Diagnostic performance of GeneXpert MTB/RIF in detecting MTB in smear-negative presumptive TB patients.

BACKGROUND: Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Control of TB is lingering by the lack of diagnostic tests that are simple, rapid, yet accurate. Thus, smear-negative pulmonary TB often misses the diagnosis. The study evaluated the performance of GeneXpert MTB/RIF assay for the detection of Mycobacterium tuberculosis (MTB). METHODS: The study was carried out from June to December 2016 in Nepal Tuberculosis Center, Bhaktapur, Nepal. A total of 173 sputum samples were collected and processed by microscopy [Auramine-O staining and Ziehl-Neelsen (ZN) staining], followed by GeneXpert MTB/RIF assay and culture in Lowenstein-Jensen (LJ) medium. RESULTS: Of 173 sputum samples, 162 (93.6%) were smear-negative. Of 162 smear-negative sputum samples, 35 (21.6%) were confirmed to have MTB by culture, and 31 (19.1%) by GeneXpert MTB/RIF assay. Of 31 GeneXpert-positive samples, 25 (80.6%) were susceptible, 4 (12.9%) were resistant, and 2 (6.45%) were intermediate to rifampicin. The sensitivity, specificity, positive predictive value, and negative predictive value of GeneXpert MTB/RIF assay for smear-negative sputum samples were 74.3%, 96.6%, 86.7%, and 92%, respectively. The GeneXpert MTB/RIF has a substantial diagnostic agreement of 90.91% with culture (Cohen's Kappa coefficient = 0.73). CONCLUSION: The diagnostic performance of GeneXpert MTB/RIF assay was almost on par with culture, and thus can be relied upon for MTB detection in smear-negative sputum samples.

Development of a scale to evaluate negative menstrual attitudes among Nepalese women.

BACKGROUND: Menstrual attitudes include negative and positive perceptions of menstruation and are associated with reproductive health, underscoring the importance of its evaluation in women. Given that social and cultural factors affect menstrual attitudes, developing evaluation methods specific to distinct societal cultures is necessary. METHODS: We developed a scale based on the menstrual attitude questionnaire, which is the most widely used measure of menstrual attitudes, to evaluate negative menstrual attitudes among Nepalese women in consideration of Nepalese cultural backgrounds and literacy. In total, 352 Nepalese women aged 20-45 years living in urban or suburban areas in Nepal were enrolled in the study. Participants were randomly divided into two groups using the permuted block method. Using the prototype, Group 1 (n = 176) was evaluated with an exploratory factor analysis to develop a reliable scale, and Group 2 (n = 176) was evaluated with a confirmatory factor analysis to confirm the structure of negative menstrual attitudes. Further, we preliminarily examined the relationship of negative menstrual attitudes with the frequency of traditional menstrual practices and intensity of menstrual pain, which are significant reproductive health issues in Nepal, in Group 2. RESULTS: We developed a 15-item, 3-factor scale to evaluate negative menstrual attitudes among Nepalese women using exploratory factor analysis. The first, second, and third factors were "Natural event" (α = 0.82), "Debilitating event" (α = 0.76), and "Bothersome event" (α = 0.69), respectively. Confirmatory factor analysis revealed that negative menstrual attitudes comprised a 3-factor structure. Participants who performed three traditional menstrual practices ("stay in their own house", "go to public spaces", and "contact with others generally") demonstrated significantly higher scores for negative menstrual attitudes than did non-performers. Negative menstrual attitudes were positively correlated with the intensity of menstrual pain (r = 0.558). CONCLUSIONS: This study is the first to evaluate menstrual attitudes and analyse its factor structure among Nepalese women. In the future, the relationship between accurately evaluated negative menstrual attitudes and reproductive health issues should be examined more comprehensively.

For sexually mature women, menstruation may be considered burdensome, but its absence may also be a source of concern. Perceptions regarding menstruation are collectively referred to as menstrual attitudes. Previous studies have reported that negative menstrual attitudes are associated with various reproductive health issues in women and vary according to social and cultural backgrounds. To adequately evaluate negative menstrual attitudes among Nepalese women (20­45 years old), we developed a 15-item scale that considers their cultural background and literacy. We observed that attitudes toward menstruation among Nepalese women consisted of three emotions: one positive emotion, including "menstruation is a natural phenomenon", and two negative emotions, including "menstruation is debilitating" and "menstruation is bothersome". In several developing countries, menstruating women are considered unclean, and various restrictions are imposed on women during menstruation (traditional menstrual practices). Nepalese women with strong negative menstrual attitudes, as assessed by our scale, engaged in several traditional menstrual practices with higher frequency. We also observed that women with more negative menstrual attitudes presented with more severe menstrual pain. Our scale was developed to be compatible with the values and literacy of Nepalese women and was easy to answer. We believe that reducing negative emotions toward menstruation will contribute to reducing the frequency of traditional menstrual practices and menstrual pain, thereby facilitating school attendance and entry into the workforce among women in Nepal.

Changing Trend of Infectious Diseases in Nepal.

Many infectious/communicable diseases (IDs) are endemic in Nepal. Until a decade and half ago, IDs were the major cause of both morbidity and mortality accounting 70% for both. However, as a result of various preventive measures implemented by both the state and non-state actors, the overall IDs have shown a changing (declining) trend. The most impressive decline has been seen in the intestinal helminth infection. Though the overall burden of IDs is decreasing, several newer infectious diseases (emerging infections) namely, dengue fever, scrub typhus, influenza (H5N1 and H1N1), and others are posing a great public health problem. On the other hand, though sporadic, outbreaks of endemic diseases together with HIV-TB coinfection and infection with drug resistance microbes during recent years have constituted a serious public health as well as medical problem. On the contrary, with the decline of IDs, noninfectious diseases (noncommunicable disease, NCD) namely, diabetes, cancer (and cancer therapy), and others are on the rise particularly in urban areas. Hence, currently Nepal is trapped in "double burden" of diseases. Risk of opportunistic infection has increased in immunocompromised person with NCD. To address the present situation, the multi-sectoral plan and strategies developed must be implemented effectively.

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.

Assessment of Antibiotic Resistance among Clinical Isolates of Enterobacteriaceae in Nepal.

Clinicians face a global challenge treating infections caused by Enterobacteriaceae because of the high rate of antibiotic resistance. This cross-sectional study from the Nepal Armed Police Force Hospital, Kathmandu, Nepal, characterized resistance patterns in Enterobacteriaceae across different antimicrobial classes and assessed incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections. Enterobacteriaceae from clinical samples were isolated on blood and MacConkey agar, except for urine samples on cysteine lactose electrolyte-deficient agar. To determine antimicrobial susceptibility patterns, including MDR and XDR, the Kirby-Bauer disc diffusion method was used. Statistics were performed using SPSS, v. 17.0. Members of the family were identified in 14.5% (95% CI: 16.2-12.8%) of the total samples (N = 1,617), primarily in urine (54.7%, 128/234), blood (19.7%, 46/234), and sputum (15.0%, 35/234). Escherichia coli (n = 118, 44.2%) was the most predominant bacteria, followed by Citrobacter freundii (n = 81, 30.3%). As much as 95.6% (392/410) of the isolates were penicillin-resistant, whereas only 36.2% (290/801) were carbapenem-resistant. A total of 96 (36.0%) MDR and 98 (36.7%) XDR Enterobacteriaceae were identified. Proteus mirabilis (44.4%, 8/18) predominated MDR cultures, whereas C. freundii (53.1%, 43/81) predominated XDR cultures. Multidrug resistant (38.4%, 71/154) and XDR Enterobacteriaceae (22.7%, 35/154) were chiefly uropathogens. Fluoroquinolone resistance rates in non-MDR, MDR, and XDR isolates were 19.9%, 63.2%, and 96.2%, respectively, whereas cephalosporin resistance rates were 28.6%, 72.9%, and 95.4% and penicillin resistance rates were 67.0%, 97.4%, and 98.0%. One-seventh of patients visiting the hospital were found to be infected with Enterobacteriaceae, and of these patients, at least one-fourth were infected with MDR strains.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Analysis of altered level of blood-based biomarkers in prognosis of COVID-19 patients.

INTRODUCTION: Immune and inflammatory responses developed by the patients with Coronavirus Disease 2019 (COVID-19) during rapid disease progression result in an altered level of biomarkers. Therefore, this study aimed to analyze levels of blood-based biomarkers that are significantly altered in patients with COVID-19. METHODS: A cross-sectional study was conducted among COVID-19 diagnosed patients admitted to the tertiary care hospital. Several biomarkers-biochemical, hematological, inflammatory, cardiac, and coagulatory-were analyzed and subsequently tested for statistical significance at P<0.01 by using SPSS version 17.0. RESULTS: A total of 1,780 samples were analyzed from 1,232 COVID-19 patients (median age 45 years [IQR 33-57]; 788 [63.96%] male). The COVID-19 patients had significantly (99% Confidence Interval, P<0.01) elevated levels of glucose, urea, alanine transaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), white blood cell (WBC), C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), ferritin, D-Dimer, and creatinine phosphokinase-MB (CPK-MB) compared to the control group. However, the levels of total protein, albumin, and platelets were significantly (P<0.01) lowered in COVID-19 patients compared to the control group. The elevated levels of glucose, urea, WBC, CRP, D-Dimer, and LDH were significantly (P<0.01) associated with in-hospital mortality in COVID-19 patients. CONCLUSIONS: Assessing and monitoring the elevated levels of glucose, urea, ALT, AST, ALP, WBC, CRP, PCT, IL-6, ferritin, LDH, D-Dimer, and CPK-MB and the lowered levels of total protein, albumin, and platelet could provide a basis for evaluation of improved prognosis and effective treatment in patients with COVID-19.

Assessment of four in vitro phenotypic biofilm detection methods in relation to antimicrobial resistance in aerobic clinical bacterial isolates.

INTRODUCTION: The lack of standardized methods for detecting biofilms continues to pose a challenge to microbiological diagnostics since biofilm-mediated infections induce persistent and recurrent infections in humans that often defy treatment with common antibiotics. This study aimed to evaluate diagnostic parameters of four in vitro phenotypic biofilm detection assays in relation to antimicrobial resistance in aerobic clinical bacterial isolates. METHODS: In this cross-sectional study, bacterial strains from clinical samples were isolated and identified following the standard microbiological guidelines. The antibiotic resistance profile was assessed through the Kirby-Bauer disc diffusion method. Biofilm formation was detected by gold standard tissue culture plate method (TCPM), tube method (TM), Congo red agar (CRA), and modified Congo red agar (MCRA). Statistical analyses were performed using SPSS version 17.0, with a significant association considered at p<0.05. RESULT: Among the total isolates (n = 226), TCPM detected 140 (61.95%) biofilm producers, with CoNS (9/9) (p<0.001) as the predominant biofilm former. When compared to TCPM, TM (n = 119) (p<0.001) showed 90.8% sensitivity and 70.1% specificity, CRA (n = 88) (p = 0.123) showed 68.2% sensitivity and 42% specificity, and MCRA (n = 86) (p = 0.442) showed 65.1% sensitivity and 40% specificity. Juxtaposed to CRA, colonies formed on MCRA developed more intense black pigmentation from 24 to 96 hours. There were 77 multi-drug-resistant (MDR)-biofilm formers and 39 extensively drug-resistant (XDR)-biofilm formers, with 100% resistance to ampicillin and ceftazidime, respectively. CONCLUSION: It is suggested that TM be used for biofilm detection, after TCPM. Unlike MCRA, black pigmentation in colonies formed on CRA declined with time. MDR- and XDR-biofilm formers were frequent among the clinical isolates.

Hand and nasal carriage of Staphylococcus aureus and its rate of recolonization among healthcare workers of a tertiary care hospital in Nepal.

Background: Carriers of Staphylococcus aureus among healthcare workers (HCWs) can spread the bacteria to patients and the environment, in addition to their own risk of infection. Objectives: To determine the prevalence of S. aureus carriers among HCWs and the rate of recolonization after decolonization therapy with mupirocin. Methods: Nasal and hand swabs from HCWs of a tertiary care hospital in Nepal were cultured on mannitol salt agar and S. aureus isolated were identified using standard microbiological procedures. Detection of MRSA and mupirocin-resistant S. aureus (MupRSA) isolates were done phenotypically. Identified S. aureus carriers were decolonized with 2% mupirocin nasal ointment. Recolonization of the carriers was assessed monthly for the next 5 months. Results: Among the 213 HCWs, 18.3% were S. aureus carriers (35 nasal carriers, 4 both nasal and hand carriers, and no hand carriers). Overall, 9.4% of the HCWS were MRSA carriers and none were MupRSA carriers. After decolonization, 25.6% of them were recolonized and 50.0% of the recolonization was detected after 3 months of decolonization. All recolonized carriers had only MSSA strains (which colonized only nose), and none were recolonized with MupRSA. Conclusions: HCWs are frequent carriers of S. aureus and MRSA. Due to their continuous exposure to the hospital environment, they are at risk of colonization by this MDR organism. Regular screening and decolonization of HCWs working with high risk, vulnerable patients would reduce the risk of MRSA transmission from HCWs to patients.

Role of Tocilizumab in the Treatment of COVID-19 Patients with Cytokine Storm: A Case Series.

The in-hospital mortality in patients with COVID-19 could be correlated with severe acute respiratory syndrome coronavirus-2 induced hyper-inflammation, which is attributed to an unconstrained inflammatory cytokine storm. The pro-inflammatory cytokine, specifically, interleukin-6 plays a prominent role in the cytokine storm and may result in alveolar-capillary blood-gas exchange dysfunction. Therefore, the method to block the signal transduction pathway of interleukin-6 could be a potential treatment for severe COVID-19 patients. In this case series of three patients with severe COVID-19, we focus on the rationale for utilization of tocilizumab, an anti-interleukin-6 receptor antibody, which could block the signal transduction pathway of interleukin-6. The observations from this study allowed us to hypothesize that the infusions of tocilizumab may not reduce the elevated level of interleukin-6, and hence may not be a significant therapeutic for reducing in-hospital mortality associated with COVID-19. Additionally, it could also be speculated that interleukin-6 may not be a potentially actionable target cytokine to treat COVID-19-associated cytokine storms. Keywords: COVID-19; cytokines; interleukin-6.

Phenotypic detection of methicillin resistance, biofilm production, and inducible clindamycin resistance in Staphylococcus aureus clinical isolates in Kathmandu, Nepal.

INTRODUCTION: Methicillin resistance, inducible clindamycin resistance (ICR), biofilm production, and increased minimum inhibitory concentration (MIC) of vancomycin in Staphylococcus aureus are major causes of antibiotic treatment failure and increased morbidity and mortality. The surveillance of such isolates and the study of their antimicrobial pattern are essential in managing the infections caused by these isolates. This study aimed to determine methicillin resistance, biofilm production, and ICR in S. aureus isolates from a tertiary care hospital in Kathmandu, Nepal. MATERIALS AND METHODS: A total of 217 S. aureus isolated from different samples were processed following standard laboratory procedures. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion technique. Methicillin-resistant S. aureus (MRSA) were identified by the cefoxitin disk diffusion test, and biofilm producers were examined using the microtiter plate technique. D-test and E-test were performed to determine inducible clindamycin resistance and minimum inhibitory concentration of vancomycin, respectively. RESULTS: Among the 217 S. aureus isolates, 78.3% were multidrug-resistant (MDR), 47.0% were MRSA, 62.2% were biofilm producers, and 50.7% showed ICR. All MRSA isolates exhibited MIC levels of vancomycin within the susceptible range. Biofilm producers and MRSA isolates showed elevated antimicrobial resistance. MRSA was significantly associated with MDR. Biofilm-producing and multidrug-resistant MRSA isolates showed significantly higher MIC levels of vancomycin (p = 0.0013 and < 0.0001, respectively), while ICR was significantly higher in MDR (p = 0.0001) isolates. CONCLUSION: High multidrug resistance, MRSA, and ICR in this study call for routine evaluation of antibiotic susceptibility patterns of S. aureus. Vancomycin can be used to treat serious staphylococcal infections. Clindamycin should be prescribed only after performing the D-test. Drugs like teicoplanin, chloramphenicol, doxycycline, amikacin, and levofloxacin can treat MRSA infections.

Prevalence of multidrug-resistant and extended-spectrum beta-lactamase producing Gram-negative isolates from clinical samples in a tertiary care hospital of Nepal.

BACKGROUND: The existence of multidrug-resistant organisms, including extended-spectrum beta-lactamases (ESBLs), is on rise across the globe and is becoming a severe problem. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. This study aimed to study the prevalence of Gram-negative isolates exhibiting ESBL at a tertiary care hospital and study their antibiogram profile. METHODS: A cross-sectional study was conducted at Shahid Gangalal National Heart Centre, Kathmandu, Nepal, from June 2018 to November 2018. A total of 770 clinical samples were collected and identified using the conventional biochemical tests following the Clinical and Laboratory Standard Institute (CLSI) guidelines. Antimicrobial susceptibility testing (AST) was performed using the standardized Kirby-Bauer disk diffusion method. The screening test for ESBL producers was performed as recommended by the CLSI and the confirmatory test was performed phenotypically using the E-test. RESULTS: Out of the 92 isolates, 84 (91.3%) were multidrug-resistant, and 47 (51.1%) were found to be potential ESBL producers. Of these, 16 isolates were confirmed ESBL producers by the E-test. Escherichia coli and Klebsiella pneumoniae were the predominant isolates and were also the major ESBL producers. Besides polymyxin B (100% sensitive), meropenem and imipenem showed high efficacy against the ESBL producers. CONCLUSION: Multidrug resistance was very high; however, ESBL production was low. Polymyxin B and carbapenems are the choice of drugs against ESBL producers but should be used only as the last line drugs.

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.

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.

Distribution and pyrethroid resistance status of Aedes aegypti and Aedes albopictus populations and possible phylogenetic reasons for the recent invasion of Aedes aegypti in Nepal.

BACKGROUND: When the first systematic list of mosquitoes in Nepal was reported in 1990, there was no description of Aedes aegypti (L.), while Aedes albopictus (Skuse) has been included in the Stegomyia subgroup since the 1950s. The first record of Ae. aegypti in Nepal was reported in 2009, suggesting some coincidence between the invasion of this species and the first record of dengue fever in Nepal in 2006. RESULTS: We performed a field survey of the distribution and insecticide susceptibility of Ae. aegypti and Ae. albopictus in Nepal in 2017 and 2018. Mosquito larvae were collected from used tires located along the streets of Kathmandu, Bharatpur and Pokhara, and a simplified bioassay was used to assess the susceptibility of the larvae to pyrethroid insecticides using d-allethrin. The presence or absence of point mutations in the voltage-gated sodium channel was also detected by direct sequencing. V1016G was detected at a high frequency and a strong correlation was observed between the frequencies of V1016G and susceptibility indices in Ae. aegypti populations. F1534C was also detected at a relatively low frequency. In Ae. albopictus populations, susceptibilities to d-allethrin were high and no point mutations were detected. Analysis of the cytochrome c oxidase subunit 1 (cox1) gene was performed for assessing genetic diversity and the existence of two strains were identified in Ae. aegypti populations. One consisted of 9 globally-distributed haplotypes while the other was derived from an African haplotype. CONCLUSIONS: The high pyrethroid resistance, high V1016G frequency, and relatively low quantity of insecticides used to control dengue vectors in Nepal may have resulted in only weak selection pressure favoring insecticide resistance and could support the hypothesis that this species has recently been introduced from neighboring Asian countries where pyrethroid resistance is relatively widespread.

Prevalence of intestinal parasitosis and associated risk factors among school children of Saptari district, Nepal: a cross-sectional study.

BACKGROUND: Intestinal parasitosis, caused by both helminths and protozoans, are among the most prevalent infections, especially in developing countries. Enteric parasites continue to be a major cause of parasitic diseases which is the most common among street and school going children with poor sanitation. This cross-sectional study was carried out to determine the prevalence and potential risk factors of intestinal parasitosis among school going children of two schools of Saptari district of southern Nepal. Stool samples were collected in a clean, dry, screw-capped, and wide-mouthed plastic container, fixed with 10% formal-saline solution, and transported to the laboratory for further microscopic analysis by following concentration technique. RESULTS: Out of the 285 stool samples analysed, 94 (33%) were positive for the parasitosis. Presence of intestinal parasites was marginally more in rural school (44.6%) than in urban (30%) (P < 0.05). Giardia lamblia was highly prevalent (15.4%) followed by Entamoeba histolytica-like (7.7%), E. coli (7%), Ascaris lumbricoides (1.8%), and Hymenolepis nana (1.08%), respectively. Children of the age group 11-15 years were highly affected (44.2%) compared to younger age groups. The findings of intestinal parasitosis in the study population were statistically significant with family income, hand-washing habit, type of drinking water, and availability of a toilet facility at home (P < 0.05). Over 85% of infection was associated with parasitosis that indicated mainly waterborne infection rather than soil-borne helminths. CONCLUSIONS: Poor hygiene measures and farming occupation are identified as major risk factors of parasitic infections, so sanitation especially focusing on safe drinking water along with multi intervention strategies must be emphasized in the Saptari district of Nepal to reduce the burden of parasitic diseases in school children.

Intestinal parasitic infections among public and private schoolchildren of Kathmandu, Nepal: prevalence and associated risk factors.

OBJECTIVE: Intestinal parasitic infections (IPIs) are a major cause of morbidity among children in developing countries. Investigation about the etiological agents and socio-ecological pattern of the infection would help to design better preventive strategy. The previous studies reported high prevalence of IPIs among schoolchildren of Nepal. Though these data may be essential for the policymakers and researchers, in Kathmandu, the capital of Nepal it remains unexplored whether the types of school and socioeconomic status affect the IPIs or not. The present study is an extension of previous works to investigate causative agents and associated risk factors. We examined 508 stool samples of schoolchildren from two schools by formal-ether concentration technique and analyzed the data based on school types. RESULTS: The overall IPIs rate was 19.9% (n = 101) with the dominance of protozoans (78.4%) over helminths (21.6%). Giardia duodenalis (32.7%) and Ascaris lumbricoides (21.8%) were the most commonly detected protozoan and helminth species respectively. Prevalence of IPIs was higher among children from public school (26.1%) than private school (12.1%). Higher infection rates were found among farmer's children (29.0%) and Dalit children (36.2%). These findings reveal the different prevalence of IPIs among public and private schoolchildren and suggest the need of effective preventive measures.

Intestinal Parasitic Infections among Prison Inmates in Kathmandu Nepal.

BACKGROUND: Prison inmates are at high risk of intestinal parasitic infections. Thus, we studied intestinal parasitic infections among inmates of the Central Jail, Kathmandu, Nepal. METHODS: Morning stool samples from 400 inmates (M=282 and F=118), were collected in a clean, dry and wide-mouthed plastic container. The samples were transported to the research laboratory of Shi-Gan International College of Science and Technology, and were fixed using 10% formal saline. Then, samples were processed by formal ether sedimentation concentration technique and were observed microscopically by direct-smear technique. RESULTS: Six percent (24/400) samples were positive for intestinal parasites, with a gender ratio (M:F) 1.7:1. But, co-parasitism was not observed. Intestinal parasitic infections were higher among 21-40 years age-group, 3.5% (14/262). Similarly, intestinal parasitic infections were higher among 'Dalits' ethnic group, 21.1% (4/19). As compared to helminths, more protozoans, 62.5% (15/24), were observed. Giardia lamblia, 41.67% (10/24), was the most common protozoans while Trichuris trichiura, 25.0% (6/24), was the most common helminths. CONCLUSIONS: Intestinal parasitic infections were lower among the inmates of Central jail, but such conditions cannot be presumed in other peripheral settings. Pure and safe drinking water supply and the effective deworming campaign can further reduce this figure at this setting and peripheral jails across the country.

Diarrheal disease outbreak in Gaidatar village of Rautahat District, Nepal.

OBJECTIVE: Diarrheal diseases, including cholera, remain a major public health concern in developing countries like Nepal. This study investigated a diarrheal outbreak that affected over 1500 people in Gaidatar village of Rautahat district in central Nepal and sought to identify the source and causation of the disease. Stool samples were collected from individuals with acute diarrheal illness (n = 16) and healthy non-diarrheal children (n = 39), along with samples from local drinking water sources (n = 8) and their sewage system (n = 10). None of the individuals were sampled multiple times. Diarrheic stool and sewage samples were analysed for the presence of Vibrio cholerae, while coliforms were tested in drinking water samples following standard microbiological protocols. Enteric parasites were tested in both diarrheic and non-diarrheic stool samples. RESULTS: Vibrio cholerae O1 Ogawa serotype was isolated in 18.7% of the diarrheic stool and 20.0% of the sewage. Coliforms were found in all drinking water samples, with 87.5% testing positive for fecal coliform. Additionally, 43.6% of the stool samples (n = 55) had at least one of the intestinal parasites tested, primarily Giardia lamblia (21.8%). However, almost all parasites were found in non-diarrheal stool. Taken together, our results provide evidence that the diarrheal outbreak was associated with V. cholerae O1 Ogawa serotype, possibly transmitted through the drinking water sources contaminated with fecal matters from their sewage (drainage) system. These findings warrant regular surveillance of drinking water sources to help prevent future outbreaks.

Changes of physical and biochemical properties of Cryptosporidium oocysts with various storage conditions.

Physical and biochemical properties of Cryptosporidium parvum oocyst were examined after storage under various conditions. Oocyst-positive-fecal samples recovered from calves were either stored in a 2.0% potassium dichromate solution (Cr) or deionized water (W), or kept as a fecal pellet (P), and stored at 4 or 18 degrees C for a maximum of 100 days. When stored in Cr at 4 degrees C, the morphology of oocysts and their ability to withstand ultrasonics was not affected by the storing media or the storage period. However, when stored at 18 degees C as a fecal pellet, the specific gravity of the oocysts increased and a significant decrease in the oocysts resistance to ultrasonics occurred. These changes in oocyst properties may affect the performance of methods used to detect oocysts in water samples. When using the current test methods or when developing a new test method, it is important to take these factors into consideration.