Validation of an In-House ELISA Method in the Diagnosis of Cutaneous Leishmaniasis Caused by Leishmania donovani in Hambantota District, Sri Lanka.

Clinical diagnosis has become a challenge amidst a surge of cutaneous leishmaniasis in Southern Sri Lanka. The routine diagnostic method, slit-skin smear (SSS), has variable sensitivity, leading to undiagnosed cases. Improved diagnostics are urgently needed. We assessed a new in-house ELISA method for its diagnostic capabilities against ITS-1 nested PCR (gold standard­Gs). A cohort of 190 clinical CL cases was examined by SSS microscopy, anti-rKRP42 IgG ELISA (serum- and urine-based), and rK39-Immunochromatographic strip test. Validation was done using non-endemic sera, and cutoffs were developed using the receiver operating curve. The sensitivity of SSS for case detection was 77.9% (authors) and 76.3% (technicians). ELISA vs. Gs demonstrated sensitivity (Sn) = 94.4%; specificity (Sp) = 50.0%; positive predictive value (PPV) = 97.1%; negative predictive value (NPV) = 33.3%; Kappa agreement (Kp) = 0.39/p < 0.01. Comparison of the combination method (SSS by technicians and ELISA) vs. Gs showed: Sn = 98.9%; Sp = 30.0; PPV = 96.2; NPV 60.0%; Kp = 0.378/p < 0.01. All methods performed better compared to SSS (29.4%) where the clinical diagnosis was doubtful (PCR = 94.15%; serum ELISA = 88.2%; combination = 94.1%; p < 0.01 for all). High serum anti-rKRP42 titers were seen in those with multiple lesions. Anti-rKRP42 urine ELISA was suboptimal as a diagnostic test. A 9% rate of positivity was seen for rk39-ICT, and positives recorded high anti-rKRP42 titers. The diagnostic accuracy can be increased above the level of the Gs by combining SSS and ELISA. Advanced studies are required to understand the association between rk39-ICT positivity and high anti-rKRP42 titers.

Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka.

The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by Leishmania donovani) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (103 to 10−2) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy.

Portable infrared imaging for longitudinal limb volume monitoring in patients with lymphatic filariasis.

BACKGROUND: The Global Programme to Eliminate Lymphatic Filariasis (LF) emphasizes hygiene, exercise, and other measures to reduce morbidity and disability related to LF. We recently reported that a portable, three-dimensional, infrared imaging system (3DIS) provides accurate limb volume measurements in patients with filarial lymphedema. To assess the practical utility of repeated 3DIS measurements for longitudinal lymphedema management, we examined intraday and day-to-day leg volume changes in adults with filarial lymphedema in southern Sri Lanka. METHODOLOGY AND PRINCIPAL FINDINGS: We assessed 41 participants with lower extremity lymphedema (stages 1-6) in their homes in the mornings (6:00-9:00 AM) and afternoons (2:00-6:00 PM) of three days within one calendar week. Two examiners performed replicate 3DIS volume measurements at each visit. Median coefficient of variation among replicate volume measurements was 1.7% (IQR 1.1% - 2.3%) for left legs and 2.2% (IQR 1.6% - 2.8%) for right legs. Median intraday volume increase was 3.0%. Range among daily volume measurements tended to be lower for afternoon measurements (median 2.25%, IQR 1.4%- 5.4%) than for morning measurements (median 3.0%, IQR 1.4% - 8.4%). CONCLUSIONS AND SIGNIFICANCE: Limb volume measurements by 3DIS are accurate and reproducible, and this technique is feasible for use in patients' homes. We have developed practical suggestions for optimal outcomes with 3DIS. Duplicate measurements should be performed and repeat assessments should be done at approximately the same time of day to minimize bias. Duplicate measures that vary by more than 8% should prompt review of scanning technique with a repeat measurement. With proper training and attention to technique, 3DIS can be a valuable tool for healthcare workers who work with lymphedema patients.

Surveillance of Wuchereria bancrofti infection by anti-filarial IgG4 in urine among schoolchildren and molecular xenomonitoring in Sri Lanka: a post mass drug administration study.

BACKGROUND: Surveillance of hidden foci or resurgence of the bancroftian filariasis has high priority to maintain the elimination status in Sri Lanka. For the surveillance, two methods were applied in Matotagama, Matara, Sri Lanka; (i) molecular xenomonitoring (MX) by PCR to detect parasite DNA in the vector, Culex (Cx) quinquefasciatus and (ii) survey of anti-filarial IgG4 in urine samples from schoolchildren. RESULTS: Mosquitoes were collected monthly from index houses for 17 months (2013 to 2014) to confirm the existence of bancroftian parasite. Index houses in Matotagama had recorded microfilaria-positive cases in the recent past. Five schools were selected considering Matotagama as the catchment area and all students who presented on the day were tested for urine anti-filarial IgG4 in 2015. Wuchereria bancrofti DNA in Cx. quinquefasciatus pools were found in 14 of 17 months studied and ranged between 0 and 1.4%. The MX rate was greatly increased at least two times in the year following the driest months (March, August). A total of 735 schoolchildren were tested for urine anti-filarial IgG4. Three schools located closer to the MX area had higher positive rates, 3.4%, 3.6%, and 6.6%. Both highest positive rates of MX and urine were located in a nearer vicinity. CONCLUSION: Monthly collections to study lymphatic filariasis (LF) transmission by MX was conducted for the first time in Sri Lanka. We observed that the filarial DNA-positive rate had an association with seasonal cycle of precipitation. More than 1% filarial DNA and > 5% anti-filarial antibody rates confirmed ongoing transmission in Matotagama. The combination of two non-invasive surveys, the urine anti-filarial IgG4 levels of schoolchildren and MX of vector mosquitoes, would be a convenient package to monitor the ongoing transmission (hotspots) of LF in the surveillance.

A novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite.

The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development.