Secondary polycythaemia in a Malay girl with homozygous Hb Tak.

Hb Tak is one of more than 200 high affinity haemoglobin variants reported worldwide. It results from the insertion of two nucleotides (AC) at the termination codon, between codon 146 and codon 147 of the beta-globin gene [Beta 147 (+AC)]. Polycythaemia is the main clinical feature although affected carriers are usually asymptomatic and do not require intervention. Several case studies in this region have reported the co-inheritance of Hb Tak with Hb E, delta beta and beta thalassaemia with one case of homozygous Hb Tak in a Thai boy. In this case report, a cluster of haemoglobin Tak was found in a family of Malay ethnic origin. Cascade family screening was conducted while investigating a 4-year old girl who presented with symptomatic polycythaemia. She had 2 previous Hb analysis done, at 7-month and 2-year-old with the diagnosis of possible Hb Q Thailand and Homozygous Hb D, respectively. Both diagnosis did not fit her clinical presentations. She was plethoric, had reduced exercise tolerance as well as cardiomyopathy. Her parents were consanguineously married and later diagnosed as asymptomatic carriers of Hb Tak. Consequently, re-analysis of the girl's blood sample revealed a homozygous state of Hb Tak. In conclusion, high oxygen affinity haemoglobin like Hb Tak should be considered in the investigation of polycythaemic patients with abnormal Hb analyses. In this case, DNA analysis was crucial in determining the correct diagnosis.

Germline BRCA1 mutations increase prostate cancer risk.

BACKGROUND: Prostate cancer (PrCa) is one of the most common cancers affecting men but its aetiology is poorly understood. Family history of PrCa, particularly at a young age, is a strong risk factor. There have been previous reports of increased PrCa risk in male BRCA1 mutation carriers in female breast cancer families, but there is a controversy as to whether this risk is substantiated. We sought to evaluate the role of germline BRCA1 mutations in PrCa predisposition by performing a candidate gene study in a large UK population sample set. METHODS: We screened 913 cases aged 36­86 years for germline BRCA1 mutation, with the study enriched for cases with an early age of onset. We analysed the entire coding region of the BRCA1 gene using Sanger sequencing. Multiplex ligation-dependent probe amplification was also used to assess the frequency of large rearrangements in 460 cases. RESULTS: We identified 4 deleterious mutations and 45 unclassified variants (UV). The frequency of deleterious BRCA1 mutation in this study is 0.45%; three of the mutation carriers were affected at age 65 years and one developed PrCa at 69 years. Using previously estimated population carrier frequencies, deleterious BRCA1 mutations confer a relative risk of PrCa of ~3.75-fold, (95% confidence interval 1.02­9.6) translating to a 8.6% cumulative risk by age 65. CONCLUSION: This study shows evidence for an increased risk of PrCa in men who harbour germline mutations in BRCA1. This could have a significant impact on possible screening strategies and targeted treatments.

Enteropathogens associated with acute diarrhea in a tertiary hospital of Bangladesh.

Acute diarrheal diseases are great concern throughout the world, as they are responsible for considerable morbidity and mortality, especially in developing countries. The present study was carried out during the period from January' 2011 to December' 2011 in the Department of Microbiology, Mymensingh Medical College. A total of 300 stool specimens were examined by standard laboratory methods for identification of enteropathogens. Rotavirus was detected by Polyacrylamide Gel electrophoresis (PAGE). Different diarrheagenic E. coli (DEC) were detected by Multiplex PCR following standard methods. Of the 300 stool specimens examined, Enteropathpgens were detected in 160(53.5%) cases. Rota virus was detected in 82(27.5%) cases, followed by DEC in 54(18%), Shigella spp. in 8(2.4%), Salmonella spp. in 5(1.6%), Entameoba histolytica in 4(1.5%) and Giardia lamblia in 3(1.0%) cases. Among the DEC, the Enterotoxigenic E. coli (ETEC) was most prevalent (72%, 39/54). The present study revealed a high prevalence of rotavirus and DEC as the predominant causes of diarrhea in this region.

Non-culture diagnosis of Chlamydia trachomatis genital infection in sexually active women.

Infections caused by Chlamydia trachomatis (CT) are one of the most prevalent of all sexually transmitted diseases (STD). This cross sectional study was carried out to diagnose genital CT infection on 108 (59 pregnant and 49 non-pregnant) women attending at Department of Gynaecology and Obstetrics, Mymensingh Medical College Hospital (MMCH) during the period from January 2009 to December 2009. This non- culture technique was based on detection of CT major outer membrane protein (MOMP) by Direct Fluorescence Antibody Test (DFAT) from endocervical swab. Chlamydial inclusion bodies (IB) were looked for using Iodine stain. CT antigens were detected in 45.3% (49/108) cases by DFAT; IBs were detected in 5.5% cases (06/108) by Iodine staining technique. Majority of CT positive cases (65%) were found in the younger age group (15 to 25 years). The CT infection was found 47.2 % (35/74) in symptomatic cases, 41.1% (14/34) in asymptomatic cases and 47.4% in pregnant group, 42.8% in non-pregnant group. Although high incidence of genital chlamydia infection is common both in pregnant, non-pregnant, symptomatic and asymptomatic women in Bangladesh an early and reliable diagnostic method for genital chlamydia infection in Bangladesh should be further explored.

BRCA2 is a moderate penetrance gene contributing to young-onset prostate cancer: implications for genetic testing in prostate cancer patients.

BACKGROUND: A family history of prostate cancer (PrCa) is a strong risk factor for the disease, indicating that inherited factors are important in this disease. We previously estimated that about 2% of PrCa cases diagnosed ≤ 55 years harbour a BRCA2 mutation and PrCa among BRCA2 carriers has been shown to be more aggressive, with poorer survival. METHODS: To further evaluate the role of BRCA2 in PrCa predisposition, we screened 1864 men with PrCa aged between 36 and 88 years. We analysed the BRCA2 gene using a novel high-throughput multiplex fluorescence heteroduplex detection system developed for the ABI3130xl genetic analyzer. RESULTS: We identified 19 protein-truncating mutations, 3 in-frame deletions and 69 missense variants of uncertain significance (UV) in our sample set. All the carriers of truncating mutations developed PrCa at ≤ 65 years, with a prevalence of BRCA2 mutation of 1.20% for cases in this age group. CONCLUSION: Based on the estimated frequency of BRCA2 mutations in the United Kingdom we estimate that germline mutations in the BRCA2 gene confer an ∼ 8.6-fold increased risk of PrCa by age 65, corresponding to an absolute risk of ∼ 15% by age 65. These results suggest that routine testing of early onset PrCa cases for germline BRCA2 mutations will further help to refine the prevalence and risk associated with BRCA2 mutations and may be useful for guiding management options.

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript.

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.

A comparative study among laparoscopically assisted vaginal hysterectomy, vaginal hysterectomy and abdominal hysterectomy: experience in a tertiary care hospital in Bangladesh.

The study was undertaken to compare the efficiency and outcome of laparoscopically assisted vaginal hysterectomy (LAVH), total abdominal hysterectomy (TAH) and vaginal hysterectomy (VH) in terms of operative time, cost, estimated blood loss, hospital stay, quantity of analgesia use, intra- and postoperative complications rate and patients recovery. A total of 750 patients were prospectively collected in the study period from January 2005 through January 2009 in a tertiary care hospital. The mean estimated blood loss in LAVH and VH group were significantly lower compared with the TAH group. As to postoperative pain, significantly less diclofenac was required in the LAVH and VH group vs the TAH group. LAVH, VH is clinically and economically comparable with TAH, with patients' benefits of less estimated blood loss; less analgesia use; less intra- and postoperative complication rates; less postoperative pain; rapid patient recovery and shorter hospital stay. The study concludes that thus, LAVH, VH is clinically and economically comparable with TAH.

Seroprevalence of genital Chlamydia trachomatis infection in women of reproductive age.

The genital chlamydial infection is the most common sexually transmitted diseases (STD) and major cause of infertility and ectopic pregnancy for millions of women in the world particularly in developing countries. This study was performed to find out the seroprevalence of Chlamydia trachomatis (CT) genital infection in women of reproductive age attending the Department of Obstetrics and Gynaecology, Mymensingh Medical College Hospital (MMCH) during the period from January 2009 to December 2009 through a cross sectional study. A total of 108 serum samples from symptomatic and asymptomatic pregnant and non-pregnant women were tested for CT specific IgG antibody by Enzyme Linked Immunosorbent Assay (ELISA). A total of 31(28.7%) patients were found to have antibody of which 44% (26/59) were from pregnant group and 10.2% (5/49) from non-pregnant group. The seropositivity was 21.6% (16/74) in symptomatic cases and 44.1% (15/34) in asymptomatic cases. The study shows high prevalence of Chlamydial antibody which is common in pregnant and non-pregnant, symptomatic and asymptomatic adult women in Bangladesh. So, screening for chlamydial infection should be done routinely by suitable tests in sexually active symptomatic and asymptomatic women including pregnant women to prevent serious complications.

Molecular diagnosis of genital Chlamydia trachomatis infection by polymerase chain reaction.

The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Polymerase chain reaction (PCR). A total of 70 females were investigated including 56 symptomatic and 14 asymptomatic cases. Endocervical swabs were collected and dipped in 500µl Tris buffer. Polymerase chain reaction (PCR) amplification was done for detection of endogenous plasmid-based nucleic acid. A total 17 out of 56 of the symptomatic cases (30.4%) were positive for C. trachomatis and none were found positive among the 14 asymptomatic cases. These findings suggest high prevalence of C. trachomatis infection among this group of population.

Prevalence and antimicrobial resistance of methicillin resistant Staphylococcus epidermidis isolated at Mymensingh Medical College Hospital.

The study was done to determine the drug resistance pattern of Methicillin resistant Staphylococcus epidermidis (MRSE) isolated from different clinical specimens at Mymensingh Medical College Hospital, Mymensingh during the period from July 2007 to June 2008. A total of 32 Staphylococcus epidermidis were isolated from 200 different clinical specimens by standard microbiological techniques. Antimicrobial susceptibility of all the isolates was carried out by disk diffusion method as per recommendation of Clinical and Laboratory Standard Institute 2007. Out of 32 Staphylococcus epidermidis 18(56.25%) were detected as Methicillin resistant Staphylococcus epidermidis (MRSE) by disk diffusion method. In this study, Methicillin resistant Staphylococcus epidermidis showed multidrug resistance. Resistant to penicillin, amoxycillin, oxacillin and cloxacillin was 100% followed by gentamycin (56%), erythromycin (50%), doxycycline (44%), cephradine (44%), ciprofloxacin (39%), fucidic acid (33%), cefuroxime (33%) and ceftriaxone (28%). All isolates of MRSE were susceptible to rifampicin and vancomycin.

Seroprevalence of human brucellosis among the population at risk in rural area.

This study was performed to evaluate the seroprevalence of brucellosis among the risk group of population. A cross sectional study was carried out in the Department of Microbiology, Mymensingh Medical College in collaboration with the Department of Medicine under the Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, between the period from July 2007 to June 2008. A total of 210 samples were included in this study from risk group of population. A variety of serological tests have been applied for detection of antibodies against Brucella spp. Among the 210 subjects from risk group of population, 9(4.28%), 7(3.33%) and 7(3.33%) were positive for brucellosis by slide agglutination test, rose bengal plate agglutination test (RBPA) and standard tube agglutination test (STAT) respectively. Out of total specimen 10 positive and 29 negative serum as detected by slide agglutination, rose bengal plate agglutination test (RBPT) and STAT were also examined by Enzyme linked immunosorbent assay (ELISA) for detection of IgG. Among them, 10 were positive by ELISA IgG. Seroprevalence among occupational groups were 11.11% in veterinary personnel, 6.45% in dairy workers and 4.67% in animal farmers. This study indicated that brucellosis is not uncommon at rural area of Mymensingh region in Bangladesh. It was suggested to conduct community survey and to plan properly the measures of control and prevention.

Pattern of aerobic bacteria with antimicrobial susceptibility causing community acquired urinary tract infection.

Since antibiotic resistance among uropathogens have gradually been rising, so it is important to have knowledge about the pattern and antimicrobial susceptibility to choose the correct treatment regimen. A cross sectional study was carried out in the Department of Microbiology, Mymensingh Medical College between July 2007 to June 2008 to determine the prevalence, relationship between pyuria and urine culture and antibiotic resistance pattern among the bacterial isolates of community acquired UTI (CUTI). A total of 100 urine samples were subjected to microscopy and culture. Antimicrobial susceptibility of isolates was done by disk diffusion method according to Clinical and Laboratory Standard Institute (CLSI) 2007. Of the total samples, 45(45%) were culture positive and among them female were more (71.1%) than the male (28.9%). The predominant age group was 15-29. Having pus cell >5/HPF, 93.3% culture positive patients showed significant pyuria. The isolated microorganisms were Escherichia coli (73%) followed by Staphylococcus saprophyticus (11.1%), Klebsiella spp (6.7%), Enterobacter spp (4.4%), Pseudomonas aeruginosa (2.2%) and Proteus spp (2.2%). All the bacterial isolates were sensitive to imipenem, while they showed variation in sensitivity to other commonly used antibiotics. Imipenem, nitrofurantoin and gentamicin were found to be effective for Gram-negative isolates and imipenem, azithromycin, vancomycin, ceftazidime for Gram-positive isolates. Our study emphasized over the changing etiology and emergence of drug resistance of the UTI within our country.

Fludarabine/i.v. BU conditioning regimen: myeloablative, reduced intensity or both?

In this study, we utilized a conditioning regimen with fludarabine and myeloablative dose i.v. BU (12.8 mg/kg) (FluBU) in 36 adult patients (median age: 44 years, range: 18-61) with myeloid or lymphoid malignancies at standard risk (n=10) or high risk of relapse (n=26), who received an allogeneic hematopoietic SCT (HSCT) from HLA-matched related (n=16) or unrelated (n=20) donors. The source of hematopoietic stem cells was peripheral blood in 28 and marrow in 8 cases. Rabbit-antithymocyte globulin at 7 mg/kg was utilized in 21 patients. Acute GVHD grade II-IV was observed in 19% of the patients (grade III-IV in 14% of patients) and chronic GVHD in 11 of 30 evaluable patients (37%). At median follow-up of 737 days (range: 152-1,737) for alive patients, overall survival rates in standard- and high-risk patients were 80 and 35%, respectively, and event-free survival rates were 70 and 31%, respectively. TRM was 10% in standard-risk and 19% in high-risk patients. Post transplant relapse was observed in 20% standard-risk and in 46% high-risk patients. FluBU conditioning regimen is associated with a limited hematologic and extrahematologic toxicity and with an antitumor activity comparable to other standard myeloablative regimens.

Comparable kinetics of myeloablation between fludarabine/full-dose busulfan and fludarabine/melphalan conditioning regimens in allogeneic peripheral blood stem cell transplantation.

Fludarabine was utilized in the conditioning regimen of 30 adult patients undergoing an allogeneic hematopoietic stem cell transplant. In 18 patients it was combined with full-dose busulfan (FluBu) as a myeloablative regimen and in 12 cases with melphalan (FluMel) as a reduced intensity conditioning (RIC) regimen. Patients in the FluBu group were younger than in the FluMel group (P=0.03). Of 30 patients, 24 received peripheral blood stem cells (PBSC) whereas six patients in the FluBu group received bone marrow cells. The hematological toxicity of each regimen was evaluated by analyzing the kinetics of the neutropenia induced by preparative regimens and the time to recovery of the absolute neutrophils count (ANC) and platelets post transplantation. In PBSC transplants, the median day of severe neutropenia (<500 ANC/microl) occurred on day +6 after the FluBu regimen and on day +3 after FluMel (P=ns), whereas both groups had a duration of severe neutropenia of 9 days and a comparable time for ANC and platelet engraftment. Extra-hematological toxicities were also comparable in the two groups. These findings suggest that the hematological and extra-hematological toxicities induced by fludarabine/full-dose i.v. busulfan are similar to those induced by a standard RIC regimen such as fludarabine/melphalan.

Prolonged responses after autologous stem cell transplantation in African-American patients with multiple myeloma.

Multiple myeloma (MM) has a double incidence in African-American (AA) than in non-AA patients and previous studies have shown a higher mortality in the former patient population. Here, we retrospectively analyzed the results of autologous stem cell transplantation (ASCT) in 38 AA and 32 non-AA consecutive patients. The two groups were comparable at diagnosis for age, stage of the disease, cytogenetic abnormalities, beta(2) microglobulin and albumin blood levels, and plasma cell marrow infiltration. The rates of complete and partial response observed in AA and non-AA patients after induction chemotherapy (9 and 42 vs 13 and 33%) and at 2 months (31 and 25 vs 30 and 20%) following ASCT were similar. At 6 months after ASCT, a greater relapse rate was observed in non-AA patients (P=0.009). At a median follow-up of 26 months, AA patients had a greater event-free survival (P=0.02) than non-AA patients, whereas overall survival was comparable in the two groups. The initial finding that AA patients with MM, compared to non-AA patients, had more prolonged responses and comparable survival after ASCT suggests that intensified chemotherapy is equally effective in patients of various ethnicities.

Mechanism and Physiologic Significance of the Suppression of Cholesterol Esterification in Human Interstitial Fluid.

Cholesterol esterification in high density lipoproteins (HDLs) by lecithin:cholesterol acyltransferase (LCAT) promotes unesterified cholesterol (UC) transfer from red cell membranes to plasma in vitro. However, it does not explain the transfer of UC from most peripheral cells to interstitial fluid in vivo, as HDLs in afferent peripheral lymph are enriched in UC. Having already reported that the endogenous cholesterol esterification rate (ECER) in lymph is only 5% of that in plasma, we have now explored the underlying mechanism. In peripheral lymph from 20 healthy men, LCAT concentration, LCAT activity (assayed using an optimized substrate), and LCAT specific activity averaged, respectively, 11.8, 10.3, and 84.9% of plasma values. When recombinant human LCAT was added to lymph, the increments in enzyme activity were similar to those when LCAT was added to plasma. Addition of apolipoprotein AI (apo AI), fatty acid-free albumin, Intralipid, or the d < 1.006 g/ml plasma fraction had no effect on ECER. During incubation of lymph plus plasma, the ECER was similar to that observed with buffer plus plasma. When lymph was added to heat-inactivated plasma, the ECER was 11-fold greater than with lymph plus buffer. Addition of discoidal proteoliposomes of apo AI and phosphatidycholine (PC) to lymph increased ECER 10-fold, while addition of apo AI/PC/UC disks did so by only six-fold. We conclude that the low ECER in lymph is due to a property of the HDLs, seemingly substrate inhibition of LCAT by excess cell-derived UC. This is reversed when lymph enters plasma, consequent upon redistribution of UC from lymph HDLs to plasma lipoproteins.

Stage-specific cell-cycling of hematopoietic progenitor cells.

We studied the growth of hematopoietic progenitors at different progressive stages of differentiation and focused especially on changes in cell-cycling. Hematopoietic progenitors from 5-fluorouracil (5-FU)-treated mice were separated into three groups on the basis of differentiation, Stages I, II, and III, and have studied their cell-cycling. Primary marrow cells collected from 5-FU-treated mice were categorized as Stage I progenitors. Stages II and III progenitors are early and late progenies of Stage I progenitors, respectively. The rate of growth of hematopoietic progenitors supported by interleukin-3 (IL-3) and steel factor (SF) was estimated by sequential analysis of colony formation and studying replating efficiency of individual colonies. The time required for hematopoietic progenitors to go through the cell-cycle shortened as their stage of differentiation progressed. Similar results were obtained with other growth factor combinations. The analysis of DNA content of cells suggests that shortening of cell-cycling is mainly due to a decrease in the time of G1 phase of the cell-cycle. Our results demonstrate that in early hematopoiesis, the cell-cycling of hematopoietic progenitors accelerates as they differentiate.

Low BCL-2 expression in acute leukemia with t(8;21) chromosomal abnormality.

In de novo t(8;21) AML which shows terminal neutrophilic differentiation, the BCL-2 expression was found to be significantly lower than that in types of other AML regardless of the phenotypic differentiation status. An inverse correlation between BCL-2 expression and the S/G2/M population cells was observed in AML. The S/G2/M population in t(8;21)AML was larger than in the other types of AML. In t(8;21)AML, spontaneous apoptosis after a 12-h liquid culture was prominent, and the autonomous DNA synthesis after a 72-h liquid culture was low. G-CSF and IL-5 promoted the colony formation of t(8;21)AML cells. The data suggest that, in vivo, the low BCL-2 in t(8;21)AML induced entry of cells from the G0/G1 phase to S phase, but the cells easily die by apoptosis, in vitro. The low BCL-2 expression and the supportive effects of G-CSF and IL-5 in t(8;21)AML is thought to be a key phenomenon which might be related to the formation of the in vivo blood picture, such as prominent neutrophilic differentiation and eosinophilia. Cellular extracts from t(8;21)AML cell line Kasumi-1 bound to both the AML1 and CRE binding sites in the bcl-2 promoter, but none of the cellular extracts from de novo t(8;21)AML bound to either of these sites. The DNA binding activity of transactivators in de novo t(8;21)AML is different from that in Kasumi-1 cells probably due to the phosphorylation status.