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Sushma Gurung,1 Sonali Kafle,2 Binod Dhungel,1 Nabaraj Adhikari,1 Upendra Thapa Shrestha,1 Bipin Adhikari,3 Megha Raj Banjara,1 Komal Raj Rijal,1 Prakash Ghimire1 1Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal; 2Alka Hospital, Lalitpur, Nepal; 3Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UKCorrespondence: Komal Raj RijalCentral Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, NepalEmail rijalkomal@gmail.comIntroduction: Resistance to carbapenem in Gram-negative bacteria is attributable to their ability to produce carbapenemase enzymes. The main objective of this study was to detect the presence of blaOXA-48 genes in carbapenem-resistant uropathogenic Escherichia coli and Klebsiella pneumoniae isolated from urine samples from patients attending Alka Hospital, Jawalakhel, Lalitpur, Nepal.Methods: A total of 1013 mid-stream urine samples were collected from patients with suspected urinary tract infection (UTI) between April and September 2018. The identified isolates underwent antibiotic susceptibility testing using the modified Kirby–Bauer disc-diffusion method. Phenotypic carbapenemase production was confirmed by the modified Hodge test, and the blaOXA-48 gene was detected using conventional polymerase chain reaction.Results: Out of 1013 urine samples, 15.2% (154/1013) had bacterial growth. Among the isolates, 91.5% (141/154) were Gram-negative bacteria, and E. coli was the most common bacterial isolate (62.9%; 97/154), followed by K. pneumoniae 15.6% (24/154). Among 121 bacterial isolates (97 E. coli isolates and 24 K. pneumoniae isolates), 70.3% (52/121) were multidrug-resistant E. coli and 29.7% (22/121) were multidrug-resistant K. pneumoniae. In addition, 9.1% (11/121) were carbapenem resistant (both imipenem and meropenem resistant). Development of multidrug resistance and development of carbapenem resistance were significantly associated (p< 0.05). Of the 11 carbapenem-resistant isolates, only seven were carbapenemase producers; of these, 28.6% (2/7) were E. coli, 72.4% (5/7) were K. pneumoniae and 42.8% (3/7) had the blaOXA-48 gene. Of the three bacterial isolates with the blaOXA-48 gene, 33.3% (1/3) were E. coli and 66.7% (2/3) were K. pneumoniae.Conclusion: One in ten isolates of E. coli and K. pneumoniae were carbapenem resistant. Among carbapenem-resistant isolates, one-third of E. coli and two-thirds of K. pneumoniae had the blaOXA-48 gene. OXA-48 serves as a potential agent to map the distribution of resistance among clinical isolates.Keywords: antimicrobial resistance, AMR, carbapenem, carbapenemase, modified Hodge test, MHT, blaOXA-48 gene

Shristi Raut,1 Komal Raj Rijal,2 Sulochana Khatiwada,1 Subash Karna,1 Rita Khanal,1 Janak Adhikari,3 Bipin Adhikari4 1Department of Microbiology, Universal College of Medical Sciences, Bhairahawa, Nepal; 2Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal; 3B.P. Koirala Institute of Health Sciences, Dharan, Nepal; 4Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UKCorrespondence: Shristi Raut Email rautshristi@gmail.comBackground: Acinetobacter baumannii is one of the major organisms causing nosocomial infections and is intrinsically resistant to multiple classes of antibiotics. The main objective of this study was to investigate the trend and characteristics of A. baumannii infections including its resistance pattern among patients attending Universal College of Medical Sciences, Teaching Hospital (UCMSTH) in Western Nepal, between January and December 2018.Patients and Methods: A total of 4862 clinical samples received at the microbiology laboratory of UCMSTH over a period of a year were analyzed. Following bacterial culture on the samples, culture-positive isolates were tested for antibiotic susceptibility using a modified Kirby–Bauer method. The demographic profile of the patient, information about samples, and the antibiotic profile of the A. baumannii isolated from different samples were recorded and analyzed.Results: A total of 1180 (24.2%; 1180/4862) organisms were isolated from the total samples. Acinetobacter baumannii (12.4%; 147/1180) was the third most common organism. Prevalence of A. baumannii was found to be high in late summer/early winter (July: 15.9%; 18/113 and December: 18.8%; 13/69). The majority 71.4% (n=105) of A. baumannii isolates were multidrug resistant (MDR). None of the isolate was pan-drug resistant. Colistin, polymyxin B, and tigecycline were 100% sensitive to A. baumannii. MDR bacteria were significantly associated with the gender of the patients [female: 51.4% (54/105) versus male: 48.6% (51/105); p=0.05], clinical specimens [swab: 40% (42/105) sputum: 21.9% (23/105) and urine: 10.5% (11/105); p=0.02] and different wards of the hospital [surgery: 30.5% (32/105); ICU: 21.9% (23/105) and medicine: 19.0% (20/105); p< 0.03].Conclusion: The high burden of MDR Acinetobacter isolates in clinical specimens shows an alarming presence of antimicrobial resistance. Two-thirds of the specimens showed MDR and were associated with demographic and clinical characteristics of the patients. In the management of infectious diseases at UCMSTH, there should be a high suspicion of Acinetobacter infection, and isolation and treatment should be carried out based on an antibiotic susceptibility test.Keywords: Acinetobacter baumannii, culture, antibiotic profile, multidrug resistance &nbsp

Upendra Thapa Shrestha,1,2,* Sabnum Shrestha,1,* Nabaraj Adhikari,1,2 Komal Raj Rijal,2 Basudha Shrestha,3 Bipin Adhikari,4 Megha Raj Banjara,2 Prakash Ghimire2 1Kantipur College of Medical Science, Tribhuvan University, Sitapaila, Kathmandu, Nepal; 2Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal; 3Department of Microbiology, Kathmandu Model Hospital, Kathmandu, Nepal; 4Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK*These authors contributed equally to this workCorrespondence: Komal Raj RijalCentral Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, NepalEmail rijalkomal@gmail.comIntroduction: Extended-spectrum β-lactamases (ESBL) among Gram-negative bacteria, predominantly Escherichia coli (E. coli), in Nepal, have been rising. The main objectives of this study were to determine the prevalence of uropathogenic E. coli, antibiotic resistance, ESBLs, ABLs (AmpC type β-lactamases), MBLs (metallo-β-lactamases) and KPCs (Klebsiella pneumoniae carbapenemases) and their correlation with plasmid profiling patterns among patients with urinary tract infections in a tertiary hospital in Kathmandu, Nepal.Methods: The mid-stream urine samples collected from patients were inoculated in cystine–lactose–electrolyte-deficient (CLED) agar plates. E. coli producing ESBLs, ABLs, MBLs/KPC were identified phenotypically using standard microbiological methods. Plasmids were extracted by alkaline lysis method from E. coli isolates and profiled using agarose gel electrophoresis.Results: Out of the total 2661 urine samples, E. coli were isolated in 64.34% (507/788), among which 170 (33.53%) were multidrug-resistant (MDR) isolates. All MDR isolates were resistant to amoxicillin and third-generation cephalosporins but were highly sensitive to imipenem (94.12%, 160/170), amikacin (92.94%, 158/170) and nitrofurantoin (86.47%, 147/170). Among 170 MDR isolates, 78.2% (133/170) were ESBLs, 46.3% (50/170) were AmpC, 11.2% (19/170) were MBL and 0.6% (1/170) were KPC producers. Coproduction of β-lactamases was detected in 24.12% (41/170) of isolates. E. coli isolates showed one plasmid (> 33.5 kb), which was present in all the isolates. Overall, 44 different plasmid profile groups were identified based on molecular weight and number of plasmids. β-Lactamase producers were relatively resistant to the higher number of antibiotics tested (≤ 10) than non-producers (≤ 8), and the number of plasmids were higher in β-lactamase producers (≤ 7) than those in non-producers (≤ 5).Conclusion: The higher prevalence of the ESBLs, AmpCs, KPCs and MBLs along with their coproduction in E. coli isolates highlights the importance of routine surveillance of ESBLs, AmpCs, KPCs and MBLs in microbiology laboratories using various phenotypic methods.Keywords: uropathogenic Escherichia coli, antibiotic resistance, ESBL, extended-spectrum β-lactamases, AmpC type β-lactamases, MBL, metallo-β-lactamases, KPC, Klebsiella pneumoniae carbapenemases

Keshav Lamichhane,1,* Nabaraj Adhikari,1,* Anup Bastola,2 Lina Devkota,2 Parmananda Bhandari,2 Binod Dhungel,1 Upendra Thapa Shrestha,1 Bipin Adhikari,3 Megha Raj Banjara,1 Komal Raj Rijal,1 Prakash Ghimire1 1Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal; 2Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, Nepal; 3Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK*These authors contributed equally to this workCorrespondence: Komal Raj RijalCentral Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, NepalEmail rijalkomal@gmail.comIntroduction: Oropharyngeal candidiasis are the commonest fungal infections among HIV-positive patients. The main objective of this study was to explore biofilm-producing Candida species causing oropharyngeal infections among HIV patients attending Sukraraj Tropical and Infectious Diseases Hospital (STIDH) in Kathmandu, Nepal.Methods: Oropharyngeal swabs were collected from the HIV-positive patients between July and December 2019. A total of 174 oropharyngeal swabs were cultured on Sabouraud Dextrose Agar (SDA). All samples were inoculated on SDA slants supplemented with chloramphenicol and underwent incubation at 37°C for 24– 48 hours. Any visible growth reported was processed for the identification of the species. Candida species were differentiated based on the growth and colour of the isolates on CHROM agar candida. Biofilm production in Candida species was determined by the microtiter plate method (MPM). Antifungal susceptibility testing was performed using the disc diffusion method.Results: Among 174 oropharyngeal samples, 23.6% (n=41/174) of them had oropharyngeal infections and 36.6% of the oropharyngeal infections (15/41) had CD4 T-lymphocytes count below 200 cells/mm3 who were also active tobacco users (p< 0.05). Among Candidial growth, 61% (25/41) were Candida albicans and 39% (16/41) were non-albicans. Of 41 Candida spp., 65% (27/41) were biofilm producers. An equal proportion of Candida albicans (4 isolates) and non-albicans (4 isolates) were strong biofilm producers. C. albicans isolates were sensitive towards clotrimazole (96%; 24/25) and fluconazole (92%; 23/25), whereas sensitivity towards ketoconazole was only 48% (12/25). Non-albicans Candida was highly sensitive to amphotericin-B (62.5%; 10/16) followed by clotrimazole (56.2%; 9/16). The biofilm-producing Candida isolates showed the highest resistivity (51.9%; 14/27) to ketoconazole and lowest (22.2%; 6/27) to clotrimazole.Conclusion: Oropharyngeal candidiasis is a common opportunistic infection among HIV-infected individuals. The majority of cases of oropharyngeal candidiasis are caused by biofilm producers Candida albicans and non-albicans Candida. Biofilm producers Candida were more resistant towards commonly used antifungal drugs.Keywords: oral candidiasis, HIV, Candida albicans, biofilm, antifungal susceptibility test

Rooku KC,1,* Sadiksha Adhikari,1,* Anup Bastola,2 Lina Devkota,2 Parmananda Bhandari,2 Prabina Ghimire,3 Bipin Adhikari,4 Komal Raj Rijal,1 Megha Raj Banjara,1 Prakash Ghimire1 1Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal; 2Sukraraj Tropical and Infectious Diseases Hospital, Kathmandu, Nepal; 3Nepal Medical College, Jorpati, Kathmandu, Nepal; 4Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK*These authors contributed equally to this workCorrespondence: Komal Raj RijalCentral Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, NepalEmail rijalkomal@gmail.comIntroduction: Opportunistic bacterial and fungal infections are the major cause of morbidity and mortality among immune suppressed HIV-positive patients. The main objective of this study was to determine bacterial and fungal organisms causing respiratory infections and their susceptibility to commonly prescribed antimicrobials among HIV patients attending a tertiary infectious disease hospital in Kathmandu.Methods: Sputum samples were collected from the HIV-positive patients attending Sukraraj Tropical and Infectious Disease Hospital (STIDH) from August 2017 to March 2018. A total of 100 sputum samples were cultured on conventional bacterial and fungal culture media. Bacterial and fungal isolates were identified based on their colony characteristics, microscopic morphology and various biochemical tests. Antibiotic susceptibility test (AST) of bacterial isolates was performed by modified Kirby Bauer disc diffusion method.Results: Out of 100 sputum samples cultured, 24% (n=24) showed bacterial growth, 42% (n=42) showed fungal growth and 10% (n=10) had both bacterial and fungal growth. Among bacteria, 91.6% (n=22) were monomicrobial and 8.4% (n=2) were polymicrobial in growth, of which, Klebsiella pneumoniae (37.5%) were predominant isolates, followed by Pseudomonas aeruginosa (29.2%), and Escherichia coli (16.7%). The antibiotic susceptibility test (AST) showed 68% (17/25) of bacterial isolates were multi-drug resistant (MDR) and among them 41.2% (7/17) were found to be extended spectrum β lactamase (ESBL) producers. Fungal growth was observed in 42% of samples (42/100). A total of six different species of Candida and four different genera of molds were identified. On species differentiation, Candida albicans (20%) were followed by Candida parapsilosis (4%), and Candida dubliniensis (3%); and various molds were Aspergillus fumigatus (4%), Aspergillus flavus (2%), and Penicillium species (5%). CD4 count was inversely associated with bacterial and fungal infections. Fifty percent of the patients with the fungal infections had a CD4 count below 200. No fungal organisms were isolated from HIV-positive patients under antifungal drug treatment.Conclusion: HIV-positive patients with a CD4 count less than 200 cells/μL are more vulnerable to opportunistic infections of bacterial and fungal origin. Early isolation, identification and appropriate treatment can reduce mortality due to co-infections. Routine screening of opportunistic pathogens is critical to contain the disease progression.Keywords: HIV, CD4, bacterial and fungal infection, opportunistic infections