Validation and Application of a Commercial Quantitative Real-Time Reverse Transcriptase-PCR Assay in Investigation of a Large Dengue Virus Outbreak in Southern Taiwan.

BACKGROUND: Accurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015. METHODS: Sera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an "in-house" qualitative DENV-specific RT-PCR assay. RESULTS: A total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 102 and 106 copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 106-109 copies/mL, which was markedly higher than the rate observed in the other age groups. CONCLUSIONS: The LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates.

The molecular signature for urothelial carcinoma of the upper urinary tract.

PURPOSE: Frequent loss of heterozygosity of microsatellites on a specific chromosomal region has been reported in various types of human cancer. The same loss of heterozygosity has also been identified in matched serum or urine DNA. We determined a urine microsatellite marker profile specific to urothelial carcinoma of the upper urinary tract. MATERIALS AND METHODS: We analyzed the loss of heterozygosity of primary tumors and their matched urine DNA samples from 30 patients with urothelial carcinoma of the upper urinary tract. We investigated a total of 77 markers from 25 chromosomal regions and a total of 53 from 23 chromosomal regions for their preferential loss in urothelial carcinoma and renal cell carcinoma of the kidney, respectively. Specificity was then confirmed in a cohort of 22 renal cell carcinoma cases. RESULTS: Of 30 patients with urothelial carcinoma 25 (83.3%) were detected using the molecular urine test. Of 48 markers detected as loss of heterozygosity in urine 20 (41.7%) were localized at the chromosomal loci reported for renal cell carcinoma. The diagnostic specificity of the remaining 31 markers was cross-validated in a renal cell carcinoma cohort. With the cutoff set at 100% specificity urothelial carcinoma was accurately diagnosed in 24 of 30 patients (80.0%) using 13 markers, including D9S195, D18S67, GSN, D1S162, D8S261, D3S1300, D21S1436, D16S310, D3S1307, FABP2, D11S907, D15S1007 and D13S133. CONCLUSIONS: The marker panel may permit a high throughput differential diagnosis of urothelial carcinoma of the upper urinary tract from that of renal cell carcinoma at an early stage of disease. The accurate diagnosis of renal cancer may help determine appropriate treatment planning and minimizing intraoperative frozen consultation.

Immunohistochemical detection of prostate-specific antigen expression in primary urothelial carcinoma of the urinary bladder.

BACKGROUND: Prostate-specific antigen (PSA) is a 33-kDa serine protease that is secreted by prostatic epithelium and non-prostate tissues. Ectopic expression of PSA has also been reported in a variety of non-prostatic epithelial tumors. PATIENTS AND METHODS: Expression of PSA and its clinical implication were examined in a total of 422 cases of primary urothelial carcinoma of the bladder. RESULTS: Cytoplasmic reactivity to PSA was observed in 6 cases (1.4%), with the staining being to a low degree in 3 and a high degree in the remaining 3 cases. Expression of PSA was positively related to multiplicity, large tumors (> or = 3 cm) and muscle invasion (> or = pT2) (p = 0.0008, 0.004 and 0.03, respectively). Patients with tumors of low PSA expression had a better survival than those with tumors of high PSA expression (p = 0.03). CONCLUSION: Focal expression of PSA can occasionally be detected in some large, invasive urothelial cancer cells. This phenotypic change should be considered in the differential diagnosis of poorly differentiated carcinoma of the lower urinary tract.

Prevalence of RHD 1227A and hybrid Rhesus box in the general Chinese population.

The Rhesus (Rh) blood group is the most polymorphic blood group system. Serological analyses revealed that only 0.1% to 0.4% of the Chinese population exhibited a D-negative phenotype. In contrast to D-negative Caucasian persons, who are mostly associated with a deletion of RHD between the upstream and downstream Rhesus boxes, approximately 30% of apparently D-negative Chinese were actually DEL, potentially harboring a grossly intact RHD gene. The authors' previous detection of the RHD 1227A allele in all DEL samples prompted the conclusion that the RHD 1227A gene is highly prevalent in DEL populations of Chinese ethnicity, which indicates that it may be a marker for the rapid detection of DEL. Currently, random surveys for RHD 1227 A allele and for hybrid Rhesus box were conducted among 399 Han Chinese in Taiwan. The estimated frequencies of RHD 1227G/G, RHD 1227A/G, RHD A/A, RHD 1227G/-, RHD 1227A/-, and RHD-/- were 0.89054, 0.0118, 0.00016, 0.0414, 0.00055, and 0.00192, respectively. These results were consistent with the prevalence of D-negative and DEL in persons of Chinese ethnicity. The authors conclude that detection of RHD 1227A and hybrid Rhesus box alleles is predictive of Rh phenotypes in the Chinese population.

Detection of RhD(el) in RhD-negative persons in clinical laboratory.

The Rhesus (Rh) blood group is the most polymorphic human blood group system, and it is clinically significant in transfusion medicine. About 15% of Caucasoid people are RhD-negative, whereas in the Asian population, the RhD-negative blood type only occurs in 0.1% to 0.5%. However, approximately 30% of apparently RhD-negative Taiwanese people actually were RhD(el). Traditionally, we verify RhD(el) by a serologically adsorption-elution procedure with polyclonal anti-D. In our recent report, RhC phenotype is highly associated with RhD(el), and RHD1227A is a useful genetic marker for RhD(el). For setting up a rapid protocol to detect RhD(el) in clinical laboratory, a total number of 395 Taiwanese serological RhD-negative blood samples, those with RhC (+) phenotypes as selected by serological tests, were further screened by adsorption/elution tests and RHD1227A allele by specific sequence primer-polymerase chain reaction (SSP-PCR) for RhD(el). Among 395 blood samples collected from RhD-negative subjects, the incidence of RhC (+) was 43% (171/395). One hundred and twenty six of the 171 RhC (+) samples were positive for both adsorption/elution for RhD detection and SSP-PCR assay for RHD1227A. The sensitivity and specificity were 96.9% and 97.5%, respectively, for RHD1227A detection as compared with the traditional adsorption/elution test. Our results also indicated that RHD1227A was highly linked to Ce haplotypes (95.2%). In conclusion, combined RhC (+) phenotyping and RHD1227A analysis can be a simple and accurate laboratory screening protocol for RhD(el) detection in RhD-negative population.

Nasopharyngeal carcinoma-susceptibility locus is localized to a 132 kb segment containing HLA-A using high-resolution microsatellite mapping.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor uniquely prevalent in southern Chinese. HLA-A2 is associated with NPC. In a previous study, we showed that the genes associated with susceptibility to NPC are primarily located within the HLA-A locus in Taiwanese NPC patients. However, the pathogenic genes causing NPC susceptibility remain unknown. Here, 8 polymorphic microsatellite markers distributed over a 1 megabase region surrounding the HLA-A locus were subjected to genetic analysis for the NPC-susceptibility locus. Statistical studies of associated alleles detected on each microsatellite locus showed that the NPC- susceptibility genes are most likely located between the D6S510 and D6S211 markers within a 132 kb segment containing the HLA-A locus. These results undoubtedly would facilitate the further positional cloning of the NPC-susceptibility locus, which has been elusive for the past 30 years.

RHD 1227A is an important genetic marker for RhD(el) individuals.

Approximately 30% of apparently Rh- Taiwanese people actually were RhD(el), a rare variant of the Rh system that might carry a grossly intact RHD gene. Several studies have indicated that the RhD(el) trait might be generated by multiple molecular mechanisms. In this study, a total of 294 Taiwanese serologically RhD- blood donors were tested for Rh phenotypes and RHD genotypes. Among them, total RHD deletion, partial RHD gene, and RhD(el) were found in 185 (62.9%), 15 (5.1%), and 94 (32.0%), respectively. The 1227A allele and exon 9 of the RHD gene were found in all 94 RhD(el) donors. The Ccee was the most prevalent phenotype in the RhD(el) group (78/94 [83%]), and the ccee phenotype was highly prevalent in the true D- group (87.6%). RHD 1227A can be used as an important and useful genetic marker for RhD(el). It can be detected easily by a simple, rapid, specific sequence primer-polymerase chain reaction method.

Genetic susceptibility to nasopharyngeal carcinoma within the HLA-A locus in Taiwanese.

NPC is an epithelial tumor that is highly prevalent among the southern Chinese. Numerous studies have indicated that specific HLA haplotypes and genes within the HLA complex are associated with NPC. As a first effort to localize the gene responsible for susceptibility, the HLA-A, -B, and -A2 subtypes were examined for their association to NPC. Consistent with previous reports, frequencies of HLA-A2 [OR = 2.50, pc = 0.020 (study population); OR = 3.73, pc = 0.0030 (> or =40 years old)] were significantly higher in patients with NPC than in healthy controls. Two-locus analysis indicated that A2(+)B46(+) individuals are at greater risk for NPC than A2(-)B46(-) individuals in both the population studied and the > or =40-year-old group. This, however, may be due to the close linkage of these 2 genes. Moreover, A2(+)B38(+) individuals were at higher risk than A2(-)B38(-) individuals in both the population studied and the > or =40-year-old group; A2 and B38 are not genetically linked. These findings suggest that B38 or B46 alone cannot confer a high risk of NPC but that, in conjunction with A2, B38 or B46 positivity greatly increases risk. None of 5 A2 subtypes identified from studied populations was significantly associated with NPC. Microsatellite marker D6S211, located 97 kb telomeric to HLA-A, was analyzed for its association with NPC. Allele 4 of D6S211 was significantly associated with NPC (OR = 3.97, pc = 0.0042). These results strongly support the hypothesis that genes associated with susceptibility to NPC in the HLA region are within the HLA-A locus.