Using a mobile nanopore sequencing lab for end-to-end genomic surveillance of Plasmodium falciparum: A feasibility study.

Genomic epidemiology holds promise for malaria control and elimination efforts, for example by informing on Plasmodium falciparum genetic diversity and prevalence of mutations conferring anti-malarial drug resistance. Limited sequencing infrastructure in many malaria-endemic areas prevents the rapid generation of genomic data. To address these issues, we developed and validated assays for P. falciparum nanopore sequencing in endemic sites using a mobile laboratory, targeting key antimalarial drug resistance markers and microhaplotypes. Using two multiplexed PCR reactions, we amplified six highly polymorphic microhaplotypes and ten drug resistance markers. We developed a bioinformatics workflow that allows genotyping of polyclonal malaria infections, including minority clones. We validated the panels on mock dried blood spot (DBS) and rapid diagnostic test (RDT) samples and archived DBS, demonstrating even, high read coverage across amplicons (range: 580x to 3,212x median coverage), high haplotype calling accuracy, and the ability to explore within-sample diversity of polyclonal infections. We field-tested the feasibility of rapid genotyping in Zanzibar in close collaboration with the local malaria elimination program using DBS and routinely collected RDTs as sample inputs. Our assay identified haplotypes known to confer resistance to known antimalarials in the dhfr, dhps and mdr1 genes, but no evidence of artemisinin partial resistance. Most infections (60%) were polyclonal, with high microhaplotype diversity (median HE = 0.94). In conclusion, our assays generated actionable data within a few days, and we identified current challenges for implementing nanopore sequencing in endemic countries to accelerate malaria control and elimination.

Risk of imported malaria infections in Zanzibar: a cross-sectional study.

BACKGROUND: Zanzibar has made substantial progress in malaria control with vector control, improved diagnosis, and artemisinin-based combination therapy. Parasite prevalence in the population has remained around 1% but imported infections from mainland Tanzania contribute to sustained local transmission. Understanding travel patterns between mainland Tanzania and Zanzibar, and the risk of malaria infection, may help to control malaria importation to Zanzibar. METHODS: A rolling cross-sectional survey linked to routine reactive case detection of malaria was carried out in Zanzibar between May 2017 and October 2018. Households of patients diagnosed with malaria at health facilities were surveyed and household members were tested for malaria using rapid diagnostic tests and a sub-sample by quantitative PCR (qPCR). Interviews elicited a detailed travel history of all household members who had travelled within the past two months, including trips within and outside of Zanzibar. We estimated the association of malaria infection with travel destinations in pre-defined malaria endemicity categories, trip duration, and other co-variates using logistic regression. RESULTS: Of 17,891 survey participants, 1177 (7%) reported a recent trip, of which 769 (65%) visited mainland Tanzania. Among travellers to mainland Tanzania with travel destination details and a qPCR result available, 241/378 (64%) reported traveling to districts with a 'high' malaria endemicity and for 12% the highest endemicity category was 'moderate'. Travelers to the mainland were more likely to be infected with malaria parasites (29%, 108/378) than those traveling within Zanzibar (8%, 16/206) or to other countries (6%, 2/17). Among travellers to mainland Tanzania, those visiting highly endemic districts had a higher odds of being qPCR-positive than those who travelled only to districts where malaria-endemicity was classified as low or very low (adjusted odd ratio = 7.0, 95% confidence interval: 1.9-25.5). Among travellers to the mainland, 110/378 (29%) never or only sometimes used a mosquito net during their travel. CONCLUSIONS: Strategies to reduce malaria importation to Zanzibar may benefit from identifying population groups traveling to highly endemic areas in mainland Tanzania. Targeted interventions to prevent and clear infections in these groups may be more feasible than attempting to screen and treat all travellers upon arrival in Zanzibar.

Multiplexed ddPCR-amplicon sequencing reveals isolated Plasmodium falciparum populations amenable to local elimination in Zanzibar, Tanzania.

Zanzibar has made significant progress toward malaria elimination, but recent stagnation requires novel approaches. We developed a highly multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug-resistance loci, and successfully sequenced 290 samples from five districts covering both main islands. Here, we elucidate fine-scale Plasmodium falciparum population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. Despite high genetic diversity, we observe pronounced fine-scale spatial and temporal parasite genetic structure. Clusters of near-clonal infections on Pemba indicate persistent local transmission with limited parasite importation, presenting an opportunity for local elimination efforts. Furthermore, we observe an admixed parasite population on Unguja and detect a substantial fraction (2.9%) of significantly related infection pairs between Zanzibar and the mainland, suggesting recent importation. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and provides actionable insights for prioritizing malaria elimination efforts.

Modelling the impact of interventions on imported, introduced and indigenous malaria infections in Zanzibar, Tanzania.

Malaria cases can be classified as imported, introduced or indigenous cases. The World Health Organization's definition of malaria elimination requires an area to demonstrate that no new indigenous cases have occurred in the last three years. Here, we present a stochastic metapopulation model of malaria transmission that distinguishes between imported, introduced and indigenous cases, and can be used to test the impact of new interventions in a setting with low transmission and ongoing case importation. We use human movement and malaria prevalence data from Zanzibar, Tanzania, to parameterise the model. We test increasing the coverage of interventions such as reactive case detection; implementing new interventions including reactive drug administration and treatment of infected travellers; and consider the potential impact of a reduction in transmission on Zanzibar and mainland Tanzania. We find that the majority of new cases on both major islands of Zanzibar are indigenous cases, despite high case importation rates. Combinations of interventions that increase the number of infections treated through reactive case detection or reactive drug administration can lead to substantial decreases in malaria incidence, but for elimination within the next 40 years, transmission reduction in both Zanzibar and mainland Tanzania is necessary.

The impact of reactive case detection on malaria transmission in Zanzibar in the presence of human mobility.

Malaria persists at low levels on Zanzibar despite the use of vector control and case management. We use a metapopulation model to investigate the role of human mobility in malaria persistence on Zanzibar, and the impact of reactive case detection. The model was parameterized using survey data on malaria prevalence, reactive case detection, and travel history. We find that in the absence of imported cases from mainland Tanzania, malaria would likely cease to persist on Zanzibar. We also investigate potential intervention scenarios that may lead to elimination, especially through changes to reactive case detection. While we find that some additional cases are removed by reactive case detection, a large proportion of cases are missed due to many infections having a low parasite density that go undetected by rapid diagnostic tests, a low rate of those infected with malaria seeking treatment, and a low rate of follow up at the household level of malaria cases detected at health facilities. While improvements in reactive case detection would lead to a reduction in malaria prevalence, none of the intervention scenarios tested here were sufficient to reach elimination. Imported cases need to be treated to have a substantial impact on prevalence.

High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy.

Most rapid diagnostic tests for Plasmodium falciparum malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the hrp2 and hrp3 genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of hrp2/hrp3 deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies hrp2, hrp3, and a control gene with very high accuracy. The theoretical limit of detection was 0.33 parasites/µl. The deletion was reliably detected in mixed infections with wild-type and hrp2-deleted parasites at a density of >100 parasites/reaction. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR hrp2 deletion was observed in 8% of samples. The ddPCR assay was applied to screen 830 samples from Kenya, Zanzibar/Tanzania, Ghana, Ethiopia, Brazil, and Ecuador. Pronounced differences in the prevalence of deletions were observed among sites, with more hrp3 than hrp2 deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e., hrp2 deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.

Malaria infection prevalence and sensitivity of reactive case detection in Zanzibar.

BACKGROUND: Reactive case detection (RCD) is a commonly used strategy for malaria surveillance and response in elimination settings. Many approaches to RCD assume detectable infections are clustered within and around homes of passively detected cases (index households), which has been evaluated in a number of settings with disparate results. METHODS: Household questionnaires and diagnostic testing were conducted following RCD investigations in Zanzibar, Tanzania, including the index household and up to 9 additional neighboring households. RESULTS: Of 12,487 participants tested by malaria rapid diagnostic test (RDT), 3·2% of those residing in index households and 0·4% of those residing in non-index households tested positive (OR = 8·4; 95%CI: 5·7, 12·5). Of 6,281 participants tested by quantitative polymerase chain reaction (qPCR), 8·4% of those residing in index households and 1·3% of those residing in non-index households tested positive (OR = 7·1; 95%CI: 6·1, 10·9). Within households of index cases defined as imported, odds of qPCR-positivity amongst members reporting recent travel were 1·4 times higher than among those without travel history (95%CI: 0·2, 4·4). Amongst non-index households, odds of qPCR-detectable infection were no different between households located within 50 m of the index household as compared with those located farther away (OR = 0·8, 95%CI: 0·5, 1·4). Sensitivity of RDT to detect qPCR-detectable infections was 34% (95%CI: 26·4, 42·3). CONCLUSIONS: Malaria prevalence in index households in Zanzibar is much higher than in non-index households, in which prevalence is very low. Travelers represent a high-risk population. Low sensitivity of RDTs due to a high prevalence of low-density infections results in an RCD system missing a large proportion of the parasite reservoir.

Stature estimation in the South-East Asian population: A systematic review.

INTRODUCTION: Stature estimation is population dependent, and population-specific regression equations should be generated for accurate anthropological assessments. Nevertheless, stature estimation data was inaccessible and limited in some of the South-East Asian countries. The systematic review was conducted to analyse the regression equations of stature estimations developed in South-East Asian region. MATERIALS AND METHODS: A systematic literature search was performed through SCOPUS database and Google Scholar from January till March 2018. All published articles which developed stature estimation from different types of bone, methods and type of statures (i.e. living stature, forensic stature and cadaveric stature) were included in this study. Risks of biases were also assessed. Population studies with no regression equations were excluded from the study. RESULTS: Seven studies that met the inclusion criteria were identified. In the South-East Asia region, regression equations for stature estimation were developed in Thailand and Malaysia. In these studies, bone measurements were done either by radiography, direct bone measurement, or palpation on body surface for anatomical bony prominence. All of these studies used various parts of bones for stature estimation. CONCLUSION: The most widely used regression equations for stature estimation in South-East Asian population were from the Thailand population. Further research is recommended to develop regression equations for other South-East Asian countries.

Ultrastructural changes of cell morphology and viral morphogenesis of two ecotypes of dengue virus infection in human monocytic U-937 cell.

Dengue virus (DENV) is maintained and circulated in both sylvatic/enzootic and endemic/human cycles and spill over infection of sylvatic DENV into human populations has been reported. Extensive deforestation and increase human activities in forest may increase the risk of human exposure to sylvatic dengue infection and this may become a threat to human. Present study investigated the changes in cell morphology and viral morphogenesis upon infection with sylvatic and endemic ecotypes in human monocytic U-937 cells using transmission electron microscopy. Autophagy, a process that is either pro-viral or anti-viral, was observed in U-937 cells of both infections, however only the replication of endemic DENV was evidenced. An insight into the infection responses of sylvatic progenitors of DENV in susceptible host cells may provide better understanding on dengue emergence in human populations.

Ultrastructural changes of cell morphology and viral morphogenesis of two ecotypes of dengue virus infection in human monocytic U-937 cell

@#Dengue virus (DENV) is maintained and circulated in both sylvatic/enzootic and endemic/human cycles and spill over infection of sylvatic DENV into human populations has been reported. Extensive deforestation and increase human activities in forest may increase the risk of human exposure to sylvatic dengue infection and this may become a threat to human. Present study investigated the changes in cell morphology and viral morphogenesis upon infection with sylvatic and endemic ecotypes in human monocytic U-937 cells using transmission electron microscopy. Autophagy, a process that is either pro-viral or anti-viral, was observed in U-937 cells of both infections, however only the replication of endemic DENV was evidenced. An insight into the infection responses of sylvatic progenitors of DENV in susceptible host cells may provide better understanding on dengue emergence in human populations.

Stature Estimation from Lower Limb Anthropometry using Linear Regression Analysis: A Study on the Malaysian Population.

INTRODUCTION: Stature is one of the characteristics that could be used to identify human, besides age, sex and racial affiliation. This is useful when the body found is either dismembered, mutilated or even decomposed, and helps in narrowing down the missing person's identity. AIM: The main aim of the present study was to construct regression functions for stature estimation by using lower limb bones in the Malaysian population. MATERIALS AND METHODS: The sample comprised 87 adult individuals (81 males, 6 females) aged between 20 to 79 years. The parameters such as thigh length, lower leg length, leg length, foot length, foot height and foot breadth were measured. They were measured by a ruler and measuring tape. Statistical analysis involved independent t-test to analyse the difference between lower limbs in male and female. The Pearson's correlation test was used to analyse correlations between lower limb parameters and stature, and the linear regressions were used to form equations. The paired t-test was used to compare between actual stature and estimated stature by using the equations formed. RESULTS: Using independent t-test, there was a significant difference (p< 0.05) in the measurement between males and females with regard to leg length, thigh length, lower leg length, foot length and foot breadth. The thigh length, leg length and foot length were observed to have strong correlations with stature with p= 0.75, p= 0.81 and p= 0.69, respectively. Linear regressions were formulated for stature estimation. Paired t-test showed no significant difference between actual stature and estimated stature. CONCLUSIONS: It is concluded that regression functions can be used to estimate stature to identify skeletal remains in the Malaysia population.

Microscopic studies on postmortem vermiform appendix of the adult males of Bangladesh.

BACKGROUND: The anatomy of the vermiform appendix shows variations in its macroscopic dimensions and microscopic features, some of which have potentials of influencing the clinical aspects of the appendix. AIM: The aim of this study was to find out some microscopic features of appendix and evaluate the correlation between the microscopic features of the appendix and the age of the subjects and to determine whether these findings should influence the clinical implications of appendix. METHODS: In this cross-sectional observational study, thirty adult males' (age from 18 to 67 years) postmortem appendices and adnexa from Bangladeshi victims of road traffic accidents were sectioned at the base, midzone and tip stained with H+E stain and examined under microscope. Measurements were taken at the base, at the midzone and at the tip of the appendix, and the mean of the three measurements was considered as the overall value. RESULTS: The overall number of mucosal glands in a section ranged from 42.33 to 130.00 and the number of the germinal centres varied between 2.33 and 10.00. The overall luminal diameter ranged between 1764.58 and 3208.33 µm. The overall luminal diameter in more than 52 % of cases was between 2700.00 and 3299.99 µm with a median value of 2750 µm. CONCLUSION: The overall number of mucosal glands showed a tendency towards a positive correlation with age. The overall luminal diameter and the overall number of germinal centres showed a tendency towards a negative correlation with the age. However, none of the tendencies of correlation reached statistically any significant level.

Immobilisation of Cu, Pb and Zn in Scrap Metal Yard Soil Using Selected Waste Materials.

Immobilisation of heavy metals in a 30-year old active scrap metal yard soil using three waste materials, namely coconut tree sawdust (CTS), sugarcane bagasse (SB) and eggshell (ES) was investigated. The contaminated soil was amended with amendments at application rates of 0 %, 1 % and 3 % (w/w). The effects of amendments on metal accumulation in water spinach (Ipomoea aquatica) and soil metal bioavailability were studied in a pot experiment. All amendments increased biomass yield and reduced metal accumulation in the plant shoots. The bioconcentration factor and translocation factor values of the metals were in the order of Zn > Cu > Pb. The addition of ES, an alternative source of calcium carbonate (CaCO3), has significantly increased soil pH and resulted in marked reduction in soil metal bioavailability. Therefore, CTS, SB and ES are promising low-cost immobilising agents to restore metal contaminated land.

A Heuristic Automatic and Robust ROI Detection Method for Medical Image Warermarking.

This paper presents an automatic region of interest (ROI) segmentation method for application of watermarking in medical images. The advantage of using this scheme is that the proposed method is robust against different attacks such as median, Wiener, Gaussian, and sharpening filters. In other words, this technique can produce the same result for the ROI before and after these attacks. The proposed algorithm consists of three main parts; suggesting an automatic ROI detection system, evaluating the robustness of the proposed system against numerous attacks, and finally recommending an enhancement part to increase the strength of the composed system against different attacks. Results obtained from the proposed method demonstrated the promising performance of the method.

High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions.

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.

Watermarking techniques used in medical images: a survey.

The ever-growing numbers of medical digital images and the need to share them among specialists and hospitals for better and more accurate diagnosis require that patients' privacy be protected. As a result of this, there is a need for medical image watermarking (MIW). However, MIW needs to be performed with special care for two reasons. Firstly, the watermarking procedure cannot compromise the quality of the image. Secondly, confidential patient information embedded within the image should be flawlessly retrievable without risk of error after image decompressing. Despite extensive research undertaken in this area, there is still no method available to fulfill all the requirements of MIW. This paper aims to provide a useful survey on watermarking and offer a clear perspective for interested researchers by analyzing the strengths and weaknesses of different existing methods.

Association of beta 2 adrenoceptor gene polymorphisms in Malaysian hypertensive subjects.

The sympathetic nervous system plays a major role in blood pressure regulation. Beta 2 (ß2) adrenoceptor gene polymorphisms have been associated with hypertension in different populations with conflicting results. We examined the association of three common polymorphisms, Arg16Gly, Gln27Glu, and Thr164Ile, of the ß2 adrenoceptor gene in Malaysian hypertensive subjects. A total of 160 hypertensive and control subjects were recruited. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and anthropometric measurements were obtained from each subject. Biochemical analyses of lipid profiles were conducted with an autoanalyzer. DNA samples were extracted from blood and buccal cells. Genotyping was accomplished with polymerase chain reaction-restriction fragment length polymorphism. SBP, DBP, body mass index, and biochemical factors all differed significantly between case and control subjects (P < 0.05). The genotype frequencies of Arg16Arg, Arg16Gly, and Gly16Gly were 22.5, 70, and 7.5% among cases and 33.1, 63.1, and 3.8% among controls, respectively. The genotype frequencies of Gln27Gln, Gln27Glu, and Glu27Glu among cases were 41.1, 50, and 1.9% compared to 77.5, 20.6, and 1.9% among controls, respectively. In this study, the Gln27Glu polymorphism was significantly associated with Malaysian hypertensive subjects (P < 0.05). Therefore, the Gln27Glu polymorphism of the ß2 adrenoceptor could be a risk factor associated with hypertension among Malaysians.

Applying a statistical PTB detection procedure to complement the gold standard.

This paper investigates a novel statistical discrimination procedure to detect PTB when the gold standard requirement is taken into consideration. Archived data were used to establish two groups of patients which are the control and test group. The control group was used to develop the statistical discrimination procedure using four vectors of wavelet coefficients as feature vectors for the detection of pulmonary tuberculosis (PTB), lung cancer (LC), and normal lung (NL). This discrimination procedure was investigated using the test group where the number of sputum positive and sputum negative cases that were correctly classified as PTB cases were noted. The proposed statistical discrimination method is able to detect PTB patients and LC with high true positive fraction. The method is also able to detect PTB patients that are sputum negative and therefore may be used as a complement to the gold standard.