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Human genetic determinants of severe Plasmodium falciparum malaria in Vietnam
Project: Consortial Project 1

Location: Vietnam (VN).

Human

About this study

The last decade has seen a decline in the number of reported malaria cases in Vietnam from 187,994 in 1991 to 54,297 in 2010, with the number of deaths dramatically reduced from 4,646 in 1991 to 21 in 2010 (National Institute of Malariology Paristology and Epidemiology, 2010). This is a great achievement for malaria control programs in Vietnam, with the availability of highly effective therapy, namely artemisinin derivatives, playing a key role in this success (Hung le Q et al, 2002; Barat LM, 2006). However, malaria still exists within Vietnam, and it is believed by some that the number of malaria cases is under-reported (Erhart A et al, 2007). Many of the provinces in the central region of Vietnam, which provide the forested areas that support the mosquito vector (Anopheles dirus and A. minimus) border Cambodia where it has been reported that there is a decline in the effectiveness of artemisinin-based combination therapy (ACT) and artesunate monotherapy (Dondorp AM et al, 2009).

Acquisition of Plasmodium falciparum with resistance to ACT poses a real threat to the successful control of malaria in Vietnam and within the South-East Asian region. To be adequately empowered to combat malaria we need to completely understand the molecular mechanisms of protective immunity to enable the development of an efficacious multi-stage malaria vaccine capable of providing life-long protection. As part of the MalariaGEN Consortial Project 1 (CP1), the aim of our study is to identify mechanisms of protective immunity against malaria using a human genetics approach, which may lead to novel vaccine development.

Summary

Over the past 20 years the Oxford University Clinical Research Unit (OUCRU) in Ho Chi Minh City (HCMC), Vietnam, in collaboration with the Hospital for Tropical Diseases, has been performing clinical studies of malaria. These diverse clinical studies have led to a large collection of clinically well-defined severe malaria patients that are available for large scale genetics studies. This valuable resource includes archived genetic material and clinical characterisation of over 1,100 severe malaria patients and over 2,500 Vietnamese population controls.

Clinical data and DNA samples from many of these Vietnamese severe malaria cases and controls were contributed to the MalariaGEN Consortial Project 1 (CP1) along with those of 11 other case-control studies from a total of 11 malaria-endemic countries. As part of the sample handling process, baseline genotyping data was generated for a number of malaria–associated single nucleotide polymorphisms (SNPs) and the appropriate data has been returned to each site for site-specific analysis. A total of 69 SNPs at candidate genes (selection based on previous reports of association with severe malaria or on their likely biological role in malaria infection/disease) will be included in our analysis.

Study site description

All studies were conducted in the southern provinces of Vietnam. The main study site was the Hospital for Tropical Disease in Ho Chi Minh City which is a 550 bed tertiary referral hospital for infectious diseases for all of southern Vietnam. Southern Vietnam is divided into coastal lowlands, extensive forests and highland regions in south central Vietnam (West Highland). The hospital is situated in a large urban city (population over 7 million), however a significant number of the severe malaria patients are referred from surrounding provinces. Malaria cases are generally found in the rural highland region of south central Vietnam that tend to be impoverished, forested and less accessible to effective health systems (Trung HD et al, 2004). Approximately 75% of patients recruited for these genetics studies were enrolled at the Hospital for Tropical Diseases. Secondary study sites in Binh Phuoc province (population of 874,961 in 2009) are in the low highland regions of south central Vietnam. Phuoc Long district hospital and Dong Xoai Provincial Hospital within Binh Phuoc province recruited the remaining 25% of severe malaria patients for these genetics studies.

The southern area of Vietnam has a monsoon tropical climate. The seasons are divided into wet (May to November) and dry (December to April). In the southern plains around Ho Chi Minh City the temperature does not vary significantly throughout the year with an average temperature of approximately 32oC, which is slightly lower in the elevated low highland regions of Binh Phuoc province. This area has low seasonal transmission of P. falciparum and P. vivax mainly in the forested rural areas and entomological inoculation rates are very low (in most areas <1).

Methods

Since 1991 the Oxford University Clinical Research Unit, in collaboration with the Hospital for Tropical Diseases in Ho Chi Minh City (HCMC) has been performing clinical studies of malaria. During this time the collection of severe malaria patients for genetics studies has been part of 4 distinct clinical studies. The first two studies were randomised controlled trials for the treatment of adults with severe P. falciparum malaria undertaken at the Hospital for Tropical Diseases during the 1990s (1991-1995 Artemether versus Quinine (Phu NH et al, 2010) and 1996-2001 Artesunate versus Artemether (Tran TH et al, 1996)). The third study was a matched case-control epidemiological study in adults and children conducted between 2000-2005 at the Hospital of Tropical Diseases and two provincial hospitals in Binh Phuoc province, Phuoc Long and Dong Xoai district hospitals. The forth study started in 2006 is an ongoing collection of adult severe malaria patients specifically for genetics studies based at the Hospital for Tropical Diseases, HCMC.

Severe malaria cases were defined as those who had asexual forms of P. falciparum in their peripheral blood smear and had at least 1 of the following; impaired consciousness (Glasgow coma score <11 or Blantyre coma score <5), pulmonary oedema, acute renal failure (oliguria and serum creatinine >265 μmol/L), jaundice (serum bilirubin >51 μmol/L with parasite count >100,000/μL or with serum creatinine >250 μmol/L), hypoglycaemia (blood glucose <2.2 mmol/L), anaemia (haematocrit <20% with parasite count >100,000/μL), hyperparasitaemia (parasite count >500,000/μL), hyperlactataemia (plasma lactate > 4 mmol/L), metabolic acidosis (standard base excess > – 5 mmol/L, base deficit <10 mmol/L), pigmented neutrophil count (>4/100) and shock (SBP< 80mmHg with cool extremities); or had parasitemia ³ 5% but none of the above features. Patients with cerebral malaria were defined as those with a Glasgow coma score <9.

The population control individuals were either cord blood controls or community controls. Cord blood control samples were collected from babies born in 2003 and between 2006-2007 at Hung Vuong Obstetric Hospital in HCMC and from babies born in 2003 at Dong Thap Hospital in Dong Thap province. In addition, community controls were recruited as part of the epidemiological study which were individually matched to a proportion of the severe malaria cases by age, gender, ethnicity and location. Potential community controls were questioned about any possible history of severe malaria or time spent in hospital. Any who had spent more than 48 hours in hospital other than for an operation, injury or known non-malaria diagnosis were excluded.

Case report forms (CRF) were specific to each clinical study. Extensive clinical data was collected on patients within the randomised controlled treatment trials, including demographic information, clinical history, clinical evaluation, laboratory testing and clinical outcome. The CRFs for the matched case-control epidemiological study included these parameters as well as information related to environmental exposures and risk factors. The forth study, specifically for human genetics, used a standardised CRF that was created by MalariaGEN CP1. It was used by all sites to collect standardised clinical data in prospective studies. The data collected on all types of CRF were uploaded onto secure web-based software developed by MalariaGEN. Here, the integrity of the data was checked and data was standardised and amalgamated.

Genomic DNA was extracted from whole blood using either the blood midi kit or maxi kit from Qiagen (http://www.qiagen.com/) [Qiagen, Crawley, UK] using manufacturer’s instructions in the laboratories of the Oxford University Clinical Research Unit, HCMC, Vietnam. Aliquots of the DNA samples were shipped to the MalariaGEN Resource Centre in Oxford for further processing and quality control for quantity, quality (by genotyping) and confirming appropriate clinical data was available. Baseline genotype data for 69 malaria-associated SNPs was generated for all contributing samples; briefly, samples underwent a primer-extension pre-amplification (PEP) step (Xu K et al, 1993; Zhang L et al, 1992) prior to genotyping on the Sequenom® MassArray® platform. Following curation, the genotype data were returned to the PIs for local analyses.

Table 1: Breakdown of samples
Number Gender: n (%) Age in years: n (%) Ethnicity: n (%)
Malaria cases: 597 Male: 452 (75)

Female: 140 (23)

Not recorded: 9 (2)

 5-15: 10 (2)

>15: 587 (98)

 Kinh: 568 (95)

Other: 8 (1)

Not recorded: 21 (4)
Healthy controls: 2219 Male: 1091 (49)

Female: 1035 (46)

Not recorded: 114 (5)

 <1: 2219 (100) Kinh: 2219 (100)

Ethics

Ethical approvals were granted by the scientific and ethical committees at either the Hospital for Tropical Diseases HCMC (proposal number: ID SECHTD 20/04/2006), Hung Vuong Hospital HCMC, Dong Thap Hospital Dong Thap Province, and the People’s Committee of Ho Chi Minh City, Department of Health. Protocols were also approved by the Oxford Tropical Research Ethics Committee, UK.

Over the previous 20 years the Hospital for Tropical Disease, in collaboration with OUCRU, performed a number of clinical trials investigating the treatment of malaria. Over this time a large number of blood samples have been archived from severe malaria patients. Ethical approval was granted to use DNA prepared from these archived samples of severe malaria patients in an anonymised fashion. Additionally, ethical approval was granted to collect new DNA samples (from cases and controls) in order to become a part of large-scale genetic studies.

Prior to study recruitment patients were informed of the risks and benefits of being in these studies. Written informed consent was obtained from each volunteer, however in one early study verbal informed consent was obtained. Where the patient was unable to consent (e.g. if unconscious), the consent of a relative was obtained. For cord blood control samples informed consent was obtained from the mother.

Additional contributors

  • Cameron P Simmons, Oxford University Clinical Research Unit, Vietnam; and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK
  • Cao Quang Thai, Hospital for Tropical Diseases, Vietnam
  • Jeremy Farrar, Oxford University Clinical Research Unit, Vietnam; and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK
  • Nguyen Hoan Phu, Hospital for Tropical Diseases, Vietnam
  • Nguyen T Hieu, Hung Vuong Hospital, Vietnam
  • Nicholas Day, Mahidol Oxford Tropical Medicine Research Unit (MORU), Thailand
  • Sean E O’Riordan, Oxford University Clinical Research Unit, Vietnam

Acknowledgements

We would like to thank all the Vietnamese individuals who agreed to provide samples for this study. We acknowledge the work of the clinical staff from the Hospital of Tropical Diseases, HCMC and Phuoc Long and Dong Xoai District Hospitals in Binh Phuoc province, Vietnam, who initially diagnosed and studied the patients with severe malaria. We would like to thank the staff from Hung Vuong Obstetric Hospital for the collection of the cord blood controls. We thank Susana Campino, Rachel Craik, Kate Rowlands, Angie Green and Christina Hubbart in the MalariaGEN Resource Centre for DNA handling and genotyping. We appreciate the contribution of the MalariaGEN ethics team (Oxford University, UK), especially Jantina de Vries.

The clinical component of this study was funded through the Wellcome Trust Major Overseas Program in Vietnam. The MalariaGEN component was funded primarily from the Wellcome Trust and from the Bill & Melinda Gates Foundation, through the Foundation for the National Institutes of Health as part of the Grand Challenges in Global Health initiative. The Wellcome Trust also provides core awards to the Wellcome Trust Centre for Human Genetics and to the Wellcome Trust Sanger Institute.

References

National Institute of Malariology Parisitology and Epidemiology. Annual Report of Malaria and Parasite Prevention in 2010. Hanoi, Vietnam, 2010.

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