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Background Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination programme is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. We assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate, and feeding behavior of vector sand flies, Phlebotomus argentipes in three VL endemic districts in two epidemiologically contrasting settings; each district with one village with and one without reported VL cases in recent years.Methods Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Sand fly species level identification was based on morphological characters and verified by the DNA barcoding method. We assessed P. argentipes abundance and seasonality based on the light trap collections, considering it as the standard method of collection. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host.Results Vector abundance varied among districts and village types and peaks in sand fly density was observed in June-July and September-November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1% — 3.7% at 95% credible interval) and 0.6% (0.2% — 1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans, 52.7% in VL villages and 74.2% in non-VL villages, followed by cattle.Conclusion Our findings highlight the risk of ongoing L. donovani transmission in VL endemic districts even in villages not reporting the presence of the disease over the past many years and emphasize the remaining threats to the VL elimination programme in Nepal.

Background: Malaria is still a major health problem in Nepal as 43.3% of population are at risk of infection. However, the national priorities have been given to achieve malaria elimination in Nepal, the question of diag­nostic accuracy could jeopardize the elimination target. Among others, multiplex PCR could be the reliable alternative tool to detect the low num­ber of parasitemia and circulating asymptomatic infection in low endemic countries like Nepal. Hence, this paper aims to analyse the diagnostic pre­cision among microscopy, rapid diagnostic test (RDT) and multiplex real time PCR assay. Methods: A total of 63 archived blood samples obtained from patient with clinical suspicion of malaria were subjected to multiplex PCR and their re­sults were compared with microscopy and rapid diagnostic test. The La­tent Class Analysis (LCA) approach was used to compare the diagnostic precision among three different assays. Results: Our study documented that PCR had 97.4% sensitivity and 83.9% specificity. Among the negative microscopy results, 10 (15.9%) were false negatives whereas 4 (6.3%) RDT results were false negatives based on PCR results. Conclusions: PCR could be the useful alternative tool to detect the low number of parasite in low endemic settings which could be crucial to rule out the false diagnosis determined by microscopy or RDT.

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

Background. Carbapenem resistance among Gram-negative isolates caused by the production of the metallo-β-lactamase (MBL) enzyme is being increasingly reported worldwide. One of the newly emerged metallo-β-lactamases is New Delhi metallo-β-lactamase. Data regarding its occurrence in hospital setting and percentage prevalence among different Gram-negative bacterial isolates are lacking in our part. This study has been undertaken for determining the presence of the bla NDM-1 gene among the clinical isolates of imipenem-resistant Gram-negative bacteria in a tertiary care center in Dharan, Nepal. Methods. A total of 75 imipenem-resistant Gram-negative isolates were studied. These were screened for metallo-β-lactamase (MBL) production by phenotypic assays such as double-disc synergy test (DDST) and combined disc diffusion test (CDDT). PCR was performed for the molecular detection of gene NDM-1. Ten-disc method was performed to detect the presence of ESBL, AmpC, carbapenamase, and K1 β-lactamase production. Results. Using the molecular method, bla NDM-1 was detected in 36% of the isolates. Phenotypically, double-disc synergy test (DDST) and combined disc diffusion test (CDST) detected MBL production in 38.7% and 37.3% of the isolates, respectively. Ten-disc method detected ESBL in 26.6% of the isolates, but none of the isolates was found to be AmpC, carbapenamase, and K1 β-lactamase producers. Conclusion. A high percentage of the NDM-1 producer was noted among imipenem-resistant GNB. Apart from performing only antimicrobial sensitivity test, phenotypic and molecular screening should be employed to find out the actual number of metallo-β-lactamase producers and the existence of the resistance gene.

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.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-spectrumA 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.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 (Drug-resistant Acinetobacter formed a substantial proportion of this hospital's samples with a large presence of the

Correction to: Scientific Reports https://doi.org/10.1038/s41598-019-46283-9, published online 09 July 2019 The original version of this Article contained an error. The information in the “Dipstick development and assay” section was incomplete. Therefore, the original text, “The assay comprises of incubation of the dipstick with diluted serum samples (1:2000) for 30 min which is followed by washing with TBST (twice). Subsequently, it is incubated with HRP conjugated anti-human IgG (1: 2000) for 30 min. Finally, after two washes in TBST and one in TBS, the strips are dipped in a freshly prepared substrate composed of 0.05% 3, 3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, USA) containing 0.05% of H2O2 in 100 mM TBS. The reaction is stopped by dipping in distilled water. The appearance of dark brown coloured bands at both the test and control line indicates VL positivity and a single band at the control line is indicative of VL negativity.” now reads: “The general assay consists of incubation of the dipsticks with diluted serum (1:2000) samples for 30 min, followed by washing with TBST (twice). Subsequently, it is incubated with HRP conjugated anti-human IgG (1:2000) for 30 min. Finally, after two washes in TBST and one in TBS, the strips are dipped in a freshly prepared substrate composed of 0.05% 3, 3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, USA), containing 0.05% of H2O2 in 100 mM TBS. The reaction is stopped by dipping in distilled water, and the strips dried at RT. In Spain, there were several modifications to the assay; the dipsticks are incubated in diluted serum (1:100), and HRP conjugated anti-human IgG, for 60 min, followed by a 10 min incubation in substrate composed of 0.07% of DAB and 0.2% of H2O2 in 60 mM TBS, before being dried at RT. The appearance of dark brown coloured bands at both the test and control line indicates VL positivity and a single band at the control line is indicative of VL negativity. Whenever this signal was hard to assess, we classified it as not clear, and therefore grouped it together with those displaying no bands. Dipsticks that did not present with a reactive band, or a control band, were excluded from further consideration.” These changes do not affect the overall conclusions of the Article. This has now been corrected in the PDF and HTML versions of the Article.

Background For ongoing malaria elimination programme, available methods like microscopy and rapid diagnostic test (RDT) are not able to detect all the cases of malaria in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load and skilled technical resources thus the study was carried out to compare performance of microscopy and RDT which are commonly used in a low resource country along with reference to real-time PCR. Methods 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. Results The test results were as follows: 5.8% positivity by Microscopy, 13.5% positivity by RDT and 27% by real time PCR. Taking into consideration of PCR as a gold standard method microscopy showed 21.4% sensitivity and 100% specificity. Likewise, RDT results revealed 28.6% sensitivity and 92.1% specificity. Conclusion Despite of various diagnostic tools available, microscopy stills remains the gold standard for the diagnosis and RDT is the user friendly to execute the test under the tree, but our preliminary results emphasized the need to implement the test with the higher sensitivity and specificity in context of malaria elimination programme which could be another important opportunity to understand the parasite circulation in case of low endemic region. However, these results should be further verified with the large study cohort in order to document the submicroscopic infection.

Background: Acinetobacter species is an important hospital acquired pathogen. Rapid development of resistance to multiple drug and ability to form biofilm make these bacteria more adaptable to survive in health care facilities, thus posing a challenge for its effective management. This study was aimed to characterize clinical isolates of Acinetobacter spp, study their antimicrobial susceptibility pattern and ability to form biofilm. Resistant Acinetobacter was further analyzed for the detection of extended spectrum β-lactamases (ESBLs), metallo β-lactamases (MBLs) production and presence of blaNDM-1 (New Delhi metallo-beta-lactamase-1) gene.Results: A total of 324 Acinetobacter species isolated from various clinical specimens submitted to Department of Microbiology, B. P. Koirala Institute of Health Sciences, Dharan, were studied Antibiotic susceptibility testing, detection of ESBL and MBL production, and biofilm formation was performed by standard microbiological methods. PCR was done to determine the presence of blaNDM-1 gene.Predominant Acinetobacter species isolated was A calcoaceticus-baumannii Complex (Acb complex) 167 (51.5%). 13 (4.0%) were ESBL producers, 61.9% (70/113) were MBL, 10.6% (12/113) were carbapenemase producers. About 40% were Multi-drug resistant (MDR). Among MDR, producers of ESBL, MBL and carbapenemase were 1.6%, 57% and 9.6%, respectively. Thirty seven percent isolates formed biofilm. A significant proportion of biofilm producers were MDR (p

The diagnosis of extra-pulmonary tuberculosis by Mycobacterium tuberculosis infection in retro­pharyngeal and thyroid abscess is challenging due to a lack of rapid, sensitive and specific diag­nostic assay. Hence, we evaluated the performance of highly sensitive polymerase chain reaction (PCR) to detect the M. tuberculosis in the retropharyngeal and thyroid abscess. The microscopy and culture were negative test result however; PCR result had demonstrated the presence of M. tuberculosis in the same specimens with retropharyngeal and thyroid abscess. Therefore, it is rec­ommended to perform PCR to detect undiagnosed cases, which were not detected by convention­al approaches for the diagnosis, of extra-pulmonary tuberculosis.

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, Author summary Visceral leishmaniasis (VL) is caused by parasitic protozoa of the Leishmania donovani complex and is lethal in the absence of treatment. Whole Genome Sequencing (WGS) of L. donovani clinical isolates revealed hitherto cryptic population structure in the Indian Sub-Continent and provided insights into the epidemiology and potential mechanisms of drug resistance. However, several biases are likely introduced during the culture step. We report here the development of a method that allows determination of parasite genomes directly in clinical samples, and validate it on bone marrow and splenic aspirates of VL patients in Nepal. Our study sheds a new light on the biology of Leishmania in the human host: we found that intracellular parasites of the patients had very low levels of aneuploidy, in sharp contrast to the situation in cultivated isolates. Moreover, the observed differences in genomes between intracellular amastigotes of the patient and the derived cultured parasites suggests polyclonality of infections, with different clones dominating in clinical samples and in culture, likely due to fitness differences. We believe this method is most suitable for clinical studies and for molecular tracking in the context of elimination programs.

Author Correction: A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain. Scientifc Reports. 2021; 11:3967. DOI: 10.1038/s41598-021-83332-8. PMID: 33574485 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. Sí

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. Electronic supplementary material The online version of this article (10.1186/s12879-017-2883-x) contains supplementary material, which is available to authorized users.

Background We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010. Methodology / Principle findings As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002–2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution. Conclusions / Significance Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region., Author summary Visceral Leishmaniasis (VL) or Kala-azar is a life-threatening neglected tropical disease that annually affects half a million people worldwide. In the Indian subcontinent (India, Nepal, Bangladesh), the disease is caused by infection with the protozoan parasite Leishmania donovani, which is transmitted by female sand flies. Currently, the Kala-azar elimination program aims at reducing the number of VL cases in the region to less than 1 in 10.000 at upazila, sub-district and district level in Bangladesh, India, and Nepal respectively. In support of this program, tools for tracking L. donovani populations are essential, because these allow monitoring geographic spread over time. However, the parasite populations in the region are highly homogeneous, requiring sequencing of the entire genome to gather sufficient information for discriminating them. Because whole genome sequencing (WGS) is impractical for large-scale use, we designed a simple alternative to identify the WGS-genotypes. Our method is based on PCR amplification followed by sequencing of one particular locus, diagnostic of each population. We provide proof-of-principle that our method can be used to track parasite populations over time, and to correlate them with clinical parameters. We believe that our assay can support the Kala-azar control efforts in the Indian subcontinent.

The isolation by distance analysis showed the linear correlation between genetic diversity and geographic distance. Three populations were defined such as Sri Lanka, India and Nepal. However, this correlation was not statistically significant. (DOC 175Â kb)

Background. Miltefosine (MIL), the only oral drug for visceral leishmaniasis (VL), is currently the first-line therapy in the VL elimination program of the Indian subcontinent. Given the paucity of anti-VL drugs and the looming threat of resistance, there is an obvious need for close monitoring of clinical efficacy of MIL. Methods. In a cohort study of 120 VL patients treated with MIL in Nepal, we monitored the clinical outcomes up to 12 months after completion of therapy and explored the potential role of drug compliance, parasite drug resistance, and reinfection. Results. The initial cure rate was 95.8% (95% confidence interval [CI], 92.2-99.4) and the relapse rate at 6 and 12 months was 10.8% (95% CI, 5.2-16.4) and 20.0% (95% CI, 12.8-27.2), respectively. No significant clinical risk factors of relapse apart from age

Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector- borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment., SCOPUS: ar.j, info:eu-repo/semantics/published

Background In the Indian subcontinent, Visceral leishmaniasis is endemic in a geographical area coinciding with the Lower Gangetic Plain, at low altitude. VL occurring in residents of hill districts is therefore often considered the result of Leishmania donovani infection during travel. Early 2014 we conducted an outbreak investigation in Okhaldhunga and Bhojpur districts in the Nepal hills where increasing number of VL cases have been reported. Methodology/Principal Findings A house-to-house survey in six villages documented retrospectively 35 cases of Visceral Leishmaniasis (VL). Anti-Leishmania antibodies were found in 22/23 past-VL cases, in 40/416 (9.6%) persons without VL and in 12/155 (7.7%) domestic animals. An age- and sex- matched case-control study showed that exposure to known VL-endemic regions was no risk factor for VL, but having a VL case in the neighbourhood was. SSU-rDNA PCR for Leishmania sp. was positive in 24 (5%) of the human, in 18 (12%) of the animal samples and in 16 (14%) bloodfed female Phlebotomus argentipes sand flies. L. donovani was confirmed in two asymptomatic individuals and in one sand fly through hsp70-based sequencing. Conclusions/Significance This is epidemiological and entomological evidence for ongoing local transmission of L. donovani in villages at an altitude above 600 meters in Nepal, in districts considered hitherto non-endemic for VL. The VL Elimination Initiative in Nepal should therefore consider extending its surveillance and control activities in order to assure VL elimination, and the risk map for VL should be redesigned., Author Summary Visceral leishmaniasis is a neglected but deadly disease occurring in north-eastern India, the south-eastern lowland of Nepal, and the Ganges delta in Bangladesh; all part of the Lower Gangetic plains. Districts at higher altitude, such as those situated in the foothills of the Himalaya in Nepal, are considered non-endemic. Consequently, diagnostic, therapeutic and surveillance facilities are not available, and sporadic cases of VL occurring in residents of these districts are considered the result of Leishmania donovani infection during travel. This parasite is transmitted from man to man through the bite of a sand fly, Phlebotomus argentipes. To investigate the increasing number of cases reported from some villages in the Nepal hills, we interviewed former VL cases, as well as their healthy co-villagers, on their history of travel, we checked their blood for signs of infection, and we set traps to capture sand flies. We found that many residents had been infected, symptomatically as well as asymptomatically, regardless of travelling to endemic areas. Moreover, we managed to capture sand flies and found DNA of leishmania parasites inside. This proves that there is indeed ongoing local transmission in hill districts and that surveillance and control activities should be extended.

A concise linear synthesis of 9,10 secosteroid Calicoferol E 6 has been completed. Key features of the process include: (i) the preparation of hitherto unknown 9α-hydroxycholest-1, 4-diene-3- one 5 from commercially available 5α-cholestan-3β-ol using conventional chemistry; (ii) the acid catalyzed rearrangement of 5 to give 6.

Leishmania donovani is an intracellular protozoan parasite that causes leishmaniasis, which can range from a self-healing cutaneous disease to a fatal visceral disease depending on the infecting species. Miltefosine is currently the latest and only oral antileishmanial that came out of drug discovery pipelines in the past few decades, but recent reports indicate a significant decline in its efficacy against visceral leishmaniasis (also known as kala-azar) in the Indian subcontinent. This relapse rate of up to 20% within 12 months after treatment was shown not to be related to reinfection, drug quality, drug exposure, or drug-resistant parasites. We therefore aimed to assess other phenotypes of the parasite that may affect treatment outcome and found a significant association between the number of metacyclic parasites, parasite infectivity, and patient treatment outcome in the Indian subcontinent. Together with previous studies on resistance of L. donovani against pentavalent antimonials, these data suggest that the infectivity of the parasite, or related phenotypes, might be a more determinant factor for treatment failure in visceral leishmaniasis than drug susceptibility, warranting a reassessment of our current view on treatment failure and drug resistance in leishmaniasis and beyond., IMPORTANCE The high miltefosine relapse rate poses a major challenge for the current Kala-Azar Elimination Program in the Indian subcontinent and other leishmaniasis control programs worldwide. This relapse rate could not be related to reinfection, drug-resistant parasites, or reduced treatment quality. Here we report that an increased infectivity of the parasite is associated with miltefosine relapse of visceral leishmaniasis (VL) patients. These results supplement those obtained with antimonial-resistant L. donovani where an increased infectivity was also observed. This challenges the current view of Leishmania drug susceptibility being the biggest parasitic factor that contributes to treatment failure in leishmaniasis. These selected more infectious parasites may pose an additional burden to leishmaniasis control programs, highlighting the importance of multifaceted control measures to achieve leishmaniasis elimination in the Indian subcontinent and other regions where leishmaniasis is endemic.

Irradiation of 2-formylbenzylamine 7 in the presence of fumarates affords adducts corresponding to trapping of quinodimethane intermediate 9, while photolysis of 2-formylbenzamides leads to amino lactones 16.

Irradiation of 2-formylbenzylamine 7 in the presence of fumarates affords adducts corresponding to trapping of quinodimethane intermediate 9, while photolysis of 2-formylbenzamides leads to amino lactones 16.

Multiple sequence alignment. Multiple sequence alignment of the Sri Lankan, Indian and Nepalese isolates along with the reference sequences using CLUSTAL 2.1 multiple sequence alignment. Dashes(â ) represent gaps introduced to optimize alignment. Asteriks (*) represent consensus nucleotides in the sequence. Lb(P):L. braziliensis [Peru] (Skin);Ld(DD8)/Ld(IN):L. donovani [India] (Bone marrow); Li(SP):L. infantum [Spain] (Unknown); Lm(A1):L. major [Libya]; Lt(TK):L. tropica [Turkey] (Skin); Lam: L. amazonenis (Skin); Li(TK):L. infantum [Turkey] (Spleen); SL-VL (52â 55): VL Sri Lankan isolate (Bone marrow); SL_CL-S:Leishmania CL Sri Lankan isolate (Skin); SL_CL-DR:Leishmania CL Sri Lankan that failed to respond to antimonial drugs (Skin); IN-VL: L. donovani [India] (Bone marrow); IN-PKDL: Leishmania from post-kala azar dermal leishmaniasis (PKDL) [India] (Skin); NP-VL: Leishmania VL Nepalian isolate (Bone marrow); NP-PKDL:Leishmania cause PKDL [Nepal] (Skin). (DOCX 25Â kb)

Multiple sequence alignment. Multiple sequence alignment of the Sri Lankan, Indian and Nepalese isolates along with the reference sequences using CLUSTAL 2.1 multiple sequence alignment. Dashes(â ) represent gaps introduced to optimize alignment. Asteriks (*) represent consensus nucleotides in the sequence. Lb(P):L. braziliensis [Peru] (Skin);Ld(DD8)/Ld(IN):L. donovani [India] (Bone marrow); Li(SP):L. infantum [Spain] (Unknown); Lm(A1):L. major [Libya]; Lt(TK):L. tropica [Turkey] (Skin); Lam: L. amazonenis (Skin); Li(TK):L. infantum [Turkey] (Spleen); SL-VL (52â 55): VL Sri Lankan isolate (Bone marrow); SL_CL-S:Leishmania CL Sri Lankan isolate (Skin); SL_CL-DR:Leishmania CL Sri Lankan that failed to respond to antimonial drugs (Skin); IN-VL: L. donovani [India] (Bone marrow); IN-PKDL: Leishmania from post-kala azar dermal leishmaniasis (PKDL) [India] (Skin); NP-VL: Leishmania VL Nepalian isolate (Bone marrow); NP-PKDL:Leishmania cause PKDL [Nepal] (Skin). (DOCX 25Â kb)