Clinical Severity of ß-Thalassemia Pediatric Patients in Myanmar.

ß-Thalassemia (ß-thal) is highly prevalent in Myanmar, but limited data are available on the molecular basis and the clinical manifestations in Myanmar patients. In this study, we investigated the clinical features and ß-globin gene abnormalities in 15 homozygous ß-thal and 60 Hb E (HBB: c.79G>A)/ß-thal pediatric patients who attended Yangon Children Hospital, the biggest thalassemia day care unit center in Myanmar. Eight different ß0-thal mutations were identified, with four accounting for 88.9% of alleles studied (excluding the Hb E variant). A genotype-phenotype correlation was found; all homozygous ß0-thalassemias had severe clinical courses, whereas the highly variable disease severity was demonstrated among Hb E/ß0-thal patients. Interactions of IVS-I-1 (G>T) (HBB: c0.92+1G>T) ß0-thal with Hb E are associated with milder clinical symptoms. The number of mildly affected Hb E/ß-thal patients was lower than expected, suggesting that there may be a considerable number of patients in the population who have either not been admitted to hospital or diagnosed with carrying the disease. Although the clinical severity in the Myanmar ß-thal patients seems to be similar to that in other populations, the levels of hemoglobin (Hb) appears to be very low. These findings indicate the need for the improvement of patient management and the development of prevention and control programs for ß-thal in Myanmar.

Ultrastructural Features of Human Liver Specimens from Patients Who Died of Dengue Hemorrhagic Fever.

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA-indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed.

Pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from Myanmar.

Vascular permeability, thrombocytopenia, liver pathology, complement activation, and altered hemostasis accompanying a febrile disease are the hallmarks of the dengue hemorrhagic fever/dengue shock syndrome, a major arthropod-borne viral disease that causes significant morbidity and mortality throughout tropical countries. We studied tissues from 13 children who died of acute dengue hemorrhagic fever/dengue shock syndrome at the Childrens' Hospital, Yangon, Myanmar. Dengue viral RNA from each of the 4 dengue viruses (DENVs) was detected by reverse transcriptase polymerase chain reaction in 11 cases, and dengue viral proteins (envelope, NS1, or NS3) were detected in 1 or more tissues from all 13 cases. Formalin-fixed and frozen tissues were studied for evidence of virus infection using monoclonal antibodies against DENV structural and nonstructural antigens (E, NS1, and nonsecreting NS3). In the liver, DENV infection occurred in hepatocytes and Kupffer cells but not in endothelial cells. Liver damage was associated with deposition on hepatocytes of complement components of both classical and alternative pathways. Evidence of dengue viral replication was observed in macrophage-like cells in spleens and lymph nodes. No dengue antigens were detected in endothelial cells in any organ. Germinal centers of the spleen and lymph nodes showed a marked reduction in the number of lymphocytes that were replaced by eosinophilic deposits, which contained dengue antigens as well as immunoglobulins, and complement components (C3, C1q, and C9). The latter findings had previously been reported but overlooked as a diagnostic feature.

Rare influenza A (H3N2) variants with reduced sensitivity to antiviral drugs.

In 2007 and 2008 in Myanmar, we detected influenza viruses A (H3N2) that exhibited reduced sensitivity to both zanamivir and amantadine. These rare and naturally occurring viruses harbored a novel Q136K mutation in neuraminidase and S31N mutation in M2.

Detection of group B rotavirus in an adult with acute gastroenteritis in Yangon, Myanmar.

In Yangon, Myanmar, a human group B rotavirus was first detected in 2007 in a stool specimen from a sporadic case of acute gastroenteritis in an adult. The strain was designated as MMR-B1. The full-length sequences of the MMR-B1 genes encoding VP7, VP4 (VP5* and VP8*), VP6, and NSP4 were determined for genetic characterization. These four MMR-B1 genes showed considerable higher sequence identities (97.2-98.4%) to those of group B rotaviruses detected in India (CAL-1 in 1998) and Bangladesh (Bang373 and Bang544 in 2000 and 2001, respectively) than to those of Chinese strains (90.7-93.6%) (ADRV and WH-1 in 1982 and 2002, respectively). Phylogenetically, the four genes of MMR-B1 were clustered into the Indian-Bangladeshi lineage. Although the deduced amino acid sequences of MMR-B1 were similar to those of strains CAL-1 and Bang373, several amino acids in VP8* were found to be different from those of the group B rotaviruses described previously. The first detection in Myanmar of a human group B rotavirus suggested endemic distribution or expansion of the group B rotavirus of the Indian-Bangladeshi lineage in Southeast Asia.

BAMBI gene is epigenetically silenced in subset of high-grade bladder cancer.

The bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) is a transmembrane TGFRI/BMPRI-related pseudoreceptor, antagonizes transforming growth factor (TGF)-beta/BMP signaling by inhibiting the formation of functional authentic receptor complexes (TGFRI/BMPRI and TGFRII/BMPRII). On the assumption that BAMBI gene expression is epigenetically altered during human bladder cancer progression, we screened the expression of BAMBI protein by immunohistochemistry and the methylation status of the BAMBI promoter. In the normal or reactive urothelium, BAMBI expression was mostly overlapped with that of BMPRI, and a similar colocalization pattern was noted in low-grade papillary cancers. In high-grade and invasive cancers, however, mainly two reciprocal immunohistochemical expression patterns were observed: BAMBI-low/BMPRI-high, and BAMBI-high/BMPRI-low, indicating that BAMBI expression is controlled such that it does not interfere with the responsiveness of high-grade cancer cells to TGF-beta/BMP signaling. Moreover, methylation of the BAMBI gene correlated significantly with negative BAMBI expression in bladder tumors. Although BAMBI overexpression significantly increased the number of apoptotic cells in T24 line, knock-down small interfering RNA showed no remarkable change. Cell motility assay revealed that on treatment with either TGF-beta1 or BMP2, T24 and HTB9 lines showed a marked increase in the number of migrated cells which, however, decreased significantly through the forced expression of BAMBI. Since certain subsets of aggressive tumors often promote cell motility, invasion and survival by inducing epithelial-to-mesenchymal transition through TGF-beta/BMP in an autocrine and paracrine manner, hypermethylation of the BAMBI gene promoter that leads to BAMBI gene suppression may be one of the epigenetic events affecting the invasiveness or aggressiveness of bladder cancers.

Human papillomavirus infection and its possible correlation with p63 expression in cervical cancer in Japan, Mongolia, and Myanmar.

Although human papillomavirus (HPV) 16 is the cause of cervical cancer in most countries including Japan, the involvement of cervical cancer with HPV types in Mongolian and Myanmar populations is largely unknown. We examined the expression of HPV in formalin-fixed and paraffin-embedded cervical tissues from 40 Japanese, 32 Mongolian, and 30 Myanmar cervical cancer patients. We performed immunohistochemistry using anti-HPV16 and anti-HPV 1, 6, 11, 16, 18 and 31 cocktail and then correlated it with the expression of Ki-67 and p63. HPV 16 was detected in 72%, 65% and 50% of Japanese, Mongolian and Myanmar cervical cancer patients, respectively, whereas 5 (13%) of the 40 patients, 8 (25%) of the 32 patients and 7 (23%) of the 30 patients in HPV 16-negative cancers were positive for other HPV types included in the cocktail, respectively. Ki-67 labeling index (LI) as well as p63 LI was significantly higher in HPV 16-positive patients than in HPV 16-negative ones in the Japanese and Mongolian samples. p63 expression was significantly associated with stage III and IV in Japan and Mongolia. These findings suggest that HPV 16 may be associated with cell proliferative activity and tumor progression, possibly depending upon the expression of p63 in the cervical cancer. In addition, immunohistochemical detection for distinguishing the type of HPV may also be useful for cervical cancer in the clinical setting.

Hepatitis C virus genotype distribution in Myanmar: Predominance of genotype 6 and existence of new genotype 6 subtype.

AIM: This study was performed to determine the prevalence and distribution of hepatitis C virus (HCV) genotypes in Myanmar. METHODS: A total of 1333 peripheral blood samples were collected from four different border cities of Myanmar. The anti-HCV antibody-positive serum samples were identified. HCV was genotyped by reverse transcriptase polymerase chain reaction, direct DNA sequencing and phylogenetic analysis on the partial core genome. RESULTS: The overall prevalence of HCV infection was 11.6% (154/1333). Regionally, it was 13.5% (47/349) in the north-eastern city, 12.8% (64/501) in the north-western city, 4.2% (16/380) in the southern city and 26.2% (27/103) in the western city. HCV was genotyped in 145/154 (94.2%) samples. Genotype 6 was the most prevalent genotype in this study (71/145, 49%), followed by genotype 3 (57/145, 39.3%), genotype 1 (16/145, 11%), and genotype 2 (1/145, 0.7%). Genotype 6 was mostly found in the northern cities and genotype 3 in the southern and western cities of Myanmar. Multiple HCV genotypes/subtypes were successfully characterized as 1a, 1b, 2a, 3a, 3b, 6m, 6n, and a new 6 subtype. Among them, subtype 6n was the most predominant subtype (38.6%), followed by subtype 3b (29.7%), 3a (9.6%), 6m (9%), 1b (6.9%), 1a (4.1%), new 6 subtype (1.4%) and 2a (0.7%). Subtype 6n was more widely distributed in the northern cities whereas subtype 3b was more common in the western city. The newly discovered genotype 6 subtype was from the northern cities. CONCLUSIONS: The results indicate there are regional differences of HCV genotype distribution in Myanmar. There is a distinct geographic variation from other South-East Asian countries in terms of the existence of the new genotype 6 subtype.

Possible correlation between iron deposition and enhanced proliferating activity in hepatitis C virus-positive hepatocellular carcinoma in Myanmar (Burma).

BACKGROUND: The aim of this study was to survey the effect of deposited iron on the cell kinetics of hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) in Myanmar (Burmese) patients. METHODS: Formalin-fixed and paraffin-embedded liver tissues from 34 Myanmar patients with HCC were used. To detect iron deposition, Prussian blue staining was performed. Cell proliferation and apoptosis were assessed by Ki-67 staining and by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay, respectively. HCV RNA was detected by in situ hybridization, and HCV protein, Fas and Fas ligand (FasL) were localized by immunohistochemistry. To identify the subtype of lymphocytes, CD8 was used as a surface marker. RESULTS: Iron deposition was found in 43% of the HCC cases, and was heavier in moderately differentiated HCC than in well-differentiated HCC. The Ki-67 labeling index (LI) in cancer cells was higher in Prussian blue-positive-HCC than in -negative HCC (3.8 +/- 2.2 vs 1.5 +/- 1.7, mean +/- SD; P=0.0067), whereas there was no significant difference between these groups in TUNEL LI. HCV protein was localized in cancer cells, and was found in 89% of the patients. In addition, Fas was expressed in HCC cells, and FasL was localized in HCC cells as well as in infiltrating CD8+ T lymphocytes. The frequency of apoptosis of HCC cells was correlated significantly with the population density of infiltrating CD8+ T lymphocytes. CONCLUSIONS: Our results indicated that, in Myanmar patients with HCC, iron deposition might accelerate hepatocarcinogenesis, by promoting cancer cell proliferation, without affecting the Fas/FasL apoptotic system.

Hemoglobin E prevalence in malaria-endemic villages in Myanmar.

The population of Myanmar comprises 8 major indigenous races (Bamar, Kayin, Kachin, Shan, Rakhine, Mon, Chin, and Kayah). The Bamar reside in the 7 central divisions of the country, and the others reside in the 7 peripheral states that border neighboring countries, including China, Laos, and Thailand in the east and India and Bangladesh in the west. Both malaria and HbE are endemic in Myanmar, although the actual prevalence of the latter in the different indigenous races is not yet known. Hemoglobin electrophoresis was performed in 4 malaria-endemic villages, each having a different predominating indigenous race. The overall prevalence of HbE was 11.4% (52/456 villagers), ranging from 2-6% in the Kayin-predominant villages to 13.1-24.4% in the Bamar-predominant villages. Although the overall HbE prevalence in the villages studied was not significantly different from that of the general Myanmar population, this study strongly documented the influence of racial differences on the prevalence of HbE in Myanmar. To prevent and control severe thalassemia syndromes in Myanmar, extensive prevalence studies of the country?s indigenous races are suggested.

A wider molecular spectrum of beta-thalassaemia in Myanmar.

Two hundred and nine beta-thalassaemia (beta-Thal) alleles of 158 unrelated Myanmar patients (107 HbE-beta-Thal; 51 beta-Thal major) were analysed for beta-globin gene mutations. Amplification refractory mutation system (ARMS) characterized six beta-thal mutations known to Myanmar [betaIVSI-1(G-->T), codon 41/42(-TCTT), betaIVSI-5(G-->C), codon 17(A-->T), betaIVS II-654(C-->T), and -28 Cap (A-->G)] in 166/209 (79.4%) alleles. DNA sequencing of 24 alleles from 43 ARMS-negative samples (20.6%) identified an additional 12 new mutations, to produce a total of 18 different mutations. Nineteen alleles (9.1%) remained for further characterization. The molecular spectrum of Myanmar beta-Thal is wider and more heterogeneous than previously reported.

Evaluation of tumor markers for the detection of hepatocellular carcinoma in Yangon General Hospital, Myanmar.

Levels of alpha-fetoprotein (AFP), its glycoforms AFP-L3 and AFP-P4, and proteins induced by vitamin K absence or antagonist-II (PIVKA-II) were determined in sera obtained from patients in Yangon General Hospital (20 with hepatocellular carcinoma (HCC), 29 with chronic liver diseases, including 3 with chronic hepatitis and 26 with cirrhosis of the liver, and 9 with other hepatobiliary diseases). Forty-five percent of the patients with HCC had serum AFP levels above 10,000 ng/ml, indicating that nearly half of the HCC patients were at an advanced stage of the disease. Thus, the AFP sensitivity was as high as 70% with 100% specificity for a cutoff level of 200 ng/ml. The sensitivity of AFP-L3 was 75% and a specificity 90% for a cutoff level of 15%. AFP-P4 showed a higher sensitivity of 80% and a similar specificity of 86% for a cutoff level of 12%. Combined evaluation of AFP-L3 and/or AFP-P4 increased the sensitivity to 90% with the same specificity of 86%, indicating that AFP-L3 and AFP-P4 are useful as adjuncts for diagnosis of HCC in the present population. PIVKA-II had a high sensitivity of 90%, although the specificity was lower than 45%, probably due to the low cutoff level, as some cholestatic patients were included in the control group.

Hemoglobinopathies, Iron Overload and Chronic Viral Hepatitis in Patients with Hepatocellular Carcinoma in Myanmar.

To assess the relevance of altered iron metabolism, hemoglobin electrophoresis by isoelectric focusing was performed for 16 cases of hepatocellular carcinoma (HCC) from the Liver Unit, Yangon General Hospital. Serum iron, total iron binding capacity, serum ferritin and free iron were also determined. Hemoglobin A (HbA) was found in all of the cases. Four cases had one extra band, hemoglobin A(2) in three cases, and hemoglobin F in one case. No abnormal hemoglobin was detected. Anemias due to chronic disorders or associated with liver disease were observed in all of the cases. Iron overload was documented in 83% and free iron was detected in all cases. Viral markers like HBsAg, AntiHBc, and AntiHCV singly or in combination were found in all cases. HCC occurring at young age was seen in this study; the youngest patient was 23 years old and four cases (25%) were under 40 years, with a mean age of 49 years. The findings support the hypothesis that free iron and iron overload is a potential promoter of the development of HCC, especially if underlying chronic viral infection is present.