Strategies for the prevention of antimalarial drug resistance: rationale for combination chemotherapy for malaria

Among the several 'tropical' diseases that affect humans, malarin poses special control problems due to the increasing population at risk from the disease, the difficulties in eradicating the mosquito vector in the tropics and the emergence and spread of parasite resistance to commonly used antimalarial drugs. There is both clinical experience and experimental evidence that, however effective when first introduced, the lifespan of drugs is inevitably curtailed by the emergence of resistant parasites. Resistance is the most important factor in determining the useful lifespan of antimalarial drugs. In this review, Nick White and Piero Olliaro discuss the rationale for combination chemotherapy.     

Practical progress and new drugs for changing patterns of leishmaniasis

The problems surrounding leishmaniasis are changing. An increase in travel, the Indian and Sudanese epidemics of visceral leishmaniasis, parasite resistance to antimony and the emergence of AIDS-related leishmaniasis have all increased the urgency for new drugs, and led to reappraisals of the old ones, as discussed here by Piero Olliaro and Anthony Bryceson.     

The plasmodium digestive vacuole: metabolic headquarters and choice drug target

The Plasmodium digestive (food) vacuole is an acidic proteolytic compartment central to the metabolism of the parasite. Here haemoglobin is degraded, haem is polymerized, amino acid are transported, oxygen radicals are detoxified, drugs are accumulated, acidification is maintained and free iron may be generated. Despite these crucial roles in parasite development, a number of questions about the digestive vacuole and the haemoglobin ingestion pathway remain unanswered; in consequence, a number of attractive drug targets remain to be exploited. Piero Olliaro and Daniel Goldberg here review the morphology, metabolism and pharmacological disruption of this specialized organelle.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.

     

Time to reconsider the role of ribavirin in Lassa fever

Ribavirin is the only available Lassa fever treatment. The rationale for using ribavirin is based on one clinical study conducted in the early 1980s. However, reanalysis of previous unpublished data reveals that ribavirin may actually be harmful in some Lassa fever patients. An urgent reevaluation of ribavirin is therefore needed.

Fifty years after its discovery, Lassa fever remains uncontrolled, and mortality remains unacceptably high. Since 2015, Nigeria has been experiencing increasingly large outbreaks of Lassa fever, with new peaks reached in 2016, 2017, and 2018. In 1987, McCormick and colleagues reported a case fatality rate (CFR) of 16.5% among 441 patients hospitalized in Sierra Leone [1]. In Nigeria in 2019, 124 deaths were recorded among 554 laboratory-confirmed cases for a CFR of 22% [2].Ribavirin is the only available Lassa fever–specific treatment and has been used routinely for over 25 years. However, intravenous ribavirin is not licensed for Lassa fever. Its mechanism of action is unclear, it is expensive and hard to source, and it has well-known toxicities [3]. Therefore, the evidence for using ribavirin in Lassa fever deserves careful scrutiny. The emergence of potential new therapeutics for Lassa fever, such as favipiravir and monoclonal antibodies, adds further weight to the case for reconsidering the role of ribavirin since the evaluation of new drugs in clinical trials requires a comparison against existing treatments with a known efficacy and safety profile [4,5].The rationale for using ribavirin in Lassa fever is primarily based on one clinical study conducted in Sierra Leone in the late 1970s and early 1980s. McCormick and colleagues [6] reported that in Lassa fever patients with a serum aspartate aminotransferase (AST) level of ≥150 IU/L, the use of intravenous ribavirin within the first 6 days of illness reduced the fatality rate from 61% (11/18) with no ribavirin to 5% (1/20) (p = 0.002). These authors concluded that ribavirin is effective in the treatment of Lassa fever. However, there are long-standing concerns about the methods used in this study. Although randomization was used to assign patients to treatment groups, the comparisons presented were not according to original randomized groups, and we have reconstructed their derivation (Fig 1). Serious limitations to the comparisons presented include the use of historic controls, inclusion of pregnant women in the control group but their exclusion from the ribavirin group (case fatality is around 2-fold higher in pregnant women than nonpregnant patients), and post hoc merging of treatment groups. Despite this and the fact that the results only supported the use of ribavirin in nonpregnant adult patients with AST ≥150 IU/L, this study is the basis upon which ribavirin is now used in all patients with Lassa fever, including children, pregnant women, and people with normal liver function.Open in a separate windowFig 1Reconstruction of the McCormick et al. data.AST, aspartate aminotransferase; PW, pregnant women. † Discrepancy within McCormick et al, with 39 patients reported treated with oral ribavirin but only 38 (14+24) outcomes reported. ‡ Discrepancy within McCormick et al, with table 1 reporting 12/63 but text reporting 13/62.It has been well known among Lassa specialists that the McCormick study reports a subset of a much larger dataset assembled by the Lassa treatment unit in Sierra Leone and that a report on the full dataset was commissioned by the United States Army Medical Research and Development Command. One of us (PH) therefore submitted a freedom of information (FOI) request to access this report. The full report and an accompanying memo are available, and we encourage readers to access and read the materials [7,8]. The memo states that some of the original trial records were unavailable, and the data should be “interpreted with extreme caution.” Nonetheless, the report presents data from 1977 through to 1991 on 807 Lassa fever patients with a known outcome that were assigned to different ribavirin treatment regimens. These newly available data raise important questions about the safety and efficacy of ribavirin for the treatment of Lassa fever.The original data were lost during the civil war in Sierra Leone, but the report contains tables showing the distribution of characteristics of the whole population according to treatment group, an appendix showing individual data for the 405 patients who died, and results of a logistic regression analysis comparing the effect of ribavirin with no treatment for some of the ribavirin regimens, after adjusting for patient characteristics. Based on these data, we derived aggregated datasets containing the number of deaths according to treatment groups and individual characteristics. We combined groups I (“No treatment given”) and X (“Drugs were not available”) as no treatment and all groups in which ribavirin was administered (II, III, and V to IX) as ribavirin. Exhibit III-8 in the FOI report presented case fatality by treatment group and AST, from which we derived crude odds ratios (ORs) comparing ribavirin with no treatment. The logistic regression reported in Exhibit III-9 was restricted to “those treatment groups that yielded the lowest case fatality rates with respect to untreated patients in the high severity patient illness category” (groups II, III, V, and VII). It was adjusted for age, gender, time to admission, time to treatment, length of stay, and log(AST). We also reconstructed analyses by digitizing the data on individuals who died in Appendix D, calculating the number of deaths according to treatment group and AST, and subtracting these numbers from the totals presented in Exhibit III-2. These allowed us to estimate overall mortality ORs before and after adjusting for ribavirin, although the numbers did not entirely match, and so the number of deaths was reduced in some small groups.Estimates of the effect of oral and intravenous ribavirin from the McCormick study and of all ribavirin from the full report are shown in Fig 2. Based on the crude ORs derived from Exhibit III-8, ribavirin reduced mortality only in patients with serum AST ≥150 IU/L, with less benefit (OR 0.48 [95% CI 0.30 to 0.78]) than reported by McCormick and colleagues. However, ribavirin appeared to increase mortality in patients with serum AST <150 IU/L (2.90 [1.42 to 5.95]). In fact, in our analysis, the only stratum in which ribavirin appeared protective (0.38 [0.21 to 0.70]) was serum AST >300 IU/L (Table H in S1 Text). The logistic regression reported in the FOI report suggested a modest reduction in mortality, but the reasons for the choice of treatment groups compared were unclear. In the reconstructed analyses, ribavirin was associated with overall increased mortality (2.12 [1.67, 2.68]), although this was attenuated after adjustment for AST (1.48 [1.05, 2.08]).Open in a separate windowFig 2Forest plot of the OR of death in treatment and risk subgroups.AST, aspartate aminotransferase; FOI, freedom of information; OR, odds ratio.In our view, there is a compelling case to reevaluate the role of ribavirin in the care of patients with Lassa fever. The data suggest that ribavirin treatment may harm Lassa fever patients with AST <150 IU/L. The limitations revealed by the US Army report, such as large amounts of missing data, unclear treatment allocation practices, imbalances in treatment groups, and errors in coding serology results, cast further doubt on the conclusions of the McCormick study. This aligns with 2 recent systematic reviews by Eberhardt and colleagues and Cheng and colleagues, which concluded that the efficacy of ribavirin in Lassa fever was uncertain because of critical risk of bias in existing studies [9,10].Challenging a quarter of century of clinical practice is difficult. The first step is to acknowledge inadequacies in our knowledge and to ensure that treatment recommendations for Lassa fever better reflect the (weak) strength of evidence for ribavirin in different patient populations. Vigorous efforts should be made to engage clinicians and patients in designing a placebo-controlled trial to assess the safety and efficacy of ribavirin treatment in Lassa fever patients, particularly in those with milder disease (as may be indicated by an admission AST <150 IU/L) in whom the available evidence is compatible with ribavirin causing more harm than good.In conclusion, Lassa fever patients are receiving a drug that may lack efficacy or cause harm. It is incumbent on us to ensure that the next 25 years of Lassa fever treatment are built on more solid foundations.     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.     

A multicentre randomized controlled trial of the efficacy and safety of single-dose praziquantel at 40 mg/kg vs. 60 mg/kg for treating intestinal schistosomiasis in the Philippines, Mauritania, Tanzania and Brazil

Background

Praziquantel at 40 mg/kg in a single dose is the WHO recommended treatment for all forms of schistosomiasis, but 60 mg/kg is also deployed nationally.

Methodology/Principal Findings

Four trial sites in the Philippines, Mauritania, Tanzania and Brazil enrolled 856 patients using a common protocol, who were randomised to receive praziquantel 40 mg/kg (n = 428) or 60 mg/kg (n = 428). While the sites differed for transmission and infection intensities (highest in Tanzania and lowest in Mauritania), no bias or heterogeneity across sites was detected for the main efficacy outcomes. The primary efficacy analysis was the comparison of cure rates on Day 21 in the intent-to-treat population for the pooled data using a logistic model to calculate Odd Ratios allowing for baseline characteristics and study site. Both doses were highly effective: the Day 21 cure rates were 91.7% (86.6%–98% at individual sites) with 40 mg/kg and 92.8% (88%–97%) with 60 mg/kg. Secondary parameters were eggs reduction rates (ERR), change in intensity of infection and reinfection rates at 6 and 12 months. On Day 21 the pooled estimate of the ERR was 91% in both arms. The Hazard Ratio for reinfections was only significant in Brazil, and in favour of 60 mg/kg on the pooled estimate (40 mg/kg: 34.3%, 60 mg/kg: 23.9%, HR = 0.78, 95%CI = [0.63;0.96]). Analysis of safety could not distinguish between disease- and drug-related events. 666 patients (78%) reported 1327 adverse events (AE) 4 h post-dosing. The risk of having at least one AE was higher in the 60 than in the 40 mg/kg group (83% vs. 73%, p<0.001). At 24 h post-dosing, 456 patients (54%) had 918 AEs with no difference between arms. The most frequent AE was abdominal pain at both 4 h and 24 h (40% and 24%).

Conclusion

A higher dose of 60 mg/kg of praziquantel offers no significant efficacy advantage over standard 40 mg/kg for treating intestinal schistosomiasis caused by either S. mansoni or S. japonicum. The results of this study support WHO recommendation and should be used to inform policy decisions in the countries.

Trial Registration

Controlled-Trials.com ISRCTN29273316 ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00403611","term_id":"NCT00403611"}}NCT00403611