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.

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.

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.

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.

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.

Feasibility of MinION Nanopore Rapid Sequencing in the Detection of Common Diarrhea Pathogens in Fecal Specimen.

The need for fast detection of etiological agents outside the narrow target range of pathogens that may cause an event of an infectious disease epidemic necessitates rapid sequencing technologies to be implemented in routine diagnostic procedures. We tested the performance of a PCR-free rapid nanopore barcoding assay to detect microbial species by analyzing genomic contents extracted from acute diarrheal case specimens. Sequenced reads were processed in an automated analysis module for species identification, whereas pathogenic subspecies detection was aided by a sequence similarity search against a gene-specific database. Evaluation of assay and analysis parameters (e.g., run-time, sequence length, and species hit abundance level) was carried out using a standard bacterial community for assessing detection accuracy. It was observed that longer sequence length (≥500 nucleotides) along with higher species abundance level (≥1%) can be critical for exclusion of false-negative outcomes, while increased sequencing run-time can affect the proportional abundance of true-positive species. Under optimal parameters, the sensitivity of the rapid assay remained 100% for the detection of a target species in a background of nontarget fecal (diarrheal) DNA that weighed up to 64 times the DNA of the target species. The method was applied to acute diarrheal samples. Among these, 62.5% (5/8) were in agreement with target-specific traditional diagnosis methods for the presence/absence of pathogenic agent(s), 12.5% (1/8) were in disagreement, and pathogenic agents that were not targeted by the traditional methods were revealed by sequencing for 25% (2/8) of samples. These observations suggest that further optimization and evaluation of the rapid nanopore sequencing method could potentiate the widening of the range of pathogens that can be detected in acute diarrheal samples in the context of regular diagnostic needs as well as epidemics.

Gauging the skin resident Leishmania parasites through a loop mediated isothermal amplification (LAMP) assay in post-kala-azar dermal leishmaniasis.

Despite the availability of highly sensitive polymerase chain reaction (PCR)-based methods, the dearth of remotely deployable diagnostic tools circumvents the early and accurate detection of individuals with post-kala-azar dermal leishmaniasis (PKDL). Here, we evaluate a design-locked loop-mediated isothermal amplification (LAMP) assay to diagnose PKDL. A total of 76 snip-skin samples collected from individuals with probable PKDL (clinical presentation and a positive rK39 rapid diagnostic test (RDT)) were assessed by microscopy, qPCR, and LAMP. An equal number of age and sex-matched healthy controls were included to determine the specificity of the LAMP assay. The LAMP assay with a Qiagen DNA extraction (Q-LAMP) showed a promising sensitivity of 72.37% (95% CI: 60.91-82.01%) for identifying the PKDL cases. LAMP assay sensitivity declined when the DNA was extracted using a boil-spin method. Q-qPCR showed 68.42% (56.75-78.61%) sensitivity, comparable to LAMP and with an excellent agreement, whereas the microscopy exhibited a weak sensitivity of 39.47% (28.44-51.35%). When microscopy and/or qPCR were considered the gold standard, Q-LAMP exhibited an elevated sensitivity of 89.7% (95% CI: 78.83-96.11%) for detection of PKDL cases and Bayesian latent class modeling substantiated the excellent sensitivity of the assay. All healthy controls were found to be negative. Notwithstanding the optimum efficiency of the LAMP assay towards the detection of PKDL cases, further optimization of the boil-spin method is warranted to permit remote use of the assay.

Evaluation of recombinase-based isothermal amplification assays for point-of-need detection of SARS-CoV-2 in resource-limited settings.

OBJECTIVES: The democratization of diagnostics is one of the key challenges towards containing the transmission of coronavirus disease 2019 (COVID-19) around the globe. The operational complexities of existing PCR-based methods, including sample transfer to advanced central laboratories with expensive equipment, limit their use in resource-limited settings. However, with the advent of isothermal technologies, the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible at decentralized facilities. METHODS: In this study, two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples. A total of 76 real-time reverse transcription PCR (real-time RT-PCR) confirmed COVID-19 cases and 100 negative controls were evaluated to determine the diagnostic performance of the isothermal methods. RESULTS: This investigation revealed equally promising diagnostic accuracy of the two methods, with a sensitivity of 76.32% (95% confidence interval 65.18-85.32%) when the target genes were RdRP and ORF1ab for RT-RPA and RT-RAA, respectively; the combination of N and RdRP in RT-RPA augmented the accuracy of the assay at a sensitivity of 85.53% (95% confidence interval 75.58-92.55%). Furthermore, high specificity was observed for each of the methods, ranging from 94.00% to 98.00% (95% confidence interval 87.40-9.76%). CONCLUSIONS: Considering the diagnostic accuracies, both RT-RPA and RT-RAA appear to be suitable assays for point-of-need deployment for the detection of the pathogen, understanding its epidemiology, case management, and curbing transmission.

Development of Quantitative Rapid Isothermal Amplification Assay for Leishmania donovani.

Quantification of pathogen load, although challenging, is of paramount importance for accurate diagnosis and clinical management of a range of infectious diseases in a point-of-need testing (PONT) scenario such as in resource-limited settings. We formulated a quantification approach to test the standard-curve based absolute quantification ability of isothermal recombinase polymerase amplification (RPA) assay. As a test of principle, a 10-fold dilution series of Leishmania donovani (LD) genomic DNA prepared in nuclease-free-water (NFW), and from culture-spiked-blood (CSB) were tested, and a 15 min assay was performed. A modified algorithm was formulated to derive the detection outcome. The threshold-record times (Tr) in seconds thus obtained were plotted against the initial load of parasite genomes for log-linear regression analysis. The quantitative RPA (Q-RPA) assay was further evaluated against a LD quantitative (q)-PCR assay with DNA extracted from visceral and post-Kala-azar dermal leishmaniasis case specimens and stratified into different ranges of threshold cycle (Ct). The best-fitted regression models were found linear with mean r2/root mean square error (RMSE) values of residual points (in seconds) estimated as 0.996/8.063 and 0.992/7.46 for replicated series of NFW and CSB, respectively. In both series, the lower limit of detection reached less than 0.1 parasite genome equivalent DNA. Absolute agreement between Q-RPA and LD-qPCR was found for test positivity, and strong positive correlations were observed between the Tr and Ct values (r = 0.89; p < 0.0001) as well as between the absolute parasite loads (r = 0.87; p < 0.0001) quantified by respective assays. The findings in this very first Q-RPA assay for leishmaniasis are suggestive of its potential in monitoring LD load in clinical specimens, and the development of rapid Q-RPA assays for other infectious diseases.

Evaluation of molecular assays to detect Leishmania donovani in Phlebotomus argentipes fed on post-kala-azar dermal leishmaniasis patients.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor. METHODS: In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases. RESULTS: The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well. CONCLUSIONS: Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples.

Evaluation of Loopamp™ Leishmania Detection Kit and Leishmania Antigen ELISA for Post-Elimination Detection and Management of Visceral Leishmaniasis in Bangladesh.

With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™ Leishmania Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the Leishmania antigen ELISA (CLIN-TECH, UK) with different sampling techniques and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS via silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the Leishmania antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp-DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the Leishmania antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The Leishmania antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the Leishmania antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.

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.

Detection of asymptomatic Leishmania infection in Bangladesh by antibody and antigen diagnostic tools shows an association with post-kala-azar dermal leishmaniasis (PKDL) patients.

BACKGROUND: Asymptomatic Leishmania infections outnumber clinical infections on the Indian subcontinent (ISC), where disease reservoirs are anthroponotic. Diagnostics which detect active asymptomatic infection, which are suitable for monitoring and surveillance, may be of benefit to the visceral leishmaniasis (VL) elimination campaign on the ISC. METHODS: Quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), and the direct agglutination test (DAT) were carried out on blood samples, and the Leishmania antigen ELISA was carried out on urine samples collected from 720 household and neighbouring contacts of 276 VL and post-kala-azar dermal leishmaniasis (PKDL) index cases, with no symptoms or history of VL or PKDL, in endemic regions of Bangladesh between September 2016 and March 2018. RESULTS: Of the 720 contacts of index cases, asymptomatic infection was detected in 69 (9.6%) participants by a combination of qPCR (1.0%), LAMP (2.1%), DAT (3.9%), and Leishmania antigen ELISA (3.3%). Only one (0.1%) participant was detected positive by all four diagnostic tests. Poor agreement between tests was calculated using Cohen's kappa (κ) statistics; however, the Leishmania antigen ELISA and DAT in combination captured all participants as positive by more than one test. We find evidence for a moderately strong association between the index case being a PKDL case (OR 1.94, p = 0.009), specifically macular PKDL (OR 2.12, p = 0.004), and being positive for at least one of the four tests. CONCLUSIONS: Leishmania antigen ELISA on urine detects active asymptomatic infection, requires a non-invasive sample, and therefore may be of benefit for monitoring transmission and surveillance in an elimination setting in combination with serology. Development of an antigen detection test in a rapid diagnostic test (RDT) format would be of benefit to the elimination campaign.

Development of a portable reverse transcription loop-mediated isothermal amplification system to detect the E1 region of Chikungunya virus in a cost-effective manner.

Chikungunya fever is a mosquito-borne disease cause of persistent arthralgia. The current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse transcription polymerase chain reaction assay. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and simple tool used for DNA-based diagnosis of a variety of infectious diseases. In this study, we established an RT-LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene that could also detect CHIKV at a concentration of 8 PFU without incorrectly detecting other mosquito-borne viruses. The system also amplified the E1 genome in the serum of CHIKV-infected mice with high sensitivity and specificity. Moreover, we established a dry RT-LAMP system that can be transported without a cold chain, which detected the virus genome in CHIKV-infected patient samples with high accuracy. Thus, the dry RT-LAMP system has great potential to be applied as a novel CHIKV screening kit in endemic areas.

Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis.

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

An immunoinformatic approach driven by experimental proteomics: in silico design of a subunit candidate vaccine targeting secretory proteins of Leishmania donovani amastigotes.

BACKGROUND: Visceral leishmaniasis (VL) caused by dimorphic Leishmania species is a parasitic disease with high socioeconomic burden in endemic areas worldwide. Sustaining control of VL in terms of proper and prevailing immunity development is a global necessity amid unavailability of a prophylactic vaccine. Screening of experimental proteome of the human disease propagating form of Leishmania donovani (amastigote) can be more pragmatic for in silico mining of novel vaccine candidates. METHODS: By using an immunoinformatic approach, CD4+ and CD8+ T cell-specific epitopes from experimentally reported L. donovani proteins having secretory potential and increased abundance in amastigotes were screened. A chimera linked with a Toll-like receptor 4 (TLR4) peptide adjuvant was constructed and evaluated for physicochemical characteristics, binding interaction with TLR4 in simulated physiological condition and the trend of immune response following hypothetical immunization. RESULTS: Selected epitopes from physiologically important L. donovani proteins were found mostly conserved in L. infantum, covering theoretically more than 98% of the global population. The multi-epitope chimeric vaccine was predicted as stable, antigenic and non-allergenic. Structural analysis of vaccine-TLR4 receptor docked complex and its molecular dynamics simulation suggest sufficiently stable binding interface along with prospect of non-canonical receptor activation. Simulation dynamics of immune response following hypothetical immunization indicate active and memory B as well as CD4+ T cell generation potential, and likely chance of a more Th1 polarized response. CONCLUSIONS: The methodological approach and results from this study could facilitate more informed screening and selection of candidate antigenic proteins for entry into vaccine production pipeline in future to control human VL.

Bacteriophages Isolated from Stunted Children Can Regulate Gut Bacterial Communities in an Age-Specific Manner.

Stunting, a severe and multigenerational growth impairment, globally affects 22% of children under the age of 5 years. Stunted children have altered gut bacterial communities with higher proportions of Proteobacteria, a phylum with several known human pathogens. Despite the links between an altered gut microbiota and stunting, the role of bacteriophages, highly abundant bacterial viruses, is unknown. Here, we describe the gut bacterial and bacteriophage communities of Bangladeshi stunted children younger than 38 months. We show that these children harbor distinct gut bacteriophages relative to their non-stunted counterparts. In vitro, these gut bacteriophages are infectious and can regulate bacterial abundance and composition in an age-specific manner, highlighting their possible role in the pathophysiology of child stunting. Specifically, Proteobacteria from non-stunted children increased in the presence of phages from younger stunted children, suggesting that phages could contribute to the bacterial community changes observed in child stunting.

Imported cutaneous leishmaniasis: molecular investigation unveils Leishmania major in Bangladesh.

BACKGROUND: The main clinical forms of leishmaniasis in Bangladesh are visceral leishmaniasis and post-kala-azar dermal leishmaniasis, which are caused by Leishmania donovani. Imported cutaneous leishmaniasis (CL) is emerging globally due mainly to increased human mobility. In recent years, several imported CL cases have also been reported in Bangladesh. Sporadic atypical cases of CL can be challenging for diagnosis and clinical management, while occurrence of infection on a frequent basis can be alarming. We report of a case of a Bangladeshi temporary-migrant worker who, upon return, presented development of skin lesions that are characteristic of CL. METHODS: A serum sample was collected and tested with an rK39 immunochromatographic test. Nucleic acid from skin biopsy derived culture sample was extracted and screened with a real-time PCR assay which targets the conserved REPL repeat region of L. donovani complex. The internal transcribed spacer 2 region of the ribosomal RNA gene cluster was amplified and sequenced. RESULTS: The suspect had a history of travel in both CL and VL endemic areas and had a positive rK39 test result. Based on clinical presentation, travel history and demonstration of the parasite in the skin biopsy, CL was diagnosed and the patient underwent a combination therapy with Miltefosine and liposomal amphotericin B. While typical endemic species were not detected, we identified Leishmania major, a species that, to our knowledge, has never been reported in Bangladesh. CONCLUSIONS: Proper monitoring and reporting of imported cases should be given careful consideration for both clinical and epidemiological reasons. Molecular tests should be performed in diagnosis to avoid dilemma, and identification of causative species should be prioritized.

Relationship of Serum Antileishmanial Antibody With Development of Visceral Leishmaniasis, Post-kala-azar Dermal Leishmaniasis and Visceral Leishmaniasis Relapse.

INTRODUCTION: To sustain the achievement of kala-azar elimination program (KEP), early detection and treatment of the visceral leishmaniasis (VL) cases and associated modalities such as treatment failure (TF), relapse VL (RVL), and Post-kala-azar dermal leishmaniasis (PKDL) is the cornerstone. A predictive biomarker for VL development and related complications could also play a crucial role in curtailing disease incidence and transmission. Investigations to find a biomarker with prospective capabilities are, however, scarce. Using samples and known clinical outcomes generated within two previous longitudinal cohort studies, we aimed to determine if fluctuations in serum anti-rK39 antibody levels could provide such predictive value. MATERIALS AND METHODS: Serum samples collected at four different time points (Baseline, 12, 18, and 24 months) from 16 patients who had developed VL within the monitoring period and 15 of their asymptomatic healthy controls counterparts were investigated. To investigate potential prediction of VL related complications, serum samples of 32 PKDL, 10 RVL, 07 TF, and 38 cured VL from a single dose AmBisome trial were analyzed. Of this second panel, all patients were monitored for 5 years and sera were collected at four time points (Baseline then 1, 6, and 12 months after treatment). The level of anti-rK39 antibodies in archived samples was measured by a semi-quantitative ELISA. RESULTS: The mean antibody level was significantly higher in VL patients compared to their asymptomatic healthy counterparts at each time point. Likewise, we observed a trend toward elevations in antibody levels for PKDL, RVL, TF relative to the reducing levels observed in cured VL. Receiver operating characteristic (ROC) analysis found a promising predictive power of rK39 antibody levels to reveal progression from asymptomatic Leishmania donovani infection stage to VL, defined as 87.5% sensitive and 95% specific. Following treatment, rk39 antibody notably showed 100% sensitivity and 95% specificity in predicting TF. CONCLUSION: Our data indicate that the relative quantity of serum anti-rK39 antibody has promise within either a predictive or prognostic algorithm for VL and VL-related modalities. These could enable VL control programs to implement more effective measures to eliminate the disease. Further research is, however, imperative to standardize the rK39 antibody ELISA between sites prior to broader use.