Multiplex PCR for bacterial, viral and protozoal pathogens in persistent diarrhoea or persistent abdominal pain in Côte d'Ivoire, Mali and Nepal.

In contrast to acute diarrhoea, the aetiology of persistent digestive disorders (≥ 14 days) is poorly understood in low-resource settings and conventional diagnostic approaches lack accuracy. In this multi-country study, we compared multiplex real-time PCR for enteric bacterial, parasitic and viral pathogens in stool samples from symptomatic patients and matched asymptomatic controls in Côte d'Ivoire, Mali and Nepal. Among 1826 stool samples, the prevalence of most pathogens was highest in Mali, being up to threefold higher than in Côte d'Ivoire and up to tenfold higher than in Nepal. In all settings, the most prevalent bacteria were EAEC (13.0-39.9%) and Campylobacter spp. (3.9-35.3%). Giardia intestinalis was the predominant intestinal protozoon (2.9-20.5%), and adenovirus 40/41 was the most frequently observed viral pathogen (6.3-25.1%). Significantly different prevalences between symptomatic and asymptomatic individuals were observed for Campylobacter, EIEC and ETEC in the two African sites, and for norovirus in Nepal. Multiple species pathogen infection was common in Côte d'Ivoire and Mali, but rarely found in Nepal. We observed that molecular testing detected multiple enteric pathogens and showed low discriminatory accuracy to distinguish between symptomatic and asymptomatic individuals. Yet, multiplex PCR allowed for direct comparison between different countries and revealed considerable setting-specificity.

Visceral leishmaniasis in the hills of western Nepal: A transmission assessment.

In Nepal, visceral leishmaniasis (VL) has been targeted for elimination as a public health problem by 2026. Recently, increasing numbers of VL cases have been reported from districts of doubtful endemicity including hills and mountains, threatening the ongoing VL elimination program in Nepal. We conducted a multi-disciplinary, descriptive cross-sectional survey to assess the local transmission of Leishmania donovani in seven such districts situated at altitudes of up to 1,764 meters in western Nepal from March to December 2019. House-to-house surveys were performed for socio-demographic data and data on past and current VL cases. Venous blood was collected from all consenting individuals aged ≥2 years and tested with the rK39 RDT. Blood samples were also tested with direct agglutination test, and a titer of ≥1:1600 was taken as a marker of infection. A Leishmania donovani species-specific PCR (SSU-rDNA) was performed for parasite species confirmation. We also captured sand flies using CDC light traps and mouth aspirators. The house-to-house surveys documented 28 past and six new VL cases of which 82% (28/34) were without travel exposure. Overall, 4.1% (54/1320) of healthy participants tested positive for L. donovani on at least one serological or molecular test. Among asymptomatic individuals, 17% (9/54) were household contacts of past VL cases, compared to 0.5% (6/1266) among non-infected individuals. Phlebotomus argentipes, the vector of L. donovani, was found in all districts except in Bajura. L. donovani was confirmed in two asymptomatic individuals and one pool of sand flies of Phlebotomus (Adlerius) sp. We found epidemiological and entomological evidence for local transmission of L. donovani in areas previously considered as non-endemic for VL. The national VL elimination program should revise the endemicity status of these districts and extend surveillance and control activities to curb further transmission of the disease.

Clinical, Bacteriological Profile and Outcome of Neonatal Sepsis.

BACKGROUND: Sepsis is a major cause of morbidity and mortality among neonates in Nepal. This study was conducted to determine the clinical-bacteriological profile, their antibiotic susceptibility patterns, and clinical outcome of culture-positive neonatal sepsis. METHODS: This was a prospective study conducted at B.P Koirala Institute of Health Sciences from July 2018 to June 2019. Neonates with clinically diagnosed sepsis having blood culture positive were included in the study. Blood samples culture and antimicrobial susceptibility testing were performed with the standard microbiological method. Demographic, clinical information, and clinical outcomes were documented. RESULTS: The incidence of culture-positive sepsis was 10.3% (183/1773) of neonatal admissions. Poor feeding 85(46%) and fever 68(37%) were the common clinical features at presentation. The incidence of early-onset sepsis and late-onset sepsis were found to be 116 (63%) and 67(37%) respectively. Staphylococcus aureus was the common pathogen in both early-onset 61(49%) and late-onset 34(41%) sepsis. The incidence of multidrug-resistant cases was 41% (75/183) with 20% (15/75) extensively drug-resistant gram-negative bacilli, 36% (20/75) multidrug-resistant gram-negative bacilli, and 44% (33/75) Methicillin-resistant Staphylococcus aureus cases. In-hospital mortality rate was 12 (7%) with a higher frequency in multidrug-resistant sepsis 92% (11/12) than non- multidrug-resistant 8% (1/12). The median hospital days were longer in multidrug-resistant cases than non- multidrug-resistant [11(9-13) verses 3(2-5)]. CONCLUSIONS: The incidence of multidrug-resistant pathogens causing neonatal sepsis is high at our hospital and are associated with more in-hospital mortality and longer hospital stay. Implementation of effective preventive strategies to combat the emergence of antimicrobial resistance is immediately needed.

Pulmonary Paragonimiasis: A Case Series.

Paragonimiasis contributes to significant foodborne zoonosis worldwide. The major mode of transmission in humans is by consumption of uncooked or undercooked crabs and crayfish harbouring Paragonimus metacercariae. It begins with symptoms like fever and lower respiratory involvement from a few months to a year, mimicking those of tuberculosis and leading to diagnostic delay. Here, we report two cases of paragonimiasis during a period of nine months. Both cases presented with symptoms of productive cough with rusty sputum, chest pain, along with eosinophilia, and pleural effusion and had a history of consumption of smoked crab from the local river. The diagnosis was established by microscopic demonstration of Paragonimus ova in the sputum. They were treated with praziquantel and recovered. Indeed, it is challenging to diagnose paragonimiasis due to the lack of its specific symptoms but should be considered in the differential diagnosis of eosinophilia and pleural effusion in such lung diseases. Keywords: case reports; eosinophilia; paragonimiasis; pleural effusion.

A randomized, observer-blind, controlled phase III clinical trial assessing safety and immunological non-inferiority of Vi-diphtheria toxoid versus Vi-tetanus toxoid typhoid conjugate vaccine in healthy volunteers in eastern Nepal.

Typhoid remains one of the major serious health concerns for children in developing countries. With extremely drug-resistant cases emerging, preventative measures like sanitation and vaccination, including typhoid conjugate vaccines (TCV) remain the mainstay in its prevention and control. Different types of TCVs are being developed to meet the global demand. This report outlines the results from a study done to assess the immunogenicity and safety of Vi-Diphtheria toxoid (Vi-DT) TCV in Nepal. The study was a randomized, active-controlled, immunological non-inferiority and safety study. Eligible participants from Sunsari and Morang districts of eastern Nepal were randomized into 4 study groups (A-D) within 3 age strata (6 months to <2 years, 2 to <18 years, and 18 to 45 years). Groups A to C received a single dose (25 µg) of Vi-DT test vaccine from any of the 3 lots, while group D received the comparator, Typbar-TCV®, Vi-tetanus toxoid (Vi-TT) vaccine (25 µg) in 1:1:1:1 ratio and evaluated at 4 weeks postvaccination with 6 months follow-up. Amongst 400 randomized participants, anti-Vi-IgG seroconversion rates for all age strata in Vi-DT pooled groups (A+B+C) were 100.00% (97.5% CI 98.34-100.00) vs 98.99% (97.5% CI 93.99-99.85) in Vi-TT group (D) at 4 weeks. Comparable safety events were reported between the groups. Three serious adverse events (1 in Vi-DT; 2 in Vi-TT group) were reported during the 6 months follow-up, none being related to the investigational product. Thus, Vi-DT vaccine is safe, immunogenic, and immunologically non-inferior to Vi-TT when analyzed at 4 weeks postvaccination.

Multidrug-Resistant Acinetobacter: Detection of ESBL, MBL, bla NDM-1 Genotype, and Biofilm Formation at a Tertiary Care Hospital in Eastern Nepal.

Background: The Acinetobacter species is an important hospital-acquired pathogen. The rapid development of resistance to multiple drugs and the ability to form biofilm make these bacteria more adaptable to survive in healthcare facilities, thus posing a challenge to their effective management. Objective: This study aimed to characterize clinical isolates of Acinetobacter spp and to study their antimicrobial susceptibility patterns and ability to form biofilm. Resistant Acinetobacter was further analyzed for the detection of extended-spectrum ß-lactamases (ESBLs), metallo ß-lactamases (MBLs), carbapenemase production, and presence of blaNDM-1 gene. Materials and Methods: A total of 324 Acinetobacter species were isolated from various clinical specimens which were submitted to the Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal, and were studied for antibiotic susceptibility testing, detection of ESBL and MBL production, and formerly biofilm formation was performed by standard microbiological methods. PCR was carried out to determine the presence of the blaNDM-1 gene. Results: The predominant Acinetobacter species isolated was A calcoaceticus-baumannii Complex (Acb complex) 167 (51.5%). Among those, all A. species 128 (40%) were multidrug resistant (MDR). In which 13 (4.0%) were ESBL producers, 70 (61.9%) were MBL, and 12 (10.6%) were carbapenemases producers. The blaNDM1 gene was present in 33 isolates. Thirty-seven percent (121/324) of isolates formed biofilm. The majority of A. species were resistant to cefotaxime 73.8% (239) and cefepime 74.4% (241). A significant proportion of biofilm producers were MDR (p < 0.001). Conclusion: Drug-resistant Acinetobacter formed a substantial proportion of this hospital's samples with a large presence of the bla NDM-1 gene. A matter of great concern is the association of multidrug-resistant phenotype with biofilm formation. This situation warranted stringent surveillance and adherence to infection prevention and control practices.

Four layer multi-omics reveals molecular responses to aneuploidy in Leishmania.

Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between closely related aneuploid strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, as in other Eukaryotes, we observed attenuation of dosage effects for protein complex subunits and in addition, non-cytoplasmic proteins. Differentially expressed transcripts and proteins between aneuploid Leishmania strains also originated from non-aneuploid chromosomes. At protein level, these were enriched for proteins involved in protein metabolism, such as chaperones and chaperonins, peptidases, and heat-shock proteins. In conclusion, our results further support the view that aneuploidy in Leishmania can be adaptive. Additionally, we believe that the high karyotype diversity in vitro and absence of classical transcriptional regulation make Leishmania an attractive model to study processes of protein homeostasis in the context of aneuploidy and beyond.

Etiological spectrum of persistent fever in the tropics and predictors of ubiquitous infections: a prospective four-country study with pooled analysis.

BACKGROUND: Persistent fever, defined as fever lasting for 7 days or more at first medical evaluation, has been hardly investigated as a separate clinical entity in the tropics. This study aimed at exploring the frequencies and diagnostic predictors of the ubiquitous priority (i.e., severe and treatable) infections causing persistent fever in the tropics. METHODS: In six different health settings across four countries in Africa and Asia (Sudan, Democratic Republic of Congo [DRC], Nepal, and Cambodia), consecutive patients aged 5 years or older with persistent fever were prospectively recruited from January 2013 to October 2014. Participants underwent a reference diagnostic workup targeting a pre-established list of 12 epidemiologically relevant priority infections (i.e., malaria, tuberculosis, HIV, enteric fever, leptospirosis, rickettsiosis, brucellosis, melioidosis, relapsing fever, visceral leishmaniasis, human African trypanosomiasis, amebic liver abscess). The likelihood ratios (LRs) of clinical and basic laboratory features were determined by pooling all cases of each identified ubiquitous infection (i.e., found in all countries). In addition, we assessed the diagnostic accuracy of five antibody-based rapid diagnostic tests (RDTs): Typhidot Rapid IgM, Test-itTM Typhoid IgM Lateral Flow Assay, and SD Bioline Salmonella typhi IgG/IgM for Salmonella Typhi infection, and Test-itTM Leptospira IgM Lateral Flow Assay and SD Bioline Leptospira IgG/IgM for leptospirosis. RESULTS: A total of 1922 patients (median age: 35 years; female: 51%) were enrolled (Sudan, n = 667; DRC, n = 300; Nepal, n = 577; Cambodia, n = 378). Ubiquitous priority infections were diagnosed in 452 (23.5%) participants and included malaria 8.0% (n = 154), tuberculosis 6.7% (n = 129), leptospirosis 4.0% (n = 77), rickettsiosis 2.3% (n = 44), enteric fever 1.8% (n = 34), and new HIV diagnosis 0.7% (n = 14). The other priority infections were limited to one or two countries. The only features with a positive LR ≥ 3 were diarrhea for enteric fever and elevated alanine aminotransferase level for enteric fever and rickettsiosis. Sensitivities ranged from 29 to 67% for the three RDTs targeting S. Typhi and were 9% and 16% for the two RDTs targeting leptospirosis. Specificities ranged from 86 to 99% for S. Typhi detecting RDTs and were 96% and 97% for leptospirosis RDTs. CONCLUSIONS: Leptospirosis, rickettsiosis, and enteric fever accounted each for a substantial proportion of the persistent fever caseload across all tropical areas, in addition to malaria, tuberculosis, and HIV. Very few discriminative features were however identified, and RDTs for leptospirosis and Salmonella Typhi infection performed poorly. Improved field diagnostics are urgently needed for these challenging infections. TRIAL REGISTRATION: NCT01766830 at ClinicalTrials.gov.

Nepal measles outbreak response immunization during COVID-19: A risk-based intervention strategy.

In 2020, National Immunization Programme (NIP) of Nepal implemented a measles outbreak response immunization (ORI) campaign, which was additional to an ongoing preventive measles-rubella SIA campaign. Both campaigns were implemented during ongoing COVID-19 transmission. By April, 220 measles cases and two deaths were confirmed from eight districts of Nepal. The NIP triangulated information from surveillance (measles and COVID-19), measles immunization performance and immunity profile, programme capacities and community engagement and applied a logical decision-making framework to the collated data to inform 'Go/No-Go' decisions for ORI interventions. This was reviewed by the National Immunization Advisory Committee (NIAC) for endorsement. Outbreak response with non-selective immunization (ORI), vitamin-A administration and case management were implemented in affected municipalities of four districts, while in the remaining districts outbreak response without ORI were undertaken. The structure and iterative application of this logical framework has been described. ORI was implemented without interrupting the ongoing measles-rubella vaccination campaign which had targeted children from 9 to 59 months of age. The age group for ORI was same as SIA in one sub-district area, while for the other three sub-district areas it was from 6 months to 15 years of age. More than 32,000 persons (97% coverage) were vaccinated in ORI response. Overall measles incidence decreased by 98% after ORI. The daily incidence rate of measles was 94 times higher (95% confidence interval: 36.11 - 347.62) before the ORI compared to two weeks after ORI until year end. Close attention to surveillance and other data to inform actions and seamless collaboration between NIP and core immunization partners (WHO, UNICEF), with guidance from NIAC were key elements in successful implementation. This was an example of feasible application of the global framework for implementation of a mass vaccination campaign during COVID-19 through application of a simple decision-making logical framework.

Co-infection of Hepatitis B and Hepatitis C among HIV-infected patients: A cross-sectional study from tertiary care hospital of eastern Nepal.

INTRODUCTION: This study was conducted with an objective to analyze prevalence and risk factors associated with co-infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) in HIV-positive patients with reference to their CD4+ T cell status. MATERIALS AND METHODS: HIV-positive patients visiting the HIV clinic for CD4+ T cells testing at B.P. Koirala Institute of Health Sciences were tested for Hepatitis B and Hepatitis C. Data regarding age, gender, mode of HIV transmission, duration of HIV diagnosis, antiretroviral therapy status, antiretroviral therapy duration, hepatitis B or C status, and CD4+ T cells count were collected via face-to-face interview, and hospital records. The data were entered in Microsoft Excel 2019 v16.0 (Microsoft, WA, USA) and statistical analysis was performed by using statistical package for social sciences, IBM SPSS® v21 (IBM, Armonk, New York). RESULTS: Out of 474 HIV-positive patients, HIV-HBV, HIV-HCV, and HIV-HBV-HCV co-infections were seen in 2.95% (14/474), 18.14% (86/474), and 2.53% (12/474) respectively. The primary route of infection was intra-venous drug use (IVDU) in those co-infected with HBV only (8, 57.14%), HCV only (46, 53.49%), and both HBV and HCV (8, 66.67%). HIV patients infected via IVDU were 2.40 times more likely to have HIV-HCV co-infection as compared to those infected via sexual route (AOR 2.40, 95% CI: 1.49,3.86). Similarly, HIV patients with CD4+ T cells count less than 350 cells/mm3 were more likely to have HIV-HBV-HCV co-infection as compared to those with CD4 count equal to and more than 350 cells/mm3 (AOR 13.84, 95% CI: 2.90,66.10). CONCLUSION: HIV-positive patients are at high risk of hepatitis B and/or hepatitis C co-infection. Intravenous drug use, and lower CD4+T cells count are the most important risk predictors of co-infection. All HIV-positive patients should be carefully screened with hepatitis B and hepatitis C tests during their follow-up.

Microbiological and Clinicoepidemiological Profile of a Series of Patients with Infective Endocarditis at a Center in Eastern Nepal.

BACKGROUND: The microbiological and clinicoepidemiological profile of infective endocarditis (IE) has undergone significant change over time. The pattern of IE studied at local level provides broader vision in understanding the current scenario of this disease. This study aimed to depict the overall picture of IE and its changing profile by evaluating the microbiological and clinicoepidemiological features in the context of a tertiary care center of eastern Nepal. METHODS: The descriptive study was conducted from September 2017 to August 2018 among IE patients presenting to B. P. Koirala Institute of Health Sciences, Nepal. Detailed history and clinical manifestations of patients were noted. Microorganisms isolated from the blood culture were processed for identification by standard microbiological methods, and susceptibility testings were done. Each patient was assessed daily during hospital stay. RESULTS: Ten definite and 7 possible endocarditis cases were studied. The mean age was 41.4 ± 15.85 (17-70) years with predominance of male (4.7 : 1). Rheumatic heart disease (41.1%) was the most common underlying heart disease observed followed by injection drug user endocarditis (23.5%). All the cases had native valve endocarditis. Aortic valve was the most common valve involved (35.3%) followed by mitral, tricuspid, and pulmonary valves. Blood culture positivity was 53%. Staphylococcus aureus was the major causative agent responsible for 23.5% of the cases followed by Enterococcus faecium, Enterococcus faecalis, and Pseudomonas aeruginosa. Mortality of 2 cases (11.8%) was associated with S. aureus and P. aeruginosa. Majority of patients developed acute kidney injury (35.3%) and congestive cardiac failure (23.5%). CONCLUSION: IE patients in our center exhibited differences from the west in terms of age at presentation and predisposing factors but held similarity in terms of commonly isolated microorganisms. The changing patterns of IE, etiological agents, and their antimicrobial susceptibility observed in this study may be helpful for clinicians in formulating a new empirical antibiotic treatment protocol.

A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings.

With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of leishmania DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45-98.42) and 88.85% (95% CI: 85.08-91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of leishmania DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.

Pharmacovigilance of Antiretroviral Drugs at B.P. Koirala Institute of Health sciences.

BACKGROUND: Antiretroviral drugs are lifeline for patients living with HIV. Adverse drug reactions can compromise the compliance to antiretroviral therapy. The objectives of the study were to estimate the prevalence of adverse drug reactions and to assess its risk factors in patients living with HIV and receiving antiretroviral therapy. METHODS: A prospective cohort study was conducted among 496 patients living with HIV at B.P. Koirala Institute of Health Sciences for a period of one year. Adverse drug reactions were evaluated based upon clinical history, clinical examination and investigations. RESULTS: Majority of patients were of 31-45 year age group (58.1%) and on first-line antiretroviral therapy regimen (94.3%). Total of 240 adverse drug reactions were documented. Prevalence of adverse drug reaction was 34.7%. Skin rash, anemia and nausea and vomiting were the three most common adverse drug reactions. The adverse drug reactions were more common in patients having non-communicable diseases, chronic co-infections, taking more than 3 non-HIV drugs, second and third-line antiretroviral regimen and it was statistically significant (P-value < 0.05). CONCLUSIONS: Prevalence of adverse drug reaction was high in the patients living with HIV. Age, gender, co-infections, non-communicable diseases, taking more than three non-HIV drugs and second and third-line antiretroviral regimen were identified as possible risk factor for occurrence of adverse drug reactions and their prior identification is important to optimize the best suited antiretroviral regimen.

Typhoid Fever among Admitted Pediatric Patients in a Tertiary Care Center: A Descriptive Cross-sectional Study.

INTRODUCTION: Typhoid fever, an acute systemic febrile illness caused by Salmonella Typhi & Paratyphi, is an important public health problem in developing countries. It requires frequent observation regarding proper diagnostic protocol and treatment practices. The aim of the study is to find the prevalence of typhoid fever among admitted pediatric patients in a tertiary care center. METHODS: This is a descriptive cross-sectional study conducted among the admitted patients of pediatric and adolescent medicine of a tertiary care center from August 2016 to May 2018 after obtaining ethical clearance (IRC/609/015). Convenience sampling was used and data was analyzed using the Statistical Package of Social version 11.5. Point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: Among 7450 patients, 151 (2.03%) at 95% Confidence Interval (1.71-2.35) patients were diagnosed with enteric fever of which 85 (56.29%) were male and 66 (43.71%) were female. Common symptoms were fever 151 (100%), and abdominal pain 94 (62.25%). Azithromycin 54 (38.03%) was the most common antibiotic received before presenting to hospital and ceftriaxone 151 (100%) was prescribed to all the patients after admission. Two-third of the patients (96/151) was hospitalized for at least 6 days, with the longest hospital stay of 14 days and shortest of 3 days. CONCLUSIONS: Occurrence rate of Typhoid Fever was similar to other studies. Antibiotic susceptibility could not be well established; further surveillance on typhoid fever and the antimicrobial susceptibility pattern is recommended.

Challenges in Laboratory Diagnosis of Malaria in a Low-Resource Country at Tertiary Care in Eastern Nepal: A Comparative Study of Conventional vs. Molecular Methodologies.

For ongoing malaria elimination programmes, available methods such as microscopy and rapid diagnostic tests (RDTs) cannot detect all malaria cases in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load, and skilled technical resources. Our study objectives were to estimate the performance of light microscopy and a RDT as well as real-time PCR for the detection of the Plasmodium parasite. Altogether, 52 blood samples collected from patients with acute febrile illness were tested by microscopy, RDT, and real-time PCR. The results were compared in terms of sensitivity and specificity. Microscopy detected the malaria parasite in 5.8% of the blood samples whereas 13.5% were detected by the RDT and 27% by real-time PCR. Considering real-time PCR as the gold standard method, microscopy had a sensitivity of 21.4% and a specificity of 100%, and the RDT had a sensitivity of 28.6% and a specificity of 92.1%. Microscopy together with the RDT successfully detected malaria positive cases in blood samples of Ct value below 20, but both were unable to detect malaria cases between 26-40 Ct value ranges amplified by real-time PCR. Despite various diagnostic tools being available, microscopy still remains the method of choice for diagnosis, while the RDT is user-friendly when applied at the point of care. However, our preliminary results emphasize the need to implement the test with higher sensitivity and specificity in the context of a malaria elimination programme. Such programmes can be a crucial opportunity to understand the species prevalent in a low-endemic region. However, these results should be further verified with a large cohort study to document the submicroscopic infection.

Urinary Calculi: A Microbiological and Biochemical Analysis at a Tertiary Care Hospital in Eastern Nepal.

BACKGROUND: The occurrence of urinary tract infection in presence of urolithiasis is frequently noted; however, microbial agents of urolithiasis and their antimicrobial susceptibility patterns remain underinvestigated. This study aimed to identify the microorganisms isolated from urine and stone matrices to determine their antimicrobial susceptibility, to find the association between the pathogens of urine and stone matrices, and to perform the biochemical analysis of stones. METHODS: A total of 88 cases of urolithiasis admitted for elective stone removal at Department of surgery, B.P. Koirala Institute of Health Sciences (BPKIHS), were enrolled. Preoperative urine culture and postoperative stone culture were performed. Isolation, identification, and AST were done by the standard microbiological technique. Further qualitative biochemical analysis of stones was also attempted. RESULT: Among 88 stone formers recruited, culture of urine, whole stone, and nidus yielded the growth of bacteria 44, 32, and 30, respectively. Bacteria isolated from urine culture correlated with those from stone matrices with a sensitivity of 90%, specificity of 79.69%, PPV of 63.64%, and NPV of 95.45%. Escherichia coli (46.7%) was the most common bacteria followed by Klebsiella pneumoniae (16.7%) and Proteus mirabilis (13.3%) from urine and stone cultures. Almost all the uropathogens isolated were susceptible to commonly used antibiotics. Calcium oxalate (84.1%) was common biochemical constituent found in stone formers followed by calcium oxalate + phosphate (8%). CONCLUSIONS: The association of microorganism isolated from urine and nidus culture was significant that can predict the source of infective stone; however, in some cases, microorganisms and the antimicrobial susceptibility pattern from urine and nidus were different. This study emphasizes the use of appropriate antimicrobial agents to prevent the regrowth of residual stones and minimize the risk of infectious complications after surgical removal of stones.

Antibiotic Resistance and mecA Gene Characterization of Coagulase-negative Staphylococci Isolated from Clinical Samples in Nepal.

BACKGROUND: Coagulase-negative Staphylococci (CoNS) are a significant cause of hospital-acquired and foreign-body-related infections. We conducted this research to assess methicillin susceptibility of CoNS by disc diffusion, agar dilution, and polymerase chain reaction (PCR) methods and to assess the antimicrobial susceptibility pattern. METHODS: We received 123 CoNS isolates from different specimens including blood, endotracheal tube, and central venous catheter. We performed sample processing, identification, and characterization following standard guidelines. Antimicrobial susceptibility was tested based on clinical and laboratory standards institute guidelines. We detected methicillin-resistant coagulase-negative staphylococci (MRCoNS) through mecA gene, disc diffusion method, and agar dilution method and compared the accuracy with PCR as reference. RESULTS: We detected eight species of CoNS with Staphylococcus epidermidis as the most common. Most of the samples were received from the intensive care unit and blood was the dominant specimen followed by endotracheal-tube aspirate. Seventy-one percentage of isolates were methicillin-resistant by PCR method; disc diffusion and agar dilution method detected methicillin resistance with an accuracy of 96.7% and 98.3%, respectively. Antimicrobial susceptibility revealed an association between the different origins of samples, and also among the types of sample. Similarly, a comparison of the degree of resistance of antimicrobial agents between mecA gene positive and negative isolates showed significant differences. Vancomycin, linezolid, and teicoplanin are still effective for treating MRCoNS. CONCLUSION: CoNS are a crucial cause of human infections especially in an intensive care unit setup where the use of devices is common. Disc diffusion and agar dilution are reliable for the detection of MRCoNS. The degree of antimicrobial resistance is much higher in organisms obtained from intensive care unit and foreign-body-related infections.

Impact of the visceral leishmaniasis elimination initiative on Leishmania donovani transmission in Nepal: a 10-year repeat survey.

BACKGROUND: Nepal launched a visceral leishmaniasis (also known as kala-azar) elimination initiative in 2005. We primarily aimed to assess whether transmission of Leishmania donovani had decreased since the launch of the initiative. We also assessed the validity of the direct agglutination test (DAT) as a marker of infection, in view of future surveillance systems. METHODS: We did a repeat survey in a population aged 2 years and older for whom baseline serological data were available from 2006. Data were from three districts in the eastern region of Nepal. The primary outcome of interest was prevalent infection with L donovani as measured with DAT (cutoff value ≥1:3200). We compared age group-specific and cluster-specific seroprevalences in 2016 with those in 2006, using χ2 tests, with a specific focus on the comparison of seroprevalences in children born between 1996 and 2005, and those born between 2006 and 2015. To estimate the overall adjusted risk ratio for being seropositive in 2016 compared with 2006, we fitted a Poisson model controlling for age, sex, and cluster. FINDINGS: Between Oct 17, 2016, and Dec 26, 2016, we assessed 6609 individuals. DAT prevalence in children younger than 10 years was 4·1% (95% CI 3·2-5·4) in 2006 versus 0·5% (0·1-1·7) in 2016 (p<0·0001). Seroprevalence was lower in 2016 than in 2006 in all age groups and in all repeated clusters. The overall adjusted risk ratio of being seropositive was 0·44 (95% CI 0·37-0·52) for 2016 compared with 2006, and 0·04 (0·01-0·16) in children younger than 10 years. INTERPRETATION: Our findings show that transmission of L donovani in Nepal has decreased significantly between 2006 and 2016, coinciding with the elimination programme. DAT seems useful for monitoring of L donovani transmission. FUNDING: The Directorate-General for Development Cooperation of Belgium.

Application of kDNA Minicircle PCR-RFLP to Characterize Leishmania donovani Clinical Isolates Obtained from Post-Kala-Azar Dermal Leishmaniasis in Eastern Nepal.

Post-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation of visceral leishmaniasis (VL) which develops after apparent cure in some patients. PKDL is considered as the potential reservoir for the VL infection. Molecular epidemiological characterization of L. donovani isolates obtained from VL and PKDL isolates is essentially required in order to understand the transmission dynamics of the VL infection. To date, genetic variation among the VL and PKDL L. donovani isolates was not fully elucidated. Therefore, 14 clinical isolates from VL and 4 clinical isolates from PKDL were speciated by hsp70 and rDNA genes. Further characterization of L. donovani by haspB PCR demonstrates two different genotypes. All PKDL isolates have the same genetic structure. kDNA PCR-RFLP assay revealed 18 different genotypes; however, structural analysis showed the two distinct kDNA genotype population (k = 2). The kDNA fingerprint patterns of parasites from hilly districts were clustered separately from low-land districts. Therefore, further study with a large number of samples is urgently required for systematic characterization of the clinical isolates to track the molecular epidemiology of the Leishmania donovani causing VL and the role of PKDL as a reservoir.