Wanchai Wanachiwanawin

Clinical and Epidemiological Analyses of Human Pythiosis in Thailand

BACKGROUND Pythiosis is an emerging and life-threatening infectious disease in humans and animals that is caused by the pathogenic oomycete Pythium insidiosum. Human pythiosis is found mostly in Thailand, although disease in animals has been increasingly reported worldwide. Clinical information on human pythiosis is limited, and health care professionals are unfamiliar with the disease, leading to underdiagnosis, delayed treatment, and poor prognosis. METHODS To retrospectively study the clinical and epidemiological features of human pythiosis, we analyzed clinical data from patients with pythiosis diagnosed during the period of January 1985 through June 2003 at 9 tertiary care hospitals throughout Thailand. RESULTS A total of 102 cases of human pythiosis were documented nationwide. A substantial proportion (40%) of cases occurred in the last 4 years of the 18-year study interval. Clinical presentations fell into 4 groups: cutaneous/subcutaneous cases (5% of cases), vascular cases (59%), ocular cases (33%), and disseminated cases (3%). Almost all patients with cutaneous/subcutaneous, vascular, and disseminated pythiosis (85%) had underlying thalassemia-hemoglobinopathy syndrome. Most ocular cases (84%) were associated with no underlying disease. A majority of the patients were male (71%), were aged 20-60 years (86%), and reported an agricultural occupation (75%). Regarding treatment outcomes, all patients with disseminated infection died; 78% of patients with vascular disease required limb amputation, and 40% of these patients died; and 79% of patients with ocular pythiosis required enucleation/evisceration. CONCLUSIONS Here, we report, to our knowledge, the largest case study of human pythiosis. The disease has high rates of morbidity and mortality. Early diagnosis and effective treatment are urgently needed to improve clinical outcomes. Because P. insidiosum is distributed worldwide and can infect healthy individuals, an awareness of human pythiosis should be promoted in Thailand and in other countries.

Malignant lymphoma in Thailand: changes in the frequency of malignant lymphoma determined from a histopathologic and immunophenotypic analysis of 425 cases at Siriraj Hospital

BACKGROUND Analysis of malignant lymphoma in a single institution at different periods of time can determine the changing status of the disease in the region. METHODS To compare with the large series of 1095 lymphoma cases reported between 1957-1971 at Siriraj Hospital, the largest hospital in Thailand, a similar study was performed through histopathologic evaluation of 425 lymphoma cases diagnosed consecutively at the same institution between August 1993 and October 1995. Phenotypic analysis was performed by paraffin section-immunoperoxidase studies. RESULTS A striking increase in lymphoma cases was noted from 73 cases/year in the first series to 189 cases/year in the second series (an increase of 158.9%). Lymphoma occurred in all age groups, with a peak incidence at the seventh decade of life. The male to female ratio decreased from 2:1 in 1957-1971 to 1.3:1 in the more recent series. The incidence of Hodgkins disease (HD) was found to have decreased from 28.9% to 8.5%. There were 36 cases (8.5%) of HD and 389 cases (91.5%) of non-Hodgkins lymphoma (NHL) reported in the second series. The subtypes of HD included 16 cases of mixed cellularity, 13 cases of nodular sclerosis, 6 cases of lymphocyte depletion, and 1 case of lymphocyte predominance. According to the Working Formulation, the 389 NHL cases included low grade (14.1%), intermediate grade (57.3%), high grade (11.3%), and miscellaneous groups (17.2%). They were classified as small lymphocytic (9.5%), follicular (11.1%), diffuse (50.9%), immunoblastic (4.1%), small noncleaved (4.4%), lymphoblastic (2.8%), anaplastic large cell (9.0%), mycosis fungoides (1.8%), hairy cell leukemia (0.3%), true histiocytic (0.5%), and extramedullary plasmacytoma (1.0%). The immunophenotypes of the 359 NHL cases available for paraffin section-immunoperoxidase studies were B-cell (71.0%), T-cell (24.5%), histiocyte (0.6%), and undetermined phenotypes (3.9%). CONCLUSIONS The incidence of malignant lymphoma is increasing in Thailand, with a high frequency of intermediate to high grade NHL of B-cell phenotype reported.

Fatal arteritis due to Pythium insidiosum infection in patients with thalassaemia

Six thalassaemic patients had a distinct clinical syndrome characterized by progressive ischemia of the lower extremities, with ascending arteritis and thrombosis of the main arteries of the lower limbs. With periodic acid Schiff and Gomoris methenamine silver staining a large number of hyphae were revealed in the arterial wall and the outer part of the thrombus. Pythium insidiosum was isolated from 3 patients. The clinical course of the disease was progressive gangrene of the extremities and the patients invariably died when the infectious process reached the bifurcation of the aorta. There is no effective antimicrobial agent for the syndrome and radical amputation was the only method to ensure survival of the patients. P. insidiosum infection should be considered in thalassaemic patients with leg ulcers or arterial occlusion of the lower limbs.

Prevalence and Clinical Significance of Hepatitis C Virus Infection in Thai Patients with Thalassemia

Hepatitis C virus (HCV) infection is a common cause of liver disease in thalassemia major patients in Western, especially Mediterranean, countries. Its significance in thalassemic patients from Southeast Asia has not been critically evaluated. In this report, we describe our study of the prevalence of HCV infection among Thai patients with thalassemia. The relationships of the infection to blood transfusion and the infection’s effects on liver function have also been determined. Of the 104 patients studied, 21 (20.2%) tested positively by enzyme immunoassay for anti-HCV antibody, whereas only 2 patients (2%) had the hepatitis B surface antigen. There was no significant relationship between the presence of anti-HCV antibodies and the number and frequency of blood transfusions. In fact, 2 patients (10%) who tested positive for anti-HCV antibodies had never received transfusions. Patients with anti-HCV antibodies had significantly abnormal liver functions, such as higher levels of serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) and lower levels of serum albumin, compared with patients without anti-HCV antibodies (P = .021, .017, and .004, respectively). However, there were also significant correlations between iron status as indicated by transferrin saturation or serum ferritin levels and SGOT, SGPT, and γ-glutamyltransferase (GGT) levels. Moreover, abnormal liver function as represented by elevated levels of SGOT, SGPT, GGT, and serum alkaline phosphatase was observed more frequently in patients with iron overload than in patients with a lower degree of iron burden. The presence of HCV did not alter the effects of iron overload on liver function. The findings suggest that both HCV and iron overload are the main causes of abnormal liver function in Thai patients with thalassemia. The treatment of both problems, if coexisting in patients with thalassemia, is required to prevent progression to chronic liver disease.

Lymphocyte subsets and specific T-cell immune response in thalassemia.

Infection is very common in thalassemia and is one of the major causes of death. To date, it is not quite clear why these patients are susceptible to infection. In this study, lymphocyte immunophenotyping for CD3(+) (T-cells), CD3(+)CD4(+) (T-helper/inducer cells), CD3(+)CD8(+) (T-suppressor/cytotoxic cells), CD3(-)CD19(+) (B-cells), and CD3(-)CD16/56(+) (natural killer cells) subsets and expression of the activation antigen CD69 on CD3(+)CD4(+) and CD3(+)CD8(+) T-cells were determined in the whole blood of thalassemia patients, using a three-color flow cytometric technique. Results showed that only splenectomized beta-thalassemia/hemoglobin (Hb) E patients displayed a marked increase in absolute number of all lymphocytes. In addition, splenectomized beta-thalassemia/Hb E showed a significantly lower percentage of CD3(+) cells, with a corresponding increase in CD19(+) cells. These differences, when compared with normal subjects and other thalassemia patients, may be attributed to splenectomy. alpha-thalassemia patients, on the other hand, showed no significant difference from the normal group. While lymphocyte subsets in splenectomized beta-thalassemia/Hb E patients showed an abnormal distribution, T-cell activation in these patients was not different from the activation seen in normal subjects. This implies that thalassemia patients, during the steady state of disease, appear to have normal T-lymphocyte function with only moderate abnormalities of T- and B-lymphocyte subsets.

Activation of monocytes for the immune clearance of red cells in β°-thalassaernia/HbE

Summary We have recently provided evidence that IgG antibodies play a role in the destruction of red cells in thalassaemia syndromes. In order further to delineate factors involved in the clearance of thalassaemic cells, monocytes of 30 Thai patients with β°‐thal/HbE (17 non‐splenectomized and 13 splenectomized) and 16 normal controls were examined for their ability to bind and phagocytose normal red cells coated with IgG anti‐Rh(D). In β°‐thal/HbE. the mean number of red cells attached to the monocytes was approximately 3‐fold greater than in normal controls and the number ingested 30% higher. Among the non‐splenectomized patients, the number of red cells attached to and ingested by the monocytes, correlated inversely with mean basal Hb levels, suggesting that activation of mononuclear phagocytes for the immune clearance of red cells is a factor in determining the severity of the anaemia. As Fc‐gamma‐RI is of primary importance in the recognition of IgG‐coated red cells by monocytes, leucocytes from 10 β°‐thallHbE patients (four non‐splenectomized and six splenectomized) and five normal controls were investigated for their expression of Fc‐gamma‐RI by flow cytometry. In β°‐thallHbE there was an approximately 3‐fold increase in the percentage of leucocytes expressing this receptor: the receptor was up‐regulated on monocytes and induced on granulocytes. The up‐regulation of Fc‐gamma‐RI in β°‐thallHbE is likely to be an important component in the activation of monocytes and in mediating their enhanced effector function towards antibody‐coated cells.

Genotypic, immunophenotypic and clinical features of Thai patients with paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematological disorder characterized by complement‐mediated haemolytic anaemia caused by deficiency of glycosylphosphatidylinositol (GPI) anchored proteins. Somatic mutation of an X‐linked gene, PIG‐A, is responsible for the defect in biosynthesis of GPI‐anchor. It appears that frequency of PNH differs geographically, and seems to be more frequent in some Asian countries, such as Thailand and China. We studied a group of 34 Thai patients with PNH to see whether the somatic mutations in PIG‐A, extent of deficiency of GPI‐anchored proteins (complete or partial) and complication with aplastic anaemia among Thai patients are different from those in other regions. We determined 37 PIG‐A mutations in 33 patients (10 base substitutions, 14 single‐base deletions, five multiple‐base deletions, three single‐base insertions, two multiple base insertions and three others) which were found to be similar to those found in European, American and Japanese patients. Most patients had cells with a complete deficiency of CD59 (type III cells), whereas 19% and 33% of the patients with reliable data for CD59 expression had partially deficient granulocytes and erythrocytes (type II cells), respectively. Most mutations resulted in a complete loss of function of PIG‐A in accordance with the prevalent PNH III phenotype. 19 patients (51%) had aplastic anaemia; their PIG‐A mutations were not different from those without pre‐existing aplastic anaemia. These characteristics of Thai patients are similar to patients from other regions. There was some negative correlation between mean basal Hb concentration and percentage of CD59‐negative granulocytes (r = −0.374; P = 0.0476). In addition, patients with severe anaemia (basal Hb <7 g/dl) had a significantly higher percentage of affected granulocytes than those with mild anaemia (88.5 ± 9.4 v 64.9 ± 25.9; P = 0.01). The data suggest that the severity of anaemia in PNH depends partly on the size of the PNH clone.

Increased serum levels of macrophage colony-stimulating factor (M-CSF) in α- and β-thalassaemia syndromes : correlation with anaemia and monocyte activation

Abstract:  Serum levels of M–CSF were determined by an ELISA method in 29 and 34 patients with HbH disease (α1/α2 or α1/HbCS) or β0‐thal/HbE, respectively, in 28 haematologically normal subjects and in five patients with anaemia due to iron deficiency or myelodysplasia. In HbH disease and β0‐thal/HbE, M–CSF concentrations were significantly higher than those in the normal subjects [986 ± 138 and 1385 ± 133, respectively, vs. 500 ± 33 pg/ml (mean ± SEM); p <0.01, and p <0.001, respectively]. By contrast, in patients with anaemia due to iron deficiency, M–CSF levels were within the normal range. In HbH disease and in β0‐thal/HbE, M–CSF levels correlated inversely with mean basal Hb values (r = –0.39, p = 0.05 and r = –0.60, p <0.001, respectively). In addition, in some of the HbH and β0‐thal/HbE patients, monocyte ADCC activities towards red cells were tested and found to be approximately twice as high as those in normal controls [38.3±5.7 and 30.7 ± 4.6 vs. 17.8 ± 1.8 % specific lysis (mean ± SEM), respectively; p <0.01 and p <0.02, respectively]. When thalassaemic patients and normal controls were considered together there was a significant correlation between M–CSF levels and monocyte ADCC activities (r = 0.51, p <0.02). The results suggest that in HbH disease and in β0‐thal/HbE, raised serum M–CSF contributes to the anaemia by enhancing the effector function of mononuclear phagocytes towards red cells.

A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia.

Abstract:  Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the clonal expansion of blood cells, which are deficient in glycosylphosphatidylinositol anchored proteins (GPI‐APs). As PNH frequently occurs during the clinical course of acquired aplastic anemia (AA), it is likely that a process inducing bone marrow failure in AA is responsible for the selection of GPI‐AP deficient blood cells or PNH clone. Objective: To explore the nature and mutation of a PNH clone in AA. Methods: We performed regular repeated flow cytometric analyses of CD59 expression on peripheral blood cells from a cohort of 32 patients with AA. Mutation of phosphatidylinositol glycan class A (PIG‐A) was also studied. Results: Fifty‐one episodes of occurrences of CD59 negative granulocytes out of a total cohort 167 flow cytometric analyses (31%) were observed in 22 patients (69%). CD59 negative erythrocytes were less apparent than the granulocytes. Repeated occurrences of PNH clones were observed in 16 patients. Most of the emerging PNH clones were transient in nature. They were more frequently detected during episodes of lower white blood cell and platelet counts. Persistence and expansion of the GPI‐AP deficient blood cell populations to the level of clinical PNH were seen in only four patients (12.5%). Analysis of PIG‐A gene demonstrated eight mutations among the four patients, with two and four independent mutations in two patients. Conclusions: Our study indicates that PIG‐A mutations of hematopoietic stem cells with resultant PNH clones, are relatively common among AA patients. It also supports the hypothesis of selection of the PNH clone by a process or condition associated with or responsible for bone marrow failure in AA. However, there must be an additional factor favoring expansion or growth of the clone to the level of clinical or florid PNH.

Erythroblast- and erythrocyte-bound antibodies in α and β thalassaemia syndromes*

Summary. Thirty‐five Thai patients with various α‐thalassaemia (α‐thal 1/α‐thal 2, α‐thal 1/HbCS, HbCS/ HbCS) and yβ‐thalassaemia (β‐thal/HbE, severe and mild form, HbE/HbE) syndromes were examined for the presence of immunoglobulins and C3d on o‐tolidine positive erythroblasts in the bone marrow, and for the amounts of IgG of some specificities bound to circulating erythrocytes. In mild, but not in severe yβ‐thal/HbE and in α‐thalassaemia, the percentages of Ig‐positive erythroblasts were significantly higher than in controls and correlated well with the percentages of IgG‐positive erythroblasts. By contrast, the percentages of IgM and C3d positive erythroblasts were low and similar in thalassaemic and control marrows. A substantial proportion of thalassaemic patients showed more erythrocyte‐bound IgG than controls, but statistically significant elevations were seen only in severe β‐thal/HbE. Within a particular syndrome erythrocyte‐bound IgG was more abundant in splenectomized than non‐splenectomized subjects. It showed specificity for spectrin in some β‐thalassaemic patients and for band 3 protein in several individuals with α‐ or β‐thalassaemia. The results suggest that IgG antibodies play a role in the haemolysis of thalassaemia and that they are likely to be involved in the ineffective erythropoiesis in at least some of the syndromes studied.