Skin pharmacokinetics of miltefosine in the treatment of post-kala-azar dermal leishmaniasis in South Asia.

INTRODUCTION: Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL. METHODS: Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin. RESULTS: Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 µg/g (IQR: 21.94-60.65 µg/g) in skin and 33.29 µg/mL (IQR: 25.9-42.58 µg/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively. CONCLUSION: This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL.

Multiplex PCR for bacterial, viral and protozoal pathogens in persistent diarrhoea or persistent abdominal pain in Côte d'Ivoire, Mali and Nepal.

In contrast to acute diarrhoea, the aetiology of persistent digestive disorders (≥ 14 days) is poorly understood in low-resource settings and conventional diagnostic approaches lack accuracy. In this multi-country study, we compared multiplex real-time PCR for enteric bacterial, parasitic and viral pathogens in stool samples from symptomatic patients and matched asymptomatic controls in Côte d'Ivoire, Mali and Nepal. Among 1826 stool samples, the prevalence of most pathogens was highest in Mali, being up to threefold higher than in Côte d'Ivoire and up to tenfold higher than in Nepal. In all settings, the most prevalent bacteria were EAEC (13.0-39.9%) and Campylobacter spp. (3.9-35.3%). Giardia intestinalis was the predominant intestinal protozoon (2.9-20.5%), and adenovirus 40/41 was the most frequently observed viral pathogen (6.3-25.1%). Significantly different prevalences between symptomatic and asymptomatic individuals were observed for Campylobacter, EIEC and ETEC in the two African sites, and for norovirus in Nepal. Multiple species pathogen infection was common in Côte d'Ivoire and Mali, but rarely found in Nepal. We observed that molecular testing detected multiple enteric pathogens and showed low discriminatory accuracy to distinguish between symptomatic and asymptomatic individuals. Yet, multiplex PCR allowed for direct comparison between different countries and revealed considerable setting-specificity.

AmBisome Monotherapy and Combination AmBisome-Miltefosine Therapy for the Treatment of Visceral Leishmaniasis in Patients Coinfected With Human Immunodeficiency Virus in India: A Randomized Open-Label, Parallel-Arm, Phase 3 Trial.

BACKGROUND: Visceral leishmaniasis (VL) in patients with human immunodeficiency virus (HIV) presents an increasingly important patient cohort in areas where both infections are endemic. Evidence for treatment is sparce, with no high-quality studies from the Indian subcontinent. METHODS: This is a randomized, open-label, parallel-arm, phase 3 trial conducted within a single hospital in Patna, India. One hundred and fifty patients aged ≥18 years with serologically confirmed HIV and parasitologically confirmed VL were randomly allocated to 1 of 2 treatment arms, either a total 40 mg/kg intravenous liposomal amphotericin B (AmBisome; Gilead Pharmaceuticals) administered in 8 equal doses over 24 days or a total 30 mg/kg intravenous AmBisome administered in 6 equal doses given concomitantly with a total 1.4 g oral miltefosine administered through 2 daily doses of 50 mg over 14 days. The primary outcome was intention-to-treat relapse-free survival at day 210, defined as absence of signs and symptoms of VL or, if symptomatic, negative parasitological investigations. RESULTS: Among 243 patients assessed for eligibility, 150 were recruited between 2 January 2017 and 5 April 2018, with no loss to follow-up. Relapse-free survival at day 210 was 85% (64/75; 95% CI, 77-100%) in the monotherapy arm, and 96%, (72/75; 90-100%) in the combination arm. Nineteen percent (28/150) were infected with concurrent tuberculosis, divided equally between arms. Excluding those with concurrent tuberculosis, relapse-free survival at day 210 was 90% (55/61; 82-100%) in the monotherapy and 97% (59/61; 91-100%) in the combination therapy arm. Serious adverse events were uncommon and similar in each arm. CONCLUSIONS: Combination therapy appears to be safe, well tolerated, and effective, and halves treatment duration of current recommendations. CLINICAL TRIALS REGISTRATION: Clinical Trial Registry India (CTRI/2015/05/005807; the protocol is available online at https://osf.io/avz7r).

Clinical Experience of Fluroscopy Guided Percutaneous Transpedicular Biopsy of Spinal Lesion.

INTRODUCTION: The diagnosis of the spinal lesions often puts the clinician in dilemma. The definite diagnosis is obviously needed for the proper management of the disease. The wrong diagnosis not only imposes the adverse effects, but sometimes may lead to the disabling conditions and even prove to be life threatening. This study is aimed at evaluating the need of fluoroscopy guided percutaneous transpedicular biopsy for establishing the proper diagnosis and find the diagnostic yield. METHODS: This is the descriptive cross-sectional study conducted over the period of 10 years in the Upendra Devkota Memorial National Institute of Neurological and Allied Sciences among the patients who underwent transpedicular biopsy for various spinal lesions. RESULTS: Among the 77 cases, 38 (49%) of the lesions on MRI were single level whereas 39 (51%) of the lesions were multiple. Most of the lesions were diagnosed as the non-tubercular infection 30 (42%), followed by the osteoporotic fractures and malignancy in 18 (25%) and 15 (21%) respectively. The sensitivity and specificity of the radiology with the background of clinical scenario was 79.5% and 90.9% respectively. The diagnostic yield of the biopsy was 93.5%. CONCLUSIONS: The transpedicular biopsy of the spinal lesion is the must for the proper diagnosis and treatment plan of such cases. The change in the diagnosis after biopsy is often possible which will drastically alter the treatment plan.

Importance of secondary screening with clinical isolates for anti-leishmania drug discovery.

The growing drug resistance (DR) raises major concerns for the control of visceral leishmaniasis (VL), a neglected disease lethal in 95 percent of the cases if left untreated. Resistance has rendered antimonials (SSG) obsolete in the Indian Sub-Continent (ISC) and the first miltefosine-resistant Leishmania donovani were isolated. New chemotherapeutic options are needed and novel compounds are being identified by high-throughput screening (HTS). HTS is generally performed with old laboratory strains such as LdBOB and we aimed here to validate the activity of selected compounds against recent clinical isolates. In this academic/industrial collaboration, 130 compounds from the GSK "Leishbox" were screened against one SSG-sensitive and one SSG-resistant strain of L. donovani recently isolated from ISC patients, using an intracellular assay of L. donovani-infected THP1-derived macrophages. We showed that only 45% of the compounds were active in both clinical isolates and LdBOB. There were also different compound efficiencies linked to the SSG susceptibility background of the strains. In addition, our results suggested that the differential susceptibility profiles were chemical series-dependent. In conclusion, we demonstrate the potential value of including clinical isolates (as well as resistant strains) in the HTS progression cascade.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Antileishmanial activity of a series of N²,N4-disubstituted quinazoline-2,4-diamines.

A series of N(2),N(4)-disubstituted quinazoline-2,4-diamines has been synthesized and tested against Leishmania donovani and L. amazonensis intracellular amastigotes. A structure-activity and structure-property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC50 values in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 23 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg kg(-1) day(-1) for 5 consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N(2),N(4)-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents.

Clinical features and histological patterns of lupus nephritis in Eastern Nepal.

To determine the clinical profile and patterns of lupus nephritis patients in Eastern Nepal, we studied 38 patients fulfilling the 1982 revised criteria of American College of Rheumatology for systemic lupus erythematous (SLE), followed up from January 2004 to January 2008. Arthritis was a common initial feature in addition to variable cutaneous, cardiac, pulmonary and neuropsychiatric manifestations. Renal biopsy showed grade 1 changes in 5 (13.5%) patients, grade 2 changes in 13 (35.1%) patients, grade 3 changes in 9 (24.3%) patients, grade 4 changes in 7 (18.9%) patients, grade 5 changes in 2 (5.4%) patients, and grade 6 changes in 2.7% patients. Antinuclear antibody (ANA) assay and anti-ds DNA were positive in 78.4 and 81.1%, respectively. We conclude that mesangial proliferative glomerulonephritis (grade 2) was the most common pattern of lupus nephritis encountered in our study. Timely diagnosis and treatment may improve the overall patients' survival.

Effectiveness and feasibility of active and passive case detection in the visceral leishmaniasis elimination initiative in India, Bangladesh, and Nepal.

This study analyzed the effectiveness of active case detection (ACD) for new visceral leishmaniasis (VL) cases. ACD detection was carried out using house to house screening in Bangladesh and India and by neighborhood screening around index cases in Nepal. The percent increase of new VL cases through ACD compared to PCD was 6.7-17.1% in India; 38.8% in Nepal; and 60% in Bangladesh. The screening effort was high in India and Bangladesh (house to house screening) compared to Nepal (index case screening). The additional cost per new VL case detected varied: $50 to $106 in India; $172 in Bangladesh; $262 in Nepal depending on the type of screening staff, transport and training costs. The estimated annual VL incidence in the ACD arm ranged from 315-383 in India; 109 in Bangladesh, and 43 per 100,000 in Nepal. The additional effort and cost rises as disease incidence declines or PCD improves.

Leprosy coinfection with kala-azar.

Leprosy and visceral leishmanias are endemic in Nepal and are both major public health problems. Two patients of visceral leishmaniasis developed leprosy during the course of the disease. Leprosy and visceral leishmaniasis share a similar immunological spectrum and can occur concomitantly in endemic regions.

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar.

OBJECTIVE: To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala-azar endemic focus of Nepal. METHODS: The sample was composed of 184 (80%) persons without any known history of KA and not living in the same house as known kala-azar cases (HNK), 24 (10%) Healthy Household Contacts (HHC) and 23 (10%) past kala-azar cases which had been successfully treated (HPK). RESULTS: PCR and DAT positivity scores were, respectively: HNK, 17.6% and 5.6%; HHC, 12.5% and 20.8%; HPK, 26.1% and 95.7%. The ratio PCR-positives/DAT-positives was significantly higher in HNK (ratio = 3.1) than in HHC (ratio = 0.6, P = 0.036) and in HPK (ratio = 0.2, P = 0.012). The ratio PCR-positives/DAT-positives did not significantly differ between HHC (ratio = 0.6) and HPK (ratio = 0.2, P = 0.473). The positive agreement index between PCR and DAT in HNK was 5%; in HHC, 0%; in HPK, 43%. CONCLUSIONS: Our study highlights the specific character of PCR and DAT for the exploration of Leishmania asymptomatic infections. PCR is probably more informative for very recent infections among HNK, while DAT provides more information among HHC and HPK, a feature likely related to the power of serology to track less recent infections.

Serum nitrite level and adenosine deaminase activity is altered in visceral leishmaniasis.

In this study we sought to determine if there is alteration in nitric oxide (NO) production and adenosine deaminase (ADA) activity among patients with visceral leishmaniasis (VL) and the effect of four weeks of chemotherapy on these levels. Fifty-three VL patients diagnosed clinically and by direct demonstration of the LD bodies in the bone marrow smear were studied. They were treated with Sodium Stibogluconate and sampled at the baseline and four weeks. Forty-three healthy individuals coming from the same endemic area were taken as control. Total nitrite (NO2- and NO3-) as an index of NO production and ADA activity was measured spectrophotometrically. Serum nitrite level decreased significantly in patients as compared to the healthy individuals but significantly increased following 4 weeks of chemotherapy. Conversely, Increased ADA activity was observed in the beginning of treatment and decreased significantly with successive 4 weeks of chemotherapy. It seems a negative correlation between NO level and ADA activity. This result indicates parasite induced evasion of NO and activation of T lymphocytes during immunopathogenesis of VL. Therefore, assessment of NO metabolites may be useful marker in the evaluation of the effector mechanism of macrophages and clinical manifestation of patients.

Epidemiological dynamics of antimonial resistance in Leishmania donovani: genotyping reveals a polyclonal population structure among naturally-resistant clinical isolates from Nepal.

Pentavalent antimonials (SbV) are the first line drug against leishmaniasis worldwide, but drug resistance is increasingly reported, particularly in the Indian sub-continent, where it represents a major threat for the control of anthroponotic visceral leishmaniasis (VL). In order to understand the epidemiological dynamics of antimonial resistance in anthroponotic VL, we analysed here the population structure of 24 Leishmania donovani stocks isolated from anthroponotic VL-patients from Eastern Nepal: 13 SbV-naturally resistant and 11 SbV-sensitive, as demonstrated by in vitro drug susceptibility assays. The parasites were genotyped by PCR-RFLP analysis of kDNA minicircles and by microsatellite analysis and the encountered polymorphism revealed a polyclonal structure among resistant isolates. Furthermore, analysis of paired samples obtained from the same patients before treatment and after failure revealed primary as well as acquired resistance. The hypothesis of independent events of drug resistance emergence is proposed and confronted to alternative explanations. Our results show the dynamics of drug resistance epidemiology and highlight the importance of surveillance networks.

Fluorogenic assay for molecular typing of the Leishmania donovani complex: taxonomic and clinical applications.

We describe a new fluorogenic assay for the identification of species and intraspecies groups within the Leishmania donovani complex. The assay combined (1) 2 polymerase chain reactions targeting the 2 cysteine proteinase b isogenes and (2) a fluorescence-resonance energy transfer/melting curve analysis of the polymorphisms within a 31-nt region. All strains within the L. donovani complex were distinguished from L. tropica, L. major, and L. aethiopica, and 5 distinct groups were identified within the L. donovani complex. Discrepancies were observed with the present taxonomy on the basis of isoenzyme analysis and concerned East African strains, which suggests the need for a systematic reevaluation of the taxonomy. The capacity to type parasites directly from clinical samples was demonstrated with blood and bone marrow samples. This rapid and high-throughput alternative for molecular diagnosis and epidemiological studies of visceral leishmaniasis could be adapted for use with other Leishmania species.

Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex.

BACKGROUND: Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP). METHODS: We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase B (cpb), an important Leishmania antigen. The assays were applied to the characterization of 15 reference strains of the L. donovani complex, and their discriminatory power was compared with that of PCR-RFLP analysis of the gp63 gene, another Leishmania antigen, and with that of multilocus enzyme electrophoresis (MLEE), which is the reference standard for parasite typing. RESULTS: Restriction patterns of the cpb locus were polymorphic, but less so than gp63 patterns. When data for both loci were combined, differences between PCR-RFLP and MLEE results were encountered. Antigen gene analysis was more discriminatory and supported a different classification of parasites, one that fitted with their geographic origin. PCR-RFLP analysis of cpb also allowed direct genotyping of parasites in bone marrow aspirate and venous blood samples obtained from patients with VL. CONCLUSION: Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.

A comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis.

We compared the validity of pancytopenia, the formol-gel test (FGT), the indirect fluorescence antibody test (IFAT), the direct agglutination test (DAT), and the rK39 dipstick test as diagnostic criteria for visceral leishmaniasis (VL) in Nepal. Between September 2000 and January 2002, 310 clinical suspects had a bone marrow aspirate, and if negative, a spleen aspirate smear examined for Leishmania donovani. Sensitivity and specificity of all tests were determined compared with parasitology and by latent class analysis (LCA). Compared with parasitology, the sensitivities of the other tests were as follows: pancytopenia = 16.3% (95% confidence interval [CI] = 11.3-22.5%), FGT = 39.9% (95% CI = 32.7-47.4%), IFAT = 28.4% (95% CI = 22.0-35.5%), DAT = 95.1% (95% CI = 90.8-97.7%), and the rK39 dipstick test = 87.4% (95% CI = 81.7-91.9%). Sensitivity estimates obtained by LCA were similar, but specificity estimates were substantially higher (DAT = 93.7% versus 77.8%; rK39 dipstick test = 93.1% versus 77.0%). The DAT or the rK39 dipstick test can replace parasitology as the basis of a decision to treat VL in Nepalese peripheral health services.

Visceral leishmaniasis in two cases of leukemia.

Two cases of visceral leishmaniasis (VL), one in a 51-year-old man with accelerated-phase chronic myeloid leukemia and another in a 35-year-old woman with acute myeloblastic leukemia, are reported. Incidental finding of Leishman-Donovan (LD) bodies in patients with leukemia highlights VL as a potent opportunistic infection in immunosuppressed patients.