Quantitative risk assessment for the introduction of bluetongue virus into mainland Europe by long-distance wind dispersal of Culicoides spp.: A case study from Sardinia.

Europe faces regular introductions and reintroductions of bluetongue virus (BTV) serotypes, most recently exemplified by the incursion of serotype 3 in the Netherlands. Although the long-distance wind dispersal of the disease vector, Culicoides spp., is recognized as a virus introduction pathway, it remains understudied in risk assessments. A Quantitative Risk Assessment framework was developed to estimate the risk of BTV-3 incursion into mainland Europe from Sardinia, where the virus has been present since 2018. We used an atmospheric transport model (HYbrid Single-Particle Lagrangian Integrated Trajectory) to infer the probability of airborne dispersion of the insect vector. Epidemiological disease parameters quantified the virus prevalence in vector population in Sardinia and its potential first transmission after introduction in a new area. When assuming a 24h maximal flight duration, the risk of BTV introduction from Sardinia is limited to the Mediterranean Basin, mainly affecting the southwestern area of the Italian Peninsula, Sicily, Malta, and Corsica. The risk extends to the northern and central parts of Italy, Balearic archipelago, and mainland France and Spain, mostly when maximal flight duration is longer than 24h. Additional knowledge on vector flight conditions and Obsoletus complex-specific parameters could improve the robustness of the model. Providing both spatial and temporal insights into BTV introduction risks, our framework is a key tool to guide global surveillance and preparedness against epizootics.

Evaluation and validation of a PrintrLab-based LAMP assay to identify Trypanosoma cruzi in newborns in Bolivia: a proof-of-concept study.

BACKGROUND: Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of T cruzi. METHODS: In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken T cruzi-LAMP prototype to detect vertically transmitted T cruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2 months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for T cruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2 months, and by serology at 8 months. FINDINGS: Between April 23 and Nov 17, 2018, 986 mothers were screened, among whom 276 were seropositive for T cruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224 infants born to 221 seropositive mothers completed 8 months of follow-up. Congenital transmission was detected in nine of the 224 newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14 more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23 of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. INTERPRETATION: The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area. FUNDING: Inter-American Development Bank.

The need for culture in tegumentary leishmaniasis diagnosis in Bolivia: A comparative evaluation of four parasitological techniques using two sampling methods.

Leishmaniases are zoonotic diseases caused by protozoa of the genus Leishmania. In Bolivia, leishmaniasis occurs mainly in the cutaneous form (CL) followed by the mucosal or mucocutaneous form (ML or MCL), grouped as tegumentary leishmaniosis (TL), while cases of visceral leishmaniasis (VL) are rare. The cases of TL are routinely diagnosed by parasitological methods: Direct Parasitological Exam (DPE) and axenic culture, the latter being performed only by specialized laboratories. The aim of the present study was to optimize the parasitological diagnosis of TL in Bolivia, using two sampling methods. Samples from 117 patients with suspected TL, obtained by aspiration (n = 121) and scraping (n = 121) of the edge of the lesion were tested by: direct parasitological exam, culture in TSTB medium, and miniculture and microculture in Schneider's medium. A positive laboratory result by any of the four techniques evaluated using either of the two sampling methods was considered the gold standard. Of the 117 suspected patients included, TL was confirmed in 96 (82 %), corresponding 79 of the confirmed cases (82.3 %) to CL and 16 (16.7 %) to ML. Parasitological techniques specificity was 100 % and their analytical sensitivity was greater with scraping samples in TSTB culture (98 %). Scraping samples in TSTB and miniculture correlated well with the reference (Cohen's kappa coefficient=0.88) and showed good reliability (Cronbach's alpha coefficient ≥0.91). Microculture provided positive results earlier than the other culture methods (mean day 4.5). By day 14, 98 % of positive cultures had been detected. Scraping sampling and miniculture were associated with higher culture contamination (6 % and 17 %, respectively). Bacterial contamination predominated, regardless of the sampling and culture method, while filamentous fungi and mixed contamination were more frequently observed in cultures from scraping samples. In conclusion: (i) scraping samples proved more suitable for the diagnosis of TL as they increased analytical sensitivity, are less traumatic for the patient and are safer for laboratory personnel than aspirates; (ii) culture, mainly in TSBT medium, should be used for the diagnosis of TL due to its high sensitivity (doubling the number of cases diagnosed by DPE) and its low cost compared to other culture media.

Health economic evaluation of strategies to eliminate gambiense human African trypanosomiasis in the Mandoul disease focus of Chad.

Human African trypanosomiasis, caused by the gambiense subspecies of Trypanosoma brucei (gHAT), is a deadly parasitic disease transmitted by tsetse. Partners worldwide have stepped up efforts to eliminate the disease, and the Chadian government has focused on the previously high-prevalence setting of Mandoul. In this study, we evaluate the economic efficiency of the intensified strategy that was put in place in 2014 aimed at interrupting the transmission of gHAT, and we make recommendations on the best way forward based on both epidemiological projections and cost-effectiveness. In our analysis, we use a dynamic transmission model fit to epidemiological data from Mandoul to evaluate the cost-effectiveness of combinations of active screening, improved passive screening (defined as an expansion of the number of health posts capable of screening for gHAT), and vector control activities (the deployment of Tiny Targets to control the tsetse vector). For cost-effectiveness analyses, our primary outcome is disease burden, denominated in disability-adjusted life-years (DALYs), and costs, denominated in 2020 US$. Although active and passive screening have enabled more rapid diagnosis and accessible treatment in Mandoul, the addition of vector control provided good value-for-money (at less than $750/DALY averted) which substantially increased the probability of reaching the 2030 elimination target for gHAT as set by the World Health Organization. Our transmission modelling and economic evaluation suggest that the gains that have been made could be maintained by passive screening. Our analysis speaks to comparative efficiency, and it does not take into account all possible considerations; for instance, any cessation of ongoing active screening should first consider that substantial surveillance activities will be critical to verify the elimination of transmission and to protect against the possible importation of infection from neighbouring endemic foci.

Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia using hsp70 gene sequencing as a reference technique. 55 Leishmania strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the L. braziliensis complex (L. braziliensis, n = 26; L. braziliensis outlier, n = 18), one species of the L. guyanensis complex (L. guyanensis, n = 1), one species of the L. lainsoni complex (L. lainsoni, n = 2), and two species of the L. mexicana complex (L. amazonensis, n = 5; and L. garnhami, n = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the hsp70 gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one L. braziliensis corresponded to L. peruviana, two L. braziliensis corresponded to L. braziliensis/L. peruviana possible hybrids, two L. amazonensis corresponded to L. mexicana, and three L. garnhami and two L. amazonensis corresponded to L. mexicana/L. amazonensis possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of Leishmania spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the Leishmania species, and a contribution to the control of tegumentary leishmaniasis. IMPORTANCE The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia, in comparison with the sequencing of the hsp70 gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of Leishmania, when there are protocols in this regard in the area.

Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches.

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10-1 to 5 × 105 versus >1 × 10-1 to 6 × 103 parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.

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.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory.

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Update of transmission modelling and projections of gambiense human African trypanosomiasis in the Mandoul focus, Chad.

BACKGROUND: In recent years, a programme of vector control, screening and treatment of gambiense human African trypanosomiasis (gHAT) infections led to a rapid decline in cases in the Mandoul focus of Chad. To represent the biology of transmission between humans and tsetse, we previously developed a mechanistic transmission model, fitted to data between 2000 and 2013 which suggested that transmission was interrupted by 2015. The present study outlines refinements to the model to: (1) Assess whether elimination of transmission has already been achieved despite low-level case reporting; (2) quantify the role of intensified interventions in transmission reduction; and (3) predict the trajectory of gHAT in Mandoul for the next decade under different strategies. METHOD: Our previous gHAT transmission model for Mandoul was updated using human case data (2000-2019) and a series of model refinements. These include how diagnostic specificity is incorporated into the model and improvements to the fitting method (increased variance in observed case reporting and how underreporting and improvements to passive screening are captured). A side-by-side comparison of fitting to case data was performed between the models. RESULTS: We estimated that passive detection rates have increased due to improvements in diagnostic availability in fixed health facilities since 2015, by 2.1-fold for stage 1 detection, and 1.5-fold for stage 2. We find that whilst the diagnostic algorithm for active screening is estimated to be highly specific (95% credible interval (CI) 99.9-100%, Specificity = 99.9%), the high screening and low infection levels mean that some recently reported cases with no parasitological confirmation might be false positives. We also find that the focus-wide tsetse reduction estimated through model fitting (95% CI 96.1-99.6%, Reduction = 99.1%) is comparable to the reduction previously measured by the decline in tsetse catches from monitoring traps. In line with previous results, the model suggests that transmission was interrupted in 2015 due to intensified interventions. CONCLUSIONS: We recommend that additional confirmatory testing is performed in Mandoul to ensure the endgame can be carefully monitored. More specific measurement of cases, would better inform when it is safe to stop active screening and vector control, provided there is a strong passive surveillance system in place.

Tegumentary leishmaniasis by Leishmania braziliensis complex in Cochabamba, Bolivia including the presence of L. braziliensis outlier: Tegumentary leishmaniasis in Cochabamba, Bolivia: Tegumentary leishmaniasis in Cochabamba, Bolivia.

Leishmaniasis is caused by protozoans of the Leishmania genus, which includes more than 20 species capable of infecting humans worldwide. In the Americas, the most widespread specie is L. braziliensis, present in 18 countries including Bolivia. The taxonomic position of the L. braziliensis complex has been a subject of controversy, complicated further by the recent identification of a particular subpopulation named L. braziliensis atypical or outlier. The aim of this study was to carry out a systematic analysis of the L. braziliensis complex in Bolivia and to describe the associated clinical characteristics. Forty-one strains were analyzed by sequencing an amplified 1245 bp fragment of the hsp70 gene, which allowed its identification as: 24 (59%) L. braziliensis, 16 (39%) L. braziliensis outlier, and one (2%) L. peruviana. In a dendrogram constructed, L. braziliensis and L. peruviana are grouped in the same cluster, whilst L. braziliensis outlier appears in a separate branch. Sequence alignment allowed the identification of five non-polymorphic nucleotide positions (288, 297, 642, 993, and 1213) that discriminate L. braziliensis and L. peruviana from L. braziliensis outlier. Moreover, nucleotide positions 51 and 561 enable L. peruviana to be discriminated from the other two taxa. A greater diversity was observed in L. braziliensis outlier than in L. braziliensis-L. peruviana. The 41 strains came from 32 patients with tegumentary leishmaniasis, among which 22 patients (69%) presented cutaneous lesions (11 caused by L. braziliensis and 11 by L. braziliensis outlier) and 10 patients (31%) mucocutaneous lesions (eight caused by L. braziliensis, one by L. braziliensis outlier, and one by L. peruviana). Nine patients (28%) simultaneously provided two isolates, each from a separate lesion, and in each case the same genotype was identified in both. Treatment failure was observed in six patients infected with L. braziliensis and one patient with L. peruviana.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

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.

Evaluation of the Performance of the Loopamp Trypanosoma cruzi Detection Kit for the Diagnosis of Chagas Disease in an Area Where It Is Not Endemic, Spain.

In Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for Trypanosoma cruzi DNA detection showed good analytical performance and ease of use. We aimed to evaluate the performance of the Loopamp Trypanosoma cruzi detection kit (Eiken Chemical Co. Ltd., Japan) (Tcruzi-LAMP) for congenital and chronic CD diagnosis using well-characterized samples. We included samples from 39 congenital and 174 chronic CD cases and from 48 uninfected children born to infected mothers and 34 nonchagasic individuals. The sensitivity, specificity, and accuracy of Tcruzi-LAMP were estimated using standard case definitions for congenital CD (positive result by parasitological or PCR tests or serology after 9 months of age) and chronic CD (positive serology by at least two tests). The Tcruzi-LAMP results were read by visual examination and a real-time fluorimeter. For congenital CD, Tcruzi-LAMP sensitivity was 97% for both types of reading; specificity was 92% by visual examination and 94% by fluorimeter. For chronic CD, sensitivity was 47% and specificity 100%. The accuracy in congenital CD was >94% versus 56% in chronic CD. The agreement of Tcruzi-LAMP with PCR tests was better in congenital CD (kappa, 0.86 to 0.91) than in chronic CD (kappa, 0.67 to 0.83). The Loopamp Trypanosoma cruzi detection kit showed good performance for the diagnosis of congenital CD. Tcruzi-LAMP, like PCR, can be useful for the screening and early diagnosis of congenital infection.

Clinical and immunological characteristics of tegumentary leishmaniasis cases in Bolivia.

BACKGROUND: Tegumentary leishmaniasis (TL) is a parasitic disease that can present a cutaneous or mucocutaneous clinical form (CL and MCL, respectively). The disease is caused by different Leishmania species and transmitted by phlebotomine sand flies. Bolivia has one of the highest incidences of the disease in South America and the diagnosis is done by parasitological techniques. Our aim was to describe the clinical and immunological characteristics of CL and MCL patients attending the leishmaniasis reference center in Cochabamba, Bolivia, in order to gain updated clinical and epidemiological information, to evaluate the diagnostic methods used and to identify biomarkers related to clinical disease and its evolution. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted from September 2014 to November 2015 and 135 patients with lesions compatible with CL or MCL were included. Epidemiological and clinical data were collected using a semi-structured questionnaire. Two parasitological diagnostic methods were used: Giemsa-stained smears and culture of lesion aspirates. Blood samples obtained from participants were used to measure the concentrations of different cytokines. 59.2% (80/135) were leishmaniasis confirmed cases (CL: 71.3%; MCL: 28.7%). Sixty percent of the confirmed cases were positive by smears and 90.6% were positive by culture. 53.8% were primo-infections. Eotaxin and monokine induced by IFN-γ presented higher serum concentrations in the MCL clinical presentation compared to CL cases and no-cases. None of the cytokines presented different concentrations between primo-infections and secondary infections due to treatment failure. CONCLUSIONS/SIGNIFICANCE: In Bolivia, parasitological diagnosis remains the reference standard in diagnosis of leishmaniasis because of its high specificity, whereas the sensitivity varies over a wide range leading to loss of cases. Until more accurate tools are implemented, all patients should be tested by both smears and culture of lesion aspirates to minimize the risk of false negatives. Our results showed higher concentrations of several cytokines in MCL compared to CL, but no differences were observed between CL and no-cases. In addition, none of the cytokines differed between primary and secondary infections. These results highlight the need of further research to identify biomarkers of susceptibility and disease progression, in addition to looking at the local cellular immune responses in the lesions.

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 and Evaluation of a Three-Dimensional Printer-Based DNA Extraction Method Coupled to Loop Mediated Isothermal Amplification for Point-of-Care Diagnosis of Congenital Chagas Disease in Endemic Regions.

Vertical transmission of Trypanosomacruzi is the cause of congenital Chagas disease, a re-emerging infectious disease that affects endemic and nonendemic regions alike. An early diagnosis is crucial because prompt treatment achieves a high cure rate, precluding evolution to symptomatic chronic Chagas disease. However, early diagnosis involves low-sensitive parasitologic assays, making necessary serologic confirmation after 9 months of life. With the aim of implementing early diagnostic strategies suitable for minimally equipped laboratories, a T. cruzi-loop-mediated isothermal amplification (LAMP) prototype was coupled with an automated DNA-extraction device repurposed from a three-dimensional printer (PrintrLab). The whole process takes <3 hours to yield a result, with an analytical sensitivity of 0.1 to 2 parasite equivalents per milliliter, depending on the T. cruzi strain. Twenty-five blood samples from neonates born to seropositive mothers were tested blindly. In comparison to quantitative real-time PCR, the PrintrLab-LAMP dual strategy showed high agreement, while both molecular-based methodologies yielded optimal sensitivity and specificity with respect to microscopy-based diagnosis of congenital Chagas disease. PrintrLab-LAMP detected all 10 congenitally transmitted T. cruzi infections, showing promise for point-of-care early diagnosis of congenital Chagas disease.

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.