Limited effectiveness of high-dose liposomal amphotericin B (AmBisome) for treatment of visceral leishmaniasis in an Ethiopian population with high HIV prevalence.

Due to unacceptably high mortality with pentavalent antimonials, Médecins Sans Frontières in 2006 began using liposomal amphotericin B (AmBisome) for visceral leishmaniasis (VL) patients in Ethiopia who were severely ill or positive for human immunodeficiency virus (HIV)

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs)

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials

Potential for Use in Rapid Serodiagnostic Tests (RDTs)

Abstract The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials. Funding: This work was funded via a Biotechnology and Biological Sciences Research Council (BBSRC, UK) Flexible Interchange Programme (FLIP) award (grant number BB/L026279/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Author Summary The development of rapid serodiagnostic tests for African sleeping sickness and other diseases caused by kinetoplastids relies in part on the affordable production of parasite-specific recombinant antigens. The majority of cases of sleeping sickness are caused by the parasite Trypanosoma brucei gambiense (T.b. gambiense) which is transmitted when bitten by an infected tsetse fly. Existing tests rely on the utilisation of extracts from the parasite or use antigens raised in animal models. In this study we have shown that using a cell culture system devised from a parasite similar to T.b. gambiense recombinant antigens can be produced that are as effective in rapid diagnostic tests as the native antigens purified from T.b. gambiense parasites grown in laboratory rodents. We compared the diagnostic sensitivity and specificity of the antigens we produced recombinantly to native antigens currently used in diagnostic kits against a panel of human sera. The evaluation on sera from 172 T.b. gambiense patients and from 119 controls without sleeping sickness showed very high diagnostic potential of two recombinant antigens where the response was not significantly different to that from the native antigens. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests

Seasonal upsurge of pneumococcal meningitis in the Central African Republic [version 2; peer review: 1 approved, 2 approved with reservations]

A high incidence of bacterial meningitis was observed in the Central African Republic (CAR) from December 2015 to May 2017 in three hospitals in the northwest of the country that are within the African meningitis belt. The majority of cases were caused by Streptococcus pneumoniae (249/328; 75.9%), which occurred disproportionately during the dry season (November-April) with a high case-fatality ratio of 41.6% (95% confidence interval [CI] 33.0, 50.8%). High rates of bacterial meningitis during the dry season in the meningitis belt have typically been caused by Neisseria meningitidis (meningococcal meningitis), and our observations suggest that the risk of contracting S. pneumoniae (pneumococcal) meningitis is increased by the same environmental factors. Cases of meningococcal meningitis (67/328; 20.4%) observed over the same period were predominantly group W and had a lower case fatality rate of 9.6% (95% CI 3.6, 21.8%). Due to conflict and difficulties in accessing medical facilities, it is likely that the reported cases represented only a small proportion of the overall burden. Nationwide vaccination campaigns in the CAR against meningitis have been limited to the use of MenAfriVac, which targets only meningococcal meningitis group A. We therefore highlight the need for expanded vaccine coverage to prevent additional causes of seasonal outbreaks

Serum collection used in this study.

<p>Serum collection used in this study.</p

Gel filtration of purified rISG65 on a Superdex 200 10/300 GL column (GE Healthcare Lifesciences) in sodium phosphate buffer, pH7.5, O.5 M NaCl.

<p>A. Molecular weight markers (Sigma-Aldrich MWGF100) 1 = thyroglobulin 669 kDa, 2 = apoferritin 443 kDa, 3 = β-amylase 200 kDa, 4 = alcohol dehydrogenase 150 kDa, 5 = bovine serum albumin 66 kDa, 6 = carbonic anhydrase 29 kDa. B. Elution profile of rISG following one round of metal affinity chromatography purification.</p

SDS-PAGE analysis of the expression of rISG65, rLiTat1.3 and rLiTat1.5 by <i>Leishmania tarentolae</i> after purification following a single round of metal affinity chromatography.

<p>Coomassie stained 10% SDS-PAGE; Precision Plus Protein Unstained Standards, BioRad (M). Lane 1: rISG65, Lane 2: rLiTat1.3, Lane 3: rLiTat1.5. A = 1 μg and B = 10 μg protein.</p

Analysis of the deglycosylation of rISG65 using PNGase F (1.5 U) (Sigma-Aldrich, P7367) for two hours at 37°C.

<p>Coomassie blue stained pre-cast 4–12% BisTris gradient SDS-PAGE gel (Novex) using the MOPS running system; Precision Plus Protein Unstained Standards, BioRad (M). Lane 1: rISG65 (1 ug) before treatment with PNGase F, Lane 2: rISG65 (1 ug) after treatment with PNGase F.</p

Area under the curve (AUC) and its 95% confidence interval (CI), Youden index, percent sensitivity (Se %), percent specificity (Sp %) and their respective 95% CI recorded for the different antigens when tested with sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i> HAT controls and 50 non-<i>r-</i>HAT controls.

<p>Area under the curve (AUC) and its 95% confidence interval (CI), Youden index, percent sensitivity (Se %), percent specificity (Sp %) and their respective 95% CI recorded for the different antigens when tested with sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i> HAT controls and 50 non-<i>r-</i>HAT controls.</p

Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls with nLiTat 1.3 (2 μg/ml), rLiTat 1.3 (4 μg/ml), nLiTat 1.5 (2 μg/ml), rLiTat 1.5 (4 μg/ml) and rISG65 (4 μg/ml).

<p>Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls with nLiTat 1.3 (2 μg/ml), rLiTat 1.3 (4 μg/ml), nLiTat 1.5 (2 μg/ml), rLiTat 1.5 (4 μg/ml) and rISG65 (4 μg/ml).</p