Post kala-azar dermal leishmaniasis burden at the village level in selected high visceral leishmaniasis endemic upazilas in Bangladesh.

OBJECTIVES: As post kala-azar Dermal Leishmaniasis (PKDL) threatens the success of the Visceral Leishmaniasis (VL) elimination initiative, we aimed to investigate the PKDL burden, including an active search for PKDL in leprosy-negative skin lesion cases. We also investigated their health-seeking behavior and perceived level of stigma. METHODS: This was a cross-sectional survey among inhabitants in the VL-endemic villages of the five most VL-endemic upazilas. VL experts trained medical officers in Upazila Health Complexes (UHCs) and leprosy facilities in PKDL management. Frontline workers conducted house-to-house surveys, referring PKDL suspects to designated centers. Data analysis involved Epi Info version 7 and IBM SPSS Statistics 25. RESULTS: Among 472,435 screened individuals, 4022 had past VL (0.85 %). Among the screened population, 82 were PKDL suspects, and 62 PKDL cases were confirmed. The overall PKDL burden was 1.3 (95 % CI: 1.0-1.7) in the 10,000 population in the endemic villages. Male predominance and macular form of PKDL were observed. Thirty-nine PKDL patients perceived stigma of different levels. Only 27 of 62 (44 %) had received PKDL treatment. Medicine's unavailability and side effects were a major reason behind treatment interruption. Active screening among 137 leprosy-negative PKDL suspects yielded 10 (7.3 %) PKDL cases. CONCLUSION: The existence of PKDL cases in the VL endemic areas is a concern as those are inter-epidemic reservoirs. As per the WHO roadmap, the PKDL burden must be reduced by 70 % and 100 %, respectively, by 2026 and 2030. NKEP can take the current burden of 1.3 per 10,000 people in VL endemic villages as a baseline. Integrating active case detection for PKDL in leprosy hospitals and screening centers is feasible and worth deploying nationwide.

A phase II, non-comparative randomised trial of two treatments involving liposomal amphotericin B and miltefosine for post-kala-azar dermal leishmaniasis in India and Bangladesh.

BACKGROUND: In Southeast Asia, treatment is recommended for all patients with post-kala-azar dermal leishmaniasis (PKDL). Adherence to the first-line regimen, twelve weeks of miltefosine (MF), is low and ocular toxicity has been observed with this exposure period. We assessed the safety and efficacy of two shorter-course treatments: liposomal amphotericin B (LAmB) alone and combined with MF. METHODOLOGY/PRINCIPAL FINDINGS: An open-label, phase II, randomized, parallel-arm, non-comparative trial was conducted in patients with parasitologically confirmed PKDL, 6 to ≤60 years. Patients were assigned to 20 mg/kg LAmB (total dose, in five injections over 15 days) alone or combined with allometric MF (3 weeks). The primary endpoint was definitive cure at 12 months, defined as complete resolution of papular and nodular lesions and >80% re-pigmentation of macular lesions. Definitive cure at 24 months was a secondary efficacy endpoint. 118/126 patients completed the trial. Definitive cure at 12 months was observed in 29% (18/63) patients receiving LAmB and 30% (19/63) receiving LAmB/MF (mITT), increasing to 58% and 66%, respectively, at 24 months. Most lesions had resolved/improved at 12 and 24 months for patients receiving LAmB (90%, 83%) and LAmB/MF (85%, 88%) by qualitative assessment. One death, unrelated to study drugs, was reported; no study drug-related serious adverse events were observed. The most frequent adverse drug reactions were MF-related vomiting and nausea, and LAmB-related hypokalaemia and infusion reactions. Most adverse events were mild; no ocular adverse events occurred. CONCLUSIONS/SIGNIFICANCE: Both regimens are suitably safe and efficacious alternatives to long-course MF for PKDL in South Asia. TRIAL REGISTRATION: CTRI/2017/04/008421.

Skin pharmacokinetics of miltefosine in the treatment of post-kala-azar dermal leishmaniasis in South Asia.

INTRODUCTION: Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL. METHODS: Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin. RESULTS: Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 µg/g (IQR: 21.94-60.65 µg/g) in skin and 33.29 µg/mL (IQR: 25.9-42.58 µg/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively. CONCLUSION: This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL.

A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Successful treatment of recurrent visceral leishmaniasis relapse in an immunocompetent adult female with functional hypopituitarism in Bangladesh.

BACKGROUND: Currently available treatment options are mostly effective in achieving long-term cure in visceral leishmaniasis (VL) patients. However, there have been reports of recurrence of this illness in both immunosuppressed and immunocompetent patients. CASE PRESENTATION: We report the first case of recurrent VL relapse in a 19-year-old immunocompetent female with functional hypopituitarism (hypogonadotropic hypogonadism with central hypothyroidism) from Bangladesh, who has been treated three times previously with optimal dosage and duration- liposomal amphotericin B (LAmB) alone and in combination with miltefosine. We treated the patient successfully with a modified treatment regimen of 10 mg/kg body weight LAmB for two consecutive days along with oral miltefosine for seven days as loading dose. For secondary prophylaxis, the patient received 3 mg/kg body weight LAmB along with oral miltefosine for seven days monthly for five doses followed by hormonal replacement. The patient remained relapse free after 12 months of her treatment completion. CONCLUSION: In the absence of protective vaccines against Leishmania species and standard treatment regimen, this modified treatment regimen could help the management of recurrent relapse cases.

Childhood growth during recovery from acute illness in Africa and South Asia: a secondary analysis of the childhood acute illness and nutrition (CHAIN) prospective cohort.

Background: Growth faltering is well-recognized during acute childhood illness and growth acceleration during convalescence, with or without nutritional therapy, may occur. However, there are limited recent data on growth after hospitalization in low- and middle-income countries. Methods: We evaluated growth following hospitalization among children aged 2-23 months in sub-Saharan Africa and South Asia. Between November 2016 and January 2019, children were recruited at hospital admission and classified as: not-wasted (NW), moderately-wasted (MW), severely-wasted (SW), or having nutritional oedema (NO). We describe earlier (discharge to 45-days) and later (45- to 180-days) changes in length-for-age [LAZ], weight-for-age [WAZ], mid-upper arm circumference [MUACZ], weight-for-length [WLZ] z-scores, and clinical, nutritional, and socioeconomic correlates. Findings: We included 2472 children who survived to 180-days post-discharge: NW, 960 (39%); MW, 572 (23%); SW, 682 (28%); and NO, 258 (10%). During 180-days, LAZ decreased in NW (-0.27 [-0.36, -0.19]) and MW (-0.23 [-0.34, -0.11]). However, all groups increased WAZ (NW, 0.21 [95% CI: 0.11, 0.32]; MW, 0.57 [0.44, 0.71]; SW, 1.0 [0.88, 1.1] and NO, 1.3 [1.1, 1.5]) with greatest gains in the first 45-days. Of children underweight (<-2 WAZ) at discharge, 66% remained underweight at 180-days. Lower WAZ post-discharge was associated with age-inappropriate nutrition, adverse caregiver characteristics, small size at birth, severe or moderate anaemia, and chronic conditions, while lower LAZ was additionally associated with household-level exposures but not with chronic medical conditions. Interpretation: Underweight and poor linear growth mostly persisted after an acute illness. Beyond short-term nutritional supplementation, improving linear growth post-discharge may require broader individual and family support. Funding: Bill & Melinda Gates FoundationOPP1131320; National Institute for Health ResearchNIHR201813.

Portable smartphone-based molecular test for rapid detection of Leishmania spp.

PURPOSE: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL. METHODS: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user. RESULTS: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively. CONCLUSION: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden.

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

Vaccine value profile for leishmaniasis.

Leishmania infections are global, occurring in 98 countries and all World Health Organization (WHO) regions with 600 million to 1 billion people at risk of infection. Visceral leishmaniasis is associated with almost 20,000 reported deaths annually, with children under 5 years of age being at the greatest risk of mortality. Amongst WHO-recognised Neglected Tropical Diseases (NTDs), leishmaniasis is one of the most important in terms of mortality and morbidity. With an increasing global burden of disease and a growing threat from climate change, urbanisation and drug resistance, there remains an imperative to develop leishmaniasis vaccines. New tools to understand correlates of protection and to assess vaccine efficacy are being developed to ease the transition into larger scale efficacy trials or provide alternate routes to licensure. Early indications suggest a diverse portfolio of manufacturers exists in endemic countries with an appetite to develop leishmaniasis vaccines. This Vaccine Value Profile (VVP) provides a high-level, comprehensive assessment of the currently available data to inform the potential public health, economic, and societal value of leishmaniasis vaccines. The leishmaniasis VVP was developed by a working group of subject matter experts from academia, public health groups, policy organizations, and non-profit organizations. All contributors have extensive expertise on various elements of the leishmaniasis VVP and have collectively described the state of knowledge and identified the current gaps. The VVP was developed using only existing and publicly available information.

Multidrug-resistant Escherichia coli isolated from patients and surrounding hospital environments in Bangladesh: A molecular approach for the determination of pathogenicity and resistance.

Extended spectrum ß-lactamase producing Escherichia coli (ESBL E. coli) is a primary concern for hospital and community healthcare settings, often linked to an increased incidence of nosocomial infections. This study investigated the characteristics of ESBL E. coli isolated from hospital environments and clinical samples. In total, 117 ESBL E. coli isolates were obtained. The isolates were subjected to molecular analysis for the presence of resistance and virulence genes, antibiotic susceptibility testing, quantitative adherence assay, ERIC-PCR for phylogenetic analysis and whole genome sequencing of four highly drug resistant isolates. Out of the 117 isolates, 68.4% were positive for blaCTX-M, 39.3% for blaTEM, 30.8% for blaNDM-1, 13.7% for blaOXA and 1.7% for blaSHV gene. Upon screening for diarrheagenic genes, no isolates were found to harbour any of the tested genes. In the case of extraintestinal pathogenic E. coli (ExPEC) virulence factors, 7.6%, 11%, 5.9%, 4.3% and 21.2% of isolates harbored the focG, kpsMII, sfaS, afa and iutA genes, respectively. At a temperature of 25°C, 14.5% of isolates exhibited strong biofilm formation with 21.4% and 28.2% exhibiting moderate and weak biofilm formation respectively, whereas 35.9% were non-biofilm formers. On the other hand at 37°C, 2.6% of isolates showed strong biofilm formation with 3.4% and 31.6% showing moderate and weak biofilm formation respectively, whereas, 62.4% were non-biofilm formers. Regarding antibiotic susceptibility testing, all isolates were found to be multidrug-resistant (MDR), with 30 isolates being highly drug resistant. ERIC-PCR resulted in 12 clusters, with cluster E-10 containing the maximum number of isolates. Hierarchical clustering and correlation analysis revealed associations between environmental and clinical isolates, indicating likely transmission and dissemination from the hospital environment to the patients. The whole genome sequencing of four highly drug resistant ExPEC isolates showed the presence of various antimicrobial resistance genes, virulence factors and mobile genetic elements, with isolates harbouring the plasmid incompatibility group IncF (FII, FIB, FIA). The sequenced isolates were identified as human pathogens with a 93.3% average score. This study suggests that ESBL producing E. coli are prevalent in the healthcare settings of Bangladesh, acting as a potential reservoir for AMR bacteria. This information may have a profound effect on treatment, and improvements in public healthcare policies are a necessity to combat the increased incidences of hospital-acquired infections in the country.

Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era.

BACKGROUND: Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. METHODOLOGY AND PRINCIPAL FINDINGS: Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. CONCLUSION: Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.

Assessment of treatment outcomes of visceral leishmaniasis (VL) treated cases and impact of COVID-19 on VL management and control services in Bangladesh.

BACKGROUND: COVID-19 has largely impacted the management of Visceral leishmaniasis (VL), like several other Neglected Tropical Diseases. The impact was particularly evident in Lower and Middle-Income countries where the already inadequate healthcare resources were diverted to managing the COVID-19 pandemic. Bangladesh achieved the elimination target for VL in 2016. To sustain this success, early diagnosis and treatment, effective vector control, and periodic surveillance are paramount. However, the specific control measures for VL in Bangladesh that were hampered during COVID-19 and their extent are unknown. METHODS: This study aimed at identifying the gaps and challenges in the follow-up of treated VL patients by interviewing both the treated VL cases and their health service providers. We followed VL cases treated between 2019 and 2020 in five VL endemic subdistricts (upazilas) both retrospectively and prospectively to monitor clinical improvement, relapse, or other consequences. Moreover, interviews were conducted with the health service providers to assess the impact of COVID-19 on VL case detection, treatment, reporting, vector control operations, and logistic supply chain management. RESULTS: There was no added delay for VL diagnosis; however, VL treatment initiation and reporting time increased almost two-fold due to COVID-19. Indoor Residual Spraying activity was significantly hampered due to a shortage of insecticides. Out of 44 enrolled and treated VL patients, two relapsed (4.5 %), two developed Para Kala-Azar Dermal Leishmaniasis (4.5 %), and three (6.8 %) Post Kala-Azar Dermal Leishmaniasis (PKDL). The health service providers highlighted patients` unwillingness to visit the hospital, financial constraints, and distance from the hospitals as the main reasons for missed follow-up visits (20.5 %). Building good communication in the community, awareness schemes, and incentive-based approaches were suggested as possible solutions to mitigate these problems. CONCLUSION: Long-term follow-up is required for the early detection and management of VL relapse and PKDL cases. Effective vector control measures, capacity development, and identification of new VL hotspots are pivotal in the VL endemic regions to sustain the elimination goal.

Long-Term Efficacy of Insecticidal Wall Painting for Controlling Visceral Leishmaniasis Vectors in Bangladesh.

The success of the visceral leishmaniasis (VL) elimination program largely depends on cost-effective vector control measures. Our goal was to investigate the longevity of the efficacy of insecticidal wall painting (IWP), a new vector control tool, compared with a routine indoor residual spraying (IRS) program for reducing the VL vector density in Bangladesh. This study is the extension of our recent IWP study for VL vector management in Bangladesh, which was undertaken in seven highly VL endemic villages of the Mymensingh district with a 12-month follow-up. In this 24-months follow-up study, we collected sand flies additionally at 15, 18, 21, and 24 months since the interventions from the IWP and control (where the program did routine IRS) clusters to examine the longevity of the efficacy of IWP on sand fly density reduction and mortality. The difference-in-differences regression models were used to estimate the effect of IWP on sand fly reduction against Program IRS. The IWP showed excellent performance in reducing sand fly density and increasing sand fly mortality compared with Program IRS. The effect of IWP for controlling sand flies was statistically significant for up to at least 24 months. The mean female Phlebotomus argentipes density reduction ranged from -56% to -83%, and the P. argentipes sand fly mortality ranged from 81% to 99.5% during the 24-month follow-up period. Considering the duration of the efficacy of IWP for controlling VL vectors, Bangladesh National Kala-azar Elimination Program may consider IWP as the best alternative to IRS for the subsequent phases of the program.

Epidemiological, serological, and entomological aspects of visceral leishmaniasis (VL) in suspected new VL foci in Bangladesh.

The study aimed to explore epidemiological, serological, and entomological aspects of visceral leishmaniasis (VL) in suspected new VL foci and assess the knowledge, attitude, and practices of the community living in the alleged new VL foci. The study investigated new visceral leishmaniasis (VL) cases reported between 2019 and 2020 in four sub-districts (Dharmapasha, Hakimpur, Islampur and Savar) where we tested 560 members using the rK39 rapid test and conducted vector collections in six neighbouring houses of the index cases to assess sandfly density and distribution, examined sandflies' infection, and determined the spatial relationship with VL infection. Furthermore, we highlighted the importance of early detection, and community awareness in controlling the spread of the disease. The study screened 1078 people from 231 households in the four sub-districts for fever, history of visceral leishmaniasis (VL), and PKDL-like skin lesions. Among sub-districts, positivity rate for rK39 rapid test was highest (3.5 %) in Savar. Sandflies were present across all areas except in Dharmapasha, but all 21 collected female P. argentipes sandflies were negative for Leishmania parasite DNA. We found one person from Islampur with a history of VL, and one from Islampur and another one from Savar had PKDL. After the awareness intervention, more people became familiar with VL infection (91.2 %), and their knowledge concerning sandflies being the vector of the disease and the risk of having VL increased significantly (30.1 %). The study found no active case in the suspected new foci, but some asymptomatic individuals were present. As sandfly vectors exist in these areas, the National Kala-azar Elimination Programme (NKEP) should consider these areas as kala-azar endemic and initiate control activities as per national guidelines.

Genomic and functional portrait of multidrug-resistant, hydrogen sulfide (H2S)-producing variants of Escherichia coli.

Atypical Escherichia coli forms exhibit unusual characteristics compared to typical strains. The H2S-producing variants of some atypical E. coli strains cause a wide range of illnesses in humans and animals. However, there are sparse reports on such strains worldwide. We performed whole-genome sequencing (WGS) and detailed characterization of four H2S-producing E. coli variants from poultry and human clinical sources in Dhaka, Bangladesh. All four isolates were confirmed as E. coli using biochemical tests and genomic analysis, and were multidrug-resistant (MDR). WGS analysis including an additional Chinese strain, revealed diverse STs among the five H2S-producing E. coli genomes, with clonal complex ST10 being detected in 2 out of 5 genomes. The predominant phylogroup detected was group A (n = 4/5). The blaTEM1B (n = 5/5) was the most predominant extended-spectrum beta-lactamase (ESBL) gene, followed by different alleles of blaCTX-M (blaCTX-M -55,-65,-123; n = 3/5). Multiple plasmid replicons were detected, with IncX being the most common. One E. coli strain was classified as enteropathogenic E. coli. The genomes of all five isolates harbored five primary and four secondary function genes related to H2S production. These findings suggest the potential of these isolates to cause disease and spread antibiotic resistance. Therefore, such atypical E. coli forms should be included in differential diagnosis to understand the pathogenicity, antimicrobial resistance and evolution of H2S-producing E. coli.

Case report: A successfully treated case of community-acquired urinary tract infection due to Klebsiella aerogenes in Bangladesh.

Klebsiella aerogenes, a nosocomial pathogen, is increasingly associated with extensive drug resistance and virulence profiles. It is responsible for high morbidity and mortality. This report describes the first successfully treated case of community-acquired urinary tract infection (UTI) caused by Klebsiella aerogenes in an elderly housewife with Type-2 diabetes (T2D) from Dhaka, Bangladesh. The patient was empirically treated with intravenous ceftriaxone (500 mg/8 h). However, she did not respond to the treatment. The urine culture and sensitivity tests, coupled with bacterial whole-genome sequencing (WGS) and analysis, revealed the bacteria to be K. aerogenes which was extensively drug-resistant but was susceptible to carbapenems and polymyxins. Based on these findings, meropenem (500 mg/8 h) was administered to the patient, who then responded to the treatment and recovered successfully without having a relapse. This case raises awareness of the importance of diagnosis of not-so-common etiological agents, correct identification of the pathogens, and targeted antibiotic therapy. In conclusion, correctly identifying etiological agents of UTI using WGS approaches that are otherwise difficult to diagnose could help improve the identification of infectious agents and improve the management of infectious diseases.

Genome dynamics of high-risk resistant and hypervirulent Klebsiella pneumoniae clones in Dhaka, Bangladesh.

Klebsiella pneumoniae is recognized as an urgent public health threat because of the emergence of difficult-to-treat (DTR) strains and hypervirulent clones, resulting in infections with high morbidity and mortality rates. Despite its prominence, little is known about the genomic epidemiology of K. pneumoniae in resource-limited settings like Bangladesh. We sequenced genomes of 32 K. pneumoniae strains isolated from patient samples at the International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b). Genome sequences were examined for their diversity, population structure, resistome, virulome, MLST, O and K antigens and plasmids. Our results revealed the presence of two K. pneumoniae phylogroups, namely KpI (K. pneumoniae) (97%) and KpII (K. quasipneumoniae) (3%). The genomic characterization revealed that 25% (8/32) of isolates were associated with high-risk multidrug-resistant clones, including ST11, ST14, ST15, ST307, ST231 and ST147. The virulome analysis confirmed the presence of six (19%) hypervirulent K. pneumoniae (hvKp) and 26 (81%) classical K. pneumoniae (cKp) strains. The most common ESBL gene identified was blaCTX-M-15 (50%). Around 9% (3/32) isolates exhibited a difficult-to-treat phenotype, harboring carbapenem resistance genes (2 strains harbored blaNDM-5 plus blaOXA-232, one isolate blaOXA-181). The most prevalent O antigen was O1 (56%). The capsular polysaccharides K2, K20, K16 and K62 were enriched in the K. pneumoniae population. This study suggests the circulation of the major international high-risk multidrug-resistant and hypervirulent (hvKp) K. pneumoniae clones in Dhaka, Bangladesh. These findings warrant immediate appropriate interventions, which would otherwise lead to a high burden of untreatable life-threatening infections locally.

Genomic attributes of Vibrio cholerae O1 responsible for 2022 massive cholera outbreak in Bangladesh.

In 2022, one of its worst cholera outbreaks began in Bangladesh and the icddr,b Dhaka hospital treated more than 1300 patients and ca. 42,000 diarrheal cases from March-1 to April-10, 20221. Here, we present genomic attributes of V. cholerae O1 responsible for the 2022 Dhaka outbreak and 960 7th pandemic El Tor (7PET) strains from 88 countries. Results show strains isolated during the Dhaka outbreak cluster with 7PET wave-3 global clade strains, but comprise subclade BD-1.2, for which the most recent common ancestor appears to be that responsible for recent endemic cholera in India. BD-1.2 strains are present in Bangladesh since 2016, but not establishing dominance over BD-2 lineage strains2 until 2018 and predominantly associated with endemic cholera. In conclusion, the recent shift in lineage and genetic attributes, including serotype switching of BD-1.2 from Ogawa to Inaba, may explain the increasing number of cholera cases in Bangladesh.

Revisiting the diagnosis and treatment of Para Kala-azar Dermal Leishmaniasis in the endemic foci of Bangladesh.

Para Kala-azar Dermal Leishmaniasis (Para-KDL) manifests the concomitant presence of Post Kala-azar Dermal Leishmaniasis and Visceral Leishmaniasis and works as a reservoir of infection. The study discusses the cases and their management and aims to address the gaps within existing methods of diagnosis and treatment. This retrospective cross-sectional study discusses 16 Para-KDL cases with one-year follow-up data, treated between 2012-2021 at the Surya Kanta Kala-azar Research Center, Bangladesh. We collected data from hospital records and used STATA 16 to analyze and see the frequency distribution and variable means. We found five patients without any history of kala-azar infection. All the patients were treated with 20 mg/kg Liposomal Amphotericin B in 4 divided doses except one with a history of AmBisome hypersensitivity. One year after treatment, all patients were free from skin lesions, with no hepatosplenomegaly, and observed significant improvement in BMI and hemoglobin levels. The Para-KDL patients are challenging to diagnose, and the relapse and treatment failure leishmania patients might have belonged to this rare group, contributing to their poor prognosis. Therefore, developing an appropriate diagnostic workflow and a new drug regimen is essential to sustain the success of our elimination efforts.

Assessment of pan-Leishmania detection by recombinase polymerase amplification assay.

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.