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.

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.

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.

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.

Characterizing the non-linear pharmacokinetics of miltefosine in paediatric visceral leishmaniasis patients from Eastern Africa.

BACKGROUND: Conventional miltefosine dosing (2.5 mg/kg/day) for treatment of visceral leishmaniasis (VL) is less effective in children than in adults. A higher allometric dose (median 3.2 mg/kg/day) was therefore investigated in paediatric VL patients in Eastern Africa. Results of this trial showed an unforeseen, lower than dose-proportional increase in exposure. Therefore, we performed a pooled model-based analysis of the paediatric data available from both dosing regimens to characterize observed non-linearities in miltefosine pharmacokinetics (PK). METHODS: Fifty-one children with VL were included in this analysis, treated with either a conventional (n = 21) or allometric (n = 30) miltefosine dosing regimen. PK data were analysed using non-linear mixed-effects modelling. RESULTS: A two-compartment model following first-order absorption and linear elimination, with two separate effects on relative oral bioavailability, was found to fit these data best. A 69% lower bioavailability at treatment start was estimated, presumably due to initial malnourishment and malabsorption. Stagnation in miltefosine accumulation in plasma, hampering increased drug exposure, was related to the increase in cumulative dose (mg/kg/day). However, the allometric regimen increased exposure 1.7-fold in the first treatment week and reduced the time to reach the PK target by 17.4%. CONCLUSIONS: Miltefosine PK in children suffering from VL are characterized by dose-dependent non-linearities that obstruct the initially expected exposure levels. Bioavailability appeared to be affected by the cumulative dose, possibly as a consequence of impaired absorption. Despite this, allometric dosing led to a faster target achievement and increased exposure compared with conventional dosing.

Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum / Leishmania donovani.

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use.

Highly sensitive UPLC-MS/MS method for the quantification of paromomycin in human plasma.

A highly sensitive method was developed to quantitate the antileishmanial agent paromomycin in human plasma, with a lower limit of quantification of 5 ng/mL. Separation was achieved using an isocratic ion-pair ultra-high performance liquid chromatographic (UPLC) method with a minimal concentration of heptafluorobutyric acid, which was coupled through an electrospray ionization interface to a triple quadrupole - linear ion trap mass spectrometer for detection. The method was validated over a linear calibration range of 5 to 1000 ng/mL (r2≥0.997) with inter-assay accuracies and precisions within the internationally accepted criteria. Volumes of 50 µL of human K2EDTA plasma were processed by using a simple protein precipitation method with 40 µL 20 % trichloroacetic acid. A good performance was shown in terms of recovery (100 %), matrix effect (C.V. ≤ 12.0 %) and carry-over (≤17.5 % of the lower limit of quantitation). Paromomycin spiked to human plasma samples was stable for at least 24 h at room temperature, 6 h at 35 °C, and 104 days at -20 °C. Paromomycin adsorbs to glass containers at low concentrations, and therefore acidic conditions were used throughout the assay, in combination with polypropylene tubes and autosampler vials. The assay was successfully applied in a pharmacokinetic study in visceral leishmaniasis patients from Eastern Africa.

Patient costs of diabetes mellitus care in public health care facilities in Kenya.

OBJECTIVE: To estimate the direct and indirect costs of diabetes mellitus care at five public health facilities in Kenya. METHODS: We conducted a cross-sectional study in two counties where diabetes patients aged 18 years and above were interviewed. Data on care-seeking costs were obtained from 163 patients seeking diabetes care at five public facilities using the cost-of-illness approach. Medicines and user charges were classified as direct health care costs while expenses on transport, food, and accommodation were classified as direct non-health care costs. Productivity losses due to diabetes were classified as indirect costs. We computed annual direct and indirect costs borne by these patients. RESULTS: More than half (57.7%) of sampled patients had hypertension comorbidity. Overall, the mean annual direct patient cost was KES 53 907 (95% CI, 43 625.4-64 188.6) (US$ 528.5 [95% CI, 427.7-629.3]). Medicines accounted for 52.4%, transport 22.6%, user charges 17.5%, and food 7.5% of total direct costs. Overall mean annual indirect cost was KES 23 174 (95% CI, 20 910-25 438.8) (US$ 227.2 [95% CI, 205-249.4]). Patients reporting hypertension comorbidity incurred higher costs compared with diabetes-only patients. The incidence of catastrophic costs was 63.1% (95% CI, 55.7-70.7) and increased to 75.4% (95% CI, 68.3-82.1) when transport costs were included. CONCLUSION: There are substantial direct and indirect costs borne by diabetic patients in seeking care from public facilities in Kenya. High incidence of catastrophic costs suggests diabetes services are unaffordable to majority of diabetic patients and illustrate the urgent need to improve financial risk protection to ensure access to care.

Diagnostic Accuracy of Unattended Automated Office Blood Pressure Measurement in Screening for Hypertension in Kenya.

Despite increasing adoption of unattended automated office blood pressure (uAOBP) measurement for determining clinic blood pressure (BP), its diagnostic performance in screening for hypertension in low-income settings has not been determined. We determined the validity of uAOBP in screening for hypertension, using 24-hour ambulatory BP monitoring as the reference standard. We studied a random population sample of 982 Kenyan adults; mean age, 42 years; 60% women; 2% with diabetes mellitus; none taking antihypertensive medications. We calculated sensitivity using 3 different screen positivity cutoffs (≥130/80, ≥135/85, and ≥140/90 mm Hg) and other measures of validity/agreement. Mean 24-hour ambulatory BP monitoring systolic BP was similar to mean uAOBP systolic BP (mean difference, 0.6 mm Hg; 95% CI, -0.6 to 1.9), but the 95% limits of agreement were wide (-39 to 40 mm Hg). Overall discriminatory accuracy of uAOBP was the same (area under receiver operating characteristic curves, 0.66-0.68; 95% CI range, 0.64-0.71) irrespective of uAOBP cutoffs used. Sensitivity of uAOBP displayed an inverse association (P<0.001) with the cutoff selected, progressively decreasing from 67% (95% CI, 62-72) when using a cutoff of ≥130/80 mm Hg to 55% (95% CI, 49-60) at ≥135/85 mm Hg to 44% (95% CI, 39-49) at ≥140/90 mm Hg. Diagnostic performance was significantly better (P<0.001) in overweight and obese individuals (body mass index, >25 kg/m2). No differences in results were present in other subanalyses. uAOBP misclassifies significant proportions of individuals undergoing screening for hypertension in Kenya. Additional studies on how to improve screening strategies in this setting are needed.

Disability-Adjusted Life-Years Due to Stroke in Kenya.

BACKGROUND: There is little information on stroke morbidity in Kenya to inform health care planning. The disability-adjusted life-years (DALYs) are a time-based measure of health status that incorporates both disability and mortality. METHODS: This was a multicenter prospective study in Kenya's public tertiary hospitals conducted in 2015-2017. Data on sex, age, and global disability outcome were collected and used to calculate the sum of years of life lost prematurely due to stroke (YLL), the years of healthy life lost due to disability (YLD), and the DALYs. RESULTS: Up to 719 adult stroke patients participated in the study. The peak age group for stroke was 60-64 years, with ischemic stroke accounting for 56.1% of the stroke cases. After 1-year follow-up, the YLD were 2,402.50, YLL were 5,335.99, and the DALYs were 7,738.49. YLD contributed 31% of the total DALYs. The DALYs varied by sex (male: 2,835.79; female: 4,902.70 years) and by stroke type (ischemic stroke: 4,652.98; hemorrhagic stroke: 3,085.51). The young age group (< 45 years) bore a greater burden accounting for 35.6% of the total DALYs. CONCLUSION: The YLD, YLL, and DALYs observed reinforce the need for targeted prevention of risk factors and comprehensive stroke care initiatives in Kenya.

Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

Patient costs of hypertension care in public health care facilities in Kenya.

BACKGROUND: Hypertension in low- and middle-income countries, including Kenya, is of economic importance due to its increasing prevalence and its potential to present an economic burden to households. In this study, we examined the patient costs associated with obtaining care for hypertension in public health care facilities in Kenya. METHODS: We conducted a cross-sectional study among adult respondents above 18 years of age, with at least 6 months of treatment in two counties. A total of 212 patients seeking hypertension care at five public facilities were interviewed, and information on care seeking and the associated costs was obtained. We computed both annual direct and indirect costs borne by these patients. RESULTS: Overall, the mean annual direct cost to patients was US$ 304.8 (95% CI, 235.7-374.0). Medicines (mean annual cost, US$ 168.9; 95% CI, 132.5-205.4), transport (mean annual cost, US$ 126.7; 95% CI, 77.6-175.9), and user charges (mean annual cost, US$ 57.7; 95% CI, 43.7-71.6) were the highest direct cost categories. Overall mean annual indirect cost was US$ 171.7 (95% CI, 152.8-190.5). The incidence of catastrophic health care costs was 43.3% (95% CI, 36.8-50.2) and increased to 59.0% (95% CI, 52.2-65.4) when transport costs were included. CONCLUSIONS: Hypertensive patients incur substantial direct and indirect costs. High rates of catastrophic costs illustrate the urgency of improving financial risk protection for these patients and strengthening primary care to ensure affordability of hypertension care.

Pharmacokinetics, Safety, and Efficacy of an Allometric Miltefosine Regimen for the Treatment of Visceral Leishmaniasis in Eastern African Children: An Open-label, Phase II Clinical Trial.

BACKGROUND: Convenient, safe, and effective treatments for visceral leishmaniasis in Eastern African children are lacking. Miltefosine, the only oral treatment, failed to achieve adequate efficacy, particularly in children, in whom linear dosing (2.5 mg/kg/day for 28 days) resulted in a 59% cure rate, with lower systemic exposure than in adults. METHODS: We conducted a Phase II trial in 30 children with visceral leishmaniasis, aged 4-12 years, to test whether 28 days of allometric miltefosine dosing safely achieves a higher systemic exposure than linear dosing. RESULTS: Miltefosine accumulated during treatment. Median areas under the concentration time curve from days 0-210 and plasma maximum concentration values were slightly higher than those reported previously for children on linear dosing, but not dose-proportionally. Miltefosine exposure at the start of treatment was increased, with higher median plasma concentrations on day 7 (5.88 versus 2.67 µg/mL). Concentration-time curves were less variable, avoiding the low levels of exposure observed with linear dosing. The 210-day cure rate was 90% (95% confidence interval, 73-98%), similar to that previously described in adults. There were 19 treatment-related adverse events (AEs), but none caused treatment discontinuation. There were 2 serious AEs: both were unrelated to treatment and both patients were fully recovered. CONCLUSIONS: Allometric miltefosine dosing achieved increased and less-variable exposure than linear dosing, though not reaching the expected exposure levels. The new dosing regimen safely increased the efficacy of miltefosine for Eastern African children with visceral leishmaniasis. Further development of miltefosine should adopt allometric dosing in pediatric patients. CLINICAL TRIALS REGISTRATION: NCT02431143.

Demographic, psychosocial and clinical factors associated with postpartum depression in Kenyan women.

BACKGROUND: Few longitudinal studies have examined associations between risk factors during pregnancy and mental health outcomes during the postpartum period. We used a cohort study design to estimate the prevalence, incidence and correlates of significant postpartum depressive symptoms in Kenyan women. METHODS: We recruited adult women residing in an urban, resource-poor setting and attending maternal and child health clinics in two public hospitals in Nairobi, Kenya. A translated Kiswahili Edinburgh Postpartum Depression Scale was used to screen for depressive symptoms at baseline assessment in the 3rd trimester and follow up assessment at 6-10 weeks postpartum. Information was collected on potential demographic, psychosocial and clinical risk variables. Potential risk factors for postpartum depression were evaluated using multivariate logistic regression analysis. RESULTS: Out of the 171 women who were followed up at 6-10 weeks postpartum, 18.7% (95% CI: 13.3-25.5) were found to have postpartum depression using an EPDS cut off of 10. In multivariate analyses, the odds of having postpartum depression was increased more than seven-fold in the presence of conflict with partner (OR = 7.52, 95% CI: 2.65-23.13). The association between antepartum and postpartum depression was quite strong but did not reach statistical significance (OR = 3.37, 95% CI: 0.98-11.64). CONCLUSIONS: The high prevalence of significant postnatal depressive symptoms among Kenyan women underscores the need for addressing this public health burden. Depression screening and psychosocial support interventions that address partner conflict resolution should be offered as part of maternal health care.

Stroke Mortality in Kenya's Public Tertiary Hospitals: A Prospective Facility-Based Study.

BACKGROUND: Despite the increasing global burden of stroke, there are limited data on stroke from Kenya to guide in decision-making. Stroke occurrence in sub-Saharan Africa has been associated with poor health outcomes. This study sought to establish the stroke incidence density and mortality in Kenya's leading public tertiary hospitals for purposes of informing clinical practice and policy. METHODS: This is a prospective study conducted at Kenya's leading referral hospitals, namely, Kenyatta National Hospital (KNH) and Moi Teaching and Referral Hospital (MTRH). Adult patients with confirmed cases of stroke were recruited from February 2015 to January 2016 and followed up for a minimum period of 1 year. The WHO 2006 Stroke STEPS instrument was used to collect data on incidence and mortality at days 10 and 28 and every 3 months for 24 months. The person-time of follow-up was computed from admission to death, loss to follow-up, or the end of the study. A survival regression analysis was done using the Cox proportional hazards model. RESULTS: A total of 719 patients were recruited (KNH: n = 406 [56.5%]; MTRH: n = 313 [43.5%]). The mean age was 58.6 ± 18.7 years, and the male-to-female ratio was 1: 1.4. Ischemic stroke accounted for 56.1% of the stroke cases. The peak age for stroke was between 50 and 69 years, when 36.3% of the cases occurred. Mortality at day 10 and day 28 was 18.4 and 26.7%, respectively. The inpatient mortality rate was 21.6%. The stroke incidence density was 507 deaths per 1,000 person-years of follow-up. The mean survival time was significantly different between inpatients (13.9 months; 95% CI: 13.0-14.7) and outpatients (18.6 months; 95% CI: 17.2-19.9) (p < 0.001). A 1-year increase in age increased the hazard by 1.8%. Inpatients had a 3.9-fold increase in hazard compared to outpatients. CONCLUSIONS: Mortality due to stroke is high, with poor survival observed in the first year after stroke. The risk of death increases with increasing age and duration of hospital stay. There is need for attention to quality of care and long-term needs of stroke patients to mitigate the high mortality rates observed. Public health initiatives aimed at early screening and diagnosis should be enhanced. Further research is recommended to establish the true burden of stroke at the community level to inform appropriate mitigation measures.

Stroke distribution patterns and characteristics in Kenya's leading public health tertiary institutions: Kenyatta National Hospital and Moi Teaching and Referral Hospital.

BACKGROUND: Cardiovascular diseases are the second leading cause of morbidity and mortality in Kenya. However, there is limited clinic-epidemiological data on stroke to inform decision making. This study sought to establish stroke distribution patterns and characteristics in patients seeking care at Kenyatta National Hospital (KNH) and Moi Teaching and Referral Hospital (MTRH), with the ultimate aim of establishing the first national stroke registry in Kenya. METHODS: This was a prospective multicentre cohort study among stroke patients. The study used a modified World Health Organisation STEP-wise approach to stroke surveillance tool in collecting data on incidence, major risk factors and mortality rate. The Cochran's Mantel-Haenszel chisquared test of conditional independence was used with p-value set at 0.05. RESULTS: A total of 691 patients with confirmed stroke were recruited [KNH 406 (males: 40.9%; females: 59.1%); MTRH 285 (males: 44.6%; females: 55.4%) ] and followed over a 12-month period. Overall, ischaemic stroke accounted for 55.6% of the stroke cases, with women being the most affected (57.5%). Mortality rate at day 10 was 18.0% at KNH and 15.5% at MTRH, and higher in the haemorrhagic cases (20.3%). The most common vascular risk factors were hypertension at 77.3% (males: 75.7%; females: 78.5%), smoking at 16.1% (males: 26.6% females: 8.3%) and diabetes at 14.9% (males: 15.7%; females: 14.4%). Ischaemic stroke was conditionally independent of gender after adjusting for age. CONCLUSION: To our knowledge this is the first pilot demonstration establishing a stroke registry in sub-Saharan Africa and clearly establishes feasibility for this approach. It also has utility to both inform and potentially guide public policy and public health measures on stroke in Kenya. Important and unexpected observations included the preponderance of women affected by cerebrovascular disease and that cigarette smoking was the second most common risk factor. The latter, over time, will further impact on the clinico-epidemiological profile of cerebrovascular disease in Kenya.

Evaluation of a Pan-Leishmania Spliced-Leader RNA Detection Method in Human Blood and Experimentally Infected Syrian Golden Hamsters.

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.

Validation of two rapid diagnostic tests for visceral leishmaniasis in Kenya.

BACKGROUND: Visceral leishmaniasis (VL) is a systemic parasitic disease that is fatal unless treated. In Kenya, national VL guidelines rely on microscopic examination of spleen aspirate to confirm diagnosis. As this procedure is invasive, it cannot be safely implemented in peripheral health structures, where non-invasive, accurate, easy to use diagnostic tests are needed. METHODOLOGY: We evaluated the sensitivity, specificity and predictive values of two rapid diagnostic tests (RDT), DiaMed IT LEISH and Signal-KA, among consecutive patients with clinical suspicion of VL in two treatment centres located in Baringo and North Pokot District, Rift Valley province, Kenya. Microscopic examination of spleen aspirate was the reference diagnostic standard. Patients were prospectively recruited between May 2010 and July 2011. PRINCIPAL FINDINGS: Of 251 eligible patients, 219 patients were analyzed, including 131 VL and 88 non-VL patients. The median age of VL patients was 16 years with predominance of males (66%). None of the tested VL patients were co-infected with HIV. Sensitivity and specificity of the DiaMed IT LEISH were 89.3% (95%CI: 82.7-94%) and 89.8% (95%CI: 81.5-95.2%), respectively. The Signal KA showed trends towards lower sensitivity (77.1%; 95%CI: 68.9-84%) and higher specificity (95.5%; 95%CI: 88.7-98.7%). Combining the tests did not improve the overall diagnostic performance, as all patients with a positive Signal KA were also positive with the DiaMed IT LEISH. CONCLUSION/SIGNIFICANCE: The DiaMed IT LEISH can be used to diagnose VL in Kenyan peripheral health facilities where microscopic examination of spleen aspirate or sophisticated serological techniques are not feasible. There is a crucial need for an improved RDT for VL diagnosis in East Africa.

Sodium stibogluconate (SSG) & paromomycin combination compared to SSG for visceral leishmaniasis in East Africa: a randomised controlled trial.

BACKGROUND: Alternative treatments for visceral leishmaniasis (VL) are required in East Africa. Paromomycin sulphate (PM) has been shown to be efficacious for VL treatment in India. METHODS: A multi-centre randomized-controlled trial (RCT) to compare efficacy and safety of PM (20 mg/kg/day for 21 days) and PM plus sodium stibogluconate (SSG) combination (PM, 15 mg/kg/day and SSG, 20 mg/kg/day for 17 days) with SSG (20 mg/kg/day for 30 days) for treatment of VL in East Africa. Patients aged 4-60 years with parasitologically confirmed VL were enrolled, excluding patients with contraindications. Primary and secondary efficacy outcomes were parasite clearance at 6-months follow-up and end of treatment, respectively. Safety was assessed mainly using adverse event (AE) data. FINDINGS: The PM versus SSG comparison enrolled 205 patients per arm with primary efficacy data available for 198 and 200 patients respectively. The SSG & PM versus SSG comparison enrolled 381 and 386 patients per arm respectively, with primary efficacy data available for 359 patients per arm. In Intention-to-Treat complete-case analyses, the efficacy of PM was significantly lower than SSG (84.3% versus 94.1%, difference = 9.7%, 95% confidence interval, CI: 3.6 to 15.7%, p = 0.002). The efficacy of SSG & PM was comparable to SSG (91.4% versus 93.9%, difference = 2.5%, 95% CI: -1.3 to 6.3%, p = 0.198). End of treatment efficacy results were very similar. There were no apparent differences in the safety profile of the three treatment regimens. CONCLUSION: The 17 day SSG & PM combination treatment had a good safety profile and was similar in efficacy to the standard 30 day SSG treatment, suggesting suitability for VL treatment in East Africa. CLINICAL TRIALS REGISTRATION: www.clinicaltrials.govNCT00255567.

Geographical variation in the response of visceral leishmaniasis to paromomycin in East Africa: a multicentre, open-label, randomized trial.

BACKGROUND: Visceral leishmaniasis (VL) is a major health problem in developing countries. The untreated disease is fatal, available treatment is expensive and often toxic, and drug resistance is increasing. Improved treatment options are needed. Paromomycin was shown to be an efficacious first-line treatment with low toxicity in India. METHODS: This was a 3-arm multicentre, open-label, randomized, controlled clinical trial to compare three treatment regimens for VL in East Africa: paromomycin sulphate (PM) at 15 mg/kg/day for 21 days versus sodium stibogluconate (SSG) at 20 mg/kg/day for 30 days; and the combination of both dose regimens for 17 days. The primary efficacy endpoint was cure based on parasite-free tissue aspirates taken 6 months after treatment. FINDINGS: Overall, 135 patients per arm were enrolled at five centres in Sudan (2 sites), Kenya (1) and Ethiopia (2), when the PM arm had to be discontinued due to poor efficacy. The trial has continued with the higher dose of PM as well as the combination of PM and SSG arms. These results will be reported later. Baseline patient characteristics were similar among treatment arms. The overall cure with PM was significantly inferior to that with SSG (63.8% versus 92.2%; difference 28.5%, 95%CI 18.8% to 38.8%, p<0.001). The efficacy of PM varied among centres and was significantly lower in Sudan (14.3% and 46.7%) than in Kenya (80.0%) and Ethiopia (75.0% and 96.6%). No major safety issues with PM were identified. CONCLUSION: The efficacy of PM at 15 mg/kg/day for 21 days was inadequate, particularly in Sudan. The efficacy of higher doses and the combination treatment warrant further studies.