Advances and Challenges in the Diagnosis of Leishmaniasis

Druzian A.F., França A.D., Higa Júnior M.G., Dorval M.E., Lima-Junior M.S., Pompilio M.A., Matos M.D., de Carvalho L.R., Mendes R.P., Paniago A.M.

Volpe J., Parchen G.P., Costa F.S., Silva A.D., Andrade H.M., Amaral C.D., Silva S.M., Kubota L.T., E. P. Souto D.

A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor's analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis, an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol/L); and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs' samples. For this reason, the PEP13 immunosensor was applied to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.

Kumar A., Singh V.K., Madhukar P., Tiwari R., Roy R., Rajneesh, Mehrotra S., Sundar S., Kumar R.

Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

van Dijk N.J., Hagos D.G., Huggins D.M., Carrillo E., Ajala S., Chicharro C., Kiptanui D., Solana J.C., Abner E., Wolday D., Schallig H.D.

Background Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. Methodology/Principle findings The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. Conclusions/Significance Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Singh O.P., Chaubey R., Kushwaha A.K., Fay M.P., Sacks D., Sundar S.

Abstract In an area endemic with Indian visceral leishmaniasis (VL), we performed direct xenodiagnosis to evaluate the transmission of Leishmania donovani from patients with VL–human immunodeficiency virus (HIV) coinfection to the vector sandflies, Phlebotomus argentipes. Fourteen patients with confirmed VL-HIV coinfection, with a median parasitemia of 42 205 parasite genome/mL of blood, were exposed to 732 laboratory-reared pathogen-free female P argentipes sandflies on their lower arms and legs. Microscopy revealed that 16.66% (122/732) of blood-fed flies were xenodiagnosis positive. Notably, 93% (13/14) of the VL-HIV group infected the flies, as confirmed by quantitative polymerase chain reaction and/or microscopy, and were 3 times more infectious than those who had VL without HIV.

Ferrari A.J., Santomauro D.F., Aali A., Abate Y.H., Abbafati C., Abbastabar H., Abd ElHafeez S., Abdelmasseh M., Abd-Elsalam S., Abdollahi A., Abdullahi A., Abegaz K.H., Abeldaño Zuñiga R.A., Aboagye R.G., Abolhassani H., et. al.

Detailed, comprehensive, and timely reporting on population health by underlying causes of disability and premature death is crucial to understanding and responding to complex patterns of disease and injury burden over time and across age groups, sexes, and locations. The availability of disease burden estimates can promote evidence-based interventions that enable public health researchers, policy makers, and other professionals to implement strategies that can mitigate diseases. It can also facilitate more rigorous monitoring of progress towards national and international health targets, such as the Sustainable Development Goals. For three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has filled that need. A global network of collaborators contributed to the production of GBD 2021 by providing, reviewing, and analysing all available data. GBD estimates are updated routinely with additional data and refined analytical methods. GBD 2021 presents, for the first time, estimates of health loss due to the COVID-19 pandemic.The GBD 2021 disease and injury burden analysis estimated years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries using 100 983 data sources. Data were extracted from vital registration systems, verbal autopsies, censuses, household surveys, disease-specific registries, health service contact data, and other sources. YLDs were calculated by multiplying cause-age-sex-location-year-specific prevalence of sequelae by their respective disability weights, for each disease and injury. YLLs were calculated by multiplying cause-age-sex-location-year-specific deaths by the standard life expectancy at the age that death occurred. DALYs were calculated by summing YLDs and YLLs. HALE estimates were produced using YLDs per capita and age-specific mortality rates by location, age, sex, year, and cause. 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws. Uncertainty was propagated at each step of the estimation process. Counts and age-standardised rates were calculated globally, for seven super-regions, 21 regions, 204 countries and territories (including 21 countries with subnational locations), and 811 subnational locations, from 1990 to 2021. Here we report data for 2010 to 2021 to highlight trends in disease burden over the past decade and through the first 2 years of the COVID-19 pandemic.Global DALYs increased from 2·63 billion (95% UI 2·44-2·85) in 2010 to 2·88 billion (2·64-3·15) in 2021 for all causes combined. Much of this increase in the number of DALYs was due to population growth and ageing, as indicated by a decrease in global age-standardised all-cause DALY rates of 14·2% (95% UI 10·7-17·3) between 2010 and 2019. Notably, however, this decrease in rates reversed during the first 2 years of the COVID-19 pandemic, with increases in global age-standardised all-cause DALY rates since 2019 of 4·1% (1·8-6·3) in 2020 and 7·2% (4·7-10·0) in 2021. In 2021, COVID-19 was the leading cause of DALYs globally (212·0 million [198·0-234·5] DALYs), followed by ischaemic heart disease (188·3 million [176·7-198·3]), neonatal disorders (186·3 million [162·3-214·9]), and stroke (160·4 million [148·0-171·7]). However, notable health gains were seen among other leading communicable, maternal, neonatal, and nutritional (CMNN) diseases. Globally between 2010 and 2021, the age-standardised DALY rates for HIV/AIDS decreased by 47·8% (43·3-51·7) and for diarrhoeal diseases decreased by 47·0% (39·9-52·9). Non-communicable diseases contributed 1·73 billion (95% UI 1·54-1·94) DALYs in 2021, with a decrease in age-standardised DALY rates since 2010 of 6·4% (95% UI 3·5-9·5). Between 2010 and 2021, among the 25 leading Level 3 causes, age-standardised DALY rates increased most substantially for anxiety disorders (16·7% [14·0-19·8]), depressive disorders (16·4% [11·9-21·3]), and diabetes (14·0% [10·0-17·4]). Age-standardised DALY rates due to injuries decreased globally by 24·0% (20·7-27·2) between 2010 and 2021, although improvements were not uniform across locations, ages, and sexes. Globally, HALE at birth improved slightly, from 61·3 years (58·6-63·6) in 2010 to 62·2 years (59·4-64·7) in 2021. However, despite this overall increase, HALE decreased by 2·2% (1·6-2·9) between 2019 and 2021.Putting the COVID-19 pandemic in the context of a mutually exclusive and collectively exhaustive list of causes of health loss is crucial to understanding its impact and ensuring that health funding and policy address needs at both local and global levels through cost-effective and evidence-based interventions. A global epidemiological transition remains underway. Our findings suggest that prioritising non-communicable disease prevention and treatment policies, as well as strengthening health systems, continues to be crucially important. The progress on reducing the burden of CMNN diseases must not stall; although global trends are improving, the burden of CMNN diseases remains unacceptably high. Evidence-based interventions will help save the lives of young children and mothers and improve the overall health and economic conditions of societies across the world. Governments and multilateral organisations should prioritise pandemic preparedness planning alongside efforts to reduce the burden of diseases and injuries that will strain resources in the coming decades.Bill & Melinda Gates Foundation.

Guha S.K., Sardar A.A., Saha P., Chatterjee M., Jana K., Samanta A., Maji D., Biswas P., Bhattacharya R., Maji A.K.

Background India is going through the maintenance phase of VL elimination programme which may be threatened by the persistence of hidden parasite pools among asymptomatic leishmanial infection (ALI) and PKDL. The present work was designed to determine the burden of VL, PKDL, and ALI and to assess the role of treatment of ALI in maintaining post-elimination phase. Methods and finding The study was undertaken in Malda district, West Bengal, India during October 2016 to September 2021. Study areas were divided into ‘Study’ and ‘Control’ arms. VL and PKDL cases of both the arms were diagnosed by three active mass surveys with an interval of one year and treated as per National guideline. ALI of ‘Study’ arm was treated like VL. ALI of ‘Control’ arm was followed up to determine their fate. Fed sand-fly pools were analysed for parasitic DNA. No significant difference was noted between the incidence of VL and PKDL in both the arms. Incidence of ALI declined sharply in ‘Study’ arm but an increasing trend was observed in ‘Control’ arm. Significantly higher rate of sero-conversion was noted in ‘Control’ arm and was found to be associated with untreated ALI burden. Parasitic DNA was detected in 22.8% ALI cases and 2.2% sand-fly pools. Conclusion Persistence of a significant number of PKDL and ALI and ongoing transmission, as evidenced by new infection and detection of leishmanial DNA in vector sand-flies, may threaten the maintenance of post-elimination phase. Emphasis should be given for elimination of pathogen to prevent resurgence of VL epidemics.

Monsieurs P., Cloots K., Uranw S., Banjara M.R., Ghimire P., Burza S., Hasker E., Dujardin J., Domagalska M.A.

Kobialka R.M., Ceruti A., Roy M., Roy S., Chowdhury R., Ghosh P., Hossain F., Weidmann M., Graf E., Bueno Alvarez J., Moreno J., Truyen U., Mondal D., Chatterjee M., Abd El Wahed A.

Abstract 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.

Sadeghi A., Sadeghi M., Fakhar M., Zakariaei Z., Sadeghi M.

Leishmania, a single-cell parasite prevalent in tropical and subtropical regions worldwide, can cause varying degrees of leishmaniasis, ranging from self-limiting skin lesions to potentially fatal visceral complications. As such, the parasite has been the subject of much interest in the scientific community. In recent years, advances in diagnostic techniques such as flow cytometry, molecular biology, proteomics, and nanodiagnosis have contributed to progress in the diagnosis of this deadly disease. Additionally, the emergence of artificial intelligence (AI), including its subbranches such as machine learning and deep learning, has revolutionized the field of medicine. The high accuracy of AI and its potential to reduce human and laboratory errors make it an especially promising tool in diagnosis and treatment. Despite the promising potential of deep learning in the medical field, there has been no review study on the applications of this technology in the context of leishmaniasis. To address this gap, we provide a scoping review of deep learning methods in the diagnosis of the disease, drug discovery, and vaccine development. In conducting a thorough search of available literature, we analyzed articles in detail that used deep learning methods for various aspects of the disease, including diagnosis, drug discovery, vaccine development, and related proteins. Each study was individually analyzed, and the methodology and results were presented. As the first and only review study on this topic, this paper serves as a quick and comprehensive resource and guide for the future research in this field.

Roy S., Moulik S., Chaudhuri S.J., Ghosh M.K., Goswami R.P., Saha B., Chatterjee M.

Abstract Background Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. Methods A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. Results The parasite load at disease presentation was 13 461.00 (2560.00–37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1–1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1–1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. Conclusion In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment.

Hagos D.G., Kiros Y.K., Abdulkader M., Schallig H.D., Wolday D.

The lack of accurate and feasible diagnostic tests poses a significant challenge to visceral leishmaniasis (VL) healthcare services in endemic areas. To date, various VL diagnostic tests have been or are being developed, and their diagnostic performances need to be assessed. In the present study, the diagnostic performances of rk39 RDT, the direct agglutination test (DAT), microscopy, loop-mediated isothermal amplification (LAMP), and miniature direct-on-blood polymerase chain reaction–nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) were assessed using quantitative polymerase chain reaction (qPCR) as the reference test in an endemic region of Ethiopia. In this study, 235 suspected VL cases and 104 non-endemic healthy controls (NEHCs) were recruited. Among the suspected VL cases, 144 (61.28%) tested positive with qPCR. The sensitivities for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA were 88.11%, 96.50%, 76.58%, 94.33%, and 95.80%, respectively. The specificities were 83.33%, 97.96%, 100%, 97.38%, and 98.92% for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA, respectively. In conclusion, rk39 RDT and microscopy exhibited lower sensitivities, while DAT demonstrated excellent performance. LAMP and mini-dbPCR-NALFIA showed excellent performances with feasibility for implementation in remote endemic areas, although the latter requires further evaluation in such regions.

Dondi A., Manieri E., Gambuti G., Varani S., Campoli C., Zama D., Pierantoni L., Baldazzi M., Prete A., Attard L., Lanari M., Melchionda F.

Background: Visceral leishmaniasis (VL) is a potentially fatal disease, with an increasing occurrence in northern Italy, affecting children and both immunocompetent and immunocompromised adults. Methods: This retrospective study conducted at the St. Orsola University Hospital of Bologna, Italy, evaluates the characteristics of 16 children (with a median age of 14.3 months) who were hospitalized between 2013 and 2022 for VL. Results: Seventy-five percent of patients presented with a triad of fever, cytopenia, and splenomegaly. An abdominal ultrasound examination revealed splenomegaly and hypoechoic spleen abnormalities in 93.8% and 73.3% of cases, respectively. Five VL cases were complicated by secondary hemophagocytic lymphohistiocytosis. Eleven patients were treated with a single 10 mg/kg dose of Liposomal Amphotericin B (L-AmB), while five received two doses (total of 20 mg/kg); one of the former groups experienced a recurrence. The fever generally decreased 48 h after the first L-AmB dose, and hemoglobin levels normalized within a month. The splenomegaly resolved in approximately 4.5 months. Conclusions: Pediatricians should consider VL in children with fever of an unknown origin, anemia, cytopenia, and splenomegaly. In our experience, abdominal ultrasounds and molecular tests on peripheral blood contributed to diagnosis without the need for bone marrow aspiration. The short-course therapy with two 10 mg/kg doses of L-AmB is safe and effective.

Abdelmula A.M., Mirzaei O., Güler E., Süer K.

Cutaneous leishmaniasis (CL) is a common illness that causes skin lesions, principally ulcerations, on exposed regions of the body. Although neglected tropical diseases (NTDs) are typically found in tropical areas, they have recently become more common along Africa’s northern coast, particularly in Libya. The devastation of healthcare infrastructure during the 2011 war and the following conflicts, as well as governmental apathy, may be causal factors associated with this catastrophic event. The main objective of this study is to evaluate alternative diagnostic strategies for recognizing amastigotes of cutaneous leishmaniasis parasites at various stages using Convolutional Neural Networks (CNNs). The research is additionally aimed at testing different classification models employing a dataset of ultra-thin skin smear images of Leishmania parasite-infected people with cutaneous leishmaniasis. The pre-trained deep learning models including EfficientNetB0, DenseNet201, ResNet101, MobileNetv2, and Xception are used for the cutaneous leishmania parasite diagnosis task. To assess the models’ effectiveness, we employed a five-fold cross-validation approach to guarantee the consistency of the models’ outputs when applied to different portions of the full dataset. Following a thorough assessment and contrast of the various models, DenseNet-201 proved to be the most suitable choice. It attained a mean accuracy of 0.9914 along with outstanding results for sensitivity, specificity, positive predictive value, negative predictive value, F1-score, Matthew’s correlation coefficient, and Cohen’s Kappa coefficient. The DenseNet-201 model surpassed the other models based on a comprehensive evaluation of these key classification performance metrics.

Leal J.F., Barroso D.H., Trindade N.S., Miranda V.L., Gurgel-Gonçalves R.

The polymorphism of cutaneous leishmaniasis (CL) complicates diagnosis in health care services because lesions may be confused with other dermatoses such as sporotrichosis, paracocidiocomycosis, and venous insufficiency. Automated identification of skin diseases based on deep learning (DL) has been applied to assist diagnosis. In this study, we evaluated the performance of AlexNet, a DL algorithm, to identify pictures of CL lesions in patients from Midwest Brazil. We used a set of 2458 pictures (up to 10 of each lesion) obtained from patients treated between 2015 and 2022 in the Leishmaniasis Clinic at the University Hospital of Brasilia. We divided the picture database into training (80%), internal validation (10%), and testing sets (10%), and trained and tested AlexNet to identify pictures of CL lesions. We performed three simulations and trained AlexNet to differentiate CL from 26 other dermatoses (e.g., chromomycosis, pyomerrhitis, ecthyma, venous insufficiency). We obtained an average accuracy of 95.04% (Confidence Interval 95%: 93.81–96.04), indicating an excellent performance of AlexNet in identifying pictures of CL lesions. We conclude that automated CL identification using AlexNet has the potential to assist clinicians in diagnosing skin lesions. These results contribute to the development of a mobile application to assist in the diagnosis of CL in health care services.