GENETIC SUBTYPES OF BLASTOCYSTIS ISOLATED FROM THAI HOSPITALIZED PATIENTS IN NORTHEASTERN THAILAND.

Blastocystis sp is probably the most common intestinal protozoan of humans. This taxon is known to include more than 17 subtypes, some of which likely cause human disease. We investigated the distribution of Blastocystis subtypes in Thai patients admitted for a variety of conditions at a hospital in northeastern Thailand. Fresh fecal samples, positive for Blastocystis by microscopy, were individually cultured in Jones' medium (n = 20) and each sample was used for amplification and sequencing a fragment of 18S rDNA. BLAST search and phylogenetic analysis demonstrated that Blastocystis subtypes ST1 (20%), ST3 (60%), ST6 (10%) and ST7 (10%) were present. No clear link between gastro-intestinal symptoms and any particular subtype of Blastocystis was apparent. Thus, there is a need to extend the work to evaluate clinical signs and subtypes in a larger cohort of patients.

HUMAN TRICHOSTRONGYLIASIS: A HOSPITAL CASE SERIES.

Trichostrongylus is a common nematode found to infect livestock throughout the tropics and can cause accidental zoonosis in humans. In the Lao PDR and Thailand, cases of human trichostrongyliasis have been reported sporadically but clinical data are limited. We retrospectively reviewed 41 cases of trichostrongyliasis who presented to Srinagarind Hospital, Thailand from 2005 to 2012. The diagnosis of trichostrongyliasis was made by finding their eggs in the stool of patients. Of the 41 cases reviewed, 30 were Thais and 11 from the Lao PDR; their age range was 26-86 years. Fifty-eight point five percent of the cases were male, 56.1% had a primary school or a lower education level, 56.1% were farmers or laborers, 63.4% lived in a rural area and 95.1% had underlying disease. Twenty-one patients were co-infected with Opisthorchis viverrini (14/21; 66.7%) and Strongyloides stercoralis (10/21; 47.6%) while the remaining (n = 20) had a single infection with Trichostrongylus only. All the trichostrongyliasis only patients who had underlying disease not related to the gastrointestinal (GI) tract had normal bowel habits and normal grossly appearing stool. GI symptoms, such as abdominal pain, flatulence, bloating, nausea, vomiting, anorexia, diarrhea and constipation, were not found in these patients suggesting they had a light infection. This study is the first report of the clinical features of a trichostrongyliasis case series from tertiary care hospital in Thailand.

Association of Hb Thailand [α56(E5)Lys→Thr] and Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] with α(0)-thalassemia: molecular and hematological features and differential diagnosis.

We report the molecular and hematological characteristics of two rare hemoglobin (Hb) variants found in associations with a common α(0)-thalassemia (α(0)-thal) in Thai patients. The first case (P1) was a generally healthy 27-year-old man discovered during our ongoing thalassemia screening program. Hemoglobin and DNA analyses identified a previously undescribed condition of compound heterozygosity for Hb Thailand [α56(E5)Lys→Thr] and α(0)-thal (SEA deletion). The second case (P2) was a 4-year-old boy with hypochromic microcytic anemia. Hemoglobin and DNA analyses also identified a compound heterozygosity for Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] in association with α(0)-thal (SEA deletion). Although Hb H (ß(4)) inclusion bodies were observed in both cases, Hb analysis using both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) did not show Hb H. While the two Hb variants could be recognized on Hb HPLC analysis, their corresponding Hb A(2) derivatives: the Hb A(2)-Thailand and Hb A(2)-Phnom Penh, were clearly observed on CE. Apparently, combination of these two Hb variants with α(0)-thal are not expressed as Hb H disease. The two Hb variants could be confirmed by polymerase chain restriction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR assays.

Effective screening for double heterozygosity of Hb E/alpha0-thalassemia.

One hundred and forty-one blood samples of hemoglobin E (Hb E) carriers were collected to define suitable cutoff values of Hb E level, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) as screening indicators for detecting individuals with double heterozygosity for Hb E/alpha(o)-thalassemia. Based on the values that gave 100% sensitivity, Hb E < 26%, MCV < 74 fl, and MCH < 24 pg were selected. Further validation of these selected values in additional 152 heterozygous Hb E pregnant women revealed 100% sensitivity, 86.2% specificity, and a 25.9% positive predictive value (PPV) for using Hb E cutoff point only, meanwhile, 100% sensitivity, 83.4% specificity, and 22.6% PPV were achieved for the MCV cutoff point. In addition, 100% sensitivity, 86.3% specificity, and 25.9% PPV were gained for the MCH cutoff point. Combining Hb E level with either MCV or MCH cutoff points, the specificity and PPV were increased to 95.1% and 50.0%, respectively. It is concluded that Hb E level < 26%, MCV < 74 fl, and MCH < 24 pg could be used for screening alpha(o)-thalassemia in heterozygous Hb E. However, to improve specificity and PPV of the tests, a combination of Hb E level < 26% with either MCV < 74 fl or MCH < 24 pg is recommended.

H63D mutation of the hemochromatosis gene and serum ferritin levels in Thai thalassemia carriers.

We determined the prevalence of the H63D and the IVS5#1G-A HFE mutations in 370 (169 males and 201 females) Thai thalassemia carriers and 201 normal subjects. While no IVS5#1G-A mutation was found, the H63D heterozygosity was identified in 5.5% (11/201) of normal subjects and 7.3% (27/370) of thalassemia carriers. Within the thalassemic group, the medians (ranges) of serum ferritin were 217.5 ng/ml (20.1-424.3) and 169.8 ng/ml (3.9-3,536.0) in male subjects and 30.4 ng/ml (11.9-130.7) and 49.3 ng/ml (0.6-931.0) in female subjects with (HD) and without (HH) H63D mutation, respectively. The proportions of subjects with elevated ferritin were found to be 37.5% (6/16) for HD and 14.0% (18/129) for HH in male and 0% (0/11) for HD and 3.0% (5/164) for HH in female subjects, respectively. Statistical analysis of all the data revealed no significant difference. Among 14 Hb E/beta-thalassemia patients, no difference in hematological data as well as serum ferritin levels was observed between those with (HD) and without (HH) H63D mutation. Therefore, the H63D heterozygosity has no significant effect on the serum ferritin level and screening for this HFE mutation in thalassemic patients is not recommended.

A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting.

Primary screening for thalassemia and hemoglobinopathies usually involves an accurate blood count using an expensive electronic blood cell counter A cheaper alternative method was tested by using a modified osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) test. Altogether 423 pregnant Thai women participated in this project. Hemoglobin patterns and globin genotypes were determined using an automated high-performance liquid chromatography analyzer and polymerase chain reaction analysis of alpha- and beta-globin genes. Among the 423 subjects, 264 (62.4%) carried thalassemia genes. The combined OF and DCIP tests detected all pregnant carriers of the 3 clinically important thalassemias, ie, alpha0-thalassemia, beta-thalassemia, and hemoglobin E with a sensitivity of 100.0%, specificity of 87.1%, positive predictive value of 84.5%, and negative predictive value of 100.0%, which show more effectiveness than these values for the standard method based on RBC counts. A combination of modified OF and DCIP tests should prove useful and applicable to prenatal screening programs for thalassemia and hemoglobinopathies in communities with limited facilities and economic resources.

Thalassemia and hemoglobinopathies in Southeast Asian newborns: diagnostic assessment using capillary electrophoresis system.

BACKGROUND: We have investigated the Capillarys 2 Hemoglobin testing system to assist in presumptive diagnosis of thalassemia and hemoglobinopathies commonly found in Southeast Asia. METHODS: Study was conducted on 226 newborns. Hematological parameters were recorded and Hb profiles were examined on the Capillarys 2 Hemoglobin analyzer (SEBIA). DNA analyses were used to establish the final diagnoses. RESULTS: Among 226 newborns examined, 122 had thalassemias with 17 different genotypes. The capillary electrophoresis system could provide useful data for presumptive diagnoses of cases, especially those with Hb E and α-thalassemia. Hb E was found to be 2.6-6.2% in heterozygote whereas Hb Bart's were clearly observed in cases with compound heterozygous or homozygous α(+)-thalassemia and heterozygous α(0)-thalassemia. Hb H disease and other forms of α-thalassemia could be differentiated based on the presence of Hb Bart's and its percentage. CONCLUSION: The capillary electrophoresis system is applicable to newborn screening for common forms of thalassemia in Southeast Asia.

Molecular and hematological profiles of hemoglobin EE disease with different forms of alpha-thalassemia.

We describe hematologic and DNA characterization of hemoglobin (Hb) E homozygote with various forms of alpha-thalassemia in Thai individuals. Altogether, 131 unrelated adult subjects with Hb EE at routine Hb analysis were studied. Forty-two cases were found to carry alpha-thalassemia with ten different genotypes. These included 21 cases with alpha(+)-thalassemia heterozygote (-alpha(3.7)/alphaalpha), one case with alpha(+)-thalassemia heterozygote (-alpha(4.2)/alphaalpha), six cases with Hb Constant Spring heterozygote (alpha(CS)alpha/alphaalpha), four cases with homozygous alpha(+)-thalassemia (-alpha(3.7)/-alpha(3.7)), one case with homozygous alpha(+)-thalassemia (-alpha(4.2)/-alpha(4.2)), two cases with compound alpha(+)-thalassemia/Hb Constant Spring (-alpha(3.7)/alpha(CS)alpha), one case with compound alpha(+)-thalassemia/Hb Paksé (-alpha(3.7)/alpha(PS)alpha), four cases with alpha(0)-thalassemia heterozygote (--(SEA)/alphaalpha), and, unexpectedly, two cases with compound alpha(0)-thalassemia/alpha(+)-thalassemia [(--(SEA)/-alpha(3.7)) and (--(SEA)/-alpha(4.2))]. The hematological expression of these Hb E homozygotes with various forms of alpha-thalassemia was presented comparatively with those of the 89 cases of pure Hb E homozygotes. Overlapping levels of Hb E, Hb F, and other hematological parameters were observed which did not predict clinical severity, indicating a need for alpha-globin gene analysis for accurate diagnosis and improved genetic counseling.