Tool For Molecular Diagnosis Research Articles

A Membrane-Based Flow-through Hybridization Technology: A Rapid and Versatile tool for Molecular Diagnostics

Genetic diseases remain an important cause of ailment, morbidity and death for children and adults. Despite the recent advances of tools for molecular diagnosis such as DNA chips and protein arrays which are capable of analyzing many genes, SNPs and proteins, few of these systems have been applied in clinical diagnosis because the apparatus are still expensive and many of the genetic tests are less likely to be developed commercially. Hence most genetic tests are still being performed through conventional methods such as Dot-Blot hybridization and Slot-blot hybridization in clinical laboratories worldwide. The expensive, time consuming and labor-intensive nature of these tests make them unaffordable for popular use. The availability of a low-cost diagnostic system will make molecular testing cost efficient and become readily available worldwide. In this report, we evaluated the Flow-through hybridization system. Our results show that Flow-through hybridization method produces higher sensitivity in 5 to 15 minutes compared to those obtained through conventional hybridization in hours to overnight respectively without sacrificing specificity. An apparatus can also be readily adopted for membrane-based home-brew tests, R&D development as well as other commercially available diagnostic kits, hence making the Flow-through method a useful tool for molecular diagnosis.

Using SELDI‐TOF MS to identify serum biomarkers of rheumatoid arthritis

Objectives: No satisfactory biomarkers are currently available to screen for rheumatoid arthritis (RA). We have developed and evaluated surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry (SELDI‐TOF MS) for detection and analysis of multiple proteins for distinguishing individuals with RA from control individuals.Methods: A total of 156 serum samples from 90 RA patients, 30 patients with ankylosing spondylitis (AS), and 36 healthy individuals were examined by SELDI technology. Spectral data were analysed by the support vector machine (SVM) approach and potential biomarkers were chosen for system training and were used to construct a diagnostic model.Results: Pattern 1, consisting of four protein peaks with m/z values of 3899, 4594, 7566, and 13 842, distinguished RA from the healthy samples with sensitivity of 90.0% and a specificity of 91.7%. Pattern 2, consisting of m/z peaks 4287 and 6471, distinguished RA from AS with a sensitivity of 86.7% and a specificity of 85.0%.Conclusion: The combination of SELDI‐TOF MS and SVM could facilitate the discovery of better biomarkers for RA and also provide a useful tool for molecular diagnosis in the future.

Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

BackgroundA finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.ResultsWe propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled.ConclusionOur work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

Molecular Detection and Differentiation of Infectious Bursal Disease Virus

Vaccination of hens, with the subsequent maternal immunity imparted to chicks, is the primary means of controlling infectious bursal disease virus (IBDV). Effective vaccination depends on rapid and accurate diagnosis of the subtype present in a flock because vaccines based on the classic subtype of IBDV can fail to protect against challenge with a variant subtype. This review describes the various methods available to detect and differentiate between IBDV subtypes. Serotype 1 IBDV causes economically significant immunosuppressive disease in young chickens. Within serotype 1, two subtypes, classic and variant, can be differentiated by the virus neutralization assay. Antigen capture enzyme-linked immunosorbent assay (AC-ELISA) with MAbs has been successful at differentiating the very virulent IBDV phenotype (vvIBDV) from less pathogenic types. More rapid and sensitive molecular diagnostic methods based on reverse transcription-polymerase chain reaction (RT-PCR) for amplification of the IBDV VP2 gene have been a major focus of investigation in recent years. Conventional RT-PCR has been useful in detecting IBDV serotypes and, to a lesser extent, differentiating IBDV subtypes. One of the approaches has been the use of SspI and NgoM IV restriction enzymes, for restriction endonuclease (RE) analysis of RT-PCR products (RT-PCR-RE) and BstNI and MboI for restriction fragment length polymorphism (RFLP) analysis (RT-PCR-RFLP) to find unique banding patterns associated with antigenic variation within the variable region of the IBDV VP2 protein. However, these approaches were ultimately found to be unreliable because subtypes could not be consistently distinguished with restriction enzymes. These limitations led to studies in differentiating subtypes by detection of single nucleotide differences in sequence through real-time RT-PCR or DNA sequencing of RT-PCR products. Conventional RT-PCR, amplifying the VP2 hypervariable region, in combination with DNA sequencing of the PCR product, can differentiate classic, variant, and vvIBDV strains because variant and vvIBDV have characteristic nucleotide and amino acid substitutions. Real-time RT-PCR, targeting different regions of the IBDV genome, including VP1, VP2, and VP4 genes, in conjunction with melting-curve analysis is being investigated as a promising tool for molecular diagnosis of IBDV infection. These methods potentially allow for more rapid, sensitive, and specific detection and differentiation of IBDV classic, very virulent, and variant subtypes.

Dye-doped nanoparticles for bioanalysis

Understanding biological processes at the molecular level with accurate quantitation needs advanced bioanalysis. In this review, we describe dye-doped silica nanoparticles (NPs), their synthesis, bioconjugation, and applications in different bioanalysis formats. Silica-based nanomaterials have been developed with optical-encoding capabilities for the selective tagging of a wide range of biomedically important targets, such as bacteria, cancer cells, and individual biomolecules. We also briefly review other closely related nanomaterials, such as quantum dots, Au NPs, and magnetic NPs. We envisage that further development of these NPs will provide a variety of advanced tools for molecular biology, genomics, proteomics, drug discovery, and diagnosis and therapy of infectious disease and cancer.

Molecular Identification and Genetic Analysis of Norovirus Genogroups I and II in Water Environments: Comparative Analysis of Different Reverse Transcription-PCR Assays

Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.

EDITORIAL PREAMBLE: Molecular biology tools in clinical diagnosis

The discovery of Polymerase Chain Reaction (PCR) by Kary Mullis (US Patent Number 4,683,202) triggered an unprecedented race for more specific, rapid and highly sensitive diagnosis methods in the clinical laboratory. A wide spectrum of infectious diseases and genetic disorders are now routinely tested using in-house designed or commercially available nucleic acid amplification tests and nucleic acid detection systems. Molecular biology testing can now aid in infection control interventions by accurately and rapidly determining antimicrobial resistance. Implementation of molecular diagnosis in clinics is not without the pains of labor. Experimental set-up, finding the ‘gold standard’ for results confirmation, dealing with standardization of DNA and RNA extraction and cross-contamination are among the problematic issues and logistic challenges often difficult to overcome in this process. High-throughput analytical platforms have been implemented in certain more sophisticated laboratories and lead to cost-effective testing and rapid turnaround time. The development of molecular techniques for diagnosis and also typing of potential agents of bioterrorism or emerging pathogens is also part of a larger strategy to develop rapid and sensitive testing while circumventing safety concerns related to growing these agents in culture. Acknowledging the rapid pace of application of molecular biology tools in clinical diagnosis, the Journal of Cellular and Molecular Biology is introducing a new section to highlight the present state of the art of molecular diagnostics in the clinical area. Manuscripts to be considered for this section should address the clinical application of molecular diagnosis of infectious diseases and genetic disorders including nucleic acid amplification tests, proteomics, microarray analysis, Human Genome Project, as well as clinical laboratory automation and validation methods. Kary Mullis (inventor of PCR and 1993 Nobel prize winner) and Dana Perkins (Assistant Editor, JCMM), 2003, National Inventors Hall of Fame, Akron, OH. Two papers in the field of molecular diagnosis are published in this issue [1, 2].

Circulating (cell-free) nucleic acids – A promising, non-invasive tool for early detection of several human diseases

Circulating nucleic acids (CNA) are present in small amounts in the plasma of healthy individuals. However, increased levels of plasma CNA have been reported in a number of clinical disorders like cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. CNA has received special attention because of its potential application as a non-invasive, rapid and sensitive tool for molecular diagnosis and monitoring of acute pathologies and the prenatal diagnosis of fetal genetic diseases. This review throws light on the current status of blood CNA as a diagnostic marker and its potential as a powerful tool in the future.

A simplified reverse transcriptase PCR for rapid detection ofMycobacterium lepraein skin specimens

An RNA-based assay is an additional molecular tool for leprosy diagnosis and determination of the viability of leprosy bacilli. To simplify RNA detection, a one-step reverse transcriptase PCR (RT-PCR) was established and evaluated. RNA and DNA could be isolated simultaneously. With the use of Mycobacterium leprae-specific primers targeting a 171-bp fragment of the M. leprae 16S RNA gene, RT-PCR resulted in detection of M. leprae in both slit skin smears and skin biopsy specimens. To enhance the positive signal, a digoxigenin-labeled DNA was developed, and successfully detected the amplified RT-PCR product. The method is sensitive, as it could detect one leprosy bacillus. When it was used directly on skin specimens collected from leprosy patients, 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases showed positive results. The assay was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the multidrug therapy for 6 months, resulting in bacterial clearance, 16 of 36 MB patients and three of 24 PB patients tested were still positive for the 16S rRNA gene of M. leprae, suggesting the advisability of a more prolonged treatment course. This form of RT-PCR is of value in terms of simplicity and sensitivity in identifying M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.

Application of serum protein fingerprint in diagnosis of papillary thyroid carcinoma

To find new biomarkers and establish serum protein fingerprint models for early diagnosis and preoperative staging of papillary thyroid carcinoma, we employed SELDI-TOF-MS and bioinformatics tools. A total of 116 samples were analyzed in this study. The first 80 samples were analyzed by SELDI-TOF-MS and two biomarker patterns were identified. Pattern 1 distinguishes patients with papillary thyroid carcinoma from healthy individuals. Pattern 2 distinguishes papillary thyroid carcinoma from benign thyroid nodes. The remaining 29 samples were analyzed on the second day and served as an independent test set. The analysis of this independent test set yielded a specificity of 80.0% and a sensitivity of 88.9% for pattern 1 and a specificity of 80.0% and a sensitivity of 80.0% for pattern 2. Two additional biomarker patterns were identified to distinguish different stages of the papillary thyroid carcinoma (pattern 3) with an accuracy of 77.1% and different pathological types of thyroid carcinoma (pattern 4) with an accuracy of 88.1%. Taken together, the SELDI-TOF-MS technique combined with bioinformatics approaches can not only facilitate the discovery of better biomarkers for papillary thyroid carcinoma but also provide a useful tool for molecular diagnosis in the future.

Validation of RT‐PCR Assays for Molecular Characterization of Porcine Teschoviruses and Enteroviruses

Porcine enteroviruses (PEVs) and teschoviruses (PTVs) are described as causative agents of neurological disorders, fertility disorders and dermal lesions of swine. Difficulties in the serological detection of these viruses may lead to a significant underestimation of infections with clinical symptoms. With the recent availability of genome sequence data for all the serotypes, molecular diagnosis is a possibility. The present study describes a new approach to molecular 'serotyping' of PTVs and PEV-B viruses, involving the amplification and sequencing of a genomic fragment of the VP1 coding region. A molecular characterization of Italian entero-teschovirus isolates was performed using a set of previously published and newly designed polymerase chain reaction primers. A total of 33 porcine isolates and 10 reference strains were analysed. Porcine enterovirus-B samples were first diagnosed as positive for enterovirus by amplification of the 5'-non-translated region. Samples were then typed by amplification and sequencing of a portion of the VP1 coding region. Porcine enterovirus-A and PTVs were detected by a published assay in the 5'-NC region that allows them to be differentiated according to the size of amplification product, using the same set of primers. For serotype characterization of PTV, we evaluated four different regions: the N terminus of the capsid protein VP2, the region encoding for RNA-dependent RNA polymerase, and the capsid VP1 and VP4 regions. The newly designed primers in the VP1 region was proved to be broad in range and suitable for serotype assessment and therefore constitute a useful diagnostic tool for molecular diagnosis of porcine teschovirus/enterovirus strains and for the study of molecular epidemiology and evolution of these viruses.

Combined Detection of CEA, CK-19 and c-met mRNAs in Peripheral Blood: A Highly Sensitive Panel for Potential Molecular Diagnosis of Non-Small Cell Lung Cancer

Objective: Detection of tumor-related mRNA in blood has become a potential cancer diagnostic approach. However, the sensitivity of single-marker assays is not high enough for clinical applications. The present study was aimed to evaluate the efficacy of a multimarker panel for molecular diagnosis of non-small cell lung cancer (NSCLC). Methods: Carcinoembryonic antigen (CEA), cytokeratin 19 (CK-19), c-met and heterogeneous nuclear ribonucleoprotein (hnRNP) B1 mRNAs were quantified by quantitative real-time reverse transcriptase polymerase chain reaction in 34 tumor tissues and 69 peripheral blood samples of NSCLC patients. Results: All four markers displayed high overexpression rates (range 82.3–97.1%) in NSCLC tumors. When used as single markers in blood for NSCLC diagnosis, CEA, CK-19, c-met and hnRNP B1 could only reach sensitivities of 52.2, 50.7, 42 and 17.4%, respectively. However, the sensitivity was enhanced up to 85.5% when CEA, CK-19 and c-met were combined in a 3-marker panel. Moreover, the expression of c-met and hnRNP B1 in blood was significantly correlated with patients’ pathological stages. Conclusions:The combined detection of CEA, CK-19 and c-met mRNAs in blood provided a valuable tool for molecular diagnosis of NSCLC. In addition, our results also suggested that hnRNP B1 was not a valuable diagnostic marker but a potential prognostic marker for NSCLC.

Methylation of circulating DNA in melanoma patients

8027 Background: DNA methylation is a well-known epigenetic phenomenon that occurs in higher order eukaryotes. Changes in the status of DNA methylation of tumor suppressor genes (TSG) is one of the common molecular alterations in many carcinomas. Methods: The purpose of this study was to investigate hypermethylation status of five different genes (TSG and cell cycle genes) involved in tumor suppression and DNA repair. Results: Serum before therapeutic intervention from 32 melanoma patients (stage I = 13; stage II = 9; stage III/IV = 10) was collected, cell-free DNA was isolated and sodium bisulfite conversion of genomic DNA was performed. The methylation status of CpG islands in the promotor region of SOCS1, SOCS2 (suppressors of cytokine signaling 1 and 2); RASSF1a (Ras-association domain family protein 1A); CDKN (D-type p16INK4a cyclin-dependent kinase inhibitor); and MGMT (O6-methylguanine DNA-methyltransferase) were analyzed using methylation specific-PCR (MSP). For comparison, sera from healthy controls (n = 20) and patients with other skin tumors (9 basal cell cancers, 5 Kaposi’s sarcoma) as well as different cancers (5 breast cancers, 5 colon cancers) were also analyzed. In addition, we examined if methylation was involved in silencing of these genes in 20 fresh melanoma specimens and we confirmed the hypermethylation status of SOCS2 using positional methylation analysis (pyrosequencing). Moreover, the expression of these genes was investigated cell lines (BLM, A375, MV3, and M13). Analysis of sera from cutaneous melanoma patients demonstrated circulating hypermethylated SOCS 1 in 59%, SOCS2 in 53%, RASSF1a in 53%, CDKN2a in 50%, while the MGMT did not show clear methylation pattern. Serum of healthy controls showed no methylation of any of the analyzed genes. The mRNA transcript of these genes showed a clear and significant downregulation, except for MGMT, for which an up regulation (7.3 times) was detected being consistent with the lack of promotor methylation. Detailed positional methylation analysis (pyrosequencing) has been performed for these genes and was found to correlate with mRNA expression in the tumor specimens in most cases. Conclusions: Analysis of positional methylation of circulating cell-free DNA seems to be a novel tool for molecular diagnosis in melanoma. No significant financial relationships to disclose.

Analysis of gene probes and gene amplification techniques for diagnosis and monitoring of treatment in childhood leprosy

Nucleic acid sequences of Mycobacterium leprae were detected using gene probes hybridizing with targeting ribosomal RNA (16S rRNA), ribosomal DNA (16S rDNA) and gene amplification techniques (PCR) in skin lesion of paediatric leprosy patients and the effect of treatment on the by these methods. Eighty paediatric leprosy patients were included in the study. Most cases (79%) were between 9 and 16 years of age. Cases were divided into three groups according to treatment status, viz. untreated (30), undergoing treatment (30), and at the end of treatment (20). Clinical and slit smear examination for acid fast bacilli (AFB) was performed and nucleic acids were extracted and fractionated from skin biopsies. M. leprae specific 16S rRNA and 16S rDNA was detected by hybridization with gene probes whereas the 36 kDa gene sequence was detected by a gene amplification assay (PCR). The cases were classified as paucibacillary (PB) and multibacillary (MB) by the standard criteria of WHO (1988). Positivity of 16S rRNA in PB cases decreased from 60% in untreated to 10.5% after 4-8 months of treatment whereas for 16S r DNA, it decreased from 50% to 21%, for PCR from 70% to 36.8% for the same specimen, and all became negative at 1 year. Similar trends were seen in MB cases where positivity in smear positive untreated cases decreased from 100% to 56.2% with 16S rRNA and 42.8% with 16S rDNA and PCR, respectively, after 9-12 months of treatment and all became negative at 2 years, except one case which remained positive with PCR. Similar results were observed in smear negative MB cases, 100% positivity detected by 16S r RNA and PCR, 75% detected by 16S rDNA decreased to zero after 9-12 months of therapy. This study suggests the potential usefulness of gene probes targeting 16S rRNA and 16S rDNA and PCR as supportive molecular tools for diagnosis of smear negative evolving MB disease and also monitoring the response to treatment, these observations however, needs to be validated in prospective follow up studies.

MAPK Signaling 1: Development

Cardio-facio-cutaneous (CFC) syndrome is a rare disorder characterized by a distinctive facial appearance, skin abnormalities, heart defects, and growth delays. Rodriguez-Viciana et al. show that the disorder is caused by acquired mutations in genes encoding components of the mitogen-activated protein kinase (MAPK) signaling pathway. About 90% of the 23 patients studied carried missense mutations in the BRAF , MEK1 , or MEK2 genes that functionally altered the corresponding proteins. This discovery highlights the critical role of the MAPK pathway in human development and provides a tool for molecular diagnosis of CFC syndrome. P. Rodriguez-Viciana, O. Tetsu, W. E. Tidyman, A. L. Estep, B. A. Conger, M. Santa Cruz, F. McCormick, K. A. Rauen, Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 311 , 1287-1290 (2006). [Abstract] [Full Text]

Methylation of RAS association domain family protein 1A as a biomarker of lung cancer

Promoter hypermethylation is an important mechanism for silencing tumor-suppressor genes in cancer and a promising tool for development of molecular biomarkers. This study aimed to determine the prevalence of RAS association domain family protein 1A (RASSF1A) promoter hypermethylation in bronchial aspirates of patients with suspected lung cancer and to test whether this type of methylation assay could be used as a diagnostic adjunct to conventional cytology. Two hundred three bronchial aspirates from patients with suspected lung cancer were analyzed for RASSF1A hypermethylation by using a sensitive quantitative methylation-specific polymerase chain reaction (QMSP). RASSF1A hypermethylation was found in 88% (35 of 40), 28% (31 of 111), and 100% (6 of 6) of bronchial aspirates collected from patients diagnosed with small cell lung cancer, nonsmall cell lung cancer, and combined small cell lung cancer, respectively. No hypermethylation was detected in patients diagnosed with nonneoplastic lung disease (0 of 46). Depending on histologic subtype, up to 82% of cases presenting with a negative histology showed a positive methylation assay. The QMSP analysis of RASSF1A hypermethylation enabled a highly specific distinction between patients diagnosed with lung cancer and those with nonneoplastic lung disease. These results suggested that a QMSP assay is a promising molecular tool for diagnosis of primary lung cancer.

Meningitis due to Capnocytophaga canimorsus: Contribution of 16S RNA ribosomal sequencing for species identification

We report a case of Capnocytophaga canimorsus meningitis, which is distinguishable from previous reports by the use of 16S rRNA sequencing for species identification. Our case report highlights the interest of molecular new tools for bacteriological diagnosis of human infections.

Multiclass cancer classification and biomarker discovery using GA-based algorithms

The development of microarray-based high-throughput gene profiling has led to the hope that this technology could provide an efficient and accurate means of diagnosing and classifying tumors, as well as predicting prognoses and effective treatments. However, the large amount of data generated by microarrays requires effective reduction of discriminant gene features into reliable sets of tumor biomarkers for such multiclass tumor discrimination. The availability of reliable sets of biomarkers, especially serum biomarkers, should have a major impact on our understanding and treatment of cancer. We have combined genetic algorithm (GA) and all paired (AP) support vector machine (SVM) methods for multiclass cancer categorization. Predictive features can be automatically determined through iterative GA/SVM, leading to very compact sets of non-redundant cancer-relevant genes with the best classification performance reported to date. Interestingly, these different classifier sets harbor only modest overlapping gene features but have similar levels of accuracy in leave-one-out cross-validations (LOOCV). Further characterization of these optimal tumor discriminant features, including the use of nearest shrunken centroids (NSC), analysis of annotations and literature text mining, reveals previously unappreciated tumor subclasses and a series of genes that could be used as cancer biomarkers. With this approach, we believe that microarray-based multiclass molecular analysis can be an effective tool for cancer biomarker discovery and subsequent molecular cancer diagnosis.

Applications of molecular methods for Leishmania control

This article reviews the recent advances made in the field of human leishmaniasis. Special emphasis is placed upon the application of various molecular tools for accurate and rapid diagnosis, understanding the mechanisms of drug resistance and identification of vaccine candidates. The focus will be on the major role played by recombinant antigens in the immunoserodiagnosis and progress of the Leishmania genome project, which has enabled researchers to design better PCR primers and molecular probes for microarrays. A special interest is placed on the recombinant antigen (rK39) cloned from the Leishmania chagasi kinesin gene and a very recently cloned recombinant antigen (KE16) from the Old World Leishmania donovani species with high sensitivity and specificity. Advances made in the specific PCR primer designed to diagnose and differentiate various species and strains of Leishmania causing visceral and post-kala-azar-dermal leishmaniasis have been covered. Molecular methods (e.g., DNA and protein microarrays) applied to understanding the pathobiology of the parasite, mechanism of host invasion, drug interaction and drug resistance to develop effective therapeutic molecules, gene expression profiling studies that have opened doors to understand many host–parasite relations, effective therapy and vaccine candidates are extensively covered in this review.

KAPOSI'S SARCOMA–ASSOCIATED HERPESVIRUS: Clinical, Diagnostic, and Epidemiological Aspects

Kaposi's sarcoma–associated herpesvirus (KSHV) is one of the few viruses proven to be associated with tumorigenesis in humans. Its causal association with all clinical and epidemiological variants of Kaposi's sarcoma (KS) is well established. KSHV is also involved in the pathogenesis of primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease (MCD). Possible associations of KSHV with other clinical settings have been extensively examined. The findings from several of these studies are contradictory and are yet to be resolved. Concentrated effort over the last decade, since the initial discovery of KSHV, led to the development of several experimental systems that resulted in a better comprehension of the biological characteristics of KSHV and set the stage for the understanding of mechanisms by which diseases are induced by the virus. The development of molecular, histological, and serological tools for KSHV diagnosis allowed researchers to track the transmission and to study the epidemiology of KSHV. These assays have been applied, in particular in ambiguous cases, in order to confirm clinically and pathologically based diagnoses. Here, we review the advances in the clinical, experimental, diagnostic, and epidemiological research of KSHV.