Disease-specific differences in pharmacokinetics of paromomycin and miltefosine between post-kala-azar dermal leishmaniasis and visceral leishmaniasis patients in eastern Africa.

Treatment regimens for post-kala-azar dermal leishmaniasis (PKDL) are usually extrapolated from those for visceral leishmaniasis (VL), but drug pharmacokinetics (PK) can differ due to disease-specific variations in absorption, distribution, and elimination. This study characterized PK differences in paromomycin and miltefosine between 109 PKDL and 264 VL patients from eastern Africa. VL patients showed 0.55-fold (95%CI: 0.41-0.74) lower capacity for paromomycin saturable reabsorption in renal tubules, and required a 1.44-fold (1.23-1.71) adjustment when relating renal clearance to creatinine-based eGFR. Miltefosine bioavailability in VL patients was lowered by 69% (62-76) at treatment start. Comparing PKDL to VL patients on the same regimen, paromomycin plasma exposures were 0.74-0.87-fold, while miltefosine exposure until the end of treatment day was 1.4-fold. These pronounced PK differences between PKDL and VL patients in eastern Africa highlight the challenges of directly extrapolating dosing regimens from one leishmaniasis presentation to another.

Paromomycin and Miltefosine Combination as an Alternative to Treat Patients With Visceral Leishmaniasis in Eastern Africa: A Randomized, Controlled, Multicountry Trial.

BACKGROUND: This study aimed to determine whether paromomycin plus miltefosine (PM/MF) is noninferior to sodium stibogluconate plus paromomycin (SSG/PM) for treatment of primary visceral leishmaniasis in eastern Africa. METHODS: An open-label, phase 3, randomized, controlled trial was conducted in adult and pediatric patients at 7 sites in eastern Africa. Patients were randomly assigned to either 20 mg/kg paromomycin plus allometric dose of miltefosine (14 days), or 20 mg/kg sodium stibogluconate plus 15 mg/kg paromomycin (17 days). The primary endpoint was definitive cure after 6 months. RESULTS: Of 439 randomized patients, 424 completed the trial. Definitive cure at 6 months was 91.2% (155 of 170) and 91.8% (156 of 170) in the PM/MF and SSG/PM arms in primary efficacy modified intention-to-treat analysis (difference, 0.6%; 97.5% confidence interval [CI], -6.2 to 7.4), narrowly missing the noninferiority margin of 7%. In the per-protocol analysis, efficacy was 92% (149 of 162) and 91.7% (155 of 169) in the PM/MF and SSG/PM arms (difference, -0.3%; 97.5% CI, -7.0 to 6.5), demonstrating noninferiority. Treatments were well tolerated. Four of 18 serious adverse events were study drug-related, and 1 death was SSG-related. Allometric dosing ensured similar MF exposure in children (<12 years) and adults. CONCLUSIONS: PM/MF and SSG/PM efficacies were similar, and adverse drug reactions were as expected given the drugs safety profiles. With 1 less injection each day, reduced treatment duration, and no risk of SSG-associated life-threatening cardiotoxicity, PM/MF is a more patient-friendly alternative for children and adults with primary visceral leishmaniasis in eastern Africa. CLINICAL TRIALS REGISTRATION: NCT03129646.

Population pharmacokinetics of a combination of miltefosine and paromomycin in Eastern African children and adults with visceral leishmaniasis.

OBJECTIVES: To improve visceral leishmaniasis (VL) treatment in Eastern Africa, 14- and 28-day combination regimens of paromomycin plus allometrically dosed miltefosine were evaluated. As the majority of patients affected by VL are children, adequate paediatric exposure to miltefosine and paromomycin is key to ensuring good treatment response. METHODS: Pharmacokinetic data were collected in a multicentre randomized controlled trial in VL patients from Kenya, Sudan, Ethiopia and Uganda. Patients received paromomycin (20 mg/kg/day for 14 days) plus miltefosine (allometric dose for 14 or 28 days). Population pharmacokinetic models were developed. Adequacy of exposure and target attainment of paromomycin and miltefosine were evaluated in children and adults. RESULTS: Data from 265 patients (59% ≤12 years) were available for this pharmacokinetic analysis. Paromomycin exposure was lower in paediatric patients compared with adults [median (IQR) end-of-treatment AUC0-24h 187 (162-203) and 242 (217-328) µg·h/mL, respectively], but were both within the IQR of end-of-treatment exposure in Kenyan and Sudanese adult patients from a previous study. Cumulative miltefosine end-of-treatment exposure in paediatric patients and adults [AUCD0-28 517 (464-552) and 524 (456-567) µg·day/mL, respectively] and target attainment [time above the in vitro susceptibility value EC90 27 (25-28) and 30 (28-32) days, respectively] were comparable to previously observed values in adults. CONCLUSIONS: Paromomycin and miltefosine exposure in this new combination regimen corresponded to the desirable levels of exposure, supporting the implementation of the shortened 14 day combination regimen. Moreover, the lack of a clear exposure-response and exposure-toxicity relationship indicated adequate exposure within the therapeutic range in the studied population, including paediatric patients.

Safety and immunogenicity of ChAd63-KH vaccine in post-kala-azar dermal leishmaniasis patients in Sudan.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6-180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42-120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway.

Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa.

BACKGROUND: To expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes. METHODS: Data from 3 clinical trials were combined in this study, in which Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative polymerase chain reaction (qPCR) before, during, and up to 6 months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at 6 months. RESULTS: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within 6 months of follow-up at a cutoff of 20 parasites/mL (area under the curve 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ = 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a > 80% power to detect a difference in cure rate between treatment regimens if this difference was high (> 50%) and when minimally 30 patients were included per regimen. CONCLUSIONS: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.

Visceral leishmaniasis relapse hazard is linked to reduced miltefosine exposure in patients from Eastern Africa: a population pharmacokinetic/pharmacodynamic study.

BACKGROUND: Low efficacy of miltefosine in the treatment of visceral leishmaniasis was recently observed in Eastern Africa. OBJECTIVES: To describe the pharmacokinetics and establish a pharmacokinetic/pharmacodynamic relationship for miltefosine in Eastern African patients with visceral leishmaniasis, using a time-to-event approach to model relapse of disease. METHODS: Miltefosine plasma concentrations from 95 patients (48 monotherapy versus 47 combination therapy) were included in the population pharmacokinetic model using non-linear mixed effects modelling. Subsequently a time-to-event model was developed to model the time of clinical relapse. Various summary pharmacokinetic parameters (various AUCs, Time > EC50, Time > EC90), normalized within each treatment arm to allow simultaneous analysis, were evaluated as relapse hazard-changing covariates. RESULTS: A two-compartment population model with first-order absorption fitted the miltefosine pharmacokinetic data adequately. Relative bioavailability was reduced (-74%, relative standard error 4.7%) during the first week of treatment of the monotherapy arm but only the first day of the shorter combination regimen. Time to the relapse of infection could be described using a constant baseline hazard (baseline 1.8 relapses/year, relative standard error 72.7%). Miltefosine Time > EC90 improved the model significantly when added in a maximum effect function on the baseline hazard (half maximal effect with Time > EC90 6.97 days for monotherapy). CONCLUSIONS: Miltefosine drug exposure was found to be decreased in Eastern African patients with visceral leishmaniasis, due to a (transient) initial lower bioavailability. Relapse hazard was inversely linked to miltefosine exposure. Significantly lower miltefosine exposure was observed in children compared with adults, further urging the need for implementation of dose adaptations for children.

Insights into the possible role of IFNG and IFNGR1 in Kala-azar and Post Kala-azar Dermal Leishmaniasis in Sudanese patients.

BACKGROUND: Little is known about the parasite/host factors that lead to Post Kala-azar Dermal Leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after drug-cure. Studies in Sudan provide evidence for association between polymorphisms in the gene (IFNGR1) encoding the alpha chain of interferon-γ receptor type I and risk of PKDL. This study aimed to identify putative functional polymorphisms in the IFNGR1 gene, and to determine whether differences in expression of interferon-γ (IFNG) and IFNGR1 at the RNA level are associated with pathogenesis of VL and/or PKDL in Sudan. METHODS: Sanger sequencing was used to re-sequence 841 bp of upstream, exon1 and intron1 of the IFNGR1 gene in DNA from 30 PKDL patients. LAGAN and SYNPLOT bioinformatics tools were used to compare human, chimpanzee and dog sequences to identify conserved noncoding sequences carrying putative regulatory elements. The relative expression of IFNG and IFNGR1 in paired pre- and post-treatment RNA samples from the lymph nodes of 24 VL patients, and in RNA samples from skin biopsies of 19 PKDL patients, was measured using real time PCR. Pre- versus post-treatment expression was evaluated statistically using the nonparametric Wilcoxon matched pairs signed-rank test. RESULTS: Ten variants were identified in the 841 bp of sequence, four of which are novel polymorphisms at -77A/G, +10 C/T, +18C/T and +91G/T relative to the IFNGR1 initiation site. A cluster of conserved non-coding sequences with putative regulatory variants was identified in the distal promoter of IFNGR1. Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment. IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment. IFNG expression was undetectable in the skin biopsies of PKDL cases, while IFNGR1 expression was also uniformly low. CONCLUSIONS: Uniformly low expression of IFN and IFNGR1 in PKDL skin biopsies could explain parasite persistence and is consistent with prior demonstration of genetic association with IFNGR1 polymorphisms. Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.

Leishmania blood parasite dynamics during and after treatment of visceral leishmaniasis in Eastern Africa: A pharmacokinetic-pharmacodynamic model.

BACKGROUND: With the current treatment options for visceral leishmaniasis (VL), recrudescence of the parasite is seen in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting patient relapse in cases of VL. This study aimed to characterize the kinetics of circulating Leishmania parasites in the blood, during and after different antileishmanial therapies, and to find predictors for clinical relapse of disease. METHODS: Data from three clinical trials, in which Eastern African VL patients received various antileishmanial regimens, were combined in this study. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during, and up to six months after treatment. An integrated population pharmacokinetic-pharmacodynamic model was developed using non-linear mixed effects modelling. RESULTS: Parasite proliferation was best described by an exponential growth model, with an in vivo parasite doubling time of 7.8 days (RSE 12%). Parasite killing by fexinidazole, liposomal amphotericin B, sodium stibogluconate, and miltefosine was best described by linear models directly relating drug concentrations to the parasite elimination rate. After treatment, parasite growth was assumed to be suppressed by the host immune system, described by an Emax model driven by the time after treatment. No predictors for the high variability in onset and magnitude of the immune response could be identified. Model-based individual predictions of blood parasite load on Day 28 and Day 56 after start of treatment were predictive for clinical relapse of disease. CONCLUSION: This semi-mechanistic pharmacokinetic-pharmacodynamic model adequately captured the blood parasite dynamics during and after treatment, and revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could be a useful biomarker to assess treatment efficacy of a treatment regimen in a clinical trial setting.

A randomized, double-blind phase 2b trial to evaluate efficacy of ChAd63-KH for treatment of post kala-azar dermal leishmaniasis.

In a recent phase 2a clinical trial, the candidate leishmaniasis vaccine ChAd63-KH was shown to be safe and immunogenic in Sudanese patients with post kala-azar dermal leishmaniasis (PKDL). However, its value as a stand-alone therapeutic was unknown. To assess the therapeutic efficacy of ChAd63-KH, we conducted a randomized, double-blind, placebo-controlled phase 2b trial (ClinicalTrials.gov: NCT03969134). Primary outcomes were safety and efficacy (≥90% improvement in clinical disease). Secondary outcomes were change in severity grade and vaccine-induced immune response. 86 participants with uncomplicated PKDL of ≥6 month duration were randomized to receive ChAd63-KH (7.5 × 1010 viral particles, once by the intramuscular route) or placebo. 75 participants (87%) completed the trial as per protocol. No severe or serious adverse events were observed. At day 90 post-vaccination, 6/40 (15%) and 4/35 (11%) participants in the vaccine and placebo groups, respectively, showed ≥90% clinical improvement (risk ratio [RR] 1.31 [95% confidence interval (CI), 0.40-4.28], p = 0.742). There were also no significant differences in PKDL severity grade between study arms. Whole-blood transcriptomic analysis identified transcriptional modules associated with interferon responses and monocyte and dendritic cell activation. Thus, a single vaccination with ChAd63-KH showed no therapeutic efficacy in this subset of Sudanese patients with PKDL.

Genetic and functional evidence implicating DLL1 as the gene that influences susceptibility to visceral leishmaniasis at chromosome 6q27.

BACKGROUND: Visceral leishmaniasis (VL) is caused by Leishmania donovani and Leishmania infantum chagasi. Genome-wide linkage studies from Sudan and Brazil identified a putative susceptibility locus on chromosome 6q27. METHODS: Twenty-two single-nucleotide polymorphisms (SNPs) at genes PHF10, C6orf70, DLL1, FAM120B, PSMB1, and TBP were genotyped in 193 VL cases from 85 Sudanese families, and 8 SNPs at genes PHF10, C6orf70, DLL1, PSMB1, and TBP were genotyped in 194 VL cases from 80 Brazilian families. Family-based association, haplotype, and linkage disequilibrium analyses were performed. Multispecies comparative sequence analysis was used to identify conserved noncoding sequences carrying putative regulatory elements. Quantitative reverse-transcription polymerase chain reaction measured expression of candidate genes in splenic aspirates from Indian patients with VL compared with that in the control spleen sample. RESULTS: Positive associations were observed at PHF10, C6orf70, DLL1, PSMB1, and TBP in Sudan, but only at DLL1 in Brazil (combined P = 3 × 10(-4) at DLL1 across Sudan and Brazil). No functional coding region variants were observed in resequencing of 22 Sudanese VL cases. DLL1 expression was significantly (P = 2 × 10(-7)) reduced (mean fold change, 3.5 [SEM, 0.7]) in splenic aspirates from patients with VL, whereas other 6q27 genes showed higher levels (1.27 × 10(-6) < P < .01) than did the control spleen sample. A cluster of conserved noncoding sequences with putative regulatory variants was identified in the distal promoter of DLL1. CONCLUSIONS: DLL1, which encodes Delta-like 1, the ligand for Notch3, is strongly implicated as the chromosome 6q27 VL susceptibility gene.

Development and validation of an ultra-high performance liquid chromatography coupled to tandem mass spectrometry method for the quantification of the antileishmanial drug paromomycin in human skin tissue.

Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r2 ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.

Geographical Variability in Paromomycin Pharmacokinetics Does Not Explain Efficacy Differences between Eastern African and Indian Visceral Leishmaniasis Patients.

INTRODUCTION: Intramuscular paromomycin monotherapy to treat visceral leishmaniasis (VL) has been shown to be effective for Indian patients, while a similar regimen resulted in lower efficacy in Eastern Africa, which could be related to differences in paromomycin pharmacokinetics. METHODS: Pharmacokinetic data were available from two randomized controlled trials in VL patients from Eastern Africa and India. African patients received intramuscular paromomycin monotherapy (20 mg/kg for 21 days) or combination therapy (15 mg/kg for 17 days) with sodium stibogluconate. Indian patients received paromomycin monotherapy (15 mg/kg for 21 days). A population pharmacokinetic model was developed for paromomycin in Eastern African and Indian VL patients. RESULTS: Seventy-four African patients (388 observations) and 528 Indian patients (1321 observations) were included in this pharmacokinetic analysis. A one-compartment model with first-order kinetics of absorption and elimination best described paromomycin in plasma. Bioavailability (relative standard error) was 1.17 (5.18%) times higher in Kenyan and Sudanese patients, and 2.46 (24.5%) times higher in Ethiopian patients, compared with Indian patients. Ethiopian patients had an approximately fourfold slower absorption rate constant of 0.446 h-1 (18.2%). Area under the plasma concentration-time curve for 24 h at steady-state (AUCτ,SS) for 15 mg/kg/day (median [interquartile range]) was higher in Kenya and Sudan (172.7 µg·h/mL [145.9-214.3]) and Ethiopia (230.1 µg·h/mL [146.3-591.2]) compared with India (97.26 µg·h/mL [80.83-123.4]). CONCLUSION: The developed model provides detailed insight into the pharmacokinetic differences among Eastern African countries and India, however the resulting differences in paromomycin exposure do not seem to explain the geographical differences in paromomycin efficacy in the treatment of VL patients.

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan.

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 x 10(-7); empirical p < 1 x 10(-5); lambdaS = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 x 10(-5); empirical p < 1 x 10(-4); lambdaS = 2.3) that were Y chromosome-lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.

Three-Dimensional Optical Scanning in Post-kala-azar Dermal Leishmaniasis (PKDL).

Post-kala-azar dermal leishmaniasis may occur after successful treatment of visceral leishmaniasis and is characterized by macules, papules, or nodules in the skin, with varying size. The response to antileishmanial therapy remains difficult to assess because there are presently no reliable biomarkers. To date, skin lesions are clinically assessed for decrease in size or change in color, which is invariably subjective. Novel 3-dimensional optical scanning devices offer safe and field-adapted methods to objectively assess skin lesions for changes over time in size and color that can be quantified with great accuracy.

Evaluation of the particle gel diffusion technique for the detection of haemoglobin S in Sudanese patients.

BACKGROUND: Sickle cell disease is a heterogenous disorder characterized by an abnormal haemoglobin and sickling phenomena of red cells. It is prevalent among certain nomadic tribes in Sudan. Painful, aplastic and haemolytic crises mark sickle cell anaemia. Haemoglobin S (HbS) is detected using haemoglobin electrophoresis, iso-electric focusing and/or high-performance liquid chromatography techniques with high sensitivity, but entails cost and expertise. This study aimed to determine the sensitivity, specificity and positive predictive values (PPV) of the ID-particle gel diffusion technique for screening, diagnosis and phenotyping of HbS in patients with a provisional diagnosis of abnormal haemoglobin. METHODS: Following informed consent, 100 sequential individuals who reported to a central referral haemoglobinopathy clinic were enrolled. ID-particle gel diffusion technique was compared with cellulose acetate electrophoresis to determine haemoglobin phenotypes. RESULTS: The ID-particle gel test detected HbAA with 100% sensitivity and 100% specificity. Sensitivity for HbS was 100%, whether as HbSS or as a mixed pattern. HbSS was identified in all cases where this is the only haemoglobin present. Other patterns were detected with <100% specificity and these would require further testing by other means to definitively identify abnormal haemoglobins. CONCLUSIONS: Although the ID-particle technique is a simple and cheap technique with high sensitivity, specificity and PPV compared with cellulose acetate electrophoresis in detecting HbSS, it could not differentiate HbAS from HbSS with high levels of HbF. High environmental temperatures could melt the test microtubes. Cellulose acetate electrophoresis remains the technique of choice for the screening of abnormal haemoglobins in the Sudan.

Distribution of Duffy Phenotypes among Plasmodium vivax Infections in Sudan.

Negative Duffy expression on the surface of human red blood cells was believed to be a barrier for Plasmodium vivax infection in most Africans. However, P. vivax has been demonstrated to infect Duffy-negative individuals in several Central and East African countries. In this study, we investigated the distribution of Duffy blood group phenotypes with regard to P. vivax infection and parasitemia in Sudan. Out of 992 microscopic-positive malaria samples, 190 were identified as P. vivax positive infections. Among them, 186 were P. vivax mono-infections and 4 were mixed P. vivax and Plasmodium falciparum infections. A subset of 77 samples was estimated with parasitemia by quantitative real-time PCR. Duffy codons were sequenced from the 190 P. vivax positive samples. We found that the Duffy Fy(a-b+) phenotype was the most prevalent, accounting for 67.9% of all P. vivax infections, while homozygous Duffy-negative Fy(a-b-) accounted for 17.9% of the P. vivax infections. The prevalence of infection in Fy(a-b+) and Fy(a+b-)were significantly higher than Fy(a-b-) phenotypes (p = 0.01 and p < 0.01, respectively). A significantly low proportion of P. vivax infection was observed in Duffy negative individuals Fy(a-b-). This study highlights the prevalence of P. vivax in Duffy-negatives in Sudan and indicates low parasitemia among the Duffy-negative individuals.

Macrophage Activation Marker Neopterin: A Candidate Biomarker for Treatment Response and Relapse in Visceral Leishmaniasis.

The Leishmania parasite resides and replicates within host macrophages during visceral leishmaniasis (VL). This study aimed to evaluate neopterin, a marker of macrophage activation, as possible pharmacodynamic biomarker to monitor VL treatment response and to predict long-term clinical relapse of VL. Following informed consent, 497 plasma samples were collected from East-African VL patients receiving a 28-day miltefosine monotherapy (48 patients) or 11-day combination therapy of miltefosine and liposomal amphotericin B (L-AMB, 48 patients). Neopterin was quantified with ELISA. Values are reported as median (inter-quartile range). Baseline neopterin concentrations were elevated in all VL patients at 98.8 (63.9-135) nmol/L compared to reported levels for healthy controls (<10 nmol/L). During the first treatment week, concentrations remained stable in monotherapy patients (p = 0.807), but decreased two-fold compared to baseline in the combination therapy patients (p < 0.01). In the combination therapy arm, neopterin concentrations increased significantly 1 day after L-AMB infusion compared to baseline for cured patients [137 (98.5-197) nmol/L, p < 0.01], but not for relapsing patients [84.4 (68.9-106) nmol/L, p = 0.96]. The neopterin parameter with the highest predictive power for VL relapse was a higher than 8% neopterin concentration increase between end of treatment and day 60 follow-up (ROC AUC 0.84), with a 93% sensitivity and 65% specificity. In conclusion, the identified neopterin parameter could be a potentially useful surrogate endpoint to identify patients in clinical trials at risk of relapse earlier during follow-up, possibly in a panel of biomarkers to increase its specificity.

Safety and Effectiveness of Sodium Stibogluconate and Paromomycin Combination for the Treatment of Visceral Leishmaniasis in Eastern Africa: Results from a Pharmacovigilance Programme.

INTRODUCTION: In 2010, WHO recommended a new first-line treatment for visceral leishmaniasis (VL) in Eastern Africa. The new treatment, a combination of intravenous (IV) or intramuscular (IM) sodium stibogluconate (SSG) and IM paromomycin (PM) was an improvement over SSG monotherapy, the previous first-line VL treatment in the region. To monitor the new treatment's safety and effectiveness in routine clinical practice a pharmacovigilance (PV) programme was developed. METHODS: A prospective PV cohort was developed. Regulatory approval was obtained in Sudan, Kenya, Uganda and Ethiopia. Twelve sentinel sites sponsored by the Ministries of Health, Médecins Sans Frontières (MSF) and Drugs for Neglected Diseases initiative (DNDi) participated. VL patients treated using the new treatment were consented and included in a common registry that collected demographics, baseline clinical characteristics, adverse events, serious adverse events and treatment outcomes. Six-monthly periodic safety update reports (PSUR) were prepared and reviewed by a PV steering committee. RESULTS: Overall 3126 patients were enrolled: 1962 (62.7%) from Sudan, 652 (20.9%) from Kenya, 322 (10.3%) from Ethiopia and 190 (6.1%) from Uganda. Patients were mostly male children (68.1%, median age 11 years) with primary VL (97.8%). SSG-PM initial cure rate was 95.1%; no geographical differences were noted. HIV/VL co-infected patients and patients older than 50 years had initial cure rates of 56 and 81.4%, respectively, while 1063 (34%) patients had at least one adverse event (AE) during treatment and 1.92% (n = 60) had a serious adverse event (SAE) with a mortality of 1.0% (n = 32). There were no serious unexpected adverse drug reactions. CONCLUSIONS: This first regional PV programme in VL supports SSG-PM combination as first-line treatment for primary VL in Eastern Africa. SSG-PM was effective and safe except in HIV/VL co-infected or older patients. Active PV surveillance of targeted safety, effectiveness and key VL outcomes such us VL relapse, PKDL and HIV/VL co-infection should continue and PV data integrated to national and WHO PV databases.

Antimony-induced cerebellar ataxia.

Visceral leishmaniasis VL, caused by Leishmania donovani is endemic over several parts of Sudan. The disease is fatal if not treated. Although sodium stibogluconate Pentostam, a pentavalent antimonial is not free from toxicity, it has been in use for treatment of VL for the last 50 years. Like other infectious diseases, neurological manifestations of VL and sodium stibogluconate have been documented. In this report, we present 2 cases of cerebellar ataxia most likely induced by Pentostam, and explain the probable cause.

Sudanese mucosal leishmaniasis: isolation of a parasite within the Leishmania donovani complex that differs genotypically from L. donovani causing classical visceral leishmaniasis.

Mucosal leishmaniasis, which is a sporadic disease in the Sudan, was shown by isoenzyme characterization and PCR to be caused by Leishmania donovani. However, it was not clear if the parasite was exactly the same strain as that causing visceral leishmaniasis (VL), or of a different strain. We utilized a new generation of molecular DNA markers, minisatellites and kinetoplast DNA, for rapid characterization of the parasite. The results show that the genotypes of some of the parasites causing VL are different from those causing mucosal leishmaniasis. The L. donovani isolates causing visceral disease, as well as post-kala-azar mucosal leishmaniasis (PKML), have been shown to possess characteristic haplotypes. However, sequencing of a portion of the cytochrome oxidase II (COII) gene indicates that the parasite that invades the oral mucosa is divergent from other parasites causing VL. It appears to possess features of a more ancestral parasite with pronounced sequence homology to L. major. This agrees with earlier studies where isolates of mucosal leishmaniasis have been shown to possess an isoenzyme profile distinct from L. donovani and a different geographical distribution, albeit often overlapping with that of L. donovani.