Burden of human metapneumovirus infections among children with acute respiratory tract infections attending a Tertiary Care Hospital, Kathmandu.

BACKGROUND: Acute respiratory infections (ARIs) are one of the most common causes of mortality and morbidity worldwide. Every year millions of children suffer from viral respiratory tract infections (RTIs) ranging from mild to severe illnesses. Human Metapneumovirus (HMPV) is among the most frequent viruses responsible for RTIs. However, HMPV infections and their severity among children have not been explored yet in Nepal. PURPOSE: Therefore, the study was focused on HMPV infections and other potential viral etiologies or co-infections using multiplex PCR among children attending Kanti Children's Hospital and assessed the clinical characteristics of the infections as well as found the co-infections. A hospital-based cross-sectional study was designed and a convenience sampling method was used to enroll children of less than 15 years with flu-like symptoms from both outpatients and inpatients departments over three months of the study period. RESULTS: HMPV infection (13.3%) was the most predominant infection among the different viral infections in children with ARIs in Kanti Children's Hospital. The HMPV was more prevalent in the age group less than three years (21.8%). Cough and fever were the most common clinical features present in all children infected with HMPV followed by rhinorrhea, sore throat, and wheezing. HMPV-positive children were diagnosed with pneumonia (42.9%), bronchiolitis (28.5%), upper respiratory tract infections (14.3%), and asthma (14.3%). The prevalence of HMPV was high in late winter (14.3%) followed by early spring (13.5%). CONCLUSIONS: This study provides the baseline information on HMPV and associated co-infection with other respiratory viruses for the differential diagnosis based on molecular methods and also the comparison of clinical presentations among the different respiratory syndromes.

gyrA ser83 mutation among fluoroquinolone-resistant Salmonella enterica serovars from enteric fever patients in tertiary care hospital, Kathmandu.

BACKGROUND: The management of enteric fever through antibiotics is difficult these days due to the emerging resistance of Salmonella to various antimicrobial agents. The development of antimicrobial resistance is associated with multiple factors including mutations in the specific genes. To know the current status of mutation-mediated fluoroquinolone-resistance among Salmonella enterica serovars; Typhi, Paratyphi A, B and C, this study was focused on detecting gyrA ser83 mutation by restriction digestion analysis of gyrA gene using HinfI endonuclease. RESULTS: A total of 948 blood samples were processed for isolation of Salmonella spp. and 3.4% of them were found to be positive for Salmonella growth. Out of the 32 Salmonella isolates, 2.2% were S. Typhi and 1.2% were S. Paratyphi A. More interestingly, we observed less than 5% of isolates were resistant to first-line drugs including chloramphenicol, cotrimoxazole and ampicillin. More than 80% of isolates were resistant to fluoroquinolones accounting for 84.4% to levofloxacin followed by 87.5% to ofloxacin and 100% to ciprofloxacin by disc diffusion methods. However, the minimum inhibitory concentration method using agar dilution showed only 50% of isolates were resistant to ciprofloxacin. A total of 3.1% of isolates were multidrug-resistant. Similarly, 90.6% of the Salmonella isolates showed gyrA ser83 mutation with resistance to nalidixic acid. CONCLUSIONS: The increased resistance to fluoroquinolones and nalidixic acid in Salmonella isolates in our study suggests the use of alternative drugs as empirical treatment. Rather, the treatment should focus on prescribing first-line antibiotics since we observed less than 5% of Salmonella isolates were resistant to these drugs.

Multidrug-resistant bacteria with ESBL genes: a growing threat among people living with HIV/AIDS in Nepal.

BACKGROUND: Bacterial opportunistic infections are common in people living with HIV/AIDS (PLHA). Besides HIV-TB co-infection, lower respiratory tract infections (LRTIs) due to multidrug-resistant (MDR) bacteria cause significant morbidity and mortality among PLHA. This study identified bacterial co-infection of the lower respiratory tract and detected plasmid-mediated blaTEM and blaCTX-M genes among Extended-Spectrum ß-Lactamase (ESBL) producing isolates from sputum samples in PLHA. METHODS: A total of 263 PLHA with LRTIs were enrolled in this study, out of which, 50 were smokers, 70 had previous pulmonary tuberculosis, and 21 had CD4 count < 200 cells/µl. Sputum samples collected from PLHA were processed with standard microbiological methods to identify the possible bacterial pathogens. The identified bacterial isolates were assessed for antibiotic susceptibility pattern using modified Kirby Bauer disk diffusion method following Clinical Laboratory Standard Institute (CLSI) guidelines. In addition, plasmid DNA was extracted from MDR and ESBL producers for screening of ESBL genes; blaCTX-M and blaTEM by conventional PCR method using specific primers. RESULTS: Of 263 sputum samples, 67 (25.48%) showed bacterial growth. Among different bacterial pathogens, Klebsiella pneumoniae, (17; 25.37%) was the most predominant, followed by Haemophillus influenzae, (14; 20.90%) and Escherichia coli, (12; 17.91%). A higher infection rate (4/8; 50%) was observed among people aged 61-70 years, whereas no infection was observed below 20 years. About 30.0% (15/50) of smokers, 32.86% (23/70) cases with previous pulmonary tuberculosis, and 52.38% (11/21) with CD4 count < 200 cells/µl had bacterial LRTIs. Among 53 bacterial isolates excluding H. influenzae, 28 isolates were MDR and 23 were ESBL producers. All ESBL producers were sensitive to colistin and polymyxin B. Among ESBL producers, 47.83% (11/23) possessed blaCTX-M, 8.6% (2/23) were positive for blaTEM gene, and 43.48% (10/23) possessed both ESBL genes. CONCLUSION: The increasing rate of MDR bacterial infections, mainly ESBL producers of LRTIs causes difficulty in disease management, leading to high morbidity and mortality of PLHA. Hence, it is crucial to know the antibiogram pattern of the isolates to recommend effective antimicrobial therapy to treat LRTIs in PLHA.

Detection of NDM-1 and VIM Genes in Carbapenem-Resistant Klebsiella pneumoniae Isolates from a Tertiary Health-Care Center in Kathmandu, Nepal.

BACKGROUND: Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenems are used as the last resort for the treatment of multidrug resistant Gram-negative bacterial infections. In recent years, resistance to these lifesaving drugs has been increasingly reported due to the production of carbapenemase. The main objective of this study was to detect the carbapenem-resistant genes blaNDM-1 and blaVIM in K. pneumoniae isolated from different clinical specimens. METHODS: A total of 585 clinical specimens (urine, pus, sputum, blood, catheter tips, and others) from human subjects attended at Annapurna Neurological Institute and Allied Sciences, Kathmandu were obtained in the period between July 2018 and January 2019. The specimens were isolated and identified for K. pneumoniae. All K. pneumoniae isolates were processed for antimicrobial susceptibility testing (AST) using the disk diffusion method. The isolates were further phenotypically confirmed for carbapenemase production by the modified Hodge test (MHT) using imipenem (10 µg) and meropenem (10 µg) discs. Thus, confirmed carbapenemase-producing isolates were further screened for the production of blaNDM-1 and blaVIM using conventional polymerase chain reaction (PCR). RESULTS: Among the clinical isolates tested, culture positivity was 38.29% (224/585), and the prevalence of K. pneumoniae was 25.89% (58/224). On AST, K. pneumoniae exhibited resistance toward carbapenems including ertapenem, meropenem, and imipenem, while it showed the highest susceptibility rate against to tigecycline (93.1%; 54/58). Overall, AST detected 60.34% (35/58) carbapenem-resistant isolates, while the MHT phenotypically confirmed 51.72% (30/58) isolates as carbapenemase-producers and 48.28% (28/58) as carbapenemase nonproducers. On subsequent screening for resistant genes among carbapenemase-producers by PCR assay, 80% (24/30) and 3.33% (1/30) isolates were found to be positive for blaNDM-1 and blaVIM, respectively. In the same assay among 28 carbapenem nonproducing isolates, 9 (32.14%) isolates were positive for blaNDM-1 gene while none of them were tested positive for blaVIM gene. CONCLUSIONS: Molecular detection of resistant genes provides greater specificity and sensitivity than those with conventional techniques, thus aiding in accurate identification of antimicrobial resistance and clinical management of the disease.

Prevalence of HIV, syphilis, and assessment of the social and structural determinants of sexual risk behaviour and health service utilisation among MSM and transgender women in Terai highway districts of Nepal: findings based on an integrated biological and behavioural surveillance survey using respondent driven sampling.

BACKGROUND: Men who have sex with men (MSM) and transgender people are disproportionately affected by HIV and sexually transmitted infections. MSM and transgender people in Nepal experience considerable discrimination and marginalisation, they are subject to abuse from legal authorities and suffer from mental health issues. These social and structural factors can lead to increased sexual risk behaviour, barriers to accessing health care and result in adverse health outcomes. This study aims to assess the prevalence of HIV and syphilis, and how individual and socio-structural factors influence sexual risk behaviour and health care service uptake, among MSM and transgender women in the Terai highway districts of Nepal. METHODS: A cross-sectional survey was conducted in June 2016 in eight Terai highway districts of Nepal, recruiting 340 MSM and transgender women through respondent driven sampling. The primary outcome variables were HIV and syphilis prevalence. The secondary outcome variables were sexual risk behaviour and health care service uptake. Logistic regression models were used to assess the individual and socio-structural determinants of sexual risk behaviour and health care service uptake. RESULTS: The prevalence of HIV among MSM was 5%, whereas it was 13% in transgender women. The prevalence of active syphilis was 4% in MSM and 11% among transgender women. Among transgender women, 76% were involved in sex work, and 51% had experienced discrimination in one or more settings. In multivariable analysis, having visited an outreach centre was positively associated with condom use in the last sexual encounter among both MSM (AOR: 5.37, 95% CI: 2.42-11.94, p < 0.001) and transgender women (AOR: 2.37, 95% CI: 1.12-5.02, p = 0.025). Moreover, transgender women who reported being open towards family about sexual identity/behaviour were 2.4 more likely to have visited an outreach centre (AOR: 2.40, 95% CI: 1.04-5.57, p = 0.041). CONCLUSIONS: The high prevalence of HIV and syphilis, as well as indicators of marginalisation and discrimination among transgender women, highlights the increased burden transgender women in Nepal are facing and the need for tailored interventions. Moreover, since health care service uptake is an important factor in determining sexual risk behaviour among MSM and transgender women in Nepal, outreach services should be scaled up.

Designing malaria surveillance strategies for mobile and migrant populations in Nepal: a mixed-methods study.

BACKGROUND: As malaria cases have declined throughout Nepal, imported cases comprise an increasing share of the remaining malaria caseload, yet how to effectively target mobile and migrant populations (MMPs) at greatest risk is not well understood. This formative research aimed to confirm the link between imported and indigenous cases, characterize high-risk MMPs, and identify opportunities to adapt surveillance and intervention strategies to them. METHODS: The study used a mixed-methods approach in three districts in far and mid-western Nepal, including (i) a retrospective analysis of passive surveillance data, (ii) a quantitative health facility-based survey of imported cases and their MMP social contacts recruited by peer-referral, and (iii) focus group (FG) discussions and key informant interviews (KIIs) with a subset of survey participants. Retrospective case data were summarised and the association between monthly indigenous case counts and importation rates in the previous month was investigated using Bayesian spatio-temporal regression models. Quantitative data from structured interviews were summarised to develop profiles of imported cases and MMP contacts, including travel characteristics and malaria knowledge, attitudes and practice. Descriptive statistics of the size of cases' MMP social networks are presented as a measure of potential programme reach. To explore opportunities and barriers for targeted malaria surveillance, data from FGs and KIIs were formally analysed using a thematic content analysis approach. RESULTS: More than half (54.1%) of malaria cases between 2013 and 2016 were classified as imported and there was a positive association between monthly indigenous cases (incidence rate ratio (IRR) 1.02 95% CI 1.01-1.03) and the previous month's case importation rate. High-risk MMPs were identified as predominantly adult male labourers, who travel to malaria endemic areas of India, often lack a basic understanding of malaria transmission and prevention, rarely use ITNs while travelling and tend not to seek treatment when ill or prefer informal private providers. Important obstacles were identified to accessing Nepali MMPs at border crossings and at workplaces within India. However, strong social connectivity during travel and while in India, as well as return to Nepal for large seasonal festivals, provide opportunities for peer-referral-based and venue-based surveillance and intervention approaches, respectively. CONCLUSIONS: Population mobility and imported malaria cases from India may help to drive local transmission in border areas of far and mid-western Nepal. Enhanced surveillance targeting high-risk MMP subgroups would improve early malaria diagnosis and treatment, as well as provide a platform for education and intervention campaigns. A combination of community-based approaches is likely necessary to achieve malaria elimination in Nepal.

Multidrug resistant Vibrio cholerae O1 from clinical and environmental samples in Kathmandu city.

BACKGROUND: Cholera, an infectious disease caused by Vibrio cholerae, is a major public health problem and is a particularly burden in developing countries including Nepal. Although the recent worldwide outbreaks of cholera have been due to V. cholerae El Tor, the classical biotypes are still predominant in Nepal. Serogroup O1 of the V. cholerae classical biotype was the primary cause of a cholera outbreak in Kathmandu in 2012. Thus, this study was designed to know serotypes and biotypes of V. cholerae strains causing recent outbreak with reference to drug resistant patterns. Moreover, we also report the toxigenic strains of V. cholerae from both environmental and clinical specimens by detecting the ctx gene. METHODS: Twenty four V. cholerae (n = 22 from stool samples and n = 2 from water samples) isolated in this study were subjected to Serotyping and biotyping following the standard protocols as described previously. All of the isolates were tested for antimicrobial susceptibility patterns using the modified Kirby-Bauer disk diffusion method as recommended by CLSI guidelines. The screening of the ctx genes (ctxA2-B gene) were performed by PCR method using a pair of primers; C2F (5'-AGGTGTAAAATTCCTTGACGA-3') and C2R (5'-TCCTCAGGGTATCCTTCATC-3') to identify the toxigenic strains of V. cholerae. RESULTS: Among twenty four V. cholerae isolates, 91.7% were clinical and 8.3% were from water samples. Higher rate of V. cholerae infection was found among adults of aged group 20-30 years. All isolates were serogroups O1 of the V. cholerae classical biotype and sub serotype, Ogawa. All isolates were resistant to ampicillin, nalidixic acid and cotrimoxazole. 90.9% were resistant to erythromycin however, tetracycline was found to be the most effective drug for the isolates. All isolates were multidrug resistant (MDR) and possessed a ctx gene of approximately 400 base pairs indicating the toxigenic strains. CONCLUSION: Hundred percent strains of V. cholerae were MDR possessing a ctx gene. It suggests that toxigenic strains be identified and proper antibiotic susceptibility testing be conducted. This will allow effective empirical therapy to be used to treat and control cholera.

Genotypically Confirmed Vancomycin-Resistant Staphylococcus aureus With vanB Gene Among Clinical Isolates in Kathmandu.

Purpose: Methicillin-resistant Staphylococcus aureus, a common bacterial pathogen causes various infections. The acquisition of various antimicrobial-resistant genes in S. aureus has led to the transformation of this bacterium into a superbug. Vancomycin resistance among MRSA isolates is an emerging threat in empirical therapy of various infections. The study was hence aimed to find out the susceptibility status of S. aureus isolates toward vancomycin and detect mecA, vanA, and vanB genes among the isolates. Methods: A total of 1245 clinical samples from the participants attending a tertiary care hospital in Kathmandu were processed. S. aureus isolated from the samples were subjected to antibiotic susceptibility patterns using the modified Kirby-Bauer disk diffusion method. Agar dilution method was used to determine the minimum inhibitory concentration of vancomycin. The antibiotic-resistant genes such as mecA, vanA, and vanB among S. aureus isolates were screened by a conventional polymerase chain reaction. Results: Of 1245 samples, 80 S. aureus were identified. Out of which, 47.5% (38/80) were phenotypically confirmed MRSA isolates. mecA gene was detected in 84.2% (32/38) of MRSA isolates. 10.5% (4/38) were confirmed as vancomycin-intermediate S. aureus (VISA) by MIC determination. None of the isolates was positive for the vanA gene; however, 2 isolates were found to possess the vanB gene. The 2 isolates have vancomycin MIC breakpoints of 4 to 8 µg/mL. Conclusion: There might be a spreading of vancomycin resistance among S. aureus, creating serious public health problems. Therefore, measures to limit vancomycin resistance should be considered in healthcare facilities as immediately as possible.

Use of antimicrobials during the COVID-19 pandemic: A qualitative study among stakeholders in Nepal.

The COVID-19 pandemic was a major public health threat and the pressure to find curative therapies was tremendous. Particularly in the early critical phase of the pandemic, a lot of empirical treatments, including antimicrobials, were recommended. Drawing on interviews with patients, clinicians and drug dispensers, this article explores the use of antimicrobials for the management of COVID-19 in Nepal. A total of 30 stakeholders (10 clinicians, 10 dispensers and 10 COVID-19 patients) were identified purposively and were approached for an interview. Clinicians and dispensers in three tertiary hospitals in Kathmandu assisted in the recruitment of COVID-19 patients who were undergoing follow-up at an out-patient department. Interviews were audio recorded, translated and transcribed into English, and were analyzed thematically. The respondents report that over-the-counter (OTC) use of antibiotics was widespread during the COVID-19 pandemic in Nepal. This was mostly rooted in patients' attempts to mitigate the potential severity of respiratory illnesses, and the fear of the stigmatization and social isolation linked to being identified as a COVID-19 patient. Patients who visited drug shops and physicians reportedly requested specific medicines including antibiotics. Clinicians reported uncertainty when treating COVID-19 cases that added pressure to prescribe antimicrobials. Respondents from all stakeholder groups recognized the dangers of excessive use of antimicrobials, with some referring to the development of resistance. The COVID-19 pandemic added pressure to prescribe, dispense and overuse antimicrobials, accentuating the pre-existing OTC use of antimicrobials. Infectious disease outbreaks and epidemics warrant special caution regarding the use of antimicrobials and specific policy response.

Highly resistant Salmonella enterica serovar Typhi with a novel gyrA mutation raises questions about the long-term efficacy of older fluoroquinolones for treating typhoid fever.

As a consequence of multidrug resistance, clinicians are highly dependent on fluoroquinolones for treating the serious systemic infection typhoid fever. While reduced susceptibility to fluoroquinolones, which lessens clinical efficacy, is becoming ubiquitous, comprehensive resistance is exceptional. Here we report ofloxacin treatment failure in typhoidal patient infected with a novel, highly fluoroquinolone-resistant isolate of Salmonella enterica serovar Typhi. The isolation of this organism has serious implications for the long-term efficacy of ciprofloxacin and ofloxacin for typhoid treatment.

Higher Rate of Extreme Drug-resistant Klebsiella pneumoniae Infections among Cardiac Patients.

BACKGROUND: The accelerating rate of carbapenems resistance in Klebseilla pneumoniae isolates has put the treatment option worrisome. The effective strategy to ameliorate this alarming situation is possible through enhancing the combination therapy and appropriate laboratory diagnosis. Hence, the study was focused on identifying carbapenemase-producing K. pneumoniae and their antibiogram pattern. METHODS: A total of 944 clinical samples from patients attending Sahid Gangalal National Heart Center were processed from September 2019 to March 2020 to identify the possible bacterial pathogens following the standard microbiological procedures. K. pneumonaie isolates were further subjected to antibiotic susceptibility testing by the modified Kirby Bauer disc diffusion technique. Phenotypic confirmation of carbapenemase production was done by the modified carbapenemase inactivation method. The minimum inhibitory concentration of colistin was determined by the broth microdilution method. RESULTS: Of the total 944 samples, 15.47% (146) samples showed bacterial growth, among which 23.97% (35) were K. pneumoniae. Out of 35 K. pneumoniae isolates, 45.71% (16) were multidrug-resistant followed by 42.86% (15) extensively drug-resistant. Fourteen isolates of K. pneumoniae were carbapenemase producers among which 20% (7) were serine carbapenemase while 20% (7) showed metallo-?-lactamase production. All the carbapenemase-producing K. pneumoniae were susceptible to colistin with <0.125µg/ml. Carbapenemase activity showed statistically significant with multidrug resistance (p=<0.05). CONCLUSIONS: An increasing resistance to the carbapenem drugs showed a great problem in the management of K. pneumoniae infections among immunocompromised patients especially cardiac patients however, colistin can be still an ultimate choice of drug for disease management.

Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence, Antimicrobial Susceptibility Pattern, and Detection of mecA Gene among Cardiac Patients from a Tertiary Care Heart Center in Kathmandu, Nepal.

BACKGROUND: Methicillin Resistant Staphylococcus aureus (MRSA) is a significant human pathogen associated with nosocomial infections. mecA in the S. aureus is a marker of MRSA. The main objective of this study was to detect mecA and vanA genes conferring resistance in S. aureus among cardiac patients attending Sahid Gangalal National Heart Centre (SGNHC), Kathmandu, Nepal between May and November 2019. METHODS: A total of 524 clinical samples (blood, urine, sputum) were collected and processed. Bacterial isolates were tested for antimicrobial susceptibility test (AST) and screening for MRSA was carried out by cefoxitin disc diffusion method. Minimum inhibitory concentration (MIC) of vancomycin for MRSA was established by agar dilution method and chromosomal DNA was extracted and used in polymerase chain reaction targeting the mecA and vanA genes. RESULTS: Out of 524 specimens, 27.5% (144/524) showed bacterial growth. Among 144 culture positive isolates, S. aureus (27.1%; 39/144) was the predominant bacteria. Among 39 S. aureus isolates, all isolates were found resistant to penicillin followed by erythromycin (94.9%; 37/39), gentamicin (94.9%; 37/39) and cefoxitin (87.2%; 34/39). Out of 39 S. aureus, 87.2% (34/39) were MRSA. Among 34 MRSA, 8.8% (3/34) were vancomycin intermediate S. aureus (VISA). None of the MRSA was resistant to vancomycin. All of the 3 VISA isolates were obtained from inpatients. Of 39 S. aureus, 82.1% (32/39) harbored mecA gene. Similarly, the entire VISA isolates and 94.1% (32/34) of the MRSA isolates were tested positive for mecA gene. CONCLUSIONS: High prevalence of MRSA among the cardiac patients indicates the increasing burden of drug resistance among bacterial isolates. Since infection control is the crucial step in coping with the burgeoning antimicrobial resistance in the country, augmentation of diagnostic facilities with routine monitoring of drug resistance is recommended.

Antibiotic Susceptibility, Biofilm Production, and Detection of mecA Gene among Staphylococcus aureus Isolates from Different Clinical Specimens.

The increasing incidence of methicillin-resistant and biofilm-forming S. aureus isolates in hospital settings is a gruesome concern today. The main objectives of this study were to determine the burden of S. aureus in clinical samples, assess their antibiotic susceptibility pattern and detect biofilm formation and mecA gene in them. A total of 1968 different clinical specimens were processed to isolate S. aureus following standard microbiological procedures. Antibiotic susceptibility test of the isolates was performed by Kirby-Bauer disc-diffusion method following CLSI guidelines. Biofilm was detected through tissue culture plate method. Methicillin-resistant S. aureus (MRSA) isolates were screened using cefoxitin (30 µg) discs and mecA gene was amplified by conventional polymerase chain reaction (PCR). Of 177 bacterial growth, the prevalence of S. aureus was 15.3% (n = 27). MRSA were 55.6% (15/27) and 44% (12/27) exhibited multidrug resistance (MDR). There was no significant association between methicillin resistance and MDR (p > 0.05). Both MRSA and MSSA were least sensitive to penicillin (100%, 75%) followed by erythromycin (86.6%, 66.6%). Most of the MRSA (93.4%) were susceptible to tetracycline. All S. aureus isolates were biofilm producers-19 (70%) were weak and only one (4%) was a strong biofilm producer. The strong biofilm-producing MSSA was resistant to most of the antibiotics except cefoxitin and clindamycin. None of the MSSA possessed mecA gene while 8 (53.3%) MRSA had it. More than half of S. aureus isolated were MRSA. High incidence of multidrug resistance along with capacity to form biofilm among clinical isolates of S.aureus is a matter of apprehension and prompt adoption of biosafety measures is suggested to curb their dissemination in the hospital environments.

Higher Prevalence of Extended Spectrum ß-Lactamase Producing Uropathogenic Escherichia coli Among Patients with Diabetes from a Tertiary Care Hospital of Kathmandu, Nepal.

This study aimed to determine the occurrence of antibiotic resistance genes for ß-lactamases; blaTEM and blaCTX-M in uropathogenic Escherichia coli isolates from urinary tract infection (UTI) suspected diabetic and nondiabetic patients. A hospital-based cross-sectional study was conducted in Kathmandu Model Hospital, Kathmandu, in association with the Department of Microbiology, GoldenGate International College, Kathmandu, Nepal, from June to December 2018. A total of 1,267 nonduplicate midstream urine specimens were obtained and processed immediately for isolation of uropathogens. The isolates were subjected to antibiotic susceptibility testing and extended spectrum ß-lactamase (ESBL) confirmation. In addition, blaTEM and blaCTX-M genes were detected using specific primers. The overall prevalence of UTI was 17.2% (218/1,267), of which patients with diabetes were significantly more infected; 32.3% (31/96) as compared with nonpatients with diabetes, 15.9% (187/1,171). A total of 221 bacterial isolates were obtained from 218 culture-positive specimens in which E. coli was the most predominant; 67.9% (150/221). Forty-four percent (66/150) of the total E. coli was multidrug resistant and 37.3% (56/150) were ESBL producers. Among 56 isolates, 92.3% (12/13) were from patients with diabetes, and 83.0% (44/53) were from nondiabetics. Furthermore, 84.9% of the screened ESBL producers were confirmed to possess either single or both of blaTEM and blaCTX-M genes. The blaTEM and blaCTX-M genes were detected in 53.6% and 87.5% of the phenotypically ESBL confirmed E. coli, respectively. Higher rates of ESBL producing uropathogenic E. coli are associated among patients with diabetes causing an alarming situation for disease management. However, second-line drugs with broad antimicrobial properties are still found to be effective drugs for multidrug resistance strains.

Molecular Confirmation of Vancomycin-Resistant Staphylococcus aureus with vanA Gene from a Hospital in Kathmandu.

Staphylococcus aureus, a commensal on the skin and in the nasal cavity of humans, is one of the most serious cases of nosocomial infections. Moreover, methicillin-resistant S. aureus (MRSA) is a leading cause of morbidity and mortality worldwide. For the treatment of MRSA infections, vancomycin is considered as a drug of choice. However, the emergence of vancomycin resistance among MRSA isolates has been perceived as a formidable threat in therapeutic management. To estimate the rate of vancomycin-resistant S. aureus (VRSA) and to detect the vancomycin-resistant genes, namely, vanA and vanB, among the isolates, a hospital-based cross-sectional study was conducted from July to December 2018 in Annapurna Neurological Institute and Allied Science, Kathmandu, Nepal. S. aureus was isolated and identified from different clinical samples and processed for antibiotic susceptibility testing by the modified Kirby-Bauer disc diffusion method. The screening of MRSA was performed as per Clinical and Laboratory Standard Institute (CLSI) guidelines. VRSA was confirmed by the minimum inhibitory concentration (MIC) method by employing E-test strips. All the phenotypically confirmed VRSA were further processed to detect the vanA and vanB gene by using the conventional polymerase chain reaction (PCR) method. A total of 74 (20.3%) S. aureus were isolated, and the highest percentage of S. aureus was from the wound samples (36.5%). Of 74 S. aureus isolates, the highest number (89.2%) was resistant to penicillin, and on the other hand, linezolid was found to be an effective drug. Likewise, 45 (60.81%) were found to be MRSA, five (11.11%) were VRSA, and 93.2% of S. aureus isolates showed an MAR index greater than 0.2. Two VRSA isolates (40%) were positive for the vanA gene. The higher prevalence of MRSA and significant rate of VRSA in this study recommend routine surveillance for the MRSA and VRSA in hospital settings before empirical therapy.

Antimicrobial Susceptibility Pattern of Salmonella spp. Isolated from Enteric Fever Patients in Nepal.

INTRODUCTION: Enteric fever, a systemic infection caused by Salmonella enterica Typhi and S. enterica Paratyphi is one of the most common infections in developing countries such as Nepal. Aside from irrational practices of antibiotic use, mutations in chromosomal genes encoding DNA gyrase and Topoisomerase IV and by plasmid mediated quinolone resistant (PMQR) genes are suggested mechanisms for the development of resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. Regardless of high endemicity of enteric fever in Nepal, there is paucity of studies on prevalence and drug-resistance of the pathogen. Therefore, this study aimed to assess the antibiotic susceptibility pattern of Salmonella isolates and determine the minimum inhibitory concentration of ciprofloxacin. METHODS: A total of 1298 blood samples were obtained from patients with suspected enteric fever, attending Sukraraj Tropical and Infectious Disease Hospital (STIDH) during March-August, 2019. Blood samples were inoculated immediately into BACTEC culture bottles and further processed for isolation and identification of Salmonella Typhi and S. Paratyphi. Axenic cultures of the isolates were further subjected to antimicrobial susceptibility testing (AST) by using the modified Kirby-Bauer disc diffusion method based on the guidelines by CLSI. The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by agar-dilution method. RESULTS: Out of 1298 blood cultures, 40 (3.1%) were positive for Salmonella spp. among which 29 (72.5%) isolates were S. Typhi and 11 (27.5%) isolates were S. Paratyphi A. In AST, 12.5% (5/40), 15% (6/40) and 20% (8/40) of the Salmonella isolates were susceptible to nalidixic acid, ofloxacin and levofloxacin, respectively, whereas none of the isolates were susceptible to ciprofloxacin. The MIC value for ciprofloxacin ranged from 0.06-16 µg/mL in which, respectively, 5% (2/40) and 52.5% (21/40) of the isolates were susceptible and resistant to ciprofloxacin. None of the isolates showed multidrug-resistance (MDR) in this study. CONCLUSION: This study showed high prevalence of quinolone-resistant Salmonella spp., while there was marked re-emergence of susceptibilities to traditional first option drugs. Hence, conventional first-line-drugs and third-generation cephalosporins may find potential usage as the empirical drugs for enteric fever. Although our reporting was free of MDR strains, extensive surveillance, augmentation of diagnostic facilities and treatment protocol aided by AST report are recommended for addressing the escalating drug-resistance in the country.

Use of antimicrobials and antimicrobial resistance in Nepal: a nationwide survey.

Nepal suffers from high burden of antimicrobial resistance (AMR) due to inappropriate use of antibiotics. The main objective of this study was to explore knowledge, attitude and practices of antibiotics uses among patients, healthcare workers, laboratories, drug sellers and farmers in eight districts of Nepal. A cross-sectional survey was conducted between April and July 2017. A total of 516 individuals participated in a face-to-face interview that included clinicians, private drug dispensers, patients, laboratories, public health centers/hospitals and, livestock and poultry farmers. Out of 516 respondents, 62.8% (324/516) were patients, 16.9% (87/516) were clinicians, 6.4% (33/516) were private drug dispensers. A significant proportion of patients (42.9%; 139/324) thought that fever could be treated with antibiotics. Majority (79%; 256/324) of the patients purchased antibiotics over the counter. The knowledge of antibiotics used among patients increased proportionately with the level of education: literate only [AOR = 1.4 (95% Cl = 0.6-4.4)], versus secondary education (8-10 grade) [AOR = 1.8 (95% Cl = 1.0-3.4)]. Adult patients were more aware of antibiotic resistance. Use of antibiotics over the counter was found high in this study. Knowledge, attitude and practice related to antibiotic among respondents showed significant gaps and need an urgent effort to mitigate such practice.

Efficacy of Urine Dipstick Test in Diagnosing Urinary Tract Infection and Detection of the blaCTX-M Gene among ESBL-Producing Escherichia coli.

A urine dipstick test used for prompt diagnosis of urinary tract infection (UTI) is a rapid and cost-effective method. The main objective of this study was to compare the efficacy of the urine dipstick test with culture methods in screening for UTIs along with the detection of the blaCTX-M gene in extended spectrum ß-lactamase (ESBL)-producing Escherichia coli. A total of 217 mid-stream urine samples were collected from UTI-suspected patients attending Bharatpur Hospital, Chitwan, and tested by dipstick test strip (COMBI-10SL, Germany) prior to the culture. E. coli isolates were identified by standard microbiological procedures and subjected to antimicrobial susceptibility testing by Kirby Bauer disc diffusion method following CLSI guideline. Primary screening of ESBL-producing E. coli isolates was conducted using ceftriaxone, cefotaxime and ceftazidime discs and phenotypically confirmed by combined disk diffusion test. Plasmid DNA of ESBL-producing strains was extracted by phenol-chloroform method and subjected to PCR for detection of the blaCTX-M gene. Out of 217 urine samples, 48 (22.12%) showed significant bacteriuria. Among 46 (21.20%) Gram negative bacteria recovered, the predominant one was E. coli 37 (77.08%) of which 33 (89.19%) were multidrug resistant (MDR). E. coli isolates showed a higher degree of resistance towards cefazolin (62.16%) while 81.08% of the isolates were sensitive towards amikacin followed by nitrofurantoin (70.27%). Among 14 (37.84%) phenotypically confirmed ESBL isolates, only eight (21.62%) isolates carried the blaCTX-M gene. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of urine dipstick test were 43.75%, 77.51%, 35.59% and 82.91%, respectively. Besides, the use of dipstick test strip for screening UTI was associated with many false positive and negative results as compared to the gold standard culture method. Hence, dipstick nitrite test alone should not be used as sole method for screening UTIs.

Detection of TEM and CTX-M Genes in Escherichia coli Isolated from Clinical Specimens at Tertiary Care Heart Hospital, Kathmandu, Nepal.

BACKGROUND: Antimicrobial resistance (AMR) among Gram-negative pathogens, predominantly ESBL-producing clinical isolates, are increasing worldwide. The main aim of this study was to determine the prevalence of ESBL-producing clinical isolates, their antibiogram, and the frequency of ESBL genes (blaTEM and blaCTX-M) in the clinical samples from patients. METHODS: A total of 1065 clinical specimens from patients suspected of heart infections were collected between February and August 2019. Bacterial isolates were identified on colony morphology and biochemical properties. Thus, obtained clinical isolates were screened for antimicrobial susceptibility testing (AST) using modified Kirby-Bauer disk diffusion method, while ESBL producers were identified by using a combination disk diffusion method. ESBL positive isolates were further assessed using conventional polymerase chain reaction (PCR) to detect the ESBL genes blaTEM and blaCTX-M. RESULTS: Out of 1065 clinical specimens, 17.8% (190/1065) showed bacterial growth. Among 190 bacterial isolates, 57.4% (109/190) were Gram-negative bacteria. Among 109 Gram-negative bacteria, 40.3% (44/109) were E. coli, and 30.2% (33/109) were K. pneumoniae. In AST, 57.7% (n = 63) Gram-negative bacterial isolates were resistant to ampicillin and 47.7% (n = 52) were resistant to nalidixic acid. Over half of the isolates (51.3%; 56/109) were multidrug resistant (MDR). Of 44 E. coli, 27.3% (12/44) were ESBL producers. Among ESBL producer E. coli isolates, 58.4% (7/12) tested positive for the blaCTX-M gene and 41.6% (5/12) tested positive for the blaTEM gene. CONCLUSION: Half of the Gram-negative bacteria in our study were MDR. Routine identification of an infectious agent followed by AST is critical to optimize the treatment and prevent antimicrobial resistance.

Detection of OXA-48 Gene in Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae from Urine Samples.

INTRODUCTION: 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.