A 34-year-old laboratory worker developed murine typhus after an accidental splashing of Rickettsia typhi over her right eye and lips. Indirect immunoperoxidase test showed a four-fold increase in titre to Rickettsia typhi. She responded well to doxycycline.
This case study demonstrates a 36-year-old ex-intravenous drug user (IVDU) who had been initially tested positive for human immunodeficiency virus (HIV) twice using Enzyme Immunoassay (EIA) method (Particle agglutination, PA done), but a year later he was tested HIV-negative. The patient was asymptomatic for HIV and T helper cells (CD4) count remained stable throughout this period. In light of this case, there may be a need to retest by molecular methods for high risk category patients who were initially diagnosed HIV-positive, but later showing an unexpected clinical course, such as a rising or stable CD4 titre over the years.
We successfully developed an in-house, competitive enzyme immunoassay to measure advanced glycosylation end-products (AGE) in serum. The assay involved coating microtitre wells with AGE-BSA at 8 micrograms/ml for 4 hours, followed by overnight incubation of 20 microliters sample (prediluted at 1:6) with 80 microliters antiserum (1:8000). HRP-labelled goat anti-rabbit was used as the second antibody and 3,5',5,5'-tetramethylbenzidine dihydrochloride as the substrate. Incubation was carried out at 4 degrees C. As suggested in an earlier study, we standardised the AGE units against normal human serum (NHS). Thus, one AGE unit was defined as the inhibition that resulted when the 1:6 diluted NHS was assayed. Mean (+/- SD) AGE level in normal subjects (n = 37) was significantly lower than in diabetes subjects with microalbuminuria (n = 57) (6.0 +/- 0.7 versus 10.2 +/- 4.7 units/ml, p = 0.0001). With the availability of in-house assay and by standardising the AGE unit with the other laboratories, more studies could be undertaken and results compared, and possibly, further elucidate the roles of AGE in the pathogenesis of diabetic complications.
Expression of p53 protein was investigated by immunohistochemical techniques in archival cases of 134 primary breast carcinomas comprising 13 comedo ductal carcinoma in situ (DCIS), 105 invasive ductal carcinomas, 7 contained the comedo DCIS component adjacent to the invasive ductal component, 5 invasive lobular carcinomas, three colloid carcinomas and one medullary carcinoma. Overexpression of p53 gene product was studied to determine the association with clinico-pathological parameters and also its relationship to c-erbB2. Overexpression of p53 protein was observed in 31% (4/13) of comedo DCIS, 37% (39/105) of invasive ductal carcinomas, 57% (4/7) of carcinomas containing both the in situ and invasive lesions and all medullary carcinomas. A significant relationship (p < 0.05) was observed between strong immunoreactivity of p53 protein and absence of estrogen receptor, histological grade and c-erbB2 but not with lymph node metastases or age of patient. These observations suggest that overexpression of p53 protein may play an important role in tumor progression from noninvasive to invasive in some breast carcinomas and may have potential as an indicator for poorer prognosis.
Overexpression and amplification of cyclin D1 were investigated by immunohistochemistry and differential polymerase chain reaction (dPCR) in 440 formalin-fixed primary breast carcinoma tissues. Overexpression of cyclin D1 was detected in 60% (263/440) and amplification of cyclin D1 was noted in 27% (119/440) of the primary breast carcinomas. Molecular analysis demonstrated that cyclin D1 was amplified in 30% (7/23) of the comedo DCIS, 22% (9/41) of the comedo DCIS and 32% (13/41) of the adjacent invasive ductal carcinomas, 30% (82/270) of the invasive ductal carcinomas, 27% (9/33) of the invasive lobular carcinomas, 19% (4/21) of the colloid carcinomas and 13% (2/15) of the medullary carcinomas. Cyclin D1 was amplified in 11% (2/19) of the invasive ductal carcinomas but not in the adjacent non-comedo DCIS lesions. Our observation showed that cyclin D1 was strongly positive in 61% (14/23) of the comedo subtype, 61% (11/18) of the non-comedo subtype, 59% (24/41) of the comedo DCIS and 63% (26/41) of the adjacent invasive ductal carcinomas, 53% (10/19) of the non-comedo DCIS and 58% (11/19) of the adjacent invasive lesions, 58% (157/270) of the invasive ductal carcinomas, 73% (24/33) of the invasive lobular carcinomas, 52% (11/21) of the colloid carcinomas and 27% (4/15) of the medullary carcinomas. A significant association was observed between in situ components and adjacent invasive lesions for cyclin D1 expression (p<0.05) and amplification (p<0.05). A significant relationship was noted between amplification of cyclin D1 and lymph node metastases (p<0.05) but not with histological grade (p>0.05), estrogen receptor status (p>0.05) and proliferation index (Ki-67 and PCNA) (p>0.05). However, overexpression of cyclin D1 was statistically associated with well differentiated tumors (p<0.05) and estrogen receptor positivity (p<0.05). No relationship was seen with nodal status (p>0.05) and proliferation index (Ki-67 and PCNA) (p>0.05). These observations suggest that tumors positive for cyclin D1 protein may have features of good prognosis but amplification of cyclin D1 gene could be an indicator of tumors with poor prognostic features. Although majority of the Malaysian patients belong to younger age group (<50 years old), amplification and expression of cyclin D1 was not statistically associated with patient age (p>0.05). These observations indicate that amplification and up-regulation of cyclin D1 may be independent of patient age. Moreover, overexpression and amplification of cyclin D1 in preinvasive, preinvasive and adjacent invasive lesions, and invasive carcinomas suggest that the gene may play an important role in early and late stages of breast carcinogenesis.
Human leucocyte antigen (HLA) expression is altered in oesophageal carcinomas compared with normal tissue. It is unclear, however, whether this phenotype precedes malignant transformation or results as a consequence of it.
Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.
A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) was developed for the fast-growing and cytopathic cell culture-adapted hepatitis A virus (HAV) strain HM175A.2. Infectivity titration by EIA correlated well with titration by cytopathic effects. The reliability of this assay was demonstrated by close agreement in virus infectivity titers among different assays of the same virus aliquot and between assays of different virus aliquots. HAV infected cell cultures after fixation could be stored for up to 1 week before testing without decline in virus titer.
An immunohistochemical assay was developed combining an avidin-biotin-glucose oxidase complex procedure (ABC-GO) with light microscopy to detect specific antibody against Plasmodium falciparum. Thin blood films were prepared from culture material of P. falciparum and fixed with acetone. Antibody was detected by successive incubations with test serum, biotinylated goat antihuman antibody, avidin-biotin-glucose oxidase complex, and glucose oxidase substrate. In the presence of reactive serum, a blue precipitate formed on the parasites and could be visually observed with a 40x objective. Sera from patients with single infections for P. vivax or P. ovale were unreactive. No cross-reactivity was observed with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The sensitivity of ABC-GO is comparable to that of the indirect fluorescent antibody test.
Scrub typhus is a major cause of febrile illness throughout the Asia-Pacific region. It is commonly undiagnosed, partly because of the lack of a simple, reliable diagnostic test which can be used in clinical laboratories. The indirect immunoperoxidase technique, configured into a test kit, was provided to technicians who were trained in its use. They used the kit during a 2 year field trial in their respective clinical hospital laboratories throughout Malaysia. In an evaluation using 1,722 consecutive sera tested in those laboratories, the kit was found to have a median sensitivity for IgG detection of 0.85 (range 0.33-0.95), a median specificity of 0.94 (range 0.88-1.00), reproducibility of 0.86, and efficiency of 0.92 when compared to the reference laboratory. In a proficiency survey in which 10 laboratories received 3 coded test samples, all but 2 laboratories had results within 1 dilution of the reference laboratory in quantitating specific IgG, whereas 7 laboratories were within 1 dilution in quantitating IgM. The shelf life of the kit was at least 1 year at 4 degrees C.
An indirect immunoperoxidase test was compared with an indirect fluorescent antibody test and the Weil-Felix OXK test for serodiagnosis of scrub typhus by measuring the rickettsial antigen specific activity of IgG, IgM, and whole globulin. Acute and convalescent sera from 50 Rickettsia tsutsugamushi isolate-positive scrub typhus patients and from 45 febrile patients diagnosed as having diseases other than scrub typhus were tested. The receiver operating characteristic for each test showed that the indirect immunoperoxidase and indirect fluorescent antibody tests were more sensitive and specific than the Weil-Felix test using convalescent and acute as well as paired sera. The indirect immunoperoxidase test showed no cross-reactivity when R. tsutsugamushi antigen was tested against sera collected from patients living outside the scrub typhus-endemic area with diseases other than scrub typhus. The indirect immunoperoxidase and indirect fluorescent antibody tests were comparable in measured response to R. tsutsugamushi, R. typhi, and TT-118 (spotted fever group) antigen. Thus the indirect immunoperoxidase test represents a sensitive, specific, reproducible, and practical semiquantitative test for rickettsial disease diagnosis.
Mosquito adults and larvae were collected from dengue high risk areas and transported to the laboratory for identification. Identified mosquitos were pooled according to the species, date and locality and stored at -70 degrees C. A total of 1,385 pools of Aedes albopictus and 267 pools of Ae. Aegypti were collected from major towns in 12 states in Peninsular Malaysia. Virus isolation was carried out using cell culture (C6/36 clone) of Ae. albopictus and detection of dengue virus by the peroxidase anti-peroxidase staining. All positive isolations were further re-confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the pools were negative with PAP staining and RT-PCR. However, 11 mosquito pools were positive with PAP staining. On the other hand, samples from Terengganu, Pulau Pinang and Johor were positive using both methods.
A new and rapid malaria immunoperoxidase assay using the enzyme horseradish peroxidase in place of fluorescein isothiocyanate was developed to allow the serological measurement of antimalarial antibody by light microscopy. Acetone-fixed thin blood films prepared from cultured Plasmodium falciparum were used as the source of antigen. This malaria immunoperoxidase assay is as sensitive as, and occasionally more sensitive than, the indirect fluorescent antibody assay. It is easy to perform and the antigen used does not show cross-reactivity with sera from nonmalarial diseases.
Hypertension complicates chronic pyelonephritis. Since arterial narrowing is common in the damaged kidney, activation of the renin-angiotensin system due to renal ischaemia has been suggested as a pathogenetic mechanism. We used an antiserum to human renin and an immunoperoxidase technique to study the anatomy of renin-containing cells (RCC) in 18 kidneys removed for pyeloneophritis. We independently assessed the degree of arterial narrowing and correlated these variables with the clinical findings. There was histological evidence of hyperplasia of RCC in 5 of the 6 hypertensive patients and in 7 of the 12 non-hypertensive cases. There was no difference in the apparent number or distribution of RCC between the hypertensive and the non-hypertensive cases. Also, the degree of arterial narrowing did not correlate with either the hyperplasia of RCC or the blood pressure of the patients. Our results do not support the hypothesis that narrowing of the intrarenal arteries is important in the pathogenesis of hypertension in pyelonephritis. In our cases, the renal veins were more severely damaged than the arteries and their lumina were often obliterated by organized thrombus. We suggest that such widespread obliteration of the renal venous tree could impair blood flow and contribute to the tissue damage in the pyelonephritic kidney.
Hepatocellular carcinoma (HCC) is among the ten most common cancers in Malaysian males. As cellular proliferation is an important feature of malignant transformation, we studied the proliferation pattern of normal and benign perineoplastic liver versus hepatocellular carcinoma in an attempt to further understand the tumour transformation process. 39 HCC (21 with accompanying and 18 without cirrhosis) histologically diagnosed at the Department of Pathology, University of Malaya Medical Centre between January 1992 and December 2003 were immunohistochemically studied using a monoclonal antibody to PCNA (Clone PC10: Dako). 20 livers from cases who had succumbed to traumatic injuries served as normal liver controls (NL). PCNA labeling index (PCNA-LI) was determined by counting the number of immunopositive cells in 1000 contiguous HCC, benign cirrhotic perineoplastic liver (BLC), benign perineoplastic non-cirrhotic (BLNC) and NL cells and conversion to a percentage. The PCNA-LI was also expressed as Ojanguren et al's grades. PCNA was expressed in 10% NL, 38.9% BLNC, 76.2% BLC and 71.8% HCC with BLNC, BLC and HCC showing significantly increased (p < 0.05) number of cases which expressed PCNA compared with NL. The number of BLC which expressed PCNA was also significantly increased compared with BLNC. PCNA-LI ranged from 0-2.0% (mean = 0.2%) in NL, 0-2.0% (mean = 0.3%) in BLNC, 0-3.6% (mean = 0.7%) in BLC and 0-53.8% (mean = 7.6%) in HCC with PCNA-LI significantly increased (p < 0.05) only in HCC compared with BLC, BLNC and NL. Accordingly, all NL, BLC and BLNC showed minimal (<5% cells being immunopositive) immunoreactivity on Ojanguren et al's grading system and only HCC demonstrated immunoreactivity which ranged up to grade 3 (75% of cells). From this study, there appears to be a generally increasing trend of proliferative activity from NL to BLNC to BLC and HCC. Nonetheless, BLNC and BLC, like NL, retained low PCNA-LI and only HCC had a significantly increased PCNA-LI compared with the benign categories. This is probably related to the malignant nature of HCC and may reflect the uncontrolled proliferation of the neoplastic hepatocytes.
Ki-67 expression in diffuse proliferative lupus nephritis, WHO Class IV, was compared against normal controls to establish that cellular proliferation is involved in the production of glomerular hypercellularity. Twenty-three histologically confirmed WHO Class IV lupus nephritis and 23 normal control renal tissue were immunohistochemically stained with a polyclonal antibody to Ki-67 (Dako) using the peroxidase labelled streptavidin bioitin kit (Dako). There were 20 females and 3 males, with 17 Chinese and 6 Malays in the WHO Class IV lupus nephritis group. Ages of patients ranged between 10-56 years with a mean of 31.9 years. The normal controls, 20 males and 3 females, and ethnically 9 Indians, 7 Malays, 2 Chinese, and 5 foreign nationals (4 Indonesians and 1 Bangladeshi), had an age range between 15-33 years (mean = 23.3 years). Sixteen (69.6%) WHO Class IV lupus nephritis and 8 (34.8%) normal controls demonstrated Ki-67 immunoreactivity in at least 1 glomerulus (p<0.05). Of the 256 WHO Class IV lupus nephritis non-sclerosed, glomeruli studied, 37 (14.5%) were Ki-67 immunopositive compared with normal controls where 16 (0.7%) of 2159 glomeruli demonstrated Ki-67 (p< 0.01). Cellular proliferative activity, as evidenced by Ki-67 expression, was significantly increased in WHO Class IV lupus nephritis confirming that cell proliferation contributes to glomerular hypercellularity.
Two forms of abnormal fibrillary protein deposition are considered: amyloidosis and fibrillary (immunotactoid) glomerulonephritis. Amyloid is characterised by an antiparallel, beta-pleated configuration which imparts to it a unique apple-green birefringence after Congo red staining. Inspite of its fairly constant physical properties, the chemical composition of amyloid fibrils is amazingly diverse, encomposing AA protein, light chain fragments, transthyretin, procalcitonin, islet amyloid polypeptide, atrial natriuretic peptides, beta-amyloid protein, beta-2-microglobulin, cystatin C, gelsolin, apolipoprotein A1, lyzozyme and their mutant variants. Amyloid P component and heparan sulphate proteoglycan are ubiquitous non-fibrillary amyloid components which have significant roles in the amyloidogenetic process, as do also precursor fibril proteins. Different amyloid fibril proteins relate to different amyloidosis syndromes and different histological patterns, and provide the basis for new diagnostic approaches to this disorder. Glomerular deposits in fibrillary glomerulonephritis (FGN), although often mistaken for amyloid, differ from it in its negative Congophilia, wider fibril width and highly organised, microtubular-tactoidal appearance ultrastructurally. FGN is essentially a primary glomerulopathy resulting in progressive renal failure. Despite certain differences, intriguing similarities between both entities of fibrillary deposition pose a challenge to researchers as to the mechanisms of abnormal protein crystallization and fibril formation in tissues.
A five-month-old male baby presented with an abdominal mass which was found on computerised tomography (CT) to be involving the left kidney. Nephrectomy and histopathological study showed morphological featues of a malignant rhabdoid tumour. The tumour cells stained strongly for cytokeratin and epithelial membrane antigen and less intensely for vimentin. Electron microscopy revealed concentric whorled arrays of intermediate filaments within the tumour cell cytoplasm. The child was put on post-operative chemotherapy and radiotherapy but developed bilateral lung metastases and died three months after surgery.
CD44 is a cell adhesion molecule that plays an important role in the cascade of metastasis and progression of human malignant tumours. A large family of variants or isoforms, generated by alternative splicing of a single gene, has been reported to be involved in the malignant process by conferring metastatic potential to non-metastatic cells. The objective of this study was to compare the expression of CD44 standard molecule with the International Neuroblastoma Pathology Classification (INPC) for neuroblastic tumours, a histological grading system based on the Shimada system for predicting the clinical outcome in neuroblastic tumours.
Eight histologically-confirmed cases of clear cell sarcoma of the kidney (CCSK) were studied for possible mutations in the p53 tumor suppressor gene by the immunohistochemical demonstration of mutant p53 proteins using a monoclonal (DO7: Dako) and a polyclonal (AB565: Chemicon) antibody to p53 protein. All cases exhibited p53 protein nuclear immunopositivity, although in varying numbers of tumor cells and with different staining intensities. p53 protein (DO7 or AB565) was expressed in < 25% of the tumor cells in four (50%) of the cases, including the one case with a known long term survival of 13 years from the time of diagnosis. The other tumors showed p53 protein immunopositivity in > 25% of the tumor cells when stained with either DO7 or AB565 or both. The intensity of staining, graded on visual impression into weak, moderate or strong, did not correlate well with the ratio of positive staining tumor cells. While this study is unable to clarify the relative prevalence and importance of p53 mutational events in the pathogenesis of this aggressive renal tumor of childhood, it is reasonably suggestive that alterations in the p53 tumor suppressor gene do occur in CCSK.