HCV Co-Infection and Its Genotypic Distribution in HIV-Infected Patients in Nepalese Population.

Hepatitis C Virus (HCV) co-infection and its genotypic distribution in people living with Human Immunodeficiency Virus (HIV) show global inconsistency. Therefore, the present study aimed to investigate the prevalence and genotypic distribution patterns of HCV, along with viral load, in people living with HIV. This cross-sectional study was conducted at SRL Diagnostics Nepal, Pvt. Ltd. in 203 HIV-seropositive patients attending the Tribhuvan University Teaching Hospital (TUTH), Maharajgunj, Kathmandu, Nepal from October 2021 to May 2022. The viral load and HCV genotypes were estimated from RNA extracted from the blood sample (plasma) of PLHIV by using a standard Q-PCR protocol. HCV infection was considered as a core variable, whereas covariates used for this study were duration of HIV infection, age, sex, and ART regimen. Out of total 203 PLHIV, the estimated prevalence of HCV co-infection was 115 (56.6%). Male gender was a unique characteristic associated with a high prevalence of HCV co-infection compared to females. The HCV viral load among PLHIV ranged from 34 to 3,000,000 IU/mL. Among HCV co-infected PLHIV, 56 (48.69%) had a low level of HCV viral load. Interestingly, only 3 (2.6%) patients had an HCV viral load higher than 3,000,000 IU/mL. Diverse HCV genotypes were found in the population, including genotypes 1, 1a, 3a, 5a, and 6. However, genotype 3 was the most prevalent HCV variant among HCV-co-infected PLHIV, with a distribution of 36 (61.1%) and viral load ranging from 34 to 3000 IU/mL. HCV co-infection is frequent in the Nepalese population of people living with HIV, particularly due to HCV genotypic variant 3. The findings of this study could be useful for the management and clearance of the HCV co-infection in PLHIV, aiming to provide a good quality of life.

Detection of blaoxa-23 Gene from Carbapenem-resistant Acinetobacter Baumannii.

BACKGROUND: Antibiotic resistance is a great concern for public health and Acinetobacter baumannii-associated infections are increasing in many parts of the world, including Nepal. However, limited data is available on the prevalence of A. baumannii harboring blaOXA-23 from Nepal. METHODS: A hospital-based cross-sectional study was designed to detect the blaOXA-23 gene from carbapenem-resistant A. baumannii isolates in Nepal. A total of 380 clinical specimens were collected and processed following standard microbiological procedures. Antibiotic susceptibility test was performed as per the protocol of the Kirby-Bauer disk diffusion technique and the CLSI guidelines, while screening of carbapenemase production was assessed by the Modified Hodge Test using meropenem (10µg) disc. The presence of the blaOXA-23 gene in carbapenemase-positive A. baumannii was confirmed by PCR. RESULTS: Among 380 specimens analyzed, 210 (55.3%) samples were positive for bacterial growth, where 33(15.7% of total growth) of the isolates were A. baumannii, and most of them were isolated from the ICU patients (20/33, 60.6%) and sputum (16/33, 48.5%). Thirty-two isolates (97%) were colistin sensitive, while only four (12.1%) isolates were sensitive to meropenem and imipenem. Twenty-three (69.7%) of A. baumannii were carbapenemase positive as revealed by the Modified Hodge Test test, and 19 of them (57.6% of total A. baumannii) harbored the blaOXA-23 gene. CONCLUSIONS: A high prevalence of the blaOXA-23 gene among carbapenem-resistant A. baumannii isolates were found. Systematic network surveillance should be established to check the spread of such isolates, especially in the intensive care units of tertiary care hospitals in Nepal.

Detection of NDM Variants (bla NDM-1, bla NDM-2, bla NDM-3) from Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae: First Report from Nepal.

Background: Increasing burden of carbapenem resistance among Enterobacterales is attributable to their ability to produce carbapenemase enzymes like metallo-beta-lactamase (MBL), Klebsiella pneumoniae carbapenemase (KPC), and OXA-type. This study aimed to determine the prevalence of carbapenemases and MBL genes ((bla NDM-1, bla NDM-1 and bla NDM-3) among E. coli and K. pneumoniae isolates. Methods: A total of 2474 urine samples collected during the study period (July-December 2017) were processed at the microbiology laboratory of Kathmandu Model Hospital, Kathmandu. Isolates of E. coli and K. pneumoniae were processed for antimicrobial susceptibility testing (AST) by disc diffusion method. Carbapenem-resistant isolates were subjected to Modified Hodge Test (MHT) for phenotypic confirmation, and inhibitor-based combined disc tests for the differentiation of carbapenemase (MBL and KPC). MBL-producing isolates were screened for NDM genes by polymerase chain reaction (PCR). Results: Of the total urine samples processed, 19.5% (483/2474) showed the bacterial growth. E. coli (72.6%; 351/483) was the predominant isolate followed by K. pneumoniae (12.6%; 61/483). In AST, 4.4% (18/412) isolates of E. coli (15/351) and K. pneumonia (3/61) showed resistance towards carbapenems, while 1.7% (7/412) of the isolates was confirmed as carbapenem-resistant in MHT. In this study, all (3/3) the isolates of K. pneumoniae were KPC-producers, whereas 66.7% (10/15), 20% (3/15) and 13.3% (2/15) of the E. coli isolates were MBL, KPC and MBL/KPC (both)-producers, respectively. In PCR assay, 80% (8/10), 90% (9/10) and 100% (10/10) of the isolates were positive for bla NDM-1, bla NDM-2 and bla NDM-3, respectively. Conclusion: Presence of NDM genes among carbapenemase-producing isolates is indicative of potential spread of drug-resistant variants. This study recommends the implementation of molecular diagnostic facilities in clinical settings for proper infection control, which can optimize the treatment therapies, and curb the emergence and spread of drug-resistant pathogens.

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.

Prevalence of carbapenemase-producing Klebsiella pneumoniae at a tertiary care hospital in Kathmandu, Nepal.

AIM: Although carbapenem is the last-resort drug for treating drug-resistant Gram-negative bacterial infections, prevalence of carbapenem-resistant bacteria has substantially increased worldwide owing to irrational use of antibiotics particularly in developing countries like Nepal.  Therefore, this study was aimed to determine the prevalence of carbapenemase-producing K. pneumoniae and to detect the carbapenemase genes (blaNDM-2 and blaOXA-48) in at a tertiary care hospital in Nepal. MATERIALS AND METHODS: A hospital-based cross-sectional study was carried out from June 2018 to January 2019 at the Microbiology Laboratory of Annapurna Neurological Institute and Allied Sciences, Kathmandu, Nepal. Different clinical samples were collected and cultured in appropriate growth media. Biochemical tests were performed for the identification of K. pneumoniae. Antibiotic susceptibility testing (AST) was performed by the Kirby-Bauer disc diffusion method. The modified Hodge test (MHT) was performed to detect carbapenemase producers. The plasmid was extracted by the modified alkaline hydrolysis method. Carbapenemase-producing K. pneumoniae were further confirmed by detecting blaNDM-2 and blaOXA-48 genes by PCR using specific forward and reverse primers followed by gel electrophoresis. RESULTS: Out of the total 720 samples, 38.9% (280/720) were culture positive. K. pneumoniae was the most predominant isolate 31.4% (88/280). Of 88 K. pneumoniae isolates, 56.8% (50/88) were multi-drug resistant (MDR), and 51.1% (45/88) were MHT positive. Colistin showed the highest sensitivity (100%; 88/88), followed by tigecycline (86.4%; 76/88). blaNDM-2 and blaOXA-48 genes were detected in 24.4% (11/45) and 15.5% (7/45) of carbapenemase-producing K. pneumoniae isolates, respectively. CONCLUSION: The rate of MDR and carbapenemase production was high in the K. pneumoniae isolates. Colistin and tigecycline could be the drug of choice for the empirical treatments of MDR and carbapenemase-producing K. pneumoniae. Our study provides a better understanding of antibiotic resistance threat and enables physicians to select the most appropriate antibiotics.

Prevalence of multidrug-resistance and bla VIM and bla IMP genes among gram-negative clinical isolates in tertiary care hospital, Kathmandu, Nepal.

BACKGROUND AND OBJECTIVES: Carbapenems have been the choice of antibiotics for the treatment of infections caused by multidrug-resistant bacteria. The main objective of this study was to determine the prevalence of carbapenemase (bla VIM and bla IMP ) producing isolates among Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii. MATERIALS AND METHODS: A total of 1,151 clinical samples were collected from the patients visiting Annapurna Neurological Institute and Allied Science and Annapurna Research Centre, Kathmandu, between June 2017 and January 2018. Antibiotic susceptibility testing (AST) was performed on the Enterobacteriaceae, P. aeruginosa and A. baumannii isolates using the Kirby-Bauer disk diffusion method. The modified Hodge test (MHT) was performed on the carbapenem-resistant isolates to confirm carbapenemase production. DNA was extracted and then screened for bla VIM and bla IMP genes by multiplex PCR. RESULTS: Of the total 1,151 clinical samples, 253 (22.0%) showed positive growth. Of them, 226 (89.3%) were identified as Enterobacteriaceae, P. aeruginosa, and A. baumannii. Among the 226 isolates, 106 (46.9%) were multidrug-resistant. Out of the 106, 97 (91.5%) isolates showed resistance to at least one of the carbapenem used. Among the 97 carbapenem-resistant isolates, 67 (69.1%) showed the modified Hodge test (MHT) positive results. bla VIM and bla IMP were detected in 40 and 38 isolates respectively using multiplex PCR assay. CONCLUSION: This study determined a high prevalence of MDR and carbapenem resistance among Enterobacteriaceae, P. aeruginosa, and A. baumannii as detected by the presence of bla VIM and bla IMP genes. This study recommends the use of rapid and advanced diagnostic tools along with conventional phenotypic detection methods in the clinical settings for early detection and management of drug-resistant pathogens to improve treatment strategies.

Trend and Patterns of Research Proposals Submitted for Ethical Review in Nepal Health Research Council.

BACKGROUND: The characteristics of research proposals submitted to the Nepal Health Research Council reflect the trend of health-related researches being conducted in Nepal. The objective of the study was to analyze research proposals submitted for the ethical review in Nepal Health Research Council from 2017 to 2019. METHODS: A retrospective record review of research proposals received over three years from 2017 to 2019 were analyzed. A total of 2,305 research proposals was included in the study. The number of research studies per year, types of study design, priority area of research, and rejected proposal were the intended variables for the study. RESULTS: 91.45% (503), 92.19% (748), and 755 (80.1%) of received proposals were approved from Ethical Review Board in 2017, 2018 and 2019. The number of research proposals increased every year (550 in 2017, 812 in 2018, and 943 in 2019). Among the approved proposals non-communicable disease (n=150;15.90%) in 2019} was the topmost prioritized area in all three years followed by reproductive health [93 (9.86%) in 2019] and communicable disease [67 (7.10%) in 2019] respectively. Quantitative research was more than two-third in all the years. Among the approved proposals, more than half were for an academic purpose [610 (64.69%)]. The reason for the rejection of the proposal was the conduction of research before ethical approval [2 (0.36%) in 2017, 2 (0.25%) in 2018, and none in 2019]. CONCLUSIONS: There was a rising trend of research proposals for ethical clearance being submitted to the Nepal Health Research Council. Research related to non-communicable disease followed by reproductive health was the commonest one.

Radical Cystectomy for Intradiverticular Bladder Carcinoma: A Case Report.

Herniation of bladder mucosa through the bladder wall muscle layer is known as bladder diverticulum. The incidence of bladder diverticulum is 1.7. About 0.8 to 10% of the urinary bladder diverticulum develops carcinoma. Transitional cell carcinoma is the most common. Painless hematuria is the most common clinical presentation. Different imaging modalities along with cystoscopy are the key to accurate diagnosis and staging. High grade multifocal urothelial carcinoma in the bladder diverticulum is better managed by radical cystectomy and standard pelvic lymph node dissection with an ileal conduit. Here we report a case of a 66-year old gentleman of high grade multifocal urothelial carcinoma in bladder diverticulum managed with radical cystectomy and standard pelvic lymph node dissection with an ileal conduit. Such cases have been addressed adequately in the literature, but we did not find such cases from our country.

Plasmid-Mediated AmpC ß-Lactamase CITM and DHAM Genes Among Gram-Negative Clinical Isolates.

BACKGROUND: Antibiotic resistance mediated by the production of extended-spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases is posing a serious threat in the management of the infections caused by Gram-negative pathogens. The aim of this study was to determine the prevalence of two AmpC ß-lactamases genes, bla CITM and bla DHAM, in Gram-negative bacterial isolates. MATERIALS AND METHODS: A total of 1151 clinical samples were obtained and processed at the microbiology laboratory of Annapurna Neurological Institute and Allied Science, Kathmandu between June 2017 and January 2018. Gram-negative isolates thus obtained were tested for antimicrobial susceptibility testing (AST) using Kirby-Bauer disk diffusion method. AmpC ß-lactamase production was detected by disk approximation method using phenylboronic acid (PBA). Confirmed AmpC ß-lactamase producers were further screened for bla CITM and bla DHAM genes by conventional polymerase chain reaction (PCR). RESULTS: Out of 1151 clinical specimens, 22% (253/1152) had bacterial growth. Of the total isolates, 89.3% (226/253) were Gram-negatives, with E. coli as the most predominant species (n=72) followed by Pseudomonas aeruginosa (n=41). In the AST, 46.9% (106/226) of the Gram-negative isolates were multidrug resistant (MDR). In disk diffusion test, 113 (50%) isolates showed resistance against cefoxitin, among which 91 isolates (83 by disk test and Boronic acid test, 8 by Boronic test only) were confirmed as AmpC ß-lactamase-producers. In PCR assay, 90.1% (82/91) and 87.9% (80/91) of the isolates tested positive for production of bla CITM and bla DHAM genes, respectively. CONCLUSIONS: High prevalence of AmpC ß-lactamase-producers in our study is an alarming sign. This study recommends the use of modern diagnostic facilities in the clinical settings for early detection and management which can optimize the treatment therapies, curb the growth and spread of the drug-resistant pathogens.

Molecular Characterization of Streptococcus agalactiae Isolates from Pregnant Women in Kathmandu City.

INTRODUCTION: Group B streptococci (GBS) are globally recognized as one of the leading causes of neonatal sepsis and meningitis and is also known to cause adverse pregnancy outcomes such as stillbirths and miscarriages. Thus, detailed investigation of GBS in pregnant women has special significance in public health related researches. OBJECTIVES: The present study is aimed at evaluating the prevalence of GBS colonization among pregnant women in Kathmandu city. METHODS: The study was carried out among 125 pregnant women at their trimester (35-37 weeks) (during the time period between January and June in 2018). The prevalence was determined by the culture method in HiCrome Strep B Selective Agar Base and then by using the PCR technique. The serotypes were evaluated by multiplex PCR analysis, while the antibiotics susceptibility tests were performed using the disk diffusion method. RESULTS: Among 125 samples studied, GBS were recorded in 24 samples (implying a prevalence of 19.2%). Furthermore, using the multiplex PCR, among 24 GBS-positive samples, 13 (54.17%) were found to be typeable while 11 (45.83%) were nontypeable. The most abundant serotype recorded in this study was type III (33.33) while the serotypes IV, V, VI, VII, and VIII were not found. CONCLUSION: The isolates were sensitive towards some antibiotics such as linezolid and ceftriaxone 100%, whereas penicillin 50% and vancomycin 75% but were resistant to tetracycline and ertapenem. Serotype III was found to be predominant in the samples collected during the study period. The observed prevalence was significantly associated with the gestational period, whereas no relationship was found for other risk factors.

Febrile Illness Outbreak Investigation in Sundarharicha-5 Foklan Tapu, Morang District.

BACKGROUND: On the date of 24 July 2017, the major national daily newspaper reported that there were two death cases from an unknown disease in the Morang district, Sundarharicha Municipality 5, Foklan Tapu. A team of researcher and experts were mobilized in the affected area to investigate and identify the etiological and epidemiological causes. METHODS: Both qualitative and quantitative methods was used to conduct the outbreak investigation. 83 blood samples were taken from the patients and microbiological analysis was done at National Public Health Laboratory, Kathmandu. Similarly, 2 verbal autopsies and 5 Key in-depth interviews were taken from a local community leader, local health service providers, medical officer of Koshi Zonal Hospital, district public health officer and medical director of WHO. RESULTS: Out of 83 participants, 49% and 25% of the participants were positive to IgM and IgG antibodies of Leptospira species respectively. 87% of the participants were exposed to animal living in his/her home (including pets). CONCLUSIONS: An outbreak which was existing during this investigation turned out to be leptospirosis outbreak, whose exposure was consumption of unhealthy meat during social gathering.

Microalbuminuria and Its Risk Factors in Type 2 Diabetic Patients.

BACKGROUND: Diabetic nephropathy is one of the dreaded complications of diabetes leading to chronic kidney disease and end stage renal failure globally. Microalbuminuria is the most sensitive marker of early recognition of the diabetic nephropathy. This study was carried out to find out the prevalence and potential risk factors of microalbuminuria which is the marker of diabetic nephropathy among diabetes patients in Nepal. METHODS: A cross-sectional study was conducted on a random sample of 227 in T2DM patients in private diabetic clinics and Bir hospital in Kathmandu. Data were collected using standard questionnaire format to collect demography, anthropometry, and laboratory assessment of, HbA1c, post prandial, fasting glucose and micro albumin in urine. Micro albuminuria was measured using early morning urine specimen. Micro albuminuria was considered positive when urinary albumin to creatinine ratio was found to be 30-300 mg/g creatinine in preferably an early morning or a spot urine sample. The entire lab test will be done by applying the internationally accepted standards of tools and techniques.Those that were reported >30mg/mL of micro albumin were considered as positive. RESULTS:   Out of total 217 diabetic patients, 56.2% (122/217) were male and 43.8% (95/217) were female. Among all age groups, maximum patients enrolled were between the age group 41 to 80 (95%).Of the total, 20% (44/217)) patientswereMA positive. A statistical significant association was seen between MA and BMI (p=0.029), duration of DM (p=<0.001, hypertension (p=<0.001, smoking (p=<0.001) and physical activity (p=<0.001). CONCLUSIONS: Diabetic patients in Nepal have prevalence of 20.3% microalbuminurea. Hypertension, obesity, sedentary lifestyles, duration more than 5 years of illness are found the most important risk factors for the development of microalbuminurea in diabetes.     Keywords:  Mellitus; microalbuminuria; type 2 diabetes.

Association of parental methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism in couples with unexplained recurrent pregnancy loss.

OBJECTIVE: The aim of this study was to identify the association of parental MTHFR C677T gene polymorphism in couples with and without RPL history. RESULTS: During the study, 21.4% (15/70) of Ala222Val polymorphism was observed among RPL couples while no polymorphism was seen among normal, healthy couples. Our study did not find any association between MTHFR C677T polymorphism and gender (p > 0.05), gestational period (p > 0.05), geographical region (p > 0.05) and menstrual history (p > 0.05). However, significant association was seen between MTHFR C677T polymorphism and number of losses (p < 0.05), concluding that the risk of the polymorphism increased with the increase in number of losses. Significant variation in the MTHFR C677T genotype with number of losses among RPL couples were seen but not with other study variables.

Comparative Study of GeneXpert MTB/RIF Assay and Multiplex PCR Assay for Direct Detection of Mycobacterium tuberculosis in Suspected Pulmonary Tuberculosis Patients.

Pulmonary tuberculosis (PTB) is one of the major infectious diseases in developing countries. The objective of this study was to compare rapid diagnostics technique, GeneXpert MTB/RIF (GeneXpert) and Multiplex PCR assay (MPCR) targeting IS6110 segment and mpb64 gene for direct detection of Mycobacterium tuberculosis (MTB) in suspected PTB patients. A cross sectional study was carried among 105 sputum samples from suspected PTB patients to evaluate GeneXpert and Multiplex PCR who visited National Tuberculosis Center, Nepal. The patient's sputum samples were used directly for the GeneXpert whereas DNA extraction by CTAB method was followed to process the sample for MPCR. The sensitivity and specificity of GeneXpert and MPCR in smear positive cases was 78.6, 33.3, and 100.0%, 66.7%, respectively (P = 0.125). However, in smear negative cases sensitivity and specificity of both methods exhibited 90.9, 95.2, and 100.0%, 100.0% respectively (P = 0.625). Finally, the sensitivity and specificity of GeneXpert and MPCR were 82.9, 95.3 and 100.0%, 98.5% respectively, (P = 0.549) in pulmonary cases. Comparatively, we observed higher sensitivity and specificity for MPCR than GeneXpert for both smear positive and negative samples. Thus, we recommend MPCR alongside GeneXpert for the better diagnostic accuracy of PTB in a resource-limited country where tuberculosis is endemic.

Novel limonene and citral based 2,5-disubstituted-1,3,4-oxadiazoles: a natural product coupled approach to semicarbazones for antiepileptic activity.

Two novel series of N(4)-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N(1)-(2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enylidene)semicarbazide and N(4)-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N(1)-(3,7-dimethylocta-3,6-dienylidene)-semicarbazide were synthesized to meet structural prerequisite indispensable for anticonvulsant activity. The anticonvulsant activities of the compounds were investigated using maximal electroshock seizure (MES), subcutaneous pentylenetrtrazole (scPTZ) and subcutaneous strychnine (scSTY) models. The rotorod test was conducted to evaluate neurotoxicity. Some of the selected active compounds were subjected to GABA assay to confirm their mode of action. The outcome of the present investigations proved that the four binding sites pharmacophore model is vital for anticonvulsant activity. The efforts were also made to establish structure-activity relationships among test compounds.