Haemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. Identification of mutations contributing to defective factor IX may be advantageous for precise carrier and prenatal diagnosis. We studied 16 patients from 11 families, consisting of 8 patients of the Malay ethnic group, of which 6 were siblings. Factor IX mutations have not been previously reported in the Malay ethnic group. The functional region of the factor IX gene was sequenced and mutations were identified in either the exon or intronic regions in 15 of the patients. One novel mutation, 6660_6664delTTCTT was identified in siblings with moderate form of haemophilia B. Mutations identified in our patients when linked with disease severity were similar to findings in other populations. In summary, this preliminary data will be used to build a Malaysian mutation database which would facilitate genetic counseling.
Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (alpha) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (beta) and delta (delta) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave's disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for alpha globin gene mutations showed a single -alpha(-3.7) deletion and Hb CS mutation. These findings were suggestive of compound heterozygosity of Hb CS and a single -alpha(-3.7) deletion with a concomitant heterozygous deltabeta thalassaemia. Co-inheritance of Hb CS and a single -alpha(-3.7) deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modification of alpha thalassemia presumptively as a result of co-inheritance with deltabeta chain abnormality as suggested by the high Hb F level.
Serotyping is not sufficient to differentiate between Salmonella species that cause paratyphoid fever from the strains that cause milder gastroenteritis as these organisms share the same serotype Salmonella Paratyphi B (S. Paratyphi B). Strains causing paratyphoid fever do not ferment d-tartrate and this key feature was used in this study to determine the prevalence of these strains among the collection of S. Paratyphi B strains isolated from patients in Malaysia. A total of 105 isolates of S. Paratyphi B were discriminated into d-tartrate positive (dT+) and d-tartrate negative (dT) variants by two lead acetate test protocols and multiplex PCR. The lead acetate test protocol 1 differed from protocol 2 by a lower inoculum size and different incubation conditions while the multiplex PCR utilized 2 sets of primers targeting the ATG start codon of the gene STM3356. Lead acetate protocol 1 discriminated 97.1% of the isolates as S. Paratyphi B dT+ and 2.9% as dT while test protocol 2 discriminated all the isolates as S. Paratyphi B dT+. The multiplex PCR test identified all 105 isolates as S. Paratyphi B dT+ strains. The concordance of the lead acetate test relative to that of multiplex PCR was 97.7% and 100% for protocol 1 and 2 respectively. This study showed that S. Paratyphi B dT+ is a common causative agent of gastroenteritis in Malaysia while paratyphoid fever appears to be relatively uncommon. Multiplex PCR was shown to be a simpler, more rapid and reliable method to discriminate S. Paratyphi B than the phenotypic lead acetate test.
Infection with Helicobacter pylori cagA-positive strains is associated with gastroduodenal diseases. The CagA protein is injected into gastric epithelial cells and supposedly induces morphological changes termed the 'hummingbird phenotype', which is associated with scattering and increased cell motility. The molecular mechanisms leading to the CagA-dependent morphological changes are only partially known. The present study was carried out to investigate the effect of CagA variants on the magnitude of gastric epithelial cell morphological changes. Recombinant 3' terminal domains of cagA were cloned and expressed in a gastric epithelial cell line and the hummingbird phenotype was quantified by microscopy. The 3' region of the cagA gene of Malaysian H. pylori isolates showed six sub-genotypes that differed in the structural organization of the EPIYA repeat sequences. The percentage of hummingbird cells induced by CagA increased with duration of transfection. The hummingbird phenotype was observed to be more pronounced when CagA with 4 EPIYA motifs rather than 3 or 2 EPIYA motifs was produced. The activity of different CagA variants in the induction of the hummingbird phenotype in gastric epithelial cells depends at least in part on EPIYA motif variability. The difference in CagA genotypes might influence the potential of individual CagAs to cause morphological changes in host cells. Depending on the relative exposure of cells to CagA genotypes, this may contribute to the various disease outcomes caused by H. pylori infection in different individuals.
We present a case of a papillary tumour at the cerebellopontine angle in a 41-year-old man. He presented with left-sided facial and ear pain associated with dizziness, nystagmus and hearing loss. CT scan of the temporal bone showed a destructive tumour at the left cerebellopontine angle. Surgical excision was performed and the diagnosis of the endolymphatic sac tumour was made. Endolymphatic tumour is a low grade adenocarcinoma that originates from the endolymphatic sac. The definitive diagnosis requires a combination of clinical features, radiological finding and pathological correlation.
Early pregnancy failure is a common pregnancy complication. In clinical practice, the time delay to distinguish viable from nonviable pregnancy is often distressing to patients and doctors. A highly sensitive and specific biomarker that accurately discriminates between viable and nonviable pregnancy would be useful for early intervention. Progesterone has been shown as a biomarker of early pregnancy failure. However the usefulness is still questionable due to the different cutoff values used. A study was conducted to determine the role of progesterone as a marker of early pregnancy failure and to establish the cut-off value in discriminating between viable and nonviable pregnancy. The study was carried out in the Obstetric and Gynecology Patient Admission Centre (OBPAC), Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for a period of twelve months. Ninety-five pregnant women of 13 weeks or less period of amenorrhoea (POA) were recruited. Fourteen normal pregnant women were controls. The patients with early pregnancy failure were classified according to types of abortion. Single measurement of serum progesterone was carried out during admission. The outcome of pregnancy was followed up until 22 weeks of POA to ascertain viability of the fetus. Median progesterone levels were significantly lower in women with nonviable pregnancies compared with viable pregnancy [10.7ng/ml (0.60-49.80) vs. 45.9ng/ml (15.40-127.20) respectively, p<0.001]. Progesterone levels were also significantly lower in threatened abortion patients with outcomes of nonviable pregnancy compared with pregnancies that progressed on to the viability period [23.3 +/- 12.0 vs. 89.7 +/- 33.2 respectively, p<0.001]. At cut-off value of 32.7ng/ ml, progesterone had 90% sensitivity with 75% negative predictive value and 92% specificity with 97% positive predictive value. The area under curve for progesterone was 0.95 (95% Confidence Interval, 0.903-0.990). In conclusion, these findings indicate that serum progesterone can be used as a marker for early pregnancy failure.
CD133, a marker which has been advocated to mark colorectal carcinoma "stem or tumour initiating cells" is amongst the frequently studied markers in colorectal cancer. A study was conducted at the Department of Pathology, University of Malaya Medical Centre to determine the expression of CD133 in 56 archived, formalin-fixed, paraffin-embedded colorectal adenocarcinoma in comparison with adjacent benign colorectal epithelium by immunohistochemical staining for CD133 expression. CD133 immunopositivity was determined as staining at the glandular luminal surface or in the intraluminal debris. Expression was semiquantitated for (1) proportion of CD133 immunopositivity in the malignant or adjacent benign colorectal epithelium and (2) intensity of staining. The final score of CD133 immunopositivity was arbitrarily taken as proportion of CD133 immunopositivity multiplied by intensity of staining in both the malignant and adjacent benign colorectal epithelium. CD133 expression was observed in significantly increased frequency in 49 (87.5%) colorectal adenocarcinoma compared with 15 (26.8%) of the adjacent benign colorectal epithelium (p<0.05). In terms of immunopositivity score (proportion of CD133 immunopositivity multiplied by intensity of staining), colorectal adenocarcinoma had a mean arbitrary score of 8.5 which was significantly higher than the mean immunopositivity score of 0.5 of the adjacent benign colorectal epithelium (p<0.05). In addition, the maximum immunopositivity score for the adjacent benign colorectal epithelium was 4, while 38 (67.9%) of colorectal adenocarcinoma had scores >4. This study shows that CD133 is able to mark colorectal adenocarcinoma but it is still unclear at this juncture whether CD133 is indeed a marker for colorectal adenocarcinoma "stem cells".
Induced pluripotent stem cells (iPSC) are derived from human somatic cells through ectopic expression of transcription factors. This landmark discovery has been considered as a major development towards patient-specific iPSC for various biomedical applications. Unlimited self renewal capacity, pluripotency and ease of accessibility to donor tissues contribute to the versatility of iPSC. The therapeutic potential of iPSC in regenerative medicine, cell-based therapy, disease modelling and drug discovery is indeed very promising. Continuous progress in iPSC technology provides clearer understanding of disease pathogenesis and ultimately new optimism in developing treatment or cure for human diseases.
Predictive biomarkers such as oestrogen (ER) and progesterone (PR) receptors and c-erbB-2 oncoprotein have become a staple in breast cancer reports in the country as they increasingly play an important role in the treatment and prognosis of women with breast cancers. This study reviews the practice of histopathology reporting of these biomarkers in a Malaysian tertiary hospital setting. Retrospective data on demographic, pathological and biomarker profiles of patients with invasive ductal carcinoma who had undergone mastectomy or lumpectomy with axillary node clearance from 2005 to 2006 were retrieved from the Department of Pathology, Penang Hospital and analysed. The prevalence of ER positivity (55.8%), PR positivity (52.5%), c-erbB-2 oncoprotein overexpression (24%) and triple negativity (ER negative, PR negative, c-erbB-2 negative) (15%) by immunohistochemistry were comparable with other studies. Notably, c-erbB-2 overexpression was equivocal (2+) in 15% of cases. Since about a quarter of equivocal (2+) cases usually show amplification by FISH, a small but certain percentage of patients would miss the benefit of anti-c-erbB-2 antibody therapy if FISH is not performed. New ASCO/CAP guidelines on the quantitation of ER and PR will probably increase the prevalence of ER/PR positivity, invariably leading to significant ramifications on the management of patients as more patients would be deemed eligible for endocrine therapy, as well as categorisation of triple negative breast cancers.
Brucellosis is a zoonotic disease which can be transmitted by direct or indirect contact with infected animal or their products. It is an important public health problem but little is known on brucellosis in the Malaysian population. The aim of this study was to determine the presence of Brucella antibodies using commercial Brucella IgG and IgM ELISA kits (Vircell, SL, Barcelona Spain). A total of 184 sera from suspected patients were received from 16 hospitals in Malaysia over the years 2004 to 2009. Only 10 serum samples (5.4%) were positive for Brucella antibodies in which 5 showed the presence of both IgM and IgG. Most of the positive patients were occupationally involved with animals. This study suggests the seroprevalance of brucellosis among individuals who have contact with infected animals in Malaysia is low.
A 73-year-old Chinese man was admitted to the Accident and Emergency Premorbid Ward of a local hospital in Malaysia. The patient complained of shortness of breath with cough and was in a semi-conscious state. He was later admitted to an intensive care unit (ICU) of the hospital. Six days after admission 5-6 maggots were recoverd from the nasal cavity. The maggots were identified as the third-instar larvae of Lucilia cuprina Wiedmann (Diptera: Calliphoridae) based on the morphological characteristics. This patient was classified as having nosocomial myiasis. The presence of the third instar larvae indicated that the infestation was not more than three to four days. An adult sarcophagid identified as Parasarcophaga ruficornis (Fabricius) caught in the ICU where the patient was warded provided further evidence of the potential for the nosocomial infestation.
Myeloid sarcoma (MS) is a rare extramedullary myeloid tumour. It has been reported in various sites, including lymph node, bone, skin, soft tissue, various organs and the CNS. It may precede or occur concurrently with acute myeloid leukemia. Urinary bladder involvement is extremely uncommon. We report a 70-year-old female who had MS of the urinary bladder, presented with frank and persistent hematuria associated with lower abdominal pain. She subsequently had tumour seeding in the abdominal skin via percutaneous suprapubic catheter. Tumours from both the urinary bladder and skin showed immature cells that were immunoreactive toward LCA (focal), MPO (strong), CD99 (weak) and CD117 (weak). Summary of cases in the literature is presented. The potential of its misdiagnosis and the useful markers for the diagnosis of MS are discussed.
Transfusion is an irreversible event which carries potential benefits as well as risk to the recipient. The objective of this study was to analyse all reported transfusion reactions of the year 2008 in the Blood Bank Unit of Universiti Kebangsaan Malaysia Medical Centre (UKMMC). This is a retrospective study that was carried out by retrieving data from the laboratory information system. A total of 27842 transfusions were documented and the total reported transfusion reactions were 149. The incidence of transfusion reaction was 1 in 187 of all transfusions (0.54%); in which 69 (0.25%) were allergic in nature and 61 (0.22%) were febrile non-haemolytic transfusion reactions (FNHTR). Hypotensive reactions were identified in 6 (0.02%) patients. There were 9 (0.03%) cases reported with haemoglobinuria where no serological evidence of haemolytic transfusion reaction (HTR) was found. One HTR (0.003%) was identified and this was due to an error in patient identification in the ward. Other specified reactions like transfusion-related acute lung injury (TRALI), bacterial infections, Graft verses host disease (GVHD) were not reported. The highest frequency of the reactions occurred in the red cell transfusions which accounted for 111 cases. In conclusion, the incidences of transfusion reactions are low when compared to those reported by other centres.
We investigated the usefulness of a single value of maternal HbA1c in late pregnancy as a predictor for neonatal hypoglycaemia and secondly, to find the appropriate threshold value. A prospective analysis of the HbA1c concentration between 36 to 38 weeks of gestation in 150 pregnant mothers with either pre-existing or gestational diabetes was performed. At delivery, glucose levels in the cord blood were analysed. Neonatal hypoglycaemia was defined as a blood sugar level of < 2.6 mmol/l. Receiver operator characteristic curve was constructed to evaluate the value of HbA1c concentration in predicting hypoglycaemia. There were 16 foetuses who were hypoglycaemic at delivery. The area under the ROC curve for predicting neonatal hypoglycaemia was 0.997 with a 95% confidence interval of 0.992 to 1, a very good prediction rate. The optimal threshold value for HbA1c in predicting hypoglycaemia in the foetus was 6.8% (51 mmol/mol). HbA1c level in late pregnancy is a good predictor for hypoglycaemia in the newborn.
A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.
Patients with pancreatic adenocarcinoma are known to have a high mortality rate. The 5-year survival rate still remains low even now compared to that of the 1960's despite new advances in management including surgery, chemotherapy, pathological classification and molecular diagnostic technologies. Precursors to invasive pancreatic adenocarcinoma have been identified in the last ten years that include mucinous cystic neoplasm, intraductal papillary mucinous neoplasm and pancreatic intraepithelial neoplasia. p53 protein accumulation in the nuclei is a common molecular event in most human neoplasms. Our objective is to investigate p53 expression in pancreatic adenocarcinoma and precursor lesions and their significance. The selected study material encompassed 31 invasive ductal adenocarcinoma, 15 mucinous cystic neoplasm and papillary mucinous neoplasm, and 27 cases of pancreatic intraepithelial neoplasia including grade 1, 2 and 3. Immunoscore was given for each case based on intensity of staining and percentage of cells positive and compared between precursor lesions and invasive adenocarcinoma. A score of 50 and above was considered significant. The results showed that p53 expression increased progressively and significantly with the grade of pancreatic intraepithelial neoplasia and adenocarcinoma (p-value < 0.001). These findings support the concept of multistep carcinogenesis in pancreatic adenocarcinoma and suggest that p53 inactivation occurs in the progression of precursors to pancreatic adenocarcinoma.
On integration into the host cervical keratinocyte genome, human papillomavirus (HPV) E7 protein binds pRB,releasing E2F from normally incompetent pRB-E2F complexes and allowing propagation of G1-S transition by the E2F. p16(INK4a), a tumour suppressor protein, increases in reflex response to counter this. 29 histologically re-confirmed low-grade squamous intraepithelial lesions (LSIL), 27 high-grade squamous intraepithelial lesions (HSIL) and 30 invasive cervical squamous carcinoma (SCC) were immunohistochemically stained for p16(INK4a) expression using the CINtec Histology Kit (REF 9511, mtm laboratories AG, Heidelberg, Germany) to re-affirm the notion that integration of HPV occurs predominantly in SCC and possibly HSIL and less in LSIL and normal squamous epithelium (NSqE). Implicit was also the attempt to understand the role of E2F, as indicated by p16(INK4a), in evolution of SCC from HSIL. No ethnic predilection was noted for LSIL, HSIL or SCC. Patients with SCC were significantly older by about 14-years compared with HSIL (p < 0.05) while there was no significant age difference between HSIL and LSIL. p16(INK4a) expression was significantly increased (p < 0.05) in both HSIL (88.9%) and SCC (83.3%) compared with LSIL (3.4%) and NSqE (0%); the NSqE being normal squamous epithelium noted in 17 of the LSIL, 19 HSIL and 5 SCC. From these findings there is suggestion that fundamental upstream events viz HPV integration, E7 upregulation followed by E2F activation occurs at point of transformation to HSIL and continues unrelentingly for another one to two decades before hitherto unclear factors convert a non-invasive lesion into an overtly invasive malignant counterpart. Interestingly, the occurrence of HSIL and LSIL in almost the same age group could mean that alteration from episomal to integrated form of HPV may not incur a prolonged incubation period, unlike from HSIL to SCC.
A field evaluation on the effectiveness of a modified approach of chemical fogging of insecticides against the conventional method was carried out in the Seremban district within the state of Negeri Sembilan, Malaysia from 7th February 2003 to 7th September 2003. In the 3 months period, November 2002 to January 2003, prior to institution of modified approach of chemical fogging, 27 of 42 (64.3%) dengue outbreaks were successfully controlled within the stipulated time frame of 14 days by the conventional approach of thermal chemical fogging. However, during the period when the modified approach of chemical fogging was instituted, 25 of 27 (92.6%) dengue outbreaks within the same district were successfully controlled within the 14-days time-line. Statistically, the modified approach of chemical fogging significantly improved the success rate of achieving dengue outbreak control within the stipulated time frame (chi2 = 5.65, p = 0.01745). The modified approach of chemical fogging also appeared to reduce the number of dengue cases recorded in the same district. This small pilot study shows that the modified approach of chemical fogging reduced cost in carrying out each fogging activity to control dengue outbreak. It also substantially reduced the required time taken to complete each fogging activity in comparison to the conventional approach. Thus, it enabled similar number of workers to cover more localities simultaneously affected by the outbreaks. In addition, the modified approach reduced the exposure time to hazardous insecticides for each worker doing hand-held thermal fogging.
Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplification efficiency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).
The number of requests for testosterone testing in adult males has been increasing in recent years. In this review, the biochemistry and physiology of testosterone in males relevant to the chemical pathologist or clinical biochemist is outlined. The methodology for total testosterone and various laboratory tests associated with the assessment of testosterone status including free testosterone, calculated free testosterone (CFT), bioavailable testosterone (BAT) and free androgen index (FAI) is then summarised. Clinical and laboratory criteria for the diagnosis of late-onset hypogonadism (LOH) in men are critically discussed with particular emphasis on the interpretation of laboratory test results. Finally, other indications for testosterone testing in adult men such as infertility are also reviewed.