Molecular Diagnosis Of Infectious Diseases Research Articles

Molecular detection of Coxiella burnetii in aborted bovine fetuses in Brazil

In the past decade, cases of Q fever have been reported in Brazil. Although the previous report of Coxiella burnetii in humans and animals, the knowledge about the occurrence of this pathogen in livestock in Brazil is scarce. This study aimed to search C. burnetii and possible coinfections in tissues of aborted bovine fetuses from Brazil. Tissue samples from seventy-six aborted bovine fetuses sent to the laboratory of molecular diagnosis of infectious diseases from 2013 to 2019 were evaluated by real-time PCR for C. burnetii. Overall, 9.2% (7/76) of the samples were positive for C. burnetii. Moreover, the molecular diagnostic history of our lab revealed the coinfection with Neospora spp. in three fetuses and the presence of histopathological features suggestive with fetal neosporosis in another one. The previous report of C. burnetii in humans and animals in the country, with the detection of C. burnetii from tissues of aborted bovine fetuses reported here, reinforces the neglected state of the disease in Brazil and raises the question of the role of the pathogen in reproductive disorders in national livestock.

Investigating Catechol Surface Modification of TiO2 for the Development of High Performance Photoelectrochemical Biosensors

DNA biosensors are chemical sensors which are used to detect concentrations of specific DNA sequences or register DNA related biological events. There is significant interest in research into the design and optimization of DNA biosensors as they allow for rapid detection of cancers and infectious diseases 1,2. Photoelectrochemical (PEC) techniques can be applied to achieve high performance DNA biosensors with low cost and ultra-sensitivity. In this work, we explored the surface modification of TiO2 nanoparticles (NP) with various catechol molecules and used them to fabricate photoelectrodes for signal-off PEC DNA biosensors. Catechol modification provided dual functionality to the TiO2 NP photoelectrodes: it enhanced their PEC response and provided a molecular linker for attaching probes that capture target DNA.The surface modification of TiO2 NPs was done by adding aqueous solutions of catechol molecules to a suspension of TiO2. Adsorption of catechol on TiO2 took place very rapidly, indicated by a quick change in the colour of the suspension. The catechol-modified TiO2 suspensions were dropdeposited on indium tin oxide coated polyethylene terephthalate (ITO/PET) substrates and oven baked to produce photoelectrodes for the PEC DNA biosensor. Seven catechol molecules were used for surface modification: Caffeic acid (CA), 3,4-Dihydroxybenzaldehyde (DHBA), 3,4-Dihydroxybenzoic acid (DHB), 3,4-Dihydroxy-L-phenylalanine (DOPA), 3,4-Dihydroxyphenylacetic acid (DHPL), 2,3,4-Trihydroxybenzaldehyde (THBA), 2,3,4-Trihydroxybenzoic acid (THB). The photoelectrodes were optically characterized via ultraviolet/visible (UV/vis) spectroscopy and incident photon-to-electron conversion efficiency (IPCE). Additionally, the PEC current of the photoelectrodes were measured using chronoamperometry under white light excitation.Compared against unmodified TiO2, the UV/Vis spectroscopy and IPCE displayed increased light absorption in the visible range and red-shifting for the catechol-modified TiO2 photoelectrodes. This correlated to a great improvement in photocurrent generation. CA-modified TiO2 resulted in the largest increase in photocurrent, over 20 times from the unmodified TiO2 and therefore it was used as the basis for our PEC DNA biosensor. Single stranded DNA (ssDNA) probes were deposited onto the photoelectrodes, followed by target ssDNA deposition. The difference in PEC current after probe deposition and target deposition indicated the concentration of target DNA. A calibration curve was plotted with PEC current density as a function of target DNA concentration, which decreased logarithmically and ranged from 100 nM – 100 fM. A limit of detection (LOD) of 2.38 pM was estimated at a signalto-noise ratio of 3Sx/y (where Sx/y is the root-mean-square error (RMSE)).It is evident from our investigation that functionalizing TiO2 with catechol ligands greatly improves its optical properties which lead to a greater PEC current generation. In particular, high PEC current resulting from the combination of CA and TiO2 can form the basis for high-performance signal-off PEC DNA biosensors with a high dynamic range and low LOD. 1. Beltrán, A. P. & García, M., DNA biosensors and biomarkers to cancer detection. International Journal of Biosensors and Bioelectronics, 4(1), 20-21 (2018)2. Chua, A., Yean, C. Y., Ravichandran, M., Lim, B. & Lalitha, P., A Rapid DNA Biosensor for the Molecular Diagnosis of Infectious Disease. Biosensors and Bioelectronics, 26(9), 3825-3831 (2011)

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.

Finding the unexpected: A critical view on molecular diagnosis of infectious diseases in archaeological samples

In the 1980s the polymerase chain reaction (PCR) technique was developed and opened the possibility to analyze ancient DNA (aDNA) in archaeological remains and identify the etiological agent of several infectious diseases. The main concern in analyzing aDNA is to successfully verify its authenticity and the information that it contains. Primers are often designed to target the desired DNA sequences, however they can target unknown sequences amplifying different organisms. With the development of next-generation sequencing platforms, the production of sequence data is increasing. There is a universe of unknown sequences that are to be discovered and deposited in the database. Could we then state in the future that we have species-specific primers? We analyzed bones of human and ground sloth remains for Trypanosoma cruzi by PCR. Although the samples analyzed did not amplify this parasite some bacteria were amplified. Normally this would be interpreted as a contamination and would not be reported. Yet, in this article we ask researchers to rethink about these findings, especially due to the age of these samples and consider the archaeological context for the data interpretation.

Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.

Recombinase Polymerase Amplification Assay—A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

Development of Multiple Cross Displacement Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Shigella spp.

Shigella spp., the etiological agent of shigellosis or “bacillary dysentery,” are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors) have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA) combined with a newly designed lateral flow biosensor (LFB) for visual, sensitive and specific detection of Shigella. The MCDA-LFB assay was conducted at 65°C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min), amplification (20) and detection (2–5 min), can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for ‘on-site’ diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers.

Molecular diagnosis of infectious diseases in São Miguel Island (Azores, Portugal): A hospital-based descriptive study.

We performed a descriptive analysis of molecular diagnosis of infectious agents in the São Miguel Island population, in order to address questions like what is the frequency of clinical requests, is it observable seasonality of pathogens, and what is the positive rate for the clinical diagnosis. This was a retrospective and descriptive study based on 878 individuals suspected of harboring infectious diseases during two consecutive years, 2012-2013. More than 25 different pathogens were investigated by polymerase chain reaction (PCR)-based methods. The individuals were stratified into gender, occupation, and age groups. The pathogen with more clinical requests was hepatitis C virus, investigated in 225 individuals (30.0%), followed by Leptospira spp., in 187 (24.9%). Overall, data demonstrated a gender distribution bias, where 72.9% of cases were males. The age group of 25 to 44 years was the class with more clinical requests. Regarding occupation, a predominance of construction workers (12.0%) was observed, followed by retired workers (11.0%). Patient distribution per year showed a higher number of patients in the fall months. Diagnoses of leptospirosis and respiratory virus infections presented seasonality. The present study provides a valid contribution to the knowledge of the epidemiology of infectious diseases in the São Miguel Island (Azores, Portugal) population.

Molecular diagnosis of infectious diseases using cytological specimens.

Pathologists have an important role in the diagnosis of infectious disease (ID). In many cases, a definitive diagnosis can be made using cytopathology alone. However, several ancillary techniques can be used on cytological material to reach a specific diagnosis by identifying the causative agent and consequently defining the management of the patient. This review aims to present the effectiveness of the application of molecular studies on cytological material to diagnose IDs and discuss the advantages and disadvantages of the various molecular techniques according to the type of cytological specimen and the infectious agents.

Efficacy of the FilmArray blood culture identification panel for direct molecular diagnosis of infectious diseases from samples other than blood.

Molecular-based techniques reduce the delay in diagnosing infectious diseases and therefore contribute to better patient outcomes. We assessed the FilmArray blood culture identification (BCID) panel (Biofire Diagnostics/bioMérieux) directly on clinical specimens other than blood: cerebrospinal, joint, pleural and ascitic fluids, bronchoscopy samples and abscesses. We compared the results from 88 samples obtained by culture-based techniques. The percentage of agreement between the two methods was 75 % with a Cohen κ value of 0.51. Global sensitivity and specificity using the FilmArray BCID panel were 71 and 97 %, respectively. Sensitivity was poorer in samples with a low bacterial load, such as ascitic and pleural fluids (25 %), whereas the sensitivity for abscess samples was high (89 %). These findings suggest that the FilmArray BCID panel could be useful to perform microbiological diagnosis directly from samples other than positive blood cultures, as it offers acceptable sensitivity and moderate agreement with conventional microbiological methods. Nevertheless, cost-benefit studies should be performed before introducing this method into algorithms for microbiological diagnostics.

Aplicação de teste molecular para detecção de adenovírus em pacientes pediátricos distintos

The evolution of the methodologies used in the diagnosis of acute respiratory infections of viral etiology is due to the advancement of molecular biology, immunology, genetics and biotechnology. The development of the in vitro cell culture1 resulted in the isolation of viruses associated with acute respiratory infection of the respiratory tract (respiratory syncytial virus, adenovirus and parainfluenza virus). The discovery of the molecular structure of nucleic acids2 contributed to the development of molecular genetics, responsible, among other things, for the improvement of molecular diagnosis of infectious diseases in the late twentieth century, as well as the disclosure of new viral etiologies related to acute respiratory infection in the first decades of the 21st century. By the end of the 1980s, the diagnosis of respiratory infection consisted in the isolation of the virus in cell cultures and serology. The great challenge faced by scientists at the time was to enhance diagnostic methodologies, aiming to address the appropriate strategies for disease prevention and control; i.e., to establish methodologies that would allow attaining fast and specific diagnoses.3 Considering the impact of infection by respiratory viruses in all age groups, especially among children <5 years of age, patients with chronic diseases and adults ≥60 years of age,3 , 4 a rapid diagnosis is important for: antiviral use; respiratory disease etiology clarification; considering prescription of antibiotics; knowing the natural history of the virus and its physiopathology, which helps to understand the possible complications that may occur depending on the infection characteristics; assessment of disease containment through quarantine; measuring the hospitalization period; preventing unnecessary laboratory investigations, and dispensing with inappropriate isolation of non-infected individuals.5 , 6 The development of monoclonal antibodies in the 1970s7 contributed to the development of a specific antibody panel for influenza virus types A and B, RSV, ADV, and PIV 1, 2 3, which allows the fast and specific identification by direct or indirect immunofluorescence of the seven respiratory viruses. The advent of molecular methodologies, starting in the 1980s,8 of which principle resides on the Polymerase Chain Reaction (PCR), allowing the amplification of conserved genome targets of different etiological agents, revolutionized the understanding of epidemiology, diagnosis and research of infectious diseases of viral etiology, among other approaches. Currently, there are several fast and specific diagnostic methods, using both the principle of immunofluorescence with monoclonal antibodies and molecular methods, for simultaneous identification of different viruses.9 , 10 In this issue of Revista Paulista de Pediatria, Puerari et al.11 assessed the performance of both methods, using a method that allows simultaneous identification of the seven respiratory viruses by direct immunofluorescence and molecular diagnosis using Nested Polymerase Chain Reaction nested PCR, aiming at the investigation of adenovirus in respiratory secretions collected from children with congenital heart disease and children from the community with acute respiratory infection (ARI). This phase of the research did not include the analysis of other respiratory viruses; however, it showed that the molecular methodology was more sensitive for the detection of adenovirus in different groups of children, when compared to immunofluorescence. Due to the sensitivity in the detection of nucleic acids by nested PCR reported in the present analysis, the authors recommend routine surveillance in patients with congenital heart disease by molecular methods, considering the efficiency in the etiological agent detection, especially the diagnostic flow performance. In this context, investment in research for the development of antiviral agents for noninfluenza respiratory viruses, as well as to obtain knowledge on the molecular epidemiology of different respiratory viruses in the community is of utmost importance, as it will yield important information regarding the infection of hospitalized patients and of patients with chronic diseases by different viruses that affect the respiratory tract.12 To date, no antiviral drug has been approved against adenoviruses, which are well known for their capacity to cause severe disease, particularly in immunosuppressed patients. Recent clinical studies have assessed the oral formulation of the antiviral brincidofovir as a promising treatment for patients belonging to risk groups and infected by adenoviruses.13 The evolution of diagnostic methodologies potentiates researches aiming at the creation of antiviral agents and their timely intervention, providing better quality of life and consequent advances in the field of Public Health.

Universal PCR coupled with high-resolution melting analysis for rapid detection and identification of microorganism

Technologies for the rapid and accurate diagnosis of infectious diseases are urgently needed. There were many methods developed for the diagnostic purpose, including microarrays, biosensors, multiplex PCR, combined PCR with mass spectrometry, capillary electrophoresis, and lab-on-chip tests, which have a major impact on the rapid diagnosis of infectious diseases. Each of the above methods has several unsolvable shortcomings, such as complexity and time-consuming operation, high cost requiring special equipment (electrospray ionization/mass spectrometry). Therefore, use of these methods has been limited in clinical practice. In the present article, we focus on a new method that couples broad-range PCR amplification with high-resolution melting analysis, which serves as a novel approach for molecular diagnosis of infectious diseases. The strategies and applications of this method are discussed.

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes, G3PD2 was chosen for the formation of multiplex PCR systems. The two multiplex PCR systems (mVL and mACL) were reproducible in all species evaluated. Results of test samples (sensitivity, specificity and efficiency) suggest its use in routine diagnosis, research activities in medicine and veterinary medicine. Additionally, the systems designed to detect the G3PD gene are capable of combining with other targets used for molecular diagnosis of infectious diseases. Concerning leishmaniasis, the multiplex PCR systems can be used in epidemiological studies for the detection of new and classic reservoirs, which may contribute to the reliability of results and development of actions to control the disease.

A rapid DNA biosensor for the molecular diagnosis of infectious disease

Treating patients with infectious diseases relies heavily on rapid and proper diagnosis. Molecular detection such as PCR has become increasingly important and efforts have been made to simplify these detection methods. This study reports the development of a glass fibre-based lateral flow DNA biosensor that uses capture reagents coupled to carrier beads and detector reagent bioconjugated to gold nanoparticles, for the detection of foodborne pathogen, Vibrio cholerae. The DNA biosensor contains a test line which captures target PCR amplicons, an internal amplification control (IC) line which captures IC amplicons and a control line which acts as membrane control to validate the functionality of this device. The test line captures biotin labelled DNA, while the IC line captures digoxigenin labelled DNA. The detector reagent recognizes the fluorescein haptens of the amplified DNA and produces visual red lines. Scanning electron microscopy (SEM) studies performed indicated that the capture reagents remained relatively immobile within the matrix of the membrane even after binding of the detector reagent. The DNA biosensor recorded a limit of detection (LoD) of 5 ng of target DNA. A clinical evaluation was carried out with 174 strains of V. cholerae and non V. cholerae bacteria and the DNA biosensor recorded 100% for both sensitivity and specificity when compared to conventional agarose gel detection of DNA. Thus it is a viable alternative to agarose gel analysis and is easy-to-use, disposable and do not require any specialized equipment and use of carcinogenic chemicals.

Evaluation of Four DNA Extraction Methods for the Detection of Tritrichomonas Foetus in Feline Stool Specimens by Polymerase Chain Reaction

Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >or=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.

Molecular diagnosis of infectious diseases

Application of techniques originally developed for the analyses of nucleic acids to the diagnosis and characterization of infectious diseases is increasingly practiced. This tendency has been intensified after the introduction of the polymerase chain reaction (PCR). With the PCR, the molecular diagnostic assays have achieved a high sensitivity that parallels and often exceeds the sensitivity of various culture methods. This means that we are entering a new era in which a new gold standard is to be established. In this review, we summarized the basic principle of molecular diagnostic methods, presented, as an example, the application of such techniques to the study of rotaviruses, the single most important etiological agent of acute diarrhea in children, and finally discussed briefly possible future trends of molecular diagnostics for infectious diseases at large.

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].

Detection of Opportunistic Infections by Low-density Microarrays

In molecular diagnosis of infectious diseases often more than 1 pathogen has to be considered. As a consequence, a series of labor-intensive and time-consuming polymerase chain reaction (PCR) approaches specific for different putative pathogens have to be carried out. To speed up diagnosis, we established a low-density microarray for simultaneous detection of diverse putative pathogens causing a disease such as granulomatous lymphadenitis. Nucleic acids from formalin-fixed, paraffin-embedded tissues of 68 patients with lymphadenitis were used for molecular diagnosis of individual pathogens by either nested single-assay PCR or 1-step multiplex PCR in combination with low-density microarray hybridization. Multiplex PCR amplicons hybridized to glass slides containing probes from Mycobacterium spp., Yersinia spp., Bartonella henselae, Toxoplasma gondii, and other pathogens showed specific and reproducible signals on the array. Our results show that microarray technology combined with multiplex PCR is a promising and time-saving tool in molecular pathology of infectious diseases, allowing sensitive, simultaneous analyses of different pathogens.

Next revolution in the molecular theranostics of infectious diseases: microfabricated systems for personalized medicine

The molecular diagnosis of infectious diseases is currently going through a revolution sustained by the regulatory approval of amplification tests that have been shown to be equivalent or superior to existing gold standard methods. The recent approval of a microarray system for the pharmacogenomic profiling of cytochrome P450-mediated drug metabolism is paving the way to novel, rapid, sensitive, robust and economical microfabricated systems for point-of-care diagnostics, which are utilized closer and closer to the patient’s bedside. These systems will enable the multiparametric genetic evaluation of several medical conditions, including infectious diseases. This forecoming revolution will position molecular theranostics in a broader integrated view of personalized medicine, which exploits genetic information from microbes and human hosts to optimize patient management and disease treatment.

Molecular diagnosis of infectious diseases in dermatology

The molecular diagnosis of infectious disease has been growing considerably over the past decade. Nucleic acid amplification techniques, such as polymerase chain reaction, ligase chain reaction, transcription-mediated amplification, and nucleic acid sequence-based amplification, provide highly accurate diagnosis of numerous bacterial, viral, fungal, and parasitic infections involved in a variety of dermatologic diseases. In addition, signal amplification with hybrid capture, branched-DNA assays, and in situ hybridization have been used to detect numerous viral pathogens with high degrees of sensitivity and specificity. New technology that involves the use of DNA and protein microarrays has also enabled the detection of a variety of genes and gene mutations. With time, these diagnostic assays are decreasing in cost, gaining approval of the U.S. Food and Drug Administration, and becoming easier and more efficient to use. In the future, these assays will be able to deliver rapid and accurate diagnosis of infectious diseases within a single clinic visit. At the completion of this learning activity, participants should be familiar with molecular diagnosis of infectious diseases in dermatology.