Increasingly inbred and fragmented populations of Plasmodium vivax associated with the eastward decline in malaria transmission across the Southwest Pacific

The human malaria parasite Plasmodium vivax is more resistant to malaria control strategies than Plasmodium falciparum, and maintains high genetic diversity even when transmission is low. To investigate whether declining P. vivax transmission leads to increasing population structure that would facilitate elimination, we genotyped samples from across the Southwest Pacific region, which experiences an eastward decline in malaria transmission, as well as samples from two time points at one site (Tetere, Solomon Islands) during intensified malaria control. Analysis of 887 P. vivax microsatellite haplotypes from hyperendemic Papua New Guinea (PNG, n = 443), meso-hyperendemic Solomon Islands (n = 420), and hypoendemic Vanuatu (n = 24) revealed increasing population structure and multilocus linkage disequilibrium yet a modest decline in diversity as transmission decreases over space and time. In Solomon Islands, which has had sustained control efforts for 20 years, and Vanuatu, which has experienced sustained low transmission for many years, significant population structure was observed at different spatial scales. We conclude that control efforts will eventually impact P. vivax population structure and with sustained pressure, populations may eventually fragment into a limited number of clustered foci that could be targeted for elimination

Exploring Plasmodium vivax transmission dynamics and population genetics through genetics and genomics

© 2019 Abebe Alemu FolaGlobally, malaria remains one of the most life-threatening and highest burden infectious diseases. Amongst the six Plasmodium species that cause malaria in humans, Plasmodium vivax and Plasmodium falciparum are the predominant cause of morbidity and mortality. P. falciparum is the most virulent species; however, P. vivax has a wider geographic range and is more difficult to control and eliminate. The burden of malaria in Papua New Guinea (PNG) is among the highest in the Asia Pacific region with a hyperendemic transmission of P. falciparum and P. vivax. Since 2004, PNG has intensified malaria control efforts to alleviate this malaria burden and with the ultimate goal to eliminate malaria from the country by 2030. P. vivax is recognized as a major obstacle to achieve this elimination goal as evidenced by a smaller reduction in P. vivax prevalence relative to P. falciparum after intensified control efforts throughout the country. Indeed, in many areas where P. falciparum and P. vivax are co-endemic, P. vivax is now becoming the dominant malaria parasite. The main reason for this shift in malaria epidemiology may result from the use of malaria control measures primarily targeted at P. falciparum, and the fact that P. vivax is more difficult to treat and diagnose because of its distinct biology, in particular, its ability to form dormant liver-stage infections, known as hypnozoites. Therefore, approaches to eliminate P. vivax malaria need to address this unique parasite biology through the development and adoption of novel tools and strategies. To facilitate the discovery of novel strategies to contain Vivax malaria, this PhD project aimed to investigate P. vivax biology, molecular epidemiology and transmission dynamics using parasite population genetics, and through the development of a novel barcoding tool for high resolution genotyping. To address the existing knowledge gaps around the impact of the unique biology of P. vivax on malaria epidemiology, I compared the prevalence, multiplicity of infection (MOI) and genetic diversity of sympatric P. vivax and P. falciparum parasite populations throughout PNG (Chapter 3). A total of 892 P. vivax and 758 P. falciparum isolates from 16 Provinces (including 49 villages) were obtained from a 2008/2009 PNG national malaria survey completed after the nationwide intensification of control. All P. vivax and P. falciparum isolates were genotyped using the size polymorphic markers Pvms16/Pvmsp1F3, and Pfmsp2, respectively. The comparative analyses of within host multiplicity of infection (MOI) and population level genetic diversity showed striking differences between these sympatric Plasmodium species. Approximately 70% of P. vivax infected individuals carry more than one clone (i.e. MOI>1, known as polyclonal infections) compared to 20% for P. falciparum. There was a strong association between MOI and infection prevalence for P. falciparum but not for P. vivax, consistent with the maintenance of high infection complexity for P. vivax despite the reduction in new infections resulting from control interventions, and therefore suggesting a higher effective transmission potential for this species. The results showed that both species have high genetic diversity in populations throughout PNG. However, while immune selection on the antigen loci used (Pfmsp2 and Pvmsp1F3) and unusually high diversity of Pvms16, facilitates the detection of unique clones, it may limit the resolution of the comparative genetic diversity analyses. To further explore P. vivax genetic diversity and population structure throughout PNG, I then conducted a detailed population genetic analyses of P. vivax using a panel of 10 well-validated and putatively neutral microsatellite markers (Chapter 4). I genotyped a total of 230 P. vivax isolates with low complexity infections (maximum MOI=2) from eight geographically and ecologically distinct regions (“geographic populations”) throughout PNG. Population genetic analyses of microsatellite haplotypes/genotypes revealed a spectrum of genetic diversity, which was associated with prevalence. Multilocus linkage disequilibrium (mLD) was identified in six of the eight parasite populations indicating significant inbreeding. Strong geographic population structure (clustering of isolates according to their geographic origins) was identified among regions, dividing the Mainland (lowlands), Highlands and Islands, and the genetic divergence was significantly correlated with geographic distance. Sources and sinks of vivax transmission and parasite population connectivity between regions were defined. Patterns of P. vivax gene flow appear to follow major human migration routes, implying that the human host could be the main driver for the spread of infections and potentially, drug resistance. This find suggests assessing the contribution of the P. vivax asymptomatic reservoir and imported infections for sustainable local transmission are necessary to design targeted control interventions. Small panels (~10) of microsatellite markers used have been the “gold standard” genotyping tool for population genetic analyses of P. vivax. However, because of their small number, extremely high diversity and propensity for genotyping error they may have limited resolution for identifying local population structure in high transmission areas (such as the north coast of PNG). In addition, several other technical challenges make them unsuitable for large scale malaria surveillance. Therefore, I developed a novel genotyping tool known as a ‘single nucleotide polymorphism (SNP) barcode’ for high resolution monitoring of P. vivax parasite populations in PNG (Chapter 5). The barcode was specifically designed to capture the genetic diversity of PNG parasite populations by using SNPs identified amongst 20 high quality published PNG genomes. A total of 24283 SNPs with minor allele frequencies (MAF) of more than 10% were identified in putatively neutral regions of the P. vivax parasite genome. A filtered subset of 178 evenly spaced informative SNPs were selected for development of a high throughput parallel targeted amplicon sequencing (PTAS) assay using a multiplex PCR and Illumina MiSeq platform. To validate the developed barcode, I tested the genotyping success of 20 SNPs using small number of mono- and polyclonal infections, followed by genotyping of 96 P. vivax isolates from four catchment areas on the north coast of PNG collected in 2012-2014. Compared to the available microsatellite data for the same samples (E. Kattenberg, unpublished), the SNP barcode revealed more variable genetic diversity and stronger population structure with greater resolution to assign genotypes according to their geographic origins than the microsatellite panel. The developed SNP barcode has low genotyping error, is relatively cheap (~$-26-30/isolate) and transfer of the technology to field settings like PNG is technically feasible. As transmission declines, measuring changes in parasite population structure is important to assess the impact of control on malaria transmission, and to guide future control and elimination of malaria. To explore P. vivax population genetic signals with changing transmission due to intensified control, I applied the developed SNP barcode assay to a total of 376 P. vivax isolates obtained from serial cross-sectional surveys in two north coast Provinces of PNG (East Sepik and Madang) (Chapter 6). Samples were collected between 2005 and 2016, a period of initially declining transmission and later rebound. This investigation revealed interesting spatio-temporal patterns with declining transmission including a reduction in polyclonal infections, genetic diversity and population connectedness as indicated by an increase in population structure. Significant multilocus linkage disequilibrium (mLD) and a large cluster of parasites with high genomic relatedness (identity by descent, IBD) was also detected at low transmission. These population genetic signatures signify a strong population bottleneck, highly clonal transmission and inbreeding resulted from significant reductions in prevalence. For Madang Province, I also observed some genetic signatures related to the sources of malaria rebound providing insight into malaria resurgence through increasing migration of parasites from other endemic areas in addition to expansion of the residual parasite population. Containment of P. vivax infections and drug resistance may be facilitated by the existence of distinct parasite populations at low transmission yet will be undermined by increased parasite migration due to frequent human movement between endemic areas. Overall, this thesis demonstrates how genotyping a relatively small number of parasite isolates from representative geographic areas can provide surrogate signals of malaria transmission dynamics and infection spread. Moreover, it reveals the power of molecular epidemiology and population genetics and genomics for malaria surveillance that in combination with epidemiological data will allow the prioritisation and optimisation of malaria control and elimination strategies. I also discuss potential approaches to integrate population genetics and genomics into current malaria surveillance systems and how the control efforts can be benefited from this approach

Nationwide genetic surveillance of Plasmodium vivax in Papua New Guinea reveals heterogeneous transmission dynamics and routes of migration amongst subdivided populations

The Asia Pacific Leaders in Malaria Alliance (APLMA) have committed to eliminate malaria from the region by 2030. Papua New Guinea (PNG) has the highest malaria burden in the Asia-Pacific region but with the intensification of control efforts since 2005, transmission has been dramatically reduced and Plasmodium vivax is now the dominant malaria infection in some parts of the country. To gain a better understanding of the transmission dynamics and migration patterns of P. vivax in PNG, here we investigate population structure in eight geographically and ecologically distinct regions of the country. A total of 219 P. vivax isolates (16-30 per population) were successfully haplotyped using 10 microsatellite markers. A wide range of genetic diversity (He=0.37-0.87, Rs=3.60-7.58) and significant multilocus linkage disequilibrium (LD) was observed in six of the eight populations (IAS=0.08-0.15 p-value<0.05) reflecting a spectrum of transmission intensities across the country. Genetic differentiation between regions was evident (Jost's D=0.07-0.72), with increasing divergence of populations with geographic distance. Overall, P. vivax isolates clustered into three major genetic populations subdividing the Mainland lowland and coastal regions, the Islands and the Highlands. P. vivax gene flow follows major human migration routes, and there was higher gene flow amongst Mainland parasite populations than among Island populations. The Central Province (samples collected in villages close to the capital city, Port Moresby), acts as a sink for imported infections from the three major endemic areas. These insights into P. vivax transmission dynamics and population networks will inform targeted strategies to contain malaria infections and to prevent the spread of drug resistance in PNG

Higher complexity of infection and genetic diversity of plasmodium vivax than plasmodium falciparum across all malaria transmission zones of Papua New Guinea

AbstractPlasmodium falciparum and Plasmodium vivax have varying transmission dynamics that are informed by molecular epidemiology. This study aimed to determine the complexity of infection and genetic diversity of P. vivax and P. falciparum throughout Papua New Guinea (PNG) to evaluate transmission dynamics across the country. In 2008-2009, a nationwide malaria indicator survey collected 8,936 samples from all 16 endemic provinces of PNG. Of these, 892 positive P. vivax samples were genotyped at PvMS16 and PvmspF3, and 758 positive P. falciparum samples were genotyped at Pfmsp2. The data were analyzed for multiplicity of infection (MOI) and genetic diversity. Overall, P. vivax had higher polyclonality (71%) and mean MOI (2.32) than P. falciparum (20%, 1.39). These measures were significantly associated with prevalence for P. falciparum but not for P. vivax. The genetic diversity of P. vivax (PvMS16: expected heterozygosity = 0.95, 0.85-0.98; PvMsp1F3: 0.78, 0.66-0.89) was higher and less variable than that of P. falciparum (Pfmsp2: 0.89, 0.65-0.97). Significant associations of MOI with allelic richness (rho = 0.69, P = 0.009) and expected heterozygosity (rho = 0.87, P < 0.001) were observed for P. falciparum. Conversely, genetic diversity was not correlated with polyclonality nor mean MOI for P. vivax. The results demonstrate higher complexity of infection and genetic diversity of P. vivax across the country. Although P. falciparum shows a strong association of these parameters with prevalence, a lack of association was observed for P. vivax and is consistent with higher potential for outcrossing of this species

Tuberculosis Laboratory Diagnosis Quality Assurance among Public Health Facilities in West Amhara Region, Ethiopia.

Reliable smear microscopy is an important component of Directly Observed Treatment Scheme (DOTS) strategy for TB control program in countries with limited resources. Despite external quality assessment is established in Ethiopia, there is lower TB detection rate (48%) in Amhara region compared to the World Health Organization (WHO) estimate (70%). This highlights the quality of smear microscopy needs to be evaluated. Therefore, the aim of this study was to assess the quality of sputum smear microscopy performance among health center laboratories in West Amhara region, Ethiopia.A cross sectional study was conducted from July 08, 2013 to July 07, 2014. Data were collected from 201 public health center laboratories using a structured questionnaire. Slides were collected based on Lot Quality Assurance Sampling (LQAS) method and rechecked blindly by trained laboratory technologists. The data were entered into EPI info V.7 and smear quality indicators and AFB results were analyzed by SPSS version 20.Among 201 laboratories enrolled in this study, 47 (23.4%) laboratories had major errors. Forty one (20.4%) laboratories had a total of 67 false negative and 29 (14.4%) laboratories had a total of 68 false positive results. Specimen quality, smear thickness and evenness were found poor in 134 (66.7%), 133 (66.2%) and 126 (62.7%) laboratories, respectively. Unavailability of microscope lens cleaning solution (AOR: 2.90; 95% CI: 1.25-6.75; P: 0.013) and dirty smears (AOR: 2.65; 95% CI: 1.14-6.18; P: 0.024) were correlated with false negative results whereas no previous EQA participation (AOR: 3.43; 95% CI: 1. 39-8.45; P: 0.007) was associated with false positive results.The performance of health facilities for sputum smear microscopy was relatively poor in West Amhara region. Hence, strengthening the EQA program and technical support on sputum smear microscopy are recommended to ensure quality tuberculosis diagnostic service

SNP barcodes provide higher resolution than microsatellite markers to measure <i>Plasmodium vivax</i> population genetics

BACKGROUND: Genomic surveillance of malaria parasite populations has the potential to inform control strategies and to monitor the impact of interventions. Barcodes comprising large numbers of single nucleotide polymorphism (SNP) markers are accurate and efficient genotyping tools, however may need to be tailored to specific malaria transmission settings, since 'universal' barcodes can lack resolution at the local scale. A SNP barcode was developed that captures the diversity and structure of Plasmodium vivax populations of Papua New Guinea (PNG) for research and surveillance. METHODS: Using 20 high-quality P. vivax genome sequences from PNG, a total of 178 evenly spaced neutral SNPs were selected for development of an amplicon sequencing assay combining a series of multiplex PCRs and sequencing on the Illumina MiSeq platform. For initial testing, 20 SNPs were amplified in a small number of mono- and polyclonal P. vivax infections. The full barcode was then validated by genotyping and population genetic analyses of 94 P. vivax isolates collected between 2012 and 2014 from four distinct catchment areas on the highly endemic north coast of PNG. Diversity and population structure determined from the SNP barcode data was then benchmarked against that of ten microsatellite markers used in previous population genetics studies. RESULTS: From a total of 28,934,460 reads generated from the MiSeq Illumina run, 87% mapped to the PvSalI reference genome with deep coverage (median = 563, range 56-7586) per locus across genotyped samples. Of 178 SNPs assayed, 146 produced high-quality genotypes (minimum coverage = 56X) in more than 85% of P. vivax isolates. No amplification bias was introduced due to either polyclonal infection or whole genome amplification (WGA) of samples before genotyping. Compared to the microsatellite panels, the SNP barcode revealed greater variability in genetic diversity between populations and geographical population structure. The SNP barcode also enabled assignment of genotypes according to their geographic origins with a significant association between genetic distance and geographic distance at the sub-provincial level. CONCLUSIONS: High-throughput SNP barcoding can be used to map variation of malaria transmission dynamics at sub-national resolution. The low cost per sample and genotyping strategy makes the transfer of this technology to field settings highly feasible

General characteristics of Laboratories in West Amhara region, 2014.

<p>TB: tuberculosis; EQA: External Quality Assessment; DEE: Diethyl Ether.</p><p>General characteristics of Laboratories in West Amhara region, 2014.</p

Performance of sputum smears microscopy in West Amhara Region, 2014.

<p>HFN: High False Negative; HFP: High False Positive; LFP: Low False Positive; LFN: Low False Negative; QE: Quantification Error.</p

Factors for false positive smear microscopy results in West Amhara region, 2014.

<p>AOR: Adjusted Odds Ratio; CI: Confidence Interval; COR: Crude Odds Ratio; CF: Carbol Fuchsin; EQA: External Quality Assessment.</p><p>Factors for false positive smear microscopy results in West Amhara region, 2014.</p

ANSV and SPR of smear microscopy in West Amhara region, 2014.

<p>ANSV: Annual Negative Slide Volume; SPR: Slide Positivity Rate; N: number.</p><p>ANSV and SPR of smear microscopy in West Amhara region, 2014.</p