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PubMed2004

Serial follow-up and the prognostic significance of reverse transcriptase-polymerase chain reaction--staged sentinel lymph nodes from melanoma patients.

PURPOSE: Reverse transcriptase-polymerase chain reaction (RT-PCR) may provide an extremely sensitive method for detection of occult nodal disease. We evaluated the role of a single-marker RT-PCR assay for tyrosinase mRNA in the detection of melanoma sentinel lymph node (SLN) metastases and correlated the results with long-term clinical outcome. PATIENTS AND METHODS: One hundred twelve patients who underwent SLN biopsy for melanoma were prospectively analyzed. SLNs were bivalved, with half of each specimen evaluated by histologic methods and the other half evaluated by nested RT-PCR for tyrosinase. RESULTS: Fifteen patients (13%) had histologically positive SLNs, all of whom were also positive by RT-PCR (HISTO+/PCR+). Thirty-nine patients (35%) had SLNs that were negative by both histology and RT-PCR (HISTO-/PCR-). Fifty-eight patients (52%) were histologically negative but upstaged with a positive RT-PCR result (HISTO-/PCR+). Initially, at a median follow-up of 42 months, recurrence rates among the three cohorts were statistically different (HISTO+/PCR+, 53%; HISTO-/PCR+, 14%; and HISTO-/PCR-, 0%). However, at a longer median follow-up (67 months), recurrence rates for the HISTO-/PCR+ (24%) and HISTO-/PCR- (15%) groups were no longer statistically different (P =.25). The median time to relapse between the HISTO-/PCR+ and HISTO-/PCR- groups differed by 10 months (31 v 41 months, respectively). CONCLUSION: With extended follow-up of patients with histologically negative SLNs, detection of submicroscopic disease by tyrosinase RT-PCR does not define a subgroup that is at higher recurrence risk when compared with patients with RT-PCR-negative SLNs. Future studies evaluating molecular staging will require approximately 5 years of median follow-up to accurately define outcome for patients with occult melanoma metastases.

Adolescent↗
PubMed1997

Clinical application of minimal residual neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction.

The highly sensitive method to detect neuroblastoma (NB) cells using reverse transcriptase polymerase chain reaction (RT-PCR) was applied in the practical clinics, and its efficacy was assessed in the present study. Human tyrosine hydroxylase (TH), a rate-limiting enzyme in the catecholamine biosynthesis, was used as the marker for NB cells, and the expression of THmRNA was examined in 13 samples (four peripheral blood and nine bone marrow) harvested from seven patients (four with stage IV, one with stage III, two with stage II) using RT-PCR with our original primers. The positive signals for NB cells were detected in four samples (one peripheral blood and three bone marrow) by the PCR method, but were undetectable by the conventional histological examinations. In the present series, a case that showed a positive signal for NB cells in the peripheral blood showed a remarkably unfavorable clinical course, indicating that the circulating NB cells detected by the PCR method can be a sign of the progressively advanced NB, and may define a new prognostic factor suggesting higher risk. In another case, the PCR detection for the residual NB cells in the bone marrow provided important supporting evidence to determine the necessity of the additional chemotherapy and the suitable timing for bone marrow transplantation. This detection also guaranteed the safety of the bone marrow for transplantation. The PCR method was considered to be very beneficial in the selected cases. However, some problems such as the false-negative results in the negative urinary vanillylmandelic acid secretor were also highlighted in the present study.

3-Iodobenzylguanidine↗
PubMed2001

Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA.

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis.

Adenocarcinoma↗
PubMed2001

A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells.

A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis.

Animals↗
PubMed2001

Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous.

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

Adipocytes↗
PubMed2002

Definition of three minimal deleted regions by comprehensive allelotyping and mutational screening of FHIT,p16(INK4A), and p19(ARF) genes in nasopharyngeal carcinoma.

BACKGROUND: Recurrent deletion on a chromosomal location in tumor cells can be detected by frequent allelic loss and generally is considered to be an indication of the existence of a tumor suppressor gene (TSG) in the region. In the current study, using fluorescent-labeled, high-density microsatellite markers for allelotyping, the authors pinpointed three minimal deleted regions (MDRs) and screened mutations of putative TSGs on chromosomes 3, 9, and 11 in nasopharyngeal carcinoma (NPC) cases occurring in Taiwan. METHODS: A total of 133 informative microsatellite markers were used on chromosomes 3, 9, and 11 with an average marker density of 4 centimorgans (cM) for the allelotyping of genomic DNAs isolated from NPC tissues and their corresponding lymphocytes in 48 patients. The correlation between allelic loss and the clinicopathologic parameters of NPC tissues was examined. In addition, putative TSGs including FHIT, p16(INK4a), and p19(ARF) were selected for mutation screening to investigate their potential participation in NPC tumorigenesis. RESULTS: Of 3787 informative allelotyping data, 25 frequent allelic losses (or loss of heterozygosity [LOH]) in 13 cytogenetic loci were identified based on a deletion frequency that was greater than the average of allelic loss on that particular chromosome. Several significant associations were determined after statistical analysis of the correlation between allelic loss and clinicopathologic parameters. The allelic losses by D9S318 and D11S1304 were associated with N2/N3 (P = 0.035 and P = 0.005, respectively), and those by D9S905 and D11S1304 were associated with grouped American Joint Committee on Cancer (AJCC) Stage III/IV samples (P = 0.022 and P = 0.017, respectively) of NPC tissues. In addition, three MDRs were revealed on 3p25.3-24.1 (< 19 cM), 3p23-21.31 (< 9 cM), and 11q22.1-23.2 (< 8 cM). To examine somatic mutations in previously reported TSGs located near these frequent LOH loci, three candidate genes, p16(INK4a), p19(ARF), and FHIT, were analyzed. Point mutations in the coding region of FHIT and in the intron 1 splicing acceptor site of both p16(INK4a) and p19(ARF) were detected in NPC cell lines. Sequence analysis of both p16(INK4a) and p19(ARF) transcripts revealed that the point mutation resulted in skipping of exon 2 and the generation of shorter transcripts. CONCLUSIONS: High-density allelotyping permitted the discovery of 3 MDRs on 3p25.3-24.1 (< 19 cM), 3p23-21.31 (< 9 cM), and 11q22.1-23.2 (< 8 cM) and a correlation was determined between allelic loss and clinicopathologic parameters of NPC tissues. More important, one somatic mutation in NPC cell lines on the intron 1/exon 2 splicing acceptor site of the INK4a/ARF locus was found to result in exon 2 skipping both p16(INK4a) and p19(ARF) transcripts, which presumably inactivates the functions of both the p16(INK4a) and p19(ARF) proteins.

Acid Anhydride Hydrolases↗
PubMed2004

Identification of ciliary epithelial-specific genes using subtractive libraries and cDNA arrays in the avian eye.

The ciliary epithelium of the ciliary body is derived from the anterior rim of the developing optic cup. Several recent studies have found that developmental abnormalities in this tissue can underlie congenital glaucoma. However, there is little known about the development of the ciliary epithelium. To better understand the developmental events responsible for the specification of this domain of the optic cup, we used a subtractive library, differential screening approach along with the construction of cDNA arrays to identify genes expressed in the ciliary epithelium of the chicken. We identified many genes specifically expressed in the ciliary epithelium, including a number that had been described previously as enriched in the ciliary epithelium of other species. By analyzing the expression of these genes during eye development, we were able to correlate the onset of ciliary epithelial gene expression with a reduction in mitotic activity in this region. We propose that the mechanisms that regulate the expression of ciliary epithelial genes are linked to the reduction in proliferation that results in the epithelial monolayer in this region.

Animals↗
PubMed2006

Scanning copy number and gene expression on the 16p13.3-13.2 chromosomal region by the systematic multiplex polymerase chain reaction and reverse transcription-polymerase chain reaction methods.

We developed the systematic multiplex reverse transcription-PCR (SM RT-PCR) method that is distinguishable from other multiplex RT-PCR methods by optimized PCR conditions allowing amplification of sequences that fall within a single exon of genes of similar band intensity using genomic DNA template as a calibration standard. Using an SM RT-PCR system of proto-oncogenes and tumor suppressor genes, we previously showed that the SM RT-PCR system, which was developed for cDNA expression analysis, could also be used for a more exquisite analysis of copy number changes in genomic DNA. Here we report that the SM PCR method semiquantitatively detected less than a two-fold difference in copy number. Furthermore, we also report the results of subchromosomal scanning of copy number and expression using the SM PCR and SM RT-PCR methods. We identified and characterized the novel homozygous deletion that spans over 12-plus genes on 16p13.3-13.2 in the MDA-MB-468 breast cancer cell line.

Chromosomes, Human, Pair 16↗
PubMed2002

Genomic gains and losses are similar in genetic and histologic subsets of rhabdomyosarcoma, whereas amplification predominates in embryonal with anaplasia and alveolar subtypes.

In this investigation, we selected PAX3/FKHR and PAX7/FKHR fusion transcript-positive and -negative alveolar rhabdomyosarcomas (ARMSs) and embryonal rhabdomyosarcomas (ERMSs) with and without anaplastic features, to ascertain genomic imbalance differences and/or similarities within these histopathologic and genetic rhabdomyosarcoma (RMS) variants. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies were performed on 45 rhabdomyosarcoma specimens consisting of 23 ARMSs and 22 ERMSs (12 ERMS cases were included from an earlier study). The anaplastic variant of RMS has not previously been subjected to CGH analysis. Overall, the most prominent imbalances were gain of chromosomes or chromosomal regions 2/2q (40%), 7/7q (31%), 8/8p (53%), 11/11q (31%), 12q13-15 (49%), 13q14 (22%), and 20/20p (31%), and loss of 1p36 (27%), 3p14-21 (22%), 9q21-22 (33%), 10q22-qter (18%), 16q (27%), 17p (22%), and 22 (22%). These gains and losses were distributed equally between ARMS and ERMS histologic subtypes (excluding 7/7q and 11/11q gain that were observed chiefly in ERMS), demonstrating that these entities are similar with respect to recurrent genomic imbalances. Moreover, genomic imbalances were also evenly distributed among the ARMS fusion transcript subtypes, providing evidence for a genetic kinship despite the absence of a fusion transcript in some cases. Genomic amplification was detected in 26% and 23% of the ARMS and ERMS cases, respectively (with nearly all of the latter subset exhibiting anaplastic features). One amplicon, involving 15q25-26, corresponds to the locus of the insulin-like growth factor type I receptor (IGF1R) gene. Amplification of IGF1R was confirmed molecularly in the cases exhibiting a 15q25-26 amplicon. In summary, these results indicate that genomic gains and losses involve alike chromosomes with similar frequencies within the histopathologic and genetic subtypes of rhabdomyosarcoma, that genomic amplification is frequent not only in the alveolar histologic subtype of rhabdomyosarcoma but also in ERMS with anaplasia, and that amplification of IGF1R possibly plays a role in the development or progression of a subset of rhabdomyosarcomas.

Adolescent↗
PubMed2003

Metaphase FISHing of transgenic mice recommended: FISH and SKY define BAC-mediated balanced translocation.

The evolving trend to use larger transgenes and their associated increased chance of unexpected genetic events mandates more careful characterization of transgenic mice. In characterizing our five new mouse strains transgenic for the BAC, bEMS4, we have identified the highest copy number reported to date: the stable incorporation of approximately 40 copies of a 194-kb expressed transgene in a single insertion site. We caution, however, that standard molecular techniques failed to identify a balanced translocation in another strain, and an inappropriate site of insertion in a third. Molecular cytogenetic analysis using metaphase FISH was the minimum level of characterization needed to reveal these unexpected genetic events. In addition, we combined FISH and SKY to identify the transgene at the breakpoints of the balanced translocation, t(3;9). This is the first description of a BAC-mediated chromosomal rearrangement and the first application of SKY to identify transgene-induced chromosomal rearrangements.

Animals↗
PubMed2001

Deletion mapping of chromosome 16q24 in hepatocellular carcinoma in Taiwan and mutational analysis of the 17-beta-HSD gene localized to the region.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.

17-Hydroxysteroid Dehydrogenases↗
PubMed2005

Endostatin inhibits nitric oxide and diminishes VEGF and collagen XVIII in squamous carcinoma cells.

Low pO(2) values are a common finding among oral squamous cell carcinomas (SCC). Our objective was to determine the role that oxygen tension plays on the direct tumor effect of endostatin (ES). Squamous carcinoma cell lines were grown under normoxic or hypoxic conditions and treated with endostatin (ES), nitric oxide (NO) donors, NO scavengers, NO synthase inhibitors, or transduced with AdenoVec-hEndo or AdenoVec Null vectors. The expression of vascular endothelial growth factor (VEGF) and collagen XVIII were determined by RT-PCR and protein levels assessed by Western blot analyses. Our studies demonstrated that collagen XVIII and VEGF are expressed and responsive to ES in a limited number of SCC cell lines during normoxia but were most responsive when grown under hypoxic conditions. VEGF and collagen XVIII were downregulated by both ES and transduction of cells with AdenoVec-hEndo. The effects of ES on SCC cells were enhanced by aminoguanidine (Ag), L-NAME, and diphenyleneiodonium chloride (DPI). Endostatin and transduced with ES vectors diminished the levels of NO whereas NO donors enhanced VEGF expression and collagen XVIII expression. In conclusion, the direct effect of endostatins on tumor cells is most effective under conditions of low oxygen tension and can be potentiated by the use of nitric oxide synthase inhibitors or NO scavengers.

Angiogenesis Inhibitors↗
PubMed2005

Aberrant methylation of Reprimo in human malignancies.

Reprimo is a new candidate mediator of p53-mediated cell cycle arrest at the G2 phase. Loss of Reprimo gene expression accompanied by its promoter methylation was identified in pancreatic and lung cancers. Our aim was to examine the methylation status of Reprimo in a broad range of cancers. We examined Reprimo expression by RT-PCR and the DNA methylation status of the Reprimo promoter by MSP in 39 tumor cell lines. Loss or downregulation of Reprimo expression was frequent (62%), and we confirmed that transcriptional repression of Reprimo was caused by hypermethylation (overall concordance 92%). Treatment of expression-negative cells with 5-aza-2'-deoxycytidine restored Reprimo expression. We then examined aberrant methylation of Reprimo in 645 tumors representing 16 tumor types. Promoter methylation of Reprimo was found in 79% of gastric cancers, 62% of gallbladder cancers, 57% of lymphomas, 56% of colorectal cancers, 40% of esophageal adenocarcinomas, 37% of breast cancers and 31% of leukemias. Methylation frequencies in ovarian cancers, bladder cancers, cervical cancers, brain tumors, malignant mesotheliomas and pediatric tumors were lower (0-20%). Reprimo methylation was rarely detected in nonmalignant tissues (0-11%) except for gastric epithelia. While colorectal polyps were also frequently methylated (27%), chronic cholecystitis samples were infrequently methylated (4%). Furthermore, we failed to identify Reprimo mutation in colorectal and gastric cancer cell lines and 50 primary colorectal cancers. Aberrant methylation of Reprimo with loss of expression is a common event and may contribute to the pathogenesis of some types of human malignancy.

Adult↗
PubMed2002

Profiling of retinoid mediated gene expression in synchronized human SCC cells using Atlas human cDNA expression arrays.

While retinoids have been demonstrated to inhibit growth of many tumor cells, including SCC cells, the molecular mechanism by which retinoids suppress growth has not been elucidated. We previously found that the growth of SCC cells was significantly inhibited by all-trans-retinoic acid (all-trans-RA) treatment, and this inhibition was dependent on the binding and activation of RARs. These nuclear receptors bind retinoids and alter the rate of transcription of specific genes. To identify targets of the activated RARs which mediate growth inhibition, we growth arrested SCC-25 cells in G-0 and examined the effect of all-trans-RA on synchronized SCC-25 cells. All-trans-RA inhibited G-1 progression in quiescent SCC-25 cells stimulated by FBS. More specifically, we found that the all-trans-RA execution point maps to mid/late G-1, 6 to 10 h after stimulation. Using this synchronized cell system, we examined the expression of cell cycle regulatory genes in quiescent SCC-25 cells stimulated with FBS and treated with all-trans-RA. We found few changes in expression of these genes which could account for all-trans-RA inhibition of SCC-25 cell growth. In order to compare the patterns of expression of a wider selection of genes in all-trans-RA treated and non-treated SCC-25 cells, we have used expression array technology. We successfully performed expression profiling experiments on the Atlas Human cDNA arrays which contain 1176 human genes. We have identified several up-regulated and several down-regulated gene expression changes mediated by all-trans-RA treatment in synchronized SCC-25 cells. This novel information will be useful in defining the mechanism by which retinoids suppress the growth of SCC cells.

Aged↗
PubMed2003

Genomic and phylogenetic analysis of hepatitis C virus isolates: a survey of 535 strains circulating in southern France.

The present study examines the distribution of Hepatitis C virus (HCV) genotypes in Marseille, France in 2001-2002 and evaluates the efficiency of two in house direct sequence PCR protocols based on 5'NC analysis or NS5B analysis. By 5'NC sequencing, the distribution of 535 HCV strains derived from patients attending gastroenterology and AIDS referral centers, or dialysis units was as follows: 33% were infected by genotype 1a; 26% by 1b; 7% by 2; 22% by 3a; 10.7% by 4. In univariate analysis, HCV distribution was associated with age and source of infection, whereas in multivariate analysis only injecting drug use was an independent determinant for genotype distribution. Among the 535 specimens submitted to 5'NC direct sequencing, 18% could not be classified accurately into subtypes. A subset of 187 samples was amplified efficiently and sequenced by targeting the NS5B region of the viral genome. The two methods yielded concordant results in 70% of cases. Specimens unsubtypeable or misclassified most frequently by 5'NC analysis were type 1b and subtypes 2a/2c and 4a/4c. The data show that 5'NC direct sequence analysis is a sensitive method to identify genotypes in all cases, but that it can lead to subtyping misclassification (in particular, subtype 1b and 1a) or doubtful results (in particular subtypes 2a/2c and 4a/4c). Conversely, NS5B direct sequence assay, based on phylogenetic analysis, allowed better discrimination between subtypes. These two approaches are complementary and should be made available in clinical laboratories to ensure a reliable survey of HCV strains.

5' Untranslated Regions↗
PubMed2006

Serological and molecular epidemiology of measles virus outbreaks reported in Ethiopia during 2000-2004.

Twenty-eight outbreaks in six regions and two major cities in Ethiopia from 2000 to 2004 were investigated, with the collection of 207 venous blood and/or oral fluid samples. Measles diagnosis was confirmed by detection of measles-specific IgM and/or detection of measles virus by polymerase chain reaction (PCR). Of 176 suspected cases tested for specific measles IgM, 142 (81%) were IgM positive. Suspected cases in vaccinated children were much less likely to be laboratory confirmed than in unvaccinated children (42% vs. 83%, P < 0.0001). Of 197 samples analyzed by RT-PCR measles virus genome was detected in 84 (43%). A total of 58 wild-type measles viruses were characterized by nucleic acid sequence analysis of the nucleoprotein (N) and hemagglutinin (H) genes. Two recognized genotypes (D4 and B3) were identified. Each outbreak comprised only a single genotype and outbreaks of each genotype tended to occur in distinct geographical locations. B3 was first observed in 2002, and has now been the cause of three documented outbreaks near to the border of Sudan. D4 genotype was previously observed in an outbreak in 1999 and occurs in more diverse locations throughout the country. These data yield insights into geographical and age-related sources of continued transmission. Refinement of measles control measures might include targeting older age groups (5-14 years) and strengthening routine immunization particularly where importation of cases is a concern.

Adolescent↗
PubMed2002

Detection and identification of dengue-1 virus in clinical samples by a nested-PCR followed by restriction enzyme digestion of amplicons.

Dengue viruses cause a disease with clinical findings ranging from asymptomatic infections to severe manifestations, characterised by haemorrhage and shock and known as dengue haemorrhagic fever/dengue shock syndrome. Since this fever and syndrome usually results from sequential infections by distinct dengue serotypes, rapid detection and identification of dengue viruses circulating in endemic areas are important to implement control measures, and ultimately to avoid secondary infections that could result in dengue haemorrhagic fever/dengue shock syndrome. A nested-PCR was developed followed by restriction enzyme (Kpn I) digestion of the amplicons to differentiate dengue-1 from dengue-2. Seventy-five IgM-containing samples collected from 2 to 17 days after the beginning of the symptoms were examined. These samples were submitted to nested-PCR amplification, the amplicons were digested with Kpn I, and the results compared to virus isolation in C6/36 cells and to results obtained by the standard PCR. Out of 75 tested samples, virus was isolated from 2 (2.6%), 17 (22.7%) were positive by the regular PCR protocol, and 58 (77.3%) were positive by nested-PCR. All of the amplicons digested by Kpn I identified dengue-1 virus as the infecting strain. These results indicate that the nested-PCR provided a high yield of dengue genome amplification even in the presence of IgM antibodies, and restriction enzyme digestion defined rapidly the circulating serotype. Therefore, the combination of these techniques may be useful to rapidly identify dengue viruses in countries where dengue-1 and dengue-2 circulates, and this approach can also be applied to the other two serotypes.

Dengue↗