Infectivity of Post-Kala-azar Dermal Leishmaniasis Patients to Sand Flies: Revisiting a Proof of Concept in the Context of the Kala-azar Elimination Program in the Indian Subcontinent

We compared xenodiagnosis with quantitative polymerase chain reaction in skin biopsies from 3 patients with maculopapular or nodular post-kala-azar dermal leishmaniasis (PKDL). All patients infected sand flies. Parasite loads in skin varied from 1428 to 63 058 parasites per microgram. PKDL detection and treatment are important missing components of the kala-azar elimination program.This work was supported by the World Health Organization Special Programme for Research and Training in Tropical Diseases, Switzerland; Spanish Foundation for lnternational Cooperation, Health and Social Affairs; and icddr,b core donors.S

Vascularized dorsal dartos flap to prevent fistula in tubularized incised plate urethroplasty for primary distal and mid shaft hypospadias

The aim of the present study was to evaluate the importance of neourethral covering using vascularized dorsal flap for preventing fistula in Tabularized incised plate (TIP) urethroplasty. The study included 52 children (aged 2-10 years) who had hypospadias repaired including 44  with distal and 8  with mid shaft hypospadias. In all children, a standard tabularized incised plate urethroplasty was followed by reconstruction of new surrounding urethral tissue. A longitudinal dartos flap was harvested from excessive dorsal preputial and penile hypospadiac skin and transposed to ventral side by a buttonhole maneuver. It was sutured to the glans wings and the neomeatus and to the corpora covernosa over the neo-urethra. Thus the new urethra was completely covered with well-vascularzed subcutaneous tissue. At a mean follow-up of 18 months, the result was successful with no fistula or urethral stenosis, except 2 of the mid penile hypospadias. All patients had good functional and cosmetic results with straight penis and vertical slit shaped meatus at the tip of the penis. The 2 patients developed tiny fistula, which were closed spontaneously after meatal dilatation. In conclusion, urethral covering should be part of TIP urethroplasty. A dorsal well-vascularized dartos flap, button holed ventrally is a good choice for preventing fistula for distal and mid shaft hypospadias.

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

Feasibility of a combined camp approach for vector control together with active case detection of visceral leishmaniasis, post kala-azar dermal leishmaniasis, tuberculosis, leprosy and malaria in Bangladesh, India and Nepal: an exploratory study

Background We assessed the feasibility and results of active case detection (ACD) of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and other febrile diseases as well as of bednet impregnation for vector control. Methods Fever camps were organized and analyzed in twelve VL endemic villages in Bangladesh, India, and Nepal. VL, PKDL, tuberculosis, malaria and leprosy were screened among the febrile patients attending the camps, and existing bednets were impregnated with a slow release insecticide. Results Among the camp attendees one new VL case and two PKDL cases were detected in Bangladesh and one VL case in Nepal. Among suspected tuberculosis cases two were positive in India but none in the other countries. In India, two leprosy cases were found. No malaria cases were detected. Bednet impregnation coverage during fever camps was more than 80% in the three countries. Bednet impregnation led to a reduction of sandfly densities after 2 weeks by 86% and 32%, and after 4 weeks by 95% and 12% in India and Nepal respectively. The additional costs for the control programmes seem to be reasonable. Conclusion It is feasible to combine ACD camps for VL and PKDL along with other febrile diseases, and vector control with bednet impregnatio

Genetic diversity of biovar 3 and 4 of Ralstonia solanacearum causing bacterial wilt of tomato using BOX- PCR, RAPD and hrp gene sequences

Genetic diversity of Ralstonia solanacearum, causal agent of bacterial wilt of tomato was assessed by using three different molecular methods such as random amplified polymorphism DNA (RAPD), BOX-PCR and hrp (hypersensitive response and pathogenicity) gene sequence analysis technique. Twelve isolates of Ralstonia solanacearum belonging to biovar 3 (9 isolates) and 4 (3 isolates) were collected from Northern parts of India including Himachal Pradesh and Uttarakhand states from infected tomato plants. Out of 16 primers used in RAPD fingerprinting, four primers (OPA-2, OPA-11, OPC-5, OPE-7) showed monomorphic bands and remaining 12 primers exhibited polymorphic amplified products of both the biovars of R. solanacearum. The primer OPE-10 showed the highest level of genetic diversity among the isolates. Ten isolates of R. solanacearum were classified into two clusters at 20 per cent similarity coefficient and cluster 1 represented all isolates of biovar 3 (UTT-23, UTT-10, UTT-26, HPT11, UTT-9, UTT-32, HPC-3) and cluster 2 comprised the biovar 4 (UTT-22, HPT-3, UTT-24). BOX-PCR fingerprint of R. solanacearum clearly distinguished biovar 3 and 4 grouped into two distinct clusters at 40% similarity coefficient. Cluster 1 represented all isolates of R. solanacearum biovar 3 and cluster 2 comprised the biovar 4 isolates. The isolates of R. solanacearum have genetic diversity in hrpB gene, but it could not differentiate the isolates of biovar 3 and 4. However, the biovars 3 and 4 of R. solanacearum can be genetically distinguished by using BOX- PCR and specific primer of RAPD

n vitro study of antiamoebic effect of methanol extract of mature seeds of Carica papaya on trophozoites of Entamoeba histolytica

Antiamoebic activity of methanol extract of mature seeds of Carica papaya was tested in vitro on axenic culture of Entamoeba histolytica using metronidazole as a reference amoebicidal agent. The MIC of seed extract was > 62.5 µg/mL as compared to < 0.8 µg/mL for metronidazole. The present study suggests that the mature seeds of C. papaya have antiamoebic effect but less pronounced than metronidazole

Quantifying the infectiousness of post-kala-azar dermal leishmaniasis towards sandflies

Background 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. Methods 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. Results 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<0.0001). In a multivariable model, parasite load, nodular lesions and positive skin microscopy were significantly associated with positive xenodiagnosis. Blood parasite load was the strongest predictor for VL. Compared to VL, nodular PKDL was more likely and macular PKDL less likely to result in positive xenodiagnosis, but neither difference reached statistical significance. Conclusions Nodular and macular PKDL, and VL, can be infectious to sand flies. Active PKDL case detection and prompt treatment should be instituted and maintained as an integral part of VL control and elimination programs

Cutaneous Leishmaniasis in an Immigrant Saudi Worker: A Case Report

Cutaneous leishmaniasis (CL), an uncommon disorder in South-East Asia, including Bangladesh, often presents as granulomatous plaque on the exposed areas, with a high index of suspicion required for diagnosis. Here we report the first imported case of CL caused by Leishmania tropica in a migrant Bangladeshi worker in the Kingdom of Saudi Arabia (KSA). The case, initially suspected as a case of cutaneous tuberculosis, arrived at specimens reception unit (SRU) of diagnostic labs of icddr,b being referred by the physician for ALS testing for tuberculosis. At his arrival in the SRU, one of the health personnel of the unit who used to work in KSA suspected him as a case of CL. The diagnosis was confirmed by smear microscopy which revealed plenty of amastigotes within macrophages. PCR was performed to confirm the species. He was treated with sodium stibogluconate at Shahid Suhrawardy Medical College Hospital, Dhaka