Phenotypic identification, frequency distribution and antibiogram of carbapenemase producing enterobacteriaceae in clinical isolates

Objective: To differentiate between Ambler class A, B and D of carbapenemase-producing Enterobacteriaceae by using simple phenotypic methods that can be carried out in the laboratory without requiring any specialised techniques. Study Design: Cross-sectional study. Place and Duration of Study: Microbiology Department, Army Medical College, NUST, Islamabad, from November 2015 to November 2016

Methodology: Clinical specimens were subjected to identification of Enterobacteriaceae by colony morphology and API 20 E. Carbapenem resistance was detected by applying meropenem disc [10 microg] by disc diffusion method according to CLSI [Clinical Laboratory Standard Institute] criteria. Carbapenemase production among Enterobacteriaceae was detected by Modified Hodge test. Phenotypic methods, Phenylboronic acid [for Class A KPC producing Enterobacteriaceae] and EDTA inhibition tests [for Class B MBL producing Entrobacteriaceae] were applied. Presence of OXA 48 was detected by phenotypic method using imipenem 10 microg, EDTA and PBA discs. Antibiotic susceptibility was determined by KirbyBauer disc diffusion method

Results: Forty-three out of 45 [95.45%] were carbapenemase producers. Thirty-eight out of 43 [88.3%] were KPC producers and 4 out of 43 [11.62%] were MBL producers. All KPC producers were Klebsiella pneumoniae. Among five MBL producers, one each [20%] was Enterobacter cloacae and Escherichia coli and 3 [60%] were Klebsiella pneumoniae. All MBL producers were resistant to aztreonam and amoxicillin/clavulanate. Two of the KPC producing Klebsiella pneumoniae were pan-drug resistant [resistant to colistin and tigecycline]. Two were non-carbapenemase producers

Conclusion: Enterobacteriaceae strains producing KPC-type carbapenemase were the most prevalent [88.3%] in the studied healthcare setup

Diagnostic accuracy of serum IGA anti-tissue transglutaminase antibody in the diagnosis of celiac disease

Objective: To determine the diagnostic accuracy of serum IgA anti-tissue transglutaminase antibody in the diagnosis of celiac disease taking histopathology as gold standard

Study Design: Cross-sectional survey

Place and Duration of Study: This study was conducted at the department of Pediatrics, Military Hospital Rawalpindi from April 2015 to July 2016

Patients and Methods: Ninety-five consecutive children presenting with suspicion of celiac disease were included in this study after taking written informed consent. A predesigned proforma was used to record patient's demographic details. Anti-tTG level of >/=25 U/ml was taken as diagnostic of celiac disease while results of histopathology on endoscopic biopsy were taken as gold standard

Results: The mean age of the patients was 6.48 +/- 3.20 years and majority [n=53, 55.8%] of the children were aged between 5 to 10 years. The serum anti-tTG level ranged from 8.0 U/ml to 759.0 U/ml with a mean of 298.75 +/- 225.51 U/ml. Taking a cut-off value of >/=25 U/ml for anti-tTG, 81 [85.3%] children were suspected of celiac disease. Histopathology of endoscopic biopsy confirmed celiac disease in 68 [71.6%] children with 62 true positive, 19 false positive, 6 false negative and 8 true negative cases. It yielded 91.18% sensitivity, 29.63% specificity and 73.68% accuracy for anti-tTG [>/=25 U/ml] in the diagnosis of celiac disease with positive and negative predictive values of 76.54% and 57.14% respectively

Conclusion: IgA anti-tissue transglutaminase antibody [>/=25 U/ml] was found to be highly sensitive test for the detection of celiac disease in children

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 [BioMerieux]. Antibiotic susceptibility was done by using modified Kirby-Bauer 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 baumannii susceptible to minocycline and tigecycline followed by Klebsiella pneumoniae [n=71, 16%] susceptible to tigecycline and meropenem. Others were Pseudomonas aeruginosa, Escherichia coll, Coagulase Negative Staphylococcus, Staphylococcus aureus, Enterococcus spp., Streptococcus spp., Klebsiella oxytoca, Stenotrophomonas maltophilia, and Candida spp

Conclusion: Acinetobacter baumannii was 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

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 [30microg] 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 st and ardized equal to 0.5 McFarl and 's turbidity st and ard 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 St and ards 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. Key Words: Ceftaroline. Methicillin resistant Staphylococcus aureus. Linezolid.

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

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. Validation study. Department of Microbiology, Army Medical College, Rawalpindi, from January 2013 to December 2013. 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. 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. 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

Early diagnosis of Typhoid by PCR for FliC-d gene of Salmonella Typhi in patients taking antibiotics

To compare PCR [Polymerase Chain Reaction] with blood culture, typhi-dot and Widal test for the diagnosis of typhoid in patients taking antibiotics. Cross-sectional, comparative study. National University of Sciences and Technology, Islamabad, Pakistan, from April 2013 to August 2014. One hundred and five patients were included in the study. Blood was collected and inoculated into tryptone soya broth for culture. Any growth obtained was identified by API 20 E and confirmed by Salmonella anti-sera. Typhi-dot and Widal test were also done on all the samples. DNA extraction was done and PCR was carried out. Among the 105 patients, 79 [75.2%] were males and 26 [24.8%] were females, with mean age of 20.64 +/- 14 years. Typhi-dot was positive in 58 [55.2%] and negative in 47 [44.8%] patients. Blood widal test was positive in 27 [25.7%] and negative in 78 [74.3%] patients. Salmonella Typhi was positive on blood culture in only one [1%] patient. PCR for Salmonella Typhi was positive in 102 [97.1%] and negative in 3 [2.9%] patients. Positive cases detected by PCR were significantly higher as compared to Typhi-dot [p < 0.001], blood Widal test [p < 0.001] and blood culture [p < 0.001]. Positivity rate of PCR was significantly higher as compared to blood culture, Typhi-dot or Widal test for diagnosing typhoid in patients who were already taking antibiotics

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 [micro]g/mL and MIC[90] was 32 [micro]g/mL. For Acinetobacter baumannii MIC[50] and MIC[90] was 32 [micro]g/mL

Conclusion: Doripenem is no more effective against Pseudomonas aeruginosa and Acinetobacter baumannii in our setting

Carbapenem resistant acinetobacter-A major pathogen in a Tertiary Care Hospital

Objective: To determine the frequency of carbapenem resistant Acinetobacter in the Military Hospital Rawalpindi, Pakistan

Study Design: Descriptive study

Place and Duration of Study: Department of Microbiology, Army Medical College, from Oct, 2012 to Feb, 2013

Material and Methods: Clinical specimens like naso-bronchial lavage, blood, pus, sputum and catheter tips were inoculated on blood agar and Mac Conkey agar while the urine samples were inoculated on Cystine Lactose Electrolyte Deficient [CLED] agar. Acinetobacter spp. isolated, were later subjected to antimicrobial susceptibility testing using the modified Kirby-Bauer disc diffusion method on Mueller Hinton agar as per Clinical Laboratory and Standards Institute [CLSI] guidelines

Results: Out of a total of 85 Acinetobacter spp. 62 isolates were found to be carbapenem resistant. They were also found to be 100% resistant to ciprofloxacin and ceftriaxone thus becoming multidrug resistant followed by tazobactam piperacillin [98%] and trimethoprim sulphamethoxazole [92%]. Minimum resistance was seen against tigecycline being 21%

Conclusion: It is concluded from our study that there is a high frequency [72.94%] of resistance to carbapenems in Acinetobacter spp. in our setup which is associated with increased morbidity and mortality due to limited treatment options

Coagulase negative staphylococci - a fast emerging threat

To determine the frequency of isolation of coagulase-negative staphylococci and their resistance to methicillin over a period of time. 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 30microg 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. 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%]. The frequency of isolation of coagulase-negative staphylococci and its methicillin resistance among hospitalised patients is on the rise

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

To determine the frequency of Vancomycin Resistant Enterococcus [VRE] in a tertiary care hospital of Rawalpindi, Pakistan. Observational, cross-sectional study. Department of Microbiology, Army Medical College, Rawalpindi, from May 2011 to May 2012. 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. 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. The frequency of VRE was 11.57% with the highest susceptibility to linezolid and chloramphenicol

Chronic suppurative otitis media; empiric quinolones in children

The aim of the study was to determine the most prevalent organisms in chronic otitis media in children and their susceptibility to various antibimicrobials so that an appropriate empiric antibiotic can be started promptly while awaiting the results of the culture and sensitivity. Prospective observational study. The study was conducted at Combined Military Hospital Lahore from Jan 2006 to Dec 2007. A total of 156 patients less than 15 years of age who were having discharge from one or both ears for at least 1 week with tympanic membrane perforation were included in the study. A sample of the ear discharge was collected on the swab and cultured on appropriate media. The Gram positive organisms were identified on the basis of Gram staining, catalase and coagulase test, the Gram negative organisms were identified by API 20e. A total of 156 patients were included in the study of which 96 [61.5%] were males and 59 [37.8%] were females. The organisms isolated were Staphylococcus aureus in 79 [50.6%], Pseudomonas-aeruginosa in 45 [28.8%] patients and Proteus mirabilis 17 [10.9%], Escherichia coli, Acinetobacter sp and streptococi were isolated in occasional patients. Staphylococcus aureus was the commonest organism isolated followed by Pseudomonas aeruginosa and Proteus mirabilis fifty four. [34.6%] of Staphylococcus aureus were sensitive to gentamicin, fifty two [33.3%] to ciprofloxacin and 42 [26.9%]% to both ciprofloxacin and gentamicin. Among the Pseudomonas aeruginosa isolates 40 [25.6%] were sensitive to gentamicin, 27 [17.3%] to ciprofloxacin and 22 [14.1%] to both ciprofloxacin and gentamicin. Out of 156 patients 91 [58.3%] had isolates which were sensitive to ciprofloxacin and 62 [39.7%] were resistant to it. One hundred fourteen patients [73.1%] had isolates which were sensitive to gentamicin whereas 33 [21.2%] were resistant. Similarly 35 isolates [22.4%] were sensitive to sulphamethoxazole/trimethoprim whereas 66 [42.3%] were resistant. Among Proteus mirabilis isolates 12 were sensitive to gentamicin, 11 were sensitive to ciprofloxacin and 11 were sensitive to both ciprofloxacin and gentamicin. Ciprofloxacin ear drops can be recommended to be given empirically in children with chronic discharging ears. The initial therapy can be modified and appropriate therapy started if the result of the culture and sensitivity report shows the isolate to be resistant to the antibiotic started empirically

Association between periurethral colonization with uropathogens and subsequent bacteriuria in catheterized patients

To determine the number of catheterized patients who develop bacteriuria due to the presence of organisms in their periurethral flora, which may subsequently cause Urinary Tract Infection [UTI] in these patients. Non-interventional, cohort study. This study was conducted on patients of Medical Intensive Care, Surgical and Urology Units of Combined Military Hospital, Lahore, from February to April 2006. A total of 60 hospitalized patients, who were catheterized for various underlying diseases, were included in the study. Urine samples and periurethral swabs were obtained from all patients and cultured on appropriate culture media. Various tests used for the identification of microorganisms were: Gram-staining, catalase test, coagulase test and esculin hydrolysis for the identification of Gram-positive bacteria, API 20e for Gram-negative bacilli, whereas lactophenol blue preparation and germ tube test were used for the identification of yeasts. Out of 60 patients, 41[68.3%] were males and 19 [31.7%] were females. The mean duration of catheterization was 4.5 days. In males, culture of periurethral swabs revealed coagulase negative staphylococci in 11 [40.7%], Staphylococcus aureus in 10 [37%] and Enterococcus fecalis in 3 [11.1%] patients. In females, the organisms isolated were coagulase negative staphylococci in 4 [25%], Staphylococcus aureus in 4 [25%], Enterococcus fecalis in 4 [25%], Pseudomonas aeruginosa in 2 [12.5%], Escherichia coli in 3 [18.6%] and Candida albicans in 3 [18.6%] patients. Twenty nine patients developed bacteriuria [p < 0.05]. Escherichia coli was the commonest organism causing bacteriuria in either gender followed by other Gram-negative organisms. Coagulase negative Staphylococcus was isolated in the urine of one male patient only. In males, 2 [10%] out of 20 patients with Gram-negative bacteriuria were colonized by the same organism, whereas in females, 4 [44.4%] out of 9 bacteriuric patients were colonized by the same organism. Predominantly Gram-positive organisms colonized the periurethral area in males as well as in the majority of females, whereas Gram-negative bacteria were mainly responsible for the bacteriuria in both genders. There was a significant association between periurethral colonization and subsequent bacteriuria, however, prior colonization with a particular organism is not a decisive event in the initiation of bacteriuria

Dehydrating diarrhoea in children due to vibrio cholerae

A descriptive study was carried out to determine the causative organism of diarrhea, it's possible source and the clinical outcome after therapy. Fifteen children were admitted with H/ o acute onset of watery diarrhea. Stool samples were submitted to look for motility of the organisms and cultures. Stools in 8 patients revealed the growth of Vibrio cholerae 01 biotype El Tor serotype Ogawa. All the isolates were sensitive to Doxycycline, Ciprofloxacin and resistant to Cotrimoxazole, Chloramphenicol, Nalidixic acid. Three of the isolates were resistant to Ampicillin. Cholera is endemic in Pakistan with the propensity to cause epidemics especially in summer season. Adequately chlorinated water supply and improvement in public and personal hygiene can go a long way in preventing large epidemics of Cholera

Incidence and multidrug resistance in typhoid salmonellae in Bahawalpur area

Typhoid fever is a major problem affecting milions of people all over the world specially the developing countries. Resistance of the salmonella isolates to conventional antityphoid drugs has continued to rise for the last 10-15 years. This study was done to determine the prevalence and susceptibity pattern of various salmonellae isolated in the region of Bahawalpur. A total of 107 blood cultures were performed out of which 42 were found to be positive for salmonella, 39 were Salmonella typhi and 3 were Salmonella paratyphi A. Among Salmonella typhi isolates 21 [53.8%] were resistant to ampicillin, chloramphenicol and Cotrimoxazole, one isolate was resistant to ampicillin and Chloramphenicol but sensitive to Cotrimoxazole. All the isolates were sensitive to the fluoroquinolones and 3rd generation cephalosporins. Inspite of all the drugs available, the problem of spread of typhoid continues. This study shows that the prevalence of overall resistance to Salmonella typhi is high in this area also. The continuous uninhibited use of antibiotics may lead to 100% resistance to these antibiotics and acquisition of resistance by other organisms also. The medical and public health officials must be prepared to tackle the future epidemics of multidrug resistant Salmonella typhi