Molecular Epidemiology of Hemoglobinopathies in Cambodia.

Determining the magnitude of the thalassemia problem in a country is important for implementing a national prevention and control program. In order to acquire accurate thalassemia prevalence data, the gene frequency of α- and ß-thalassemia (α- and ß-thal) in different regions of a country should be determined. The molecular basis of thalassemia in Cambodia was performed by polymerase chain reaction (PCR)-based techniques in a community-based cross-sectional survey of 1631 unrelated individuals from three regions, Battambang, Preah Vihear and Phnom Penh. Thalassemia mutations were detected in 62.7% of the three studied population of Cambodia. Hb E (HBB: c.79G > A) was the most common ß-globin gene mutation with a frequency ranging from 0.139 to 0.331, while the most frequent α-globin gene mutation was the -α(3.7) (rightward) deletion (0.098-0.255). The other frequencies were 0.001-0.003 for ß-thal, 0.008-0.011 for α-thal-1 (- -(SEA)), 0.003-0.008 for α-thal-2 [-α(4.2) (leftward deletion)], 0.021-0.044 for Hb Constant Spring (Hb CS, HBA2: c.427T > C) and 0.009-0.036 for Hb Paksé (HBA2: c.429A > T). A regional specific thalassemia gene frequency was observed. Preah Vihear had the highest prevalence of Hb E (55.9%), α-thal-2 (24.0%) and nondeletional α-thal (15.1%), whereas Phnom Penh had the lowest frequency of thalassemia genes. Interestingly, in Preah Vihear, the frequency of Hb Paksé was extremely high (0.036), almost equivalent to that of Hb CS (0.044). Our results indicate the importance of micromapping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in Cambodia.

The Homozygous Hemoglobin EE Genotype and Chronic Inflammation Are Associated with High Serum Ferritin and Soluble Transferrin Receptor Concentrations among Women in Rural Cambodia.

BACKGROUND: Ferritin and soluble transferrin receptor (sTfR) concentrations are commonly used to assess iron deficiency (ID); however, they are influenced by multiple factors. OBJECTIVES: We assessed associations between numerous variables and both ferritin and sTfR concentrations in Cambodian women and compared ID prevalence through the use of study-generated correction factors (CFs) for ferritin with those from a published meta-analysis. METHODS: Venous blood from 450 women (aged 18-45 y) was assessed for hemoglobin (Hb), ferritin, sTfR, retinol binding protein, folate, vitamin B-12, C-reactive protein, α-1 acid glycoprotein (AGP), and genetic Hb disorders. Linear regression was used to calculate geometric mean ratios (95% CIs) for ferritin and sTfR concentrations. RESULTS: The variant Hb EE genotype was associated with 50% (14%, 96%) and 51% (37%, 66%) higher geometric mean ferritin and sTfR concentrations, respectively, than was the normal Hb AA genotype; a 1-g/L increase in AGP was associated with 99% (50%, 162%) and 48% (33%, 64%) higher concentrations in the same variables, respectively. ID prevalence in nonpregnant women (n = 420) was 2% (n = 9) with the use of ferritin <15 µg/L and 18% (n = 79) with the use of sTfR >8.3 mg/L as criteria. ID prevalence with the use of sTfR was higher in women with the Hb EE genotype (n = 17; 55%) than in those with the Hb AA genotype (n = 20; 10%); and in women with the Hb AA genotype and chronic inflammation (n = 10; 18%) than in that group of women without chronic inflammation (n = 10; 7%) (P < 0.05). No differences in ID prevalence were found with the use of ferritin between women with Hb EE and AA genotypes (P = 1.0) or by chronic inflammation status (P = 0.32). There were no differences in mean ferritin concentrations among all 450 women when study-generated CFs were compared with those from the meta-analysis (P = 0.87). CONCLUSIONS: Compared with sTfR, ferritin concentrations appear to reflect more accurately true ID in rural Cambodian women. The CFs from a published meta-analysis were appropriate for use in this population with a high prevalence of Hb disorders and inflammation.

Genetic hemoglobin disorders, infection, and deficiencies of iron and vitamin A determine anemia in young Cambodian children.

In Cambodia, many factors may complicate the detection of iron deficiency. In a cross-sectional survey, we assessed the role of genetic hemoglobin (Hb) disorders, iron deficiency, vitamin A deficiency, infections, and other factors on Hb in young Cambodian children. Data on sociodemographic status, morbidity, and growth were collected from children (n = 3124) aged 6 to 59 mo selected from 3 rural provinces and Phnom Penh municipality. Blood samples were collected (n = 2695) for complete blood count, Hb type (by DNA analysis), ferritin, soluble transferrin receptor (sTfR), retinol-binding protein (RBP), C-reactive protein, and α(1)-acid glycoprotein (AGP). Genetic Hb disorders, anemia, and vitamin A deficiency were more common in rural than in urban provinces (P < 0.001): 60.0 vs. 40.0%, 58.2 vs. 32.7%, and 7.4 vs. 3.1%, respectively. Major determinants of Hb were age group, Hb type, ferritin, sTfR, RBP, AGP >1.0 g/L (P < 0.001), and rural setting (P < 0.05). Age group, Hb type, RBP, elevated AGP, and rural setting also influenced ferritin and sTfR (P < 0.02). Multiple factors affected anemia status, including the following: age groups 6-11.99 mo (OR: 6.1; 95% CI: 4.3, 8.7) and 12-23.99 mo (OR: 2.7; 95% CI: 2.1, 3.6); Hb type, notably Hb EE (OR: 18.5; 95% CI: 8.5, 40.4); low ferritin (OR: 3.2; 95% CI: 2.2, 4.7); elevated AGP (OR: 1.4; 95% CI: 1.2,1.7); rural setting (OR: 2.3; 95% CI: 1.7, 3.1); low RBP (OR: 3.6; 95% CI: 2.2, 5.9); and elevated sTfR (OR: 2.1; 95% CI: 1.7, 2.7). In Cambodia, where a high prevalence of genetic Hb disorders exists, ferritin and sTfR are of limited use for assessing the prevalence of iron deficiency. New low-cost methods for detecting genetic Hb disorders are urgently required.

The effect of oral iron with or without multiple micronutrients on hemoglobin concentration and hemoglobin response among nonpregnant Cambodian women of reproductive age: a 2 x 2 factorial, double-blind, randomized controlled supplementation trial.

Background: Despite a high prevalence of anemia among nonpregnant Cambodian women, current reports suggest that iron deficiency (ID) prevalence is low. If true, iron supplementation will not be an effective anemia reduction strategy.Objective: We measured the effect of daily oral iron with or without multiple micronutrients (MMNs) on hemoglobin concentration in nonpregnant Cambodian women screened as anemic.Design: In this 2 × 2 factorial, double-blind, randomized trial, nonpregnant women (aged 18-45 y) with hemoglobin concentrations ≤117 g/L (capillary blood) were recruited from 26 villages in Kampong Chhnang province and randomly assigned to receive 12 wk of iron (60 mg; Fe group), MMNs (14 other micronutrients; MMN group), iron plus MMNs (Fe+MMN group), or placebo capsules. A 2 × 2 factorial intention-to-treat analysis with the use of a generalized mixed-effects model was used to assess the effects of iron and MMNs and the interaction between these factors. Results: In July 2015, 809 women were recruited and 760 (94%) completed the trial. Baseline anemia prevalence was 58% (venous blood). Mean (95% CI) hemoglobin concentrations at 12 wk in the Fe, MMN, Fe+MMN, and placebo groups were 121 (120, 121), 116 (116, 117), 123 (122, 123), and 116 (116, 117) g/L, with no iron × MMN interaction (P = 0.66). Mean (95% CI) increases in hemoglobin were 5.6 g/L (3.8, 7.4 g/L) (P < 0.001) among women who received iron (n = 407) and 1.2 g/L (-0.6, 3.0 g/L) (P = 0.18) among women who received MMNs (n = 407). The predicted proportions (95% CIs) of women with a hemoglobin response (≥10 g/L at 12 wk) were 19% (14%, 24%), 9% (5%, 12%), 30% (24%, 35%), and 5% (2%, 9%) in the Fe, MMN, Fe+MMN, and placebo groups, respectively.Conclusions: Daily iron supplementation for 12 wk increased hemoglobin in nonpregnant Cambodian women; however, MMNs did not confer additional significant benefit. Overall, ∼24% of women who received iron responded after 12 wk; even fewer would be likely to respond in the wider population. This trial was registered at clinicaltrials.gov as NCT02481375.

Interaction of hemoglobin E and several forms of alpha-thalassemia in Cambodian families.

BACKGROUND AND OBJECTIVES: This study aimed to describe hematologic and molecular characterization of the interaction of hemoglobin (Hb) E and several forms of alpha-thalassemia causing complex thalassemia syndromes in two Cambodian families as well as to establish a rapid polymerase chain reaction (PCR) assay for simultaneous detection of Hb Constant Spring (CS) and Hb Pakse' (PS). DESIGN AND METHODS: Using PCR and DNA sequencing, the alpha- and beta-globin genotypes were examined. Clinical and hematologic data were assessed. A multiplex asymmetric allele-specific PCR for differential diagnosis of HbCS and HbPS was developed and validated. RESULTS: Eight genotypes including heterozygous HbCS, heterozygous HbPS, double heterozygous HbE/HbPS, double heterozygous HbE/alpha-thalassemia 2, triple heterozygous HbE/alpha-thalassemia /HbPS, homozygous HbE/alpha-thalassemia 2, compound alpha-thalassemia 2/HbCS and a hitherto undescribed compound HbCS/HbPS were found in these two families. Genotype-phenotype relationships are discussed and successful application of a multiplex PCR system for differential diagnosis of HbCS and HbPS is described. INTERPRETATION AND CONCLUSIONS: The interaction of several globin gene abnormalities in Cambodian families emphasizes the high frequencies of thalassemia and hemoglobinopathies. Identification of HbPS suggests that this mutation might be common and underestimated among South-east Asian populations. A simplified PCR assay for simultaneous detection of HbCS and HbPS would facilitate characterization of these genotypes in both the clinical setting and population screening programs in the region.

Effect of micronutrient sprinkles on reducing anemia: a cluster-randomized effectiveness trial.

OBJECTIVE: To evaluate the effectiveness of Sprinkles alongside infant and young child feeding (IYCF) education compared with IYCF education alone on anemia, deficiencies in iron, vitamin A, and zinc, and growth in Cambodian infants. DESIGN: Cluster-randomized effectiveness study. SETTING: Cambodian rural health district. PARTICIPANTS: Among 3112 infants aged 6 months, a random subsample (n = 1350) was surveyed at baseline and 6-month intervals to age 24 months. INTERVENTION: Daily micronutrient Sprinkles alongside IYCF education vs IYCF education alone for 6 months from ages 6 to 11 months. MAIN OUTCOME MEASURES: Prevalence of anemia; iron, vitamin A, and zinc deficiencies; and growth via biomarkers and anthropometry. RESULTS: Anemia prevalence (hemoglobin level <11.0 g/dL [to convert to grams per liter, multiply by 10.0]) was reduced in the intervention arm compared with the control arm by 20.6% at 12 months (95% CI, 9.4-30.2; P = .001), and the prevalence of moderate anemia (hemoglobin level <10.0 g/dL) was reduced by 27.1% (95% CI, 21.0-31.8; P < .001). At 12 and 18 months, iron deficiency prevalence was reduced by 23.5% (95% CI, 15.6-29.1; P < .001) and 11.6% (95% CI, 2.6-17.9; P = .02), respectively. The mean serum zinc concentration was increased at 12 months (2.88 µg/dL [to convert to micromoles per liter, multiply by 0.153]; 95% CI, 0.26-5.42; P = .03). There was no statistically significant difference in the prevalence of zinc and vitamin A deficiencies or in growth at any time. CONCLUSIONS: Sprinkles reduced anemia and iron deficiency and increased the mean serum zinc concentration in Cambodian infants. Anemia and zinc effects did not persist beyond the intervention period. TRIAL REGISTRATION anzctr.org.au Identifier: ACTRN12608000069358.

Impact of Infant and Young Child Feeding (IYCF) Practices on Growth in Young Children in Rural Cambodia.

Objectives: To evaluate the impact of IYCF education and practices on growth compared to an ecological control. Methods: As part of a randomized controlled effectiveness trial of micronutrient Sprinkles and IYCF education, all mothers received IYCF education. Group and individual sessions were delivered by health staff and trained village health workers. Anthropometry and IYCF were recorded at baseline (age 6 mo), endline (age 12 mo), and at two follow up points (age 18 mo and 24 mo) and compared with same aged children in the same province from the 2005 Cambodia Demographic and Health Survey (CDHS). Results: There was no treatment effect on growth so intervention and control groups were combined. At 6, 12 and 18 mo, 59%, 86% and 73% of children met minimum adequate WHO IYCF practices respectively in the study group compared to 33%, 62% and 47% for CDHS children. Stunting (HAZ <-2) prevalence at 6, 12, 18 and 24 mo was 11%, 19%, 32% and 38% in the study group compared to 11%, 37%, 62%, and 44% for CDHS children. Underweight (WAZ <-2) prevalence at 6, 12, 18 and 24 mo was 14%, 16%, 21% and 26% in the study group compared to 18%, 28%, 37% and 21% for CDHS children. Differences for stunting and underweight were significant (p<0.05) at 12 and 18 mo. Conclusions: IYCF education to study mothers improved IYCF practices and slowed stunting and underweight significantly at 12 and 18 months of age compared to CDHS children. Strengthened IYCF education and practices improved growth in children in this low-income, rural population.

Impact of Genetic Hemoglobin (Hb) Disorders on the Effectiveness of Micronutrient Sprinkles in Reducing Anemia in Young Children in Rural Cambodia.

Objectives: To evaluate the impact of genetic Hb disorders on the effectiveness of 6 months of daily micronutrient Sprinkles for infants age 6-12 months on anemia. Methods: As part of a randomized controlled effectiveness trial, subjects were screened for genetic Hb disorders at aged 18 months on whole blood using the SEBIA MINICAP analyser, HEMOGLOBIN (E) program, and IC α THAL test to detect major Hb variants and α-thalassemia. Anemia (Hb < 110 g/L) was measured at baseline (age 6 mo), endline (age 12 mo), and at two follow up points (age 18 mo and 24 mo) for children with and without genetic Hb disorders. Results: Half of all children had a genetic haemoglobin disorder (at least 15 types). At baseline, overall prevalence of anemia for children with normal Hb was 81% versus 88% for those with any genetic Hb disorder. At endline, anemia prevalence for the intervention group with normal Hb vs genetic Hb disorder was 60.2% versus 72.0% (P = 0.02) and for the control group 81.1% versus 88.8% (P = 0.03), respectively. At further follow-up, anemia decreased in both the control and intervention groups. However those with any genetic Hb disorder remained more anemic with varying levels of anemia according to the disorder type. Conclusions: Sprinkles had a similar proportional effect regardless of overall genetic Hb status indicating that children with genetic Hb disorders can effectively utilize iron and micronutrients to reduce anemia. Sprinkles can be effective in populations with a high prevalence of genetic Hb disorders.

The prevalence and molecular basis of hemoglobinopathies in Cambodia.

Blood counts, hemoglobin (Hb) high performance liquid chromatography (HPLC), and DNA analyses were performed on 260 children, aged 5 months to 16 years, at Siem Reap to assess the prevalence of thalassemia and other hemoglobinopathies in regional Cambodia. Hemoglobinopathies were present in 134 children (51.5%) with 20 abnormal genotypes identified. alpha-Thalassemia (thal) (35.4%) was the most prevalent disorder and the -alpha3.7 gene deletion was the most common alpha-globin gene abnormality. The - -SEA deletion and nondeletional forms of alpha-thal, Hb Constant Spring [Hb CS, alpha142, Term-->Gln, TAA-->CAA (alpha2)], Hb Paksé [alpha142, Term-->Tyr, TAA-->TAT (alpha2)] and triplicated alpha genes, were also present but at low frequencies. Hb E [beta26(B8)Glu-->Lys, GAG-->AAG] (28.8%) was the most common beta-globin gene abnormality, whilst beta-thal was only detected in two children (0.8% of cases). Although hemoglobinopathies were common, the majority of abnormalities detected (heterozygous -alpha3.7 and Hb E) were not clinically significant. On the basis of these findings, and with the majority of abnormalities being mild, it seems improbable that thalassemia represents a major health burden in this region of Cambodia.