Epidemiology of Uterine Cancer in Sarawak, Borneo, A 20-Year Review.

OBJECTIVE: This study aimed to determine the epidemiology of uterine cancer in Sarawak, Malaysia, using data from a population-based cancer registry. METHODS: The study population included all women diagnosed with uterine cancer in Sarawak, Malaysia between January 1996 and December 2015. Data on demographic and clinical characteristics were obtained from the Sarawak Cancer Registry. The crude incidence rate, age-standardized incidence rate (ASR), and incidence risk ratios (IRR) were calculated. Joinpoint regression analyses were performed to assess trends in incidence rates. RESULTS: A total of 811 women were diagnosed with primary uterine cancer during the study period. The overall crude incidence rate for uterine cancer in Sarawak for the period 1996-2015 was 3.7 per 100,000. The ASR was 4.4 per 100,000 with a 95% CI (4.1-4.8). The ASR in 2011-2015 is 1.6 times higher than the ASR of uterine cancer in 1996-2000. Higher incidence rates were observed in women aged 40-59 years and those aged 60 years and above. Chinese women had the highest ASR, followed by Malay and Iban women. Joinpoint regression analyses showed a significant increase in cases of uterine cancer among all ethnic groups and age groups. CONCLUSION: The incidence of primary uterine cancer in Sarawak, Malaysia, has increased over the past 20 years, with higher incidence rates observed in older age groups and among Chinese women. The findings suggest the need for continued efforts to improve the prevention, early detection, and treatment of uterine cancer in Sarawak.

Epidemiology of Nasopharyngeal Carcinoma in Sarawak, East Malaysia.

OBJECTIVES: This analysis provides an epidemiological update for nasopharyngeal carcinomas the state of Sarawak and an analysis of the trend over a 20 years period. METHOD: Data between 1996 to 2015 from a population-based cancer registry in Sarawak Malaysia was analyzed. Crude incidence rates and age-standardized rates (ASR) were calculated and compared between ethnic groups and locations (administrative division) and Joinpoint regression analysis was done to analyze trends. RESULT: A total of 3643 cases of NPC were recorded with male to female ratio of 2.5:1. Annualised age-standardized incidence rates able 2) for men is 13.2 cases per 100,000 population (95% CI: 12.6, 13.7) and for women is 5.3 cases per 100,000 population (95% CI: 5.0, 5.6). The highest incidence rates were reported among the Bidayuh population and it ranks among the highest in the world. Trend analysis noted an overall reduction of cases, with a significant decrease between 1996 and 2003 (annual percentage reduction of incidence by 3.9%). Analysis of individual ethnic groups also shows a general reduction with exception of Iban males showing an average 5.48 per cent case increase between 2009 to 2015, though not statistically significant. CONCLUSION: Comparing the incidences with other registries, the Bidayuh population in Sarawak remained among the highest in the world and warrants close attention for early screening and prevention strategies.

The epidemiology of haematological cancers in Sarawak, Malaysia (1996 to 2015).

BACKGROUND: Published epidemiological studies of haematological cancers are few. Hereby we present a 20-year epidemiological data of haematological cancers in Sarawak from a population-based cancer registry. METHODS: Haematological cancer cases with ICD-10 coded C81-C96 and ICD-O coded /3 diagnosed from 1996 to 2015 were retrieved from Sarawak Cancer Registry. Adult was defined as those 15 years and above. Incidence rate (IR) was calculated based on yearly Sarawak citizen population stratified to age, gender, and ethnic groups. Age-standardised IR (ASR) was calculated using Segi World Standard Population. RESULTS: A total of 3,947 cases were retrieved and analysed. ASR was 10 and male predominance (IR ratio 1.32, 95%CI 1.24,1.41). Haematological cancers generally had a U-shaped distribution with lowest IR at age 10-14 years and exponential increment from age 40 years onwards, except acute lymphoblastic leukaemia (ALL) with highest IR in paediatric 2.8 versus adult 0.5. There was a significant difference in ethnic and specific categories of haematological cancers, of which, in general, Bidayuh (IR ratio 1.13, 95%CI 1.00, 1.27) and Melanau (IR ratio 0.54, 95%CI 0.45, 0.65) had the highest and lowest ethnic-specific IR, respectively, in comparison to Malay. The ASR (non-Hodgkin lymphoma, acute myeloid leukaemia, ALL, chronic myeloid leukaemia, and plasma cell neoplasm) showed a decreasing trend over the 20 years, -2.09 in general, while Hodgkin lymphoma showed an increasing trend of + 2.80. There was crude rate difference between the 11 administrative divisions of Sarawak. CONCLUSIONS: This study provided the IR and ASR of haematological cancers in Sarawak for comparison to other regions of the world. Ethnic diversity in Sarawak resulted in significant differences in IR and ASR.

Profiling the imported human malaria in Sarawak, Malaysia in 2011-2019.

OBJECTIVES: Malaysia has achieved the status of zero indigenous human malaria cases. Nevertheless, imported human malaria has increasingly been reported in Sarawak, Malaysian Borneo. As zoonotic malaria caused by Plasmodium knowlesi remains a major public health problem in Sarawak, the threat of imported malaria must be addressed as it can cause human malaria reintroduction, sustain transmission, and lead to complications. The objectives of this study were to investigate the epidemiological characteristics of imported malaria cases reported in Sarawak and to underline the challenge posed by imported malaria towards malaria elimination efforts. METHODS: Imported malaria cases reported in Sarawak from 2011 to 2019 were collected from Sarawak State Health Department and analysed in this longitudinal retrospective study. RESULTS: A total of 2058 imported malaria cases were registered in all districts in Sarawak. Highest number of cases were reported in Kapit (n = 559; 27.16%), followed by Sibu (n = 424; 20.6%), and Miri (n = 166; 8.07%). Based on the demographic profile, most of the patients constituted of either male sex (98.49%), age group of 40-49 years (39.6%), Iban ethnic (57.92%), worked in logging industry (88.58%), Malaysian nationals (91.84%), contracted malaria in Papua New Guinea (46.11%), uncomplicated disease (77.89%), or hospitalised cases (97.86%). The most prominent Plasmodium species diagnosed were P. vivax (52.67%) and P. falciparum (35.81%). CONCLUSIONS: Surveillance, disease detection, and medical follow-up must be carried out thoroughly for individuals who returned from malaria-endemic countries. It is also necessary to promote pre-travel preventive education as well as chemoprophylaxis to travellers heading to endemic areas.

Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia.

Plasmodium knowlesi is a simian malaria parasite that causes significant zoonotic infections in Southeast Asia, particularly in Malaysia. The Plasmodium thrombospondin-related apical merozoite protein (TRAMP) plays an essential role in the invasion of the parasite into its host erythrocyte. The present study investigated the genetic polymorphism and natural selection of the full length PkTRAMP from P. knowlesi clinical isolates from Malaysia. Blood samples (n = 40) were collected from P. knowlesi malaria patients from Peninsular Malaysia and Malaysian Borneo. The PkTRAMP gene was amplified using PCR, followed by cloning into a plasmid vector and sequenced. Results showed that the nucleotide diversity of PkTRAMP was low (π: 0.009). Z-test results indicated negative (purifying) selection of PkTRAMP. The alignment of the deduced amino acid sequences of PkTRAMP of Peninsular Malaysia and Malaysian Borneo revealed 38 dimorphic sites. A total of 27 haplotypes were identified from the amino acid sequence alignment. Haplotype analysis revealed that there was no clustering of PkTRAMP from Peninsular Malaysia and Malaysian Borneo.

Epidemiology of indigenous Plasmodium knowlesi infection in Sarawak, 2011-2019.

OBJECTIVES: To characterise the state-wide epidemiology of indigenous knowlesi malaria in Sarawak from 2011 to 2019. METHODS: Longitudinal retrospective study was conducted based on Sarawak knowlesi malaria surveillance data recorded from 2011-2019. Only indigenous cases were included and information extracted for analysis comprised age, sex, occupation, ethnicity, case severity, hospital admission and parasite density. RESULTS: Over the 9 years, 8473 indigenous knowlesi malaria cases were recorded. Age group 40-49 years, males, plantation workers and Iban communities recorded the highest percentage of cases in each demographic variable. Most of the cases were uncomplicated (n = 7292; 86.1%) and 89.6% (n = 7589) of the total cases were reported with ≤20 000 parasites/µl of blood. Age group and ethnic group are associated with the severity of knowlesi malaria in Sarawak. Multivariable logistic regression indicated that the age group 60+ years had the highest odds of developing severe knowlesi malaria compared with other age groups (AOR 2.48; 95% CI 1.22, 5.02; p = 0.012). Bidayuh patients were more likely to develop severe knowlesi malaria than Ibans, the largest ethnic group among knowlesi malaria patients (AOR 1.97; 95% CI 1.31, 2.97; p = 0.001). CONCLUSIONS: Identification of risk groups is important for the implementation of prevention programs and treatments targeting at specific group to combat knowlesi malaria effectively.

Spatial and Temporal Patterns of Plasmodium knowlesi Malaria in Sarawak from 2008 to 2017.

Zoonotic knowlesi malaria has replaced human malaria as the most prevalent malaria disease in Malaysia. The persistence of knowlesi malaria in high-risk transmission areas or hotspots can be discouraging to existing malaria elimination efforts. In this study, retrospective data of laboratory-confirmed knowlesi malaria cases were obtained from the Sarawak Health Department to investigate the spatiotemporal patterns and clustering of knowlesi malaria in the state of Sarawak from 2008 to 2017. Purely spatial, purely temporal, and spatiotemporal analyses were performed using SaTScan software to define clustering of knowlesi malaria incidence. Purely spatial and spatiotemporal analyses indicated most likely clusters of knowlesi malaria in the northern region of Sarawak, along the Sarawak-Kalimantan border, and the inner central region of Sarawak between 2008 and 2017. Temporal cluster was detected between September 2016 and December 2017. This study provides evidence of the existence of statistically significant Plasmodium knowlesi malaria clusters in Sarawak, Malaysia. The analysis approach applied in this study showed potential in establishing surveillance and risk management system for knowlesi malaria control as Malaysia approaches human malaria elimination.

Validation of SYBR green I based closed-tube loop-mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria.

BACKGROUND: As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. METHODS: A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. RESULTS: LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. CONCLUSIONS: These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

Evaluation of WarmStart Colorimetric Loop-Mediated Isothermal Amplification Assay for Diagnosis of Malaria.

The incidence of zoonotic malaria, Plasmodium knowlesi, infection is increasing and now is the major cause of malaria in Malaysia. Here, we describe a WarmStart colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Plasmodium spp. The detection limit for this assay was 10 copies/µL for P knowlesi and Plasmodium ovale and 1 copy/µL for Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae. To test clinical sensitivity and specificity, 100 microscopy-positive and 20 malaria-negative samples were used. The WarmStart colorimetric LAMP was 98% sensitive and 100% specific. Amplification products were visible for direct observation, thereby eliminating the need for post-amplification processing steps. Therefore, WarmStart colorimetric LAMP is suitable for use in resource-limited settings.

Correction to: Evidence of asymptomatic submicroscopic malaria in low transmission areas in Belaga district, Kapit division, Sarawak, Malaysia.

Please note that an author has been erroneously omitted from the author list of the published article [1].

Case Report: Two Cases of Recurring Ovale Malaria in Sarawak, Malaysia, after Successful Treatment of Imported Plasmodium falciparum Infection.

Here are two cases of recurring ovale malaria in Sarawak, Malaysia, that are likely relapses that occurred 1-2 months after successful treatment of the initial imported falciparum malaria with artemisinin-based combined therapy. The patients have no history or recollection of previous malaria episodes. These cases add to the limited evidence on the relapsing nature of Plasmodium ovale, after a febrile episode. In regions where P. ovale is not known to be autochthonous, active follow-up of treated imported malaria patients is highly recommended following their return, particularly to areas nearing or having achieved elimination.

Distribution of the Duffy genotypes in Malaysian Borneo and its relation to Plasmodium knowlesi malaria susceptibility.

The Duffy blood group plays a key role in Plasmodium knowlesi and Plasmodium vivax invasion into human erythrocytes. The geographical distribution of the Duffy alleles differs between regions with the FY*A allele having high frequencies in many Asian populations, the FY*B allele is found predominately in European populations and the FY*Bes allele found predominantly in African regions. A previous study in Peninsular Malaysia indicated high homogeneity of the dominant FY*A/FY*A genotype. However, the distribution of the Duffy genotypes in Malaysian Borneo is currently unknown. In the present study, the distribution of Duffy blood group genotypes and allelic frequencies among P. knowlesi infected patients as well as healthy individuals in Malaysian Borneo were determined. A total of 79 P. knowlesi patient blood samples and 76 healthy donor samples were genotyped using allele specific polymerase chain reaction (ASP-PCR). Subsequently a P. knowlesi invasion assay was carried out on FY*AB/ FY*A and FY*A/ FY*A Duffy genotype blood to investigate if either genotype conferred increased susceptibility to P. knowlesi invasion. Our results show almost equal distribution between the homozygous FY*A/FY*A and heterozygous FY*A/FY*B genotypes. This is in stark contrast to the Duffy distribution in Peninsular Malaysia and the surrounding Southeast Asian region which is dominantly FY*A/FY*A. The mean percent invasion of FY*A/FY*A and FY*A/FY*B blood was not significantly different indicating that neither blood group confers increased susceptibility to P. knowlesi invasion.

Genetic characterisation of the erythrocyte-binding protein (PkßII) of Plasmodium knowlesi isolates from Malaysia.

Plasmodium knowlesi contributes to the majority of human malaria incidences in Malaysia. Its uncontrollable passage among the natural monkey hosts can potentially lead to zoonotic outbreaks. The merozoite of this parasite invades host erythrocytes through interaction between its erythrocyte-binding proteins (EBPs) and their respective receptor on the erythrocytes. The regionII of P. knowlesi EBP, P. knowlesi beta (PkßII) protein is found to be mediating merozoite invasion into monkey erythrocytes by interacting with sialic acid receptors. Hence, the objective of this study was to investigate the genetic diversity, natural selection and haplotype grouping of PkßII of P. knowlesi isolates in Malaysia. Polymerase chain reaction amplifications of PkßII were performed on archived blood samples from Malaysia and 64 PkßII sequences were obtained. Sequence analysis revealed length polymorphism, and its amino acids at critical residues indicate the ability of PkßII to mediate P. knowlesi invasion into monkey erythrocytes. Low genetic diversity (π = 0.007) was observed in the PkßII of Malaysia Borneo compared to Peninsular Malaysia (π = 0.015). The PkßII was found to be under strong purifying selection to retain infectivity in monkeys and it plays a limited role in the zoonotic potential of P. knowlesi. Its haplotypes could be clustered into Peninsular Malaysia and Malaysia Borneo groups, indicating the existence of two distinct P. knowlesi parasites in Malaysia as reported in an earlier study.

Evidence of asymptomatic submicroscopic malaria in low transmission areas in Belaga district, Kapit division, Sarawak, Malaysia.

BACKGROUND: Malaysia has declared its aim to eliminate malaria with a goal of achieving zero local transmission by the year 2020. However, targeting the human reservoir of infection, including those with asymptomatic infection is required to achieve malaria elimination. Diagnosing asymptomatic malaria is not as straightforward due to the obvious lack of clinical manifestations and often subpatent level of parasites. Accurate diagnosis of malaria is important for providing realistic estimates of malaria burden and preventing misinformed interventions. Low levels of parasitaemia acts as silent reservoir of transmission thus remains infectious to susceptible mosquito vectors. Hence, the aim of this study is to investigate the prevalence of asymptomatic submicroscopic malaria (SMM) in the District of Belaga, Sarawak. METHODS: In 2013, a total of 1744 dried blood spots (DBS) were obtained from residents of 8 longhouses who appeared healthy. Subsequently, 251 venous blood samples were collected from residents of 2 localities in 2014 based on the highest number of submicroscopic cases from prior findings. Thin and thick blood films were prepared from blood obtained from all participants in this study. Microscopic examination were carried out on all samples and a nested and nested multiplex PCR were performed on samples collected in 2013 and 2014 respectively. RESULTS: No malaria parasites were detected in all the Giemsa-stained blood films. However, of the 1744 samples, 29 (1.7%) were positive for Plasmodium vivax by PCR. Additionally, of the 251 samples, the most prevalent mono-infection detected by PCR was Plasmodium falciparum 50 (20%), followed by P. vivax 39 (16%), P. knowlesi 9 (4%), and mixed infections 20 (8%). CONCLUSIONS: This research findings conclude evidence of Plasmodium by PCR, among samples previously undetectable by routine blood film microscopic examination, in local ethnic minority who are clinically healthy. SMM in Belaga district is attributed not only to P. vivax, but also to P. falciparum and P. knowlesi. In complementing efforts of programme managers, there is a need to increase surveillance for SMM nationwide to estimate the degree of SMM that warrant measures to block new transmission of malaria.

Development of Loop-Mediated Isothermal Amplification-Based Lateral Flow Device Method for the Detection of Malaria.

The present study aims to develop a method for rapid diagnosis of malaria using loop-mediated isothermal amplification (LAMP) combined with a lateral flow device (LFD). By adding the biotin-labeled and fluorescein amidite-labeled loop primers to the LAMP reaction solution, the end product can be visualized on a LFD. The entire procedure takes approximately 42 minutes to complete, LAMP assay exhibited high sensitivity, as the detection limit was 0.01 pg/µL for all five Plasmodium species. It was demonstrated that all Plasmodium knowlesi (N = 90) and Plasmodium vivax (N = 56) were positively amplified by LAMP-LFD assay, whereas healthy donor samples (N = 8) were negative. However, not all mixed infections were positive, and other infected nonmalaria samples were negative. Loop-mediated isothermal amplification-LFD represents a robust approach with potential suitability for use in resource-constrained laboratories. We believe that LAMP-LFD has a potential to be developed as point-of-care diagnostic tool in future.

Recombinase Polymerase Amplification Combined with a Lateral Flow Strip for the Detection of Plasmodium knowlesi.

The aim of this study was to develop a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method for specific diagnosis of Plasmodium knowlesi. With incubation at 37°C, the 18S rRNA gene of P. knowlesi was successfully amplified within 12 minutes. By adding a specifically designed probe to the reaction solution, the amplified RPA product can be visualized on a LF strip. The RPA assay exhibited high sensitivity with limits of detection down to 10 parasites/µL of P. knowlesi. Nonetheless, it was demonstrated that all P. knowlesi (N = 41) and other Plasmodium sp. (N = 25) were positive while negative samples (N = 8) were negative. Therefore, a combination of RPA and LF strip detection is a highly promising approach with the potential to be suitable for use in resource-limited settings.

Rapid Detection of Plasmodium knowlesi by Isothermal Recombinase Polymerase Amplification Assay.

In this study, we developed a recombinase polymerase amplification (RPA) assay for specific diagnosis of Plasmodium knowlesi. Genomic DNA was extracted from whole blood samples using a commercial kit. With incubation at 37°C, the samples were successfully amplified within 20 minutes. The end product of RPA was further examined by loading onto agarose gel and a specific band was observed with a size of 128 bp. The RPA assay exhibited high sensitivity with limits of detection down to one copy of the plasmid. From the specificity experiments, it was demonstrated that all P. knowlesi samples (N = 45) were positive while other Plasmodium spp. (N = 42) and negative samples (N = 6) were negative. Therefore, the RPA assay is a highly promising approach with the potential to be used in resource-limited settings. This assay can be further optimized for bedside and on field application.

Over two decades of Plasmodium knowlesi infections in Sarawak: Trend and forecast.

Malaria is still of great public health concern, especially in Malaysian Borneo. The aim of this study was to determine the trends of P. knowlesi infection in Sarawak, Malaysia and to forecast the incidence of P. knowlesi until the year 2040. Data on P. knowlesi malaria cases from 1992 to the year 2014 were obtained from the Sarawak Health Department, Malaysia. ARIMA model was applied to forecast the future incidence of P. knowlesi infection. The data for the whole of Sarawak and subsequently the selected six districts which have high incidence rates of P. knowlesi infection were analyzed. Results of the analysis showed that there was an increasing trend of P. knowlesi cases from the year 1992-2014 (p<0.001). The trend in the incidence started to increase in the year 2008 (p=0.029). The incidence rate per 100,000 populations was between 4.15 in the year 1992 and 42.03 in the year 2014. High incidence of P. knowlesi infections has been detected in the districts adjacent to each other within the interior region of Sarawak. The forecasted incidence and incidence rate per 100,000 populations in the year 2020 were 1229 and 44.04, respectively, while those in the year 2040 were 2056 and 62.91, respectively. The forecasted incidence showed an upward trend highlighting an urgent need to draw up strategic and holistic prevention plans to limit further the increase in P. knowlesi morbidity and mortality in Sarawak. It is imperative that these measures are customized taking into consideration the challenges faced in the interior areas of Sarawak and the behavior of the main vector of P. knowlesi (i.e., An. latens) in Sarawak.