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.

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.

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.

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.

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.

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.

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.

Genomes of Leishmania parasites directly sequenced from patients with visceral leishmaniasis in the Indian subcontinent.

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.

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.

Bacteriological Profile and Antimicrobial Susceptibility Pattern among Isolates Obtained From Body Fluids.

BACKGROUND: Infections of the sterile body sites, when occurs typically have greater clinical urgency and these infections could be life threatening. For the appropriate management of patient, early detection and identification of organism is crucial. Hence, the present study was designed to evaluate the prevalence, organism profile and antimicrobial susceptibility pattern of isolates obtained from infection of body fluids. METHODS: Laboratory and clinical records of all the sterile body fluid except blood and cerebrospinal fluid submitted to the Department of Microbiology, B. P. Koirala Institute of Health Sciences during the period of 5 years (January 2012 to December 2016) were analyzed. RESULTS: Among 1835 samples, 196 fluids samples showed growth of organisms with an isolation rate of 10.68%. Escherichia coli (E. coli) was the most common organism followed by Staphylococcus aureus (S. aureus) and Acinetobacter calcoaceticus-baumannii complex. Antimicrobial susceptibility testing showed variable degree of resistance. Thirty percentage of organisms were multi-drug resistant, 10% were extensively-drug resistant, none was pan-drug resistant, 35% of E. coli and K. pneumoniae were extended spectrum ?-lactamase producer and 30% of S. aureus were methicillin-resistant S. aureus. The study showed increasing trends of multi-drug resistant, extensively-drug resistant, methicillin-resistant S. aureus and extended spectrum ?-lactamase over the years. CONCLUSIONS: The study showed infection rate of 10% among normally sterile body fluids. E.coli, S. aureus and Acinetobacter calcoaceticus-baumannii complex are the common organisms. The infections are associated with multi drug resistant organisms. Routine surveillance of multi-drug resistant in infection of body fluids is necessary to guide treatment.

A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain.

Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis.

Application of PCR and Microscopy to Detect Helicobacter pylori in Gastric Biopsy Specimen among Acid Peptic Disorders at Tertiary Care Centre in Eastern Nepal.

BACKGROUND: Helicobacter pylori infection is most prevalent in developing countries. It is an etiological agent of peptic ulcer, gastric adenocarcinoma, and mucosal-associated lymphoid tissue (MALT) lymphoma. Despite the development of different assays to confirm H. pylori infection, the diagnosis of infection is challenged by precision of the applied assay. Hence, the aim of this study was to understand the diagnostic accuracy of PCR and microscopy to detect the H. pylori in the gastric antrum biopsy specimen from gastric disorder patients. METHODS: A total of 52 patients with gastric disorders underwent upper gastrointestinal endoscopy with biopsy. The H. pylori infection in gastric biopsies was identified after examination by microscopy and 23S rRNA specific PCR. The agreement between two test results were analysed by McNemar's test and Kappa coefficient. RESULT: H. pylori infection was confirmed in 9 (17.30%) patients by both assays, 6.25% in antral gastritis, 22.22% in gastric ulcer, 100% in gastric ulcer with duodenitis, 50% in gastric ulcer with duodenal ulcer, and 33.33% in severe erosive duodenitis with antral gastritis. Out of nine H. pylori infection confirmed patients, 3 patients were confirmed by microscopy and 8 patients by PCR. In case of two patients, both microscopy and PCR assay confirmed the H. pylori infection. The agreement between two test results was 86.54% and disagreed by 13.46% (p value > 0.05). CONCLUSION: We found that PCR assay to detect H. pylori is more sensitive than microscopy. However, we advocate for the combination of both assays to increase the strength of diagnostic accuracy due to the absence of the gold standard assay for H. pylori infection.

Incidence and outcomes of multidrug-resistant gram-negative bacteria infections in intensive care unit from Nepal- a prospective cohort study.

Background: Infections caused by multi-drug resistant gram-negative bacterial infections are the principle threats to the critically ill patients of intensive care units. Increasing reports of these infections from the Nepalese intensive care unit underline the clinical importance of these pathogens. However, the impact of these infections on the patient's clinical outcome has not yet been clearly evaluated. The objective of our study was to determine the incidence and associated clinical outcome of multi-drug resistant gram-negative bacterial infections in intensive care unit from a tertiary care center of Nepal. Methods: A prospective cohort study was conducted among adult patients admitted in intensive care unit of B. P Koirala Institute of Health Sciences from July to December 2017. Patients infected with multi-drug resistant gram-negative bacteria, non-multi-drug resistant gram-negative bacteria and those without infection were included. Identification of gram-negative bacteria and their antibiotic susceptibility pattern was performed with standard microbiological methods. Demographic, clinical profiles and outcomes (in-hospital-mortality, intensive care unit and hospital length of stay) were documented. Results: The incidence rate of multi-drug resistant gram-negative bacteria infections was 47 per 100 admitted patients (64/137) with 128 episodes. Acinetobacter species (41%, 52/128) was the commonest followed by Klebsiella pneumoniae (28%, 36/128) and Pseudomonas spp (21%, 27/128). Patients with multi-drug resistant gram-negative bacteria in comparison to non-multi-drug resistant gram-negative bacteria had high healthcare-associated infections (95%, 61/64 versus 20%, 2/10; p = < 0.001). In-hospital-mortality was 38% (24/64), 20% (2/10) and 10% (4/41) in multi-drug resistant, non-multi-drug resistant and uninfected group respectively (p = 0.007). After adjustment for independent risk factors, compared to uninfected patients, the odds ratio (CI) for in-hospital-mortality in multi-drug resistant and non-multi-drug resistant group was (4.7[1.4-15.5], p = 0.01) and 2.60 [0.38-17.8], p = 0.32) respectively. Multi-drug resistant patients also had longer intensive care unit and hospital stay, however, it was statistically insignificant. Conclusion: The incidence of multi-drug resistant gram-negative bacterial infections was remarkably high in our intensive care unit and showed a significant association with healthcare-associated infections and in-hospital-mortality.

Comparative evaluation of methods for the detection of biofilm formation in coagulase-negative staphylococci and correlation with antibiogram.

INTRODUCTION: Coagulase-negative staphylococci (CNS) are normal commensals of human skin and mucous membranes. The objective of the study was to determine the prevalence of CNS among clinical isolates, characterize them up to species level, compare the three conventional methods for detection of biofilm formation, and study their antimicrobial susceptibility pattern. MATERIALS AND METHODS: CNS were obtained from various clinical samples including blood, urine, central venous catheter tips, endotracheal tube aspirate, and pus during a 1-year period (July 1, 2014, to June 30, 2015). Characterization up to species level was done using biochemical tests, and biofilm formation was detected by tube adherence, Congo red agar, and tissue culture plate method. Antimicrobial susceptibility testing was performed following Clinical and Laboratory Standards Institute guidelines. RESULTS: A total of 71 CNS isolates, comprising of seven species were obtained. Staphylococcus epidermidis was the most common species followed by S. saprophyticus and S. haemolyticus. We detected biofilm formation in 71.8% of isolates. Considering the fact that tissue culture plate method is the gold standard, sensitivity of tube adherence method and Congo red agar method was found as 82% and 78%, respectively. The isolates exhibited high resistance toward penicillin (90%), azithromycin (60%), co-trimoxazole (60%), and ceftriaxone (40%), while all were susceptible to vancomycin and linezolid. Biofilm former isolates showed higher resistance than the non-formers. CONCLUSION: Among 71 CNS isolated, S. epidermidis was the most common isolate followed by S. saprophyticus and S. haemolyticus. Biofilm formation was detected in 71.8% of the isolates. All of the methods were effective in detecting biofilm-producing CNS strains. The antimicrobial resistance was significantly higher in biofilm formers than non-formers.

Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent.

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context.

Visceral Leishmaniasis IgG1 Rapid Monitoring of Cure vs. Relapse, and Potential for Diagnosis of Post Kala-Azar Dermal Leishmaniasis.

Background: There is a recognized need for an improved diagnostic test to assess post-chemotherapeutic treatment outcome in visceral leishmaniasis (VL) and to diagnose post kala-azar dermal leishmaniasis (PKDL). We previously demonstrated by ELISA and a prototype novel rapid diagnostic test (RDT), that high anti-Leishmania IgG1 is associated with post-treatment relapse versus cure in VL. Methodology: Here, we further evaluate this novel, low-cost RDT, named VL Sero K-SeT, and ELISA for monitoring IgG1 levels in VL patients after treatment. IgG1 levels against L. donovani lysate were determined. We applied these assays to Indian sera from cured VL at 6 months post treatment as well as to relapse and PKDL patients. Sudanese sera from pre- and post-treatment and relapse were also tested. Results: Of 104 paired Indian sera taken before and after treatment for VL, when deemed clinically cured, 81 (77.9%) were positive by VL Sero K-SeT before treatment; by 6 months, 68 of these 81 (84.0%) had a negative or reduced RDT test line intensity. ELISAs differed in positivity rate between pre- and post-treatment (p = 0.0162). Twenty eight of 33 (84.8%) Indian samples taken at diagnosis of relapse were RDT positive. A comparison of Indian VL Sero K-SeT data from patients deemed cured and relapsed confirmed that there was a significant difference (p < 0.0001) in positivity rate for the two groups using this RDT. Ten of 17 (58.8%) Sudanese sera went from positive to negative or decreased VL Sero K-SeT at the end of 11-30 days of treatment. Forty nine of 63 (77.8%) PKDL samples from India were positive by VL Sero K-SeT. Conclusion: We have further shown the relevance of IgG1 in determining clinical status in VL patients. A positive VL Sero K-SeT may also be helpful in supporting diagnosis of PKDL. With further refinement, such as the use of specific antigens, the VL Sero K-SeT and/or IgG1 ELISA may be adjuncts to current VL control programmes.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Antibiotic resistance and biofilm formation among coagulase-negative staphylococci isolated from clinical samples at a tertiary care hospital of eastern Nepal.

BACKGROUND: Coagulase negative staphylococci were long regarded non-pathogenic as they are the commensals of human skin and mucosa but the recent changes in the medical practice and changes in underlying host populations, they are being considered significant pathogens associated with number of nosocomial infections. The objective of the study was to determine the species, antimicrobial susceptibility pattern, biofilm forming ability of the clinically significant CoNS isolates and to compare the different methods for the detection of biofilm formation. METHODS: A total of 52 clinically significant CoNS isolates obtained from different units during a year period were studied. Characterization was done using standard microbiological guidelines and antimicrobial susceptibility was done following CLSI guidelines. Biofilm formation was detected by using three methods i.e. tissue culture plate method, congo red agar method and tube adherence method. RESULTS: Among 52 isolates, S. epidermidis (52%) was the most common species which was followed by S. saprophyticus (18%) and S. haemolyticus (14%). Antimicrobial susceptibility pattern of CoNS documented resistance of 80% to ampicillin. Resistance to cefoxitin and ceftriaxone was observed in 58% of the isolates. Biofilm formation was observed in 65.38% of the isolates. The accuracy of Congo red agar and tube adherence method for the detection of biofilm formation was 82% and 76% respectively. CONCLUSION: CoNS isolates obtained from clinical samples should be processed routinely and antimicrobial susceptibility testing should be performed. Multidrug-resistant CoNS are prevalent. All the three methods i.e. tissue culture plate, Congo red agar and tube adherence method can be used in detecting biofilm formation.