatory test. RESULTS: Enterobacter cloacae were isolated from 221 samples, out of which 33 (14.93%) were ESBL producers and 188 (85.07%) were non-ESBL producers. The gender distribution of ESBL producing Enterobacter cloacae was 21 (63.6%) in males and 12 (36.4%) in females. Highest frequency (63%) of ESBL producing Enterobacter cloacae was detected in blood samples. Comparison of DDST and CLSI confirmatory test showed that 25 (75.75%) isolates were characterized by DDST and 33 (100%) using CLSI confirmatory test. CONCLUSION: The present study shows moderately high frequency of ESBL producing Enterobacter cloacae among children. DDST was found to be less efficient in ESBL detection as compared to CLSI confirmatory test.

Detection of extended-spectrum ß-lactamases in Klebsiella pneumoniae: comparison of phenotypic characterization methods.

OBJECTIVE: Extended-spectrum ß-lactamase producing K. pneumoniae is a serious threat to the patients. This manuscript shows the comparison of phenotypic characterization methods used for ESBL K. pneumoniae and frequency distribution of these isolates in various clinical samples. METHODOLOGY: Eleven different types of pathological samples collected on various time intervals were analyzed. K. pneumoniae were identified with API 20E system (bioMerieux) and initial screening of ESBL K. pneumoniae was performed using the ceftazidime antimicrobial disc. Double-disc synergy test (DDST) and CLSI confirmatory test were compared for the phenotypic detection of ESBL K. pneumoniae. RESULTS: A total number of 214 ESBL producing K. pneumoniae were isolated from various clinical samples. Frequency distribution of ESBL producing K. pneumoniae was found to be highest among blood 117 (54.7%) and urine 46 (21.5%) samples. Data regarding the use of various interventions among these patients showed most common presence of intravenous line 209 (97.7%) and urinary catheters 46 (21.5%). Comparison of DDST and CLSI confirmatory test showed that the DDST detected 145 (67.8%) isolates while 213 (99.5%) ESBL K. pneumoniae were characterized by CLSI confirmatory test. CONCLUSION: The use of CLSI confirmatory test is very efficient in the early detection of ESBL K. pneumoniae especially when the facilities for molecular characterization are not available.

Nosocomial bloodstream infection in a tertiary care paediatric intensive care unit.

OBJECTIVE: To determine the frequency, causative organisms and susceptibility pattern of nosocomial bloodstream infections in children. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Paediatric Intensive Care Unit of the Children's Hospital, Lahore, from January to December 2004. PATIENTS AND METHODS: All children admitted to the unit during the study period were daily evaluated for features suggestive of nosocomial infection. In addition to other investigations, blood cultures were done in all suspected cases for the confirmation of nosocomial bloodstream infection (BSI). Nosocomial infection was defined according to the criteria set by Centre for Disease Control and Prevention. Demographic, microbiological and other variables were carefully studied to analyze frequency, incidence rate, spectrum of isolates and susceptibility pattern. Children with and without nosocomial BSI were compared with regard to age, duration of stay in hospital, need and duration of ventilation and the outcome. RESULTS: Of the total 406 admissions, 134 children were suspected to have nosocomial infection on at least 214 occasions (episodes). Blood cultures yielded growth of pathological organisms in 62 of these episodes, giving the frequency of nosocomial BSI as 15.2 per 100 admissions (62/406 episodes). Children with nosocomial bloodstream infection were found to have younger mean age (2.1 vs. 4.1 years), longer average duration of stay (13.1 vs. 6.6 days), more frequent need for ventilation (64% vs. 34%) and longer duration of ventilation (9.7 vs. 4.8 days). Majority of isolates (77%) were gram-negative bacteria; Klebsiella being the most common isolate (n= 23). Aztreonam, Ceftiazidime, Ceforuxime and Ciprofloxacin showed high resistance pattern (33-50%). Isolates showed good sensitivity to Vancomycin (100%), Imipenem (80%), Meropenem (100%) and Co-amoxiclav (88%). CONCLUSION: The frequency of nosocomial BSI in the observed setting was quite high, having marked impact on the duration of stay and outcome. Emergence of resistant pathogens is alarming.