Publications

A change in population density can generate spatial and demographic effects that can have an impact on fluctuations in population size for many years. Although the demographic effects of time lags have been incorporated into analyses of time series data, there are few detailed descriptions of the long-term demographic consequences of a change in density. We use detailed, individual-based data from a population of red deer (Cervus elaphus) from the North Block of the Isle of Rum, Scotland, to describe long-term demographic and spatial effects of a change in density. The population was released from hunting pressure in 1972. Over the following 10 years population density doubled and, since the early 1980s, has fluctuated around ecological carrying capacity. The cessation of culling led to long-term transient spatial and demographic effects that have persisted for 30 years. Different vital rates responded to the increase in density at different rates, causing long-term changes to the demographic and spatial structure of the population. These changes altered the impact of different age- and sex-specific vital rates on annual changes in population size. These changes are still ongoing, 30 years after cessation of the cull, suggesting that a change in density may generate transient dynamics that persist for several generations.

<h2>Abstract</h2><h3>Background</h3> The malaria burden is decreasing throughout the Greater Mekong Subregion, however transmission persists in some areas. Human movement, subclinical infections and complicated transmission patterns contribute to the persistence of malaria. This research describes the micro-geographical epidemiology of both clinical malaria and subclinical <i>Plasmodium</i> infections in three villages in Western Cambodia. <h3>Methods</h3> Three villages in Western Cambodia were selected for the study based on high reported <i>Plasmodium falciparum</i> incidence. A census was conducted at the beginning of the study, including demographic information and travel history. The total population was 1766. Cross-sectional surveys were conducted every three months from June 2013 to June 2014. <i>Plasmodium</i> infections were detected using an ultra-sensitive, high-volume, quantitative polymerase chain reaction (uPCR) technique. Clinical episodes were recorded by village health workers. The geographic coordinates (latitude and longitude) were collected for all houses and all participants were linked to their respective houses using a demographic surveillance system. Written informed consent was obtained from all participants. <h3>Results</h3> Most clinical episodes and subclinical infections occurred within a single study village. Clinical <i>Plasmodium vivax</i> episodes clustered spatially in each village but only lasted for a month. In one study village subclinical infections clustered in geographic proximity to clusters of clinical episodes. The largest risk factor for clinical <i>P. falciparum</i> episodes was living in a house where another clinical <i>P. falciparum</i> episode occurred (model adjusted odds ratio (AOR): 6.9; CI: 2.3–19. 8). Subclinical infections of both <i>P. vivax</i> and <i>P. falciparum</i> were associated with clinical episodes of the same species (AOR: 5.8; CI: 1.5–19.7 for <i>P. falciparum</i> and AOR: 14.6; CI: 8.6–25.2 for <i>P. vivax</i>) and self-reported overnight visits to forested areas (AOR = 3.8; CI: 1.8–7. 7 for <i>P. falciparum</i> and AOR=2.9; CI: 1.7–4.8 for <i>P. vivax</i>). <h3>Discussion</h3> Spatial clustering within the villages was transient, making the prediction of spatial clusters difficult. Interventions that are dependent on predicting spatial clusters (such as reactive case detection) would only have detected a small proportion of cases unless the entire village was screened within a limited time frame and with a highly sensitive diagnostic test. Subclinical infections may be acquired outside of the village (particularly in forested areas) and may play an important role in transmission.

Background Plasmodium vivax malaria accounts for approximately 60% of malaria cases in Kolkata, India. There has been limited information on the genotypic polymorphism of P. vivax in this malaria endemic area. Three highly polymorphic and single copy genes were selected for a study of genetic diversity in Kolkata strains. Methods Blood from 151 patients with P. vivax infection diagnosed in Kolkata between April 2003 and September 2004 was genotyped at three polymorphic loci: the P. vivax circumsporozoite protein ( pvcs ), the merozoite surface protein 1 ( pvmsp 1) and the merozoite surface protein 3-alpha ( pvmsp 3-alpha). Results Analysis of these three genetic markers revealed that P. vivax populations in Kolkata are highly diverse. A large number of distinguishable alleles were found from three genetic markers: 11 for pvcs , 35 for pvmsp 1 and 37 for pvmsp 3-alpha. These were, in general, randomly distributed amongst the isolates. Among the 151 isolates, 142 unique genotypes were detected the commonest genotype at a frequency of less than 2% (3/151). The overall rate of mixed genotype infections was 10.6%. Conclusion These results indicate that the P. vivax parasite population is highly diverse in Kolkata, despite the low level of transmission. The genotyping protocols used in this study may be useful for differentiating re-infection from relapse and recrudescence in studies assessing of malarial drug efficacy in vivax malaria.

Testing of Cryptococcus neoformans for susceptibility to antifungal drugs by standard microtiter methods has not been shown to correlate with clinical outcomes. This report describes a modified quantitative broth macrodilution susceptibility method showing a correlation with both the patient's quantitative biological response in the cerebrospinal fluid (CSF) and the survival of 85 patients treated with amphotericin B (AMB). The Spearman rank correlation between the quantitative in vitro measure of susceptibility and the quantitative measure of the number of organisms in the patient's CSF was 0.37 (P < 0.01; 95% confidence interval [95% CI], 0.20, 0.60) for the first susceptibility test replicate and 0.46 (P < 0.001; 95% CI, 0.21, 0.62) for the second susceptibility test replicate. The median in vitro estimated response (defined as the fungal burden after AMB treatment) at 1.5 mg/liter AMB for patients alive at day 14 was 5 CFU (95% CI, 3, 8), compared to 57 CFU (95% CI, 4, 832) for those who died before day 14. These exploratory results suggest that patients whose isolates show a quantitative in vitro susceptibility response below 10 CFU/ml were more likely to survive beyond day 14.

Centre for Tropical Diseases, a 500-bed hospital for infectious diseases in Ho Chi Minh City, Vietnam.The factors that determine outcome in adults with tuberculous meningitis are poorly understood. The objective of the study was to investigate the relationship between admission clinical features, HIV infection, drug resistance, mycobacterial genotype and outcome in adults with tuberculous meningitis.Clinical and laboratory data were recorded prospectively for 56 Vietnamese adults with tuberculous meningitis confirmed by culture of cerebrospinal fluid. Variables associated with in-hospital mortality, IV infection, drug resistance and microbial genotype were assessed by univariate and multivariate analysis.Admission coma score independently predicted death in hospital (OR 0.73, 95%CI 0.61-0.87, P = 0.001). HIV-infected adults with tuberculous meningitis were more likely to be infected with Mycobacterium tuberculosis resistant to isoniazid (P = 0.011) and streptomycin (P = 0.002). Isoniazid resistance, streptomycin resistance, HIV infection and microbial genotype were not associated with increased in-hospital mortality.Treatment of tuberculous meningitis before the onset of coma saves lives. Resistance to isoniazid and/or streptomycin does not appear to affect outcome.

In 2012, the World Health Organization recommended blocking the transmission of Plasmodium falciparum with single low-dose primaquine (SLDPQ, target dose 0.25 mg base/kg body weight), without testing for glucose-6-phosphate dehydrogenase deficiency (G6PDd), when treating patients with uncomplicated falciparum malaria. We sought to develop an age-based SLDPQ regimen that would be suitable for sub-Saharan Africa. Using data on the anti-infectivity efficacy and tolerability of primaquine (PQ), the epidemiology of anaemia, and the risks of PQ-induced acute haemolytic anaemia (AHA) and clinically significant anaemia (CSA), we prospectively defined therapeutic-dose ranges of 0.15–0.4 mg PQ base/kg for children aged 1–5 years and 0.15–0.5 mg PQ base/kg for individuals aged ≥6 years (therapeutic indices 2.7 and 3.3, respectively). We chose 1.25 mg PQ base for infants aged 6–11 months because they have the highest rate of baseline anaemia and the highest risks of AHA and CSA. We modelled an anthropometric database of 661,979 African individuals aged ≥6 months (549,127 healthy individuals, 28,466 malaria patients and 84,386 individuals with other infections/illnesses) by the Box–Cox transformation power exponential and tested PQ doses of 1–15 mg base, selecting dosing groups based on calculated mg/kg PQ doses. From the Box–Cox transformation power exponential model, five age categories were selected: (i) 6–11 months (n = 39,886, 6.03%), (ii) 1–5 years (n = 261,036, 45.46%), (iii) 6–9 years (n = 20,770, 3.14%), (iv) 10–14 years (n = 12,155, 1.84%) and (v) ≥15 years (n = 328,132, 49.57%) to receive 1.25, 2.5, 5, 7.5 and 15 mg PQ base for corresponding median (1st and 99th centiles) mg/kg PQ base of: (i) 0.16 (0.12–0.25), (ii) 0.21 (0.13–0.37), (iii) 0.25 (0.16–0.38), (iv) 0.26 (0.15–0.38) and (v) 0.27 (0.17–0.40). The proportions of individuals predicted to receive optimal therapeutic PQ doses were: 73.2 (29,180/39,886), 93.7 (244,537/261,036), 99.6 (20,690/20,770), 99.4 (12,086/12,155) and 99.8% (327,620/328,132), respectively. We plan to test the safety of this age-based dosing regimen in a large randomised placebo-controlled trial (ISRCTN11594437) of uncomplicated falciparum malaria in G6PDd African children aged 0.5 − 11 years. If the regimen is safe and demonstrates adequate pharmacokinetics, it should be used to support malaria elimination.

Summary Multi‐drug resistant falciparum malaria is widespread in Asia. In Thailand, Cambodia and Vietnam the national protocols have changed largely to artesunate combined treatment regimens but elsewhere in East and South Asia chloroquine (CQ) and sulfadoxine‐pyrimethamine (SP) are still widely recommended by national malaria control programmes. In Kachin State, northern Myanmar, an area of low seasonal malaria transmission, the efficacy of CQ (25 mg base/kg) and SP (1.25/25 mg/kg), the nationally recommended treatments at the time, were compared with mefloquine alone (M; 15 mg base/kg) and mefloquine combined with artesunate (MA; 15:4 mg/kg). An open randomized controlled trial enrolled 316 patients with uncomplicated Plasmodium falciparum malaria, stratified prospectively into three age‐groups. Early treatment failures (ETF) occurred in 41% (32/78) of CQ treated patients and in 24% of patients treated with SP (18/75). In young children the ETF rates were 87% after CQ and 35% after SP. Four children (two CQ, two SP) developed symptoms of cerebral malaria within 3 days after treatment. By day 42, failure rates (uncorrected for reinfections) had increased to 79% for CQ and 81% for SP. ETF rates were 2.5% after treatment with M and 3.9% after treatment with MA ( P &gt; 0.2). Overall uncorrected treatment failure rates at day 42 following M and MA were 23% and 21%, respectively. Chloroquine and SP are completely ineffective for the treatment of falciparum malaria in northern Myanmar. Mefloquine treatment is much more effective, but three day combination regimens with artesunate will be needed for optimum efficacy and protection against resistance.

Background. The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. Methods. P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. Results. The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10−12). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. Conclusions. K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Background Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for P. malariae , an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N=37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, genetic richness index, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity ( H d ) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), genetic richness index (0.050-0.250) and absence of linkage disequilibrium ( I A S = 0.03, p-value &gt; 0.05 ) (N=37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.092, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10 -4 and was further reduced to 1.66 × 10 -4 when pmmsp1 polymorphisms were included. Conclusions Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

Humanity owes a great debt to quinine. Cinchona alkaloids have been used to treat malaria for hundreds of years after the arrival of cinchona bark in Europe in the 17th century, where it was mixed with rose leaves, lemon juice and wine to treat the malarious patients of Essex (Butler et al. 2010). While quinine has been largely replaced by more effective and better-tolerated drugs in the treatment of uncomplicated malaria, it remains the standard treatment for severe malaria in many countries. In Africa, where over 90% of the estimated 781 000 malaria-related deaths in 2009 occurred, quinine is first-line therapy for severe malaria in almost all countries (WHO 2010). Recent evidence shows that it is time to replace quinine for severe malaria as well. A large randomized trial conducted in Asia in 2005 found that parenteral artesunate reduced overall mortality by 39% compared with quinine (Dondorp et al. 2005). As a result, in 2006 WHO recommended artesunate as the treatment of choice for adults, but considered there was insufficient evidence to extend this recommendation to children in Africa. That evidence came in late 2010, from the largest ever study of severe malaria (5425 children across Africa), which found that artesunate reduced mortality by 22.5% compared with quinine (Dondorp et al. 2010). Importantly, there was no evidence in this or previous studies of an increase in neurological sequelae in survivors. These results were confirmed by a recent Cochrane meta-analysis that found an overall mortality reduction of 39% among adults and 24% among children compared to quinine (Sinclair et al. 2011), an identical finding to the meta-analysis accompanying the African trial (Dondorp et al. 2010). So what now? WHO has just changed its guidelines to put artesunate as the treatment of choice for severe malaria everywhere (WHO 2011), and these recommendations need to be disseminated to all relevant malaria actors to support national guideline change where needed. To date, only one African country (Nigeria) has revised its guidelines to include artesunate for severe malaria, and only as an alternative treatment (WHO 2010). Given the long history of quinine use, the dissemination of national guidelines will need to be accompanied by training to help shift health provider habits and personal convictions. All this needs to be properly supported, both by international donors and through technical advice from WHO’s regional offices. The challenge in translating evidence into practice should never be underestimated, particularly when it comes to malaria treatment, as recent history shows. In 2000, WHO recommended a policy shift in the management of uncomplicated malaria from chloroquine to artemisinin-based combination therapies (ACTs) because of high levels of chloroquine resistance. Yet, despite substantial evidence supporting the superior efficacy of ACTs, international donors and ministries of health in malaria-endemic countries continued to support chloroquine use for several years, mainly because chloroquine was a much cheaper drug (Attaran et al. 2004). Similarly, the higher unit price of artesunate is likely to be a barrier. Cost effectiveness studies, however, show that when mortality and associated costs such as reduced side-effect management and hospitalization are considered, artesunate is cost effective (Lubell et al. 2009, 2011). Furthermore, the cost of artesunate may fall as demand increases. Nevertheless, for policy and practice to change in the short-term, additional international funding support is needed. Another important potential concern is the limited availability of quality-assured sources of artesunate. Currently, only one source of injectable artesunate has been pre-qualified by WHO, and malaria control programmes may be reluctant to make the switch as long as consistency in supply is uncertain. In addition, quinine production represents an important economic activity in a number of malaria-endemic countries; in Burundi, for example, quinine is one of the few drugs manufactured in-country, making it more popular and more easily accessible than imported antimalarial drugs (Amuasi et al. 2011). However, artemisinin, the raw material for artesunate, is increasingly being produced in Africa, including in Kenya, Tanzania and Uganda (http://www.artepal.org), and as demand grows, supply should logically follow. Finally, it can be anticipated that calls for local evidence may be made by national governments out of concern that studies performed in other contexts may not apply to their setting. MSF faced similar challenges when trying to move from chloroquine to ACT (Guthmann et al. 2008). While this may initially appear reasonable, the evidence to date is broadly generalizable and the latest Cochrane review concludes that further research to assess the efficacy of artesunate versus quinine is unnecessary (Sinclair et al. 2011). Patients should only be subjected to experimental trials if there is real uncertainty about which drug is better (CIOMS 2002), and while operational research may help guide implementation, it would clearly be unethical to delay implementation and subject patients to further drug effectiveness studies. Global funding for malaria control is already insufficient (Snow et al. 2010), and in the current economic climate, donors may be reluctant to support a switch to a more expensive treatment. However, replacing quinine with artesunate is a clear cut intervention that has the potential to save nearly 200 000 lives each year and the total annual cost of providing artesunate for treating all cases of severe malaria worldwide would likely be less than $US 50 million (MSF 2011). For African countries to make the switch, strong international support will be required to provide additional funds to support drug procurement and training costs and send a clear message to manufacturers that quality sources of artesunate are needed.

A direct immunofluorescent antibody test (DIF) was developed for the rapid diagnosis of melioidosis, a potentially fatal infection caused by Pseudomonas pseudomallei. In a clinical evaluation of 369 sputum, pus, or urine specimens from 272 patients with suspected melioidosis, the DIF had a sensitivity of 73% and a specificity of 99% compared with culture. Using this DIF, a confident diagnosis of melioidosis can now be made within two hours of admission to hospital, compared with the delay of two to four days required for culture results. Consequent early institution of specific antimicrobial therapy may help to save lives.