Nation-wide investigation of RHD variants in Thai blood donors: Impact for molecular diagnostics.

BACKGROUND: Knowledge of the molecular determinants driving antigen expression is critical to design, optimize, and implement a genotyping approach on a population-specific basis. Although RHD gene variability has been extensively reported in Caucasians, Africans, and East-Asians, it remains to be explored in Southeast Asia. Thus the molecular basis of non-D+ blood donors was investigated in Thailand. STUDY DESIGN AND METHODS: First, 1176 blood samples exhibiting an inconclusive or negative result by automated serological testing were collected in the 12 Regional Blood Centres of the Thai Red Cross located throughout Thailand. Second, the RHD gene was analyzed in all samples by 1) quantitative multiplex PCR of short fluorescent fragments, and 2) direct sequencing, when necessary, for identifying structural variants and single nucleotide variants, respectively. RESULTS: Additional serological typing yielded 51 and 1125 samples with weak/partial D and D-negative (D-) phenotype, respectively. In the first subset, partial RHD*06.03 was the most common variant allele (allele frequency: 18.6%). In the second subset, the whole deletion of the gene is largely the most frequent (allele frequency: 84.9%), followed by the Asian DEL allele found in 15.6% of the samples. Eight novel alleles with various mutational mechanisms were identified. CONCLUSION: We report, for the first time at the national level, the molecular basis of weak/partial D and serologically D- phenotypes in Thai blood donors. The design and implementation of a dedicated diagnostic strategy in blood donors and patients are the very next steps for optimizing the management and supply of RBC units in Thailand.

Comprehensive Molecular Analysis of Serologically D-Negative and Weak/Partial D Phenotype in Thai Blood Donors.

BACKGROUND: Molecular genetics of the Rh system has been extensively studied in Caucasians, Black Africans, East Asians, and Indians more recently. In this work, we sought to investigate the molecular basis of variant D expression in the Thai population, which remains unknown. MATERIALS AND METHODS: Blood samples from 450 Thai donors showing the variant D phenotype were collected. The RHD gene was analyzed by quantitative multiplex polymerase chain reaction of short fluorescent fragments and/or Sanger sequencing. RESULTS: The most frequent alleles in 200 D-negative and 121 DEL samples were the whole RHD gene deletion and the Asian DEL alleles, respectively. In 129 weak/partial D samples, 36 variant alleles were identified, including eight novel alleles. RHD*06.03, which is common in variant D samples from South China, is the most prevalent variant allele, followed by the recently reported Indian RHD*01W.150 allele. DISCUSSION: For the first time, a comprehensive overview of the nature and distribution of variant RHD alleles in Thailand is reported. It is a milestone to pave the way towards improvement of the current screening strategy to identify DEL donors accurately. The next step will be the design and implementation of a simple molecular test for screening the most frequent alleles, specifically in this population.

The Potential Impact of Chikungunya Virus Outbreaks on Blood Transfusion.

Chikungunya virus (CHIKV) is responsible for large periodic epidemics in both endemic and nonendemic areas where competent mosquitoes are present. Transmission of CHIKV by transfusion during explosive outbreaks has never been documented, and the true impact of CHIKV infection on blood transfusion during an outbreak is unknown. Considerations include not only transfusions in the active outbreak areas but also returning travelers to nonendemic areas. Because there are no documented cases of transfusion-transmitted CHIKV, there are no standard guidelines regarding transfusion policies during a chikungunya fever outbreak. We review current information from studies during outbreaks with the goal of estimating the potential effect of different blood safety interventions (eg, querying donors for possible CHIKV exposure, chikungunya fever-related symptoms, screening for CHIKV RNA).

Risk of chikungunya virus transmission associated with European travelers returning from southern Thailand (2008-2015).

BACKGROUND: The impact of the spread of chikungunya virus (CHIKV) by autochthonous transmission and blood transfusion in nonendemic areas via travelers returning from CHIKV-affected locations is a concern. METHODS: We analyzed the risks of potential CHIKV importation and transfusion transmission from Thailand to Europe via travelers visiting southern Thailand from 2008 through 2015, using the web-based European Up-front Risk Assessment Tool. RESULTS: The risk of CHIKV importation by European travelers returning from Thailand from 2008 through 2015 varied depending on the year of travel, tourist destination, duration of stay, and time since last possible exposure. Specifically, the risks of acquiring CHIKV among travelers visiting Songkhla and Krabi for 1, 5, or 10-30 days during the highest epidemic activity in 2009 were estimated to be 74.40, 371.99, and 706.77 (Songkhla) and 1.82, 9.08, and 17.25 (Krabi) per 100,000 travelers, respectively. In contrast, such risks were estimated to be fewer than 0.099 per 100,000 travelers in nonepidemic years. The 2009 yearly average rates of expected incidence among 4,059,988 European travelers who stayed for 1 or 10-30 days in all six outbreak activity destinations were calculated to be, respectively, 4.01 × 10-6 or 1.20 × 10-4 cases per day, corresponding to the estimated rates of viremia and transfusion-transmitted CHIKV via traveling blood donations of 3.21 × 10-5 and 0.61, and 9.62 × 10-4 and 3.34, respectively. Additionally, it is probable that 18 (0.0004%) Europeans acquired CHIKV in Thailand, representing a maximum attack rate of 0.0023%. CONCLUSION: The extent of the expected risks and attack rates of CHIKV infection might reflect the travel preferences for popular destinations rather than the true risks of CHIKV transmission in travelers' home nonendemic countries. Nevertheless, preventive and blood-safety intervention measures may be applied to returning travelers at risk for infection to reduce CHIKV transfusion threats in their home countries.

Infection with Burkholderia pseudomallei - immune correlates of survival in acute melioidosis.

Melioidosis, caused by Burkholderia pseudomallei, is a potentially lethal infection with no licensed vaccine. There is little understanding of why some exposed individuals have no symptoms, while others rapidly progress to sepsis and death, or why diabetes confers increased susceptibility. We prospectively recruited a cohort of 183 acute melioidosis patients and 21 control subjects from Northeast Thailand and studied immune parameters in the context of survival status and the presence or absence of diabetes. HLA-B*46 (one of the commonest HLA class I alleles in SE Asia) and HLA-C*01 were associated with an increased risk of death (odds ratio 2.8 and 3.1 respectively). Transcriptomic analysis during acute infection in diabetics indicated the importance of interplay between immune pathways including those involved in antigen presentation, chemotaxis, innate and adaptive immunity and their regulation. Survival was associated with enhanced T cell immunity to nine of fifteen immunodominant antigens analysed including AhpC (BPSL2096), BopE (BPSS1525), PilO (BPSS1599), ATP binding protein (BPSS1385) and an uncharacterised protein (BPSL2520). T cell immunity to GroEL (BPSL2697) was specifically impaired in diabetic individuals. This characterization of immunity associated with survival during acute infection offers insights into correlates of protection and a foundation for design of an effective multivalent vaccine.

Risk of transfusion-transmitted chikungunya infection and efficacy of blood safety implementation measures: experience from the 2009 epidemic in Songkhla Province, Thailand.

BACKGROUND: To date, neither is there a standard guideline for maintaining a safe blood supply during a chikungunya fever (CHIKF) outbreak nor has a study been performed on actual transfusion-transmitted CHIKF to recipients. This study estimated the potential incidence of transfusion-transmitted CHIKF and compared the efficacies of various blood safety intervention strategies to mitigate the transfusion-transmitted CHIKF risk. STUDY DESIGN AND METHODS: A Web-based tool named the European Up-Front Risk Assessment Tool (EUFRAT) was used to estimate the risk of transfusion-transmitted CHIKF using data inputs from the 2009 Songkhla epidemic in Thailand. RESULTS: The mean and maximal risks of viremic donations during the entire epidemic period were estimated to be 0.9 (95% confidence interval [CI], 0.0-2.7) and 4.8 (95% CI, 0.5-9.1), respectively. This meant that the potential risk of transfusion-transmitted CHIKF to recipients receiving all infective end products in the absence of blood safety measures was from 10.9 (95% CI, 1.8-20.4) to 57.6 (95% CI, 36.4-79.5). Based on experience from the 2009 Thai epidemic, the proportion of 10% asymptomatic cases, for instance, with predonation screening for CHIKF-related symptoms and follow-up observation in donors at risk was estimated to be 88.4% (95% CI, 69.9%-100.0%) to 99.1% (95% CI, 79.6%-100.0%) effective in reducing this transfusion risk compared to 83.7% (95% CI, 65.8%-100.0%) to 90.7% (95% CI, 72.1%-100.0%) by predonation screening for donors at risk of chikungunya virus infection alone. CONCLUSION: This study suggests that prompt blood screening measures can reduce the risk of transfusion-transmitted CHIKF and maintain a safe blood supply during an outbreak.

The risk of blood transfusion-associated Chikungunya fever during the 2009 epidemic in Songkhla Province, Thailand.

BACKGROUND: Asymptomatic Chikungunya fever (CHIKF)-viremic blood donors could be a potential threat of spreading the disease unwittingly through contaminated blood transfusions. The relatively low prevalence of Chikungunya virus antibodies in the population and the records of more than 9000 suspected CHIKF cases raised concern about the potential transfusion-associated CHIKF during the 2009 epidemic. This study assessed the potential transfusion risk for CHIKF and the implementation of blood safety measures to mitigate this risk. STUDY DESIGN AND METHODS: A probabilistic model using key variables obtained from local information was used to estimate the weekly risk of transfusion-associated CHIKF during the 2009 epidemic. In addition, other blood safety measure-based strategies involving screening for donors at risk, donor tracing, and a 7-day quarantine of blood components at risk were implemented at the time of the epidemic. RESULTS: The risk of viremic donations per 100,000 ranged from 38.2 (95% confidence interval [CI], 36.5-39.8) to 52.3 (95% CI, 50.4-54.2). The potential risk of transfusion-associated CHIKF per 100,000 was estimated to be 1 in 2429 (0.04%; 95% CI, 1 in 6681 [0.02%]-1 in 1572 [0.06%]) to 1 in 1781 (0.06%; 95% CI, 1 in 3817 [0.03%]-1 in 1214 (0.08%]) donations. Among 26,722 donations, 11 (95% CI, 4-17) to 15 (95% CI, 7-22) donations were predicted to associate with transfusion risk. The implementation of blood safety measure-based strategies for this epidemic period suggested to deter 11 blood donations of transfusion risk. CONCLUSION: The interventions for blood safety measures applied in this study had mitigated the potential transfusion-associated CHIKF during the 2009 epidemic.

Frequencies and specificities of red cell alloantibodies in the Southern Thai population.

CONTEXT: Detailed reports of red cell alloantibody frequencies and specificities in the Thai population are limited. The aims of this study were to determine the specificity and compare the frequency of alloantibodies detected using column agglutination technology (CAT) and conventional tube techniques in blood donors and previously transfused patients. SETTINGS AND DESIGN: WE RETROSPECTIVELY REVIEWED ANTIBODY SCREENING AND IDENTIFICATION RECORDS FOR TWO TIME PERIODS: January-December 2006 during which conventional tube techniques were used and January 2008-December 2009 when CAT was used. RESULTS: The overall prevalence of alloantibodies in both patients and donors when using conventional tube techniques was 0.7%, for patients only was 0.9% and donors 0.6%. The most frequent antibodies detected in both groups were anti-Le(a), anti-Mi(a), anti-Le(b), anti-P1 and anti-E. When using CAT, alloantibodies were found in 0.8% of patients and 0.13% of donors with the five most common alloantibodies found in patients were anti-Mi(a), anti-E, anti-Le(a), anti-c and anti-Le(b) respectively. Similarly the common alloantibody specificities in donors were anti-Le(a), anti-Mi(a), anti-Le(b), anti-M and anti-D. CONCLUSIONS: One of the most commonly identified alloantibodies in the Thai population studied was anti-Mi(a) suggesting that Mia positive red cells should routinely be included in antibody screening and identification in this population. For antibody screening and identification, CAT method detected immune and warm alloantibody (ies) more frequently than that associated with conventional tube techniques.

The distribution of hepatitis B virus genotypes in Thailand.

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.

Platelet antibody screening by flow cytometry is more sensitive than solid phase red cell adherence assay and lymphocytotoxicity technique: a comparative study in Thai patients.

The objective of this study was to compare the sensitivity and specificity of lymphocytotoxicity test (LCT), solid phase red cell adherence assay (SPRCA) and flow cytometry in detecting platelet reactive antibodies against human leukocyte antigens (HLA) class I and human platelet antigens (HPA). Sera from 38 thrombocytopenic patients and 5 mothers of thrombocytopenic newborns were screened for platelet reactive antibodies by these three methods using screening platelets and/or lymphocytes panels derived from six subjects. The sensitivity and specificity of each method and levels of agreement were analysed. HLA antibodies were found in 18, 17 and 19 out of 43 patients' sera tested by LCT, SPRCA and flow cytometry, respectively. Four out of 43 patients' sera were reactive against HPA by flow cytometry, but were reactive to only 2 sera by SPRCA. Using flow cytometry as the reference method, the sensitivities/specificities of SPRCA and LCT in HLA antibody detection were 84.21/95.83% and 94.73/100%, respectively, with a good strength of agreement. SPRCA had 50% sensitivity and 100% specificity in HPA antibody detection compare to flow cytometry. Flow cytometry appeared to be the most sensitive technique compared with SPRCA and LCT for both HPA and HLA antibody screening. SPRCA sensitivity was too low for HPA antibody detection, but this might be because of the small number of samples. There was one serum from the mother of a baby suffering neonatal alloimmune thrombocytopenia (NAIT), in whom SPRCA could not detect HPA antibodies, while flow cytometry came out positive. Therefore, SPRCA should not be used in NAIT investigation and flow cytometry should be employed instead.