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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 &lt, 0.0001) as well as between the absolute parasite loads (r = 0.87, 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.

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. Open-Access-Publikationsfonds 2019

Background\ud In the Indian subcontinent, visceral leishmaniasis (VL) incidence is on track to reach elimination goals by 2020 in nearly all endemic districts. Although not included in official targets, previous data suggest post-kala-azar dermal leishmaniasis (PKDL) patients can act as an infection reservoir.\ud \ud Methods\ud We conducted xenodiagnosis on 47 PKDL patients and 15 VL patients using laboratory-reared Phlebotomus argentipes. In direct xenodiagnosis, flies were allowed to feed on the patient’s skin for 15 minutes. For indirect xenodiagnosis, flies were fed through a membrane on the patient’s blood. Five days later, blood-fed flies were dissected and examined by microscopy and/or PCR. A 3-mm skin snip biopsy (PKDL) or venous blood VL) was processed by quantitative PCR.\ud \ud Results\ud Twenty-seven PKDL patients (57.4%) had positive results by direct and/or indirect xenodiagnosis. Direct was significantly more sensitive than indirect xenodiagnosis (55.3% vs 6.4%, p 1 log10 unit higher than those with negative results (2.88 vs 1.66, p

Background Post-kala-azar dermal leishmaniasis (PKDL) is a sequel to visceral leishmaniasis (VL), which is found in VL-endemic countries including Bangladesh. Because of these enigmatic cases, the success of the National Kala-azar Elimination Program is under threat. To date, diagnostic methods for PKDL cases in endemic regions have been limited to clinical examination and rK39 test or microscopy, and a suitable and accurate alternative method is needed. In this study, we investigated the application of real-time polymerase chain reaction (PCR) as a potential method for diagnosis of PKDL in comparison with microscopy. Methods Ninety-one suspected macular PKDL cases from Mymensingh district, Bangladesh, were enrolled in the study after diagnosis by clinical examination and an rK39 strip test. All of them responded after completion of the treatment with miltefosine. During enrollment, a skin biopsy was done for each patient, and both microscopy and real-time PCR were performed for detection and quantification of Leishmania donovan body (LDB) and LD DNA, respectively. Results Real-time PCR detected 83 cases among all suspected PKDL patients, with an encouraging sensitivity of 91.2% (83.4%–96.1%), whereas microscopy showed 50.6% (39.9%–61.2%) sensitivity. Among all suspected PKDL cases, 42 cases were positive in both microscopy and qPCR, whereas 41 cases were detected as positive through qPCR only. Conclusions This study provides evidence that real-time PCR is a promising tool for diagnosis of PKDL in endemic regions. In addition to diagnosis, the quantitative ability of this method could be further exploited for after-treatment prognosis and cure assessment of PKDL cases.