Use Of Molecular Diagnostic Techniques Research Articles

Molecular Diagnostics for Ocular Infectious Diseases: LXXVIII Edward Jackson Memorial Lecture

To review the use of molecular diagnostic techniques in the management of ocular infectious disease. Retrospective review. A combination of literature review and personal recollections are used. Although the broad term molecular diagnostics may encompass techniques to identify pathogens via protein or metabolomic signatures, this review concentrates on detection of pathogen nucleic acid as an indicator of infection. The introduction of the polymerase chain reaction (PCR) in 1985 opened a new era in analysis of nucleic acids. This technique was soon applied to the detection of potential pathogen DNA and RNA, including viruses, bacteria, and parasites in infectious eye disease. Advances in PCR have allowed class-specific diagnostics (ie, pan-bacterial and pan-fungal), quantitation of pathogen DNA, and multiplexed testing. The Human Genome Project in the early 2000s greatly accelerated development of DNA sequencers, ushering in the era of "Next Generation Sequencing" and permitting pathogen-agnostic methods for the detection of potential infectious agents. Most recently, new technologies such as nanopore sequencing have reduced both cost and equipment requirements for whole-genome sequencing; when coupled with real-time sequence analysis methods, these methods offer the promise of true, real-time, point-of-service ocular infectious disease diagnostics. Molecular methods for pathogen detection have greatly advanced the diagnosis of ocular infectious disease. Further methodologic advances will have a direct impact on the management of these conditions.

Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivaxP. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India.

With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021–March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination.

The role of Kingella kingae in pre-school aged children with bone and joint infections

ObjectivesThe Pre-school Osteoarticular Infection (POI) study aimed to describe the burden of disease, epidemiology, microbiology and treatment of acute osteoarticular infections (OAI) and the role of Kingella kingae in these infections. MethodsInformation about children 3–60 months of age who were hospitalized with an OAI to 11 different hospitals across Australia and New Zealand between January 2012 and December 2016 was collected retrospectively. ResultsA total of 907 cases (73%) were included. Blood cultures grew a likely pathogen in only 18% (140/781). The peak age of presentation was 12 to 24 months (466/907, 51%) and Kingella kingae was the most frequently detected microorganism in this age group (60/466, 13%). In the majority of cases, no microorganism was detected (517/907, 57%). Addition of PCR to culture increased detection rates of K. kingae. However, PCR was performed infrequently (63/907, 7%). ConclusionsThis large multi-national study highlights the need for more widespread use of molecular diagnostic techniques for accurate microbiological diagnosis of OAI in pre-school aged children. The data from this study supports the hypothesis that a substantial proportion of pre-school aged children with OAI and no organism identified may in fact have undiagnosed K. kingae infection. Improved detection of Kingella cases is likely to reduce the average length of antimicrobial treatment.

Use of Molecular Diagnostic Techniques to Determinate the Epidemiology of Malaria Parasites in North Eastern Nigeria

The emergence of resistance to all antimalarial drugs in clinical use is now making it necessary to discover the markers responsible for the resistance. The principal aim of this research is the use of molecular diagnostic techniques to Determine the epidemiology of malaria parasites. Thirty blood samples were analyzed by microscopy and molecular techniques to monitor the relative efficiency in malaria diagnosis. Molecular analysis revealed 28 out of 30 samples as positive for malaria while Microscopic analysis revealed 27out of 30 samples as positive malaria parasite. The molecular analysis was particularly useful to unveil parasites presence in infections not detectable by blood smear analysis.
 Keywords: Molecular Diagnostic Techniques, Epidemiology, Malaria Parasites

Economics of New Molecular Targeted Personalized Radiopharmaceuticals

Nuclear medicine has come a long way since 2007 when Adrian Nunn pointed out the approval of radiopharmaceuticals was at an all-time low with all the major radiopharmaceutical agents in use having been approved over 10 years ago. Challenges being the prohibitively high cost of drug development and the large number of drugs failing in clinical trials. Proceed to today where molecular imaging is fast-tracking the drug discovery process by reducing both the time and cost to screen candidates by quantitating the drugs effect on the target and toxicity to normal tissues. Nuclear medicine is now leading medical practice in personalized medicine using the theragnostic approach. Theragnostics is defined as the use of molecular diagnostic techniques in real time to stratify patients to guide treatment decisions such as the choice of drug, the dose of administration, and the timing of drug delivery for a given patient. Enabling visualization and quantitation of in vivo function of the whole body and thus patient heterogeneity and variability informs the physician on how to treat an individual patient. Recent successes such as the Food and Drug Administration approval of Lutathera and NETSPOT have resulted in an increasing number of pharmaceutical companies pursing theragnostics further heightened by the purchase of Advanced Accelerator Applications for 3.9 billion by Novartis and Endocyte, Inc for 2.1 billion. Theragnostics are further aiding drug development by showing which agents are most viable and reducing the overall cost of bringing a drug to clinical trials and regulatory approval. This is indeed a renaissance for nuclear medicine in which the acceptance of imaging to inform and monitor therapy has been embraced and even required by the Food and Drug Administration for the clinical evaluation of targeted therapeutic radiopharmaceuticals showing there is indeed a viable business model for targeted theragnostic radiopharmaceuticals and personalized medicine.

Molecular Diagnosis of Thalassemias and Hemoglobinopathies: An ACLPS Critical Review.

To describe the use of molecular diagnostic techniques for patients with hemoglobin disorders. A clinical scenario is presented in which molecular diagnosis is important for genetic counseling. Globin disorders, techniques for their diagnosis, and the role of molecular genetic testing in managing patients with these disorders are described in detail. Hemoglobin disorders, including thalassemias and hemoglobinopathies, are among the commonest genetic diseases, and the clinical laboratory is essential for the diagnosis of patients with these abnormalities. Most disorders can be diagnosed with protein-based techniques such as electrophoresis and chromatography. Since severe syndromes can result due to inheritance of combinations of globin genetic disorders, genetic counseling is important to prevent adverse outcomes. Protein-based methods cannot always detect potentially serious thalassemia disorders; in particular, α-thalassemia may be masked in the presence of β-thalassemia. Deletional forms of β-thalassemia are also sometimes difficult to diagnose definitively with standard methods. Molecular genetic testing serves an important role in identifying individuals carrying thalassemia traits that can cause adverse outcomes in offspring. Furthermore, prenatal genetic testing can identify fetuses with severe globin phenotypes.

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria.

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.

Importance of respiratory virus in immunocompetent adult patients hospitalized with community-acquired pneumonia

Community-acquired pneumonia (CAP) is a relevant worldwide cause of morbidity and mortality in adult population, however its etiology is often not identified and therapy is empirical. To assess the etiology of CAP in immunocompetent adult hospitalized patients using conventional and molecular diagnostic methods. We prospectively studied 240 adult patients who were hospitalized for CAP to identify the microbial etiology. Sputum and blood cultures were obtained as well as serology testing for Mycoplasma pneumoniae and Chlamydophila pneumoniae, urinary antigen testing for Legionella pneumophila and Streptococcus pneumoniae, and a nasopharyngeal swab for the detection of sixteen respiratory viruses by reverse transcriptase polymerase chain reaction (RT-PCR). In 100 patients (41.7%) a single respiratory pathogen was identified. In 17 (7.1%) cases, a mixed bacterial and viral infection was detected and no pathogen was identified in 123 cases (51%). The most commonly identified pathogens identified were: influenza virus (15.4%), parainfluenza virus (10.8%), rhinovirus (5%), Streptococcus pneumoniae (5%), respiratory syncytial virus (2.9%) and Mycoplasma pneumoniae (2.5%). Infectious agent detection by RT-PCR provided greater sensitivity than conventional techniques. Viral respiratory infections were more prevalent in older patients with comorbidities and high risk patients, according to the Fine index at hospital admission. The clinical severity and outcome were independent of the etiological agents detected. The use of molecular diagnostic techniques expanded the detection of respiratory viruses in immunocompetent adults hospitalized with CAP.

Enteroviruses and Parechoviruses.

Infections with enteroviruses and human parechoviruses are highly prevalent, particularly in neonates, where they may cause substantial morbidity and mortality. Individuals with B-cell-related immunodeficiencies are at risk for severe enteroviral infections, usually a chronic and fatal meningoencephalitis. In transplant recipients and patients with malignancy, enterovirus infections typically involve the respiratory tract, but cases of severe, disseminated infection have been described. The mainstay of diagnosis for enterovirus and human parechovirus infections involves the use of molecular diagnostic techniques. However, routine nucleic acid-detection methods for enteroviruses will not detect human parechoviruses. Laboratory diagnosis of these viral infections is important in determining a patient's prognosis and guiding clinical management.

Evaluation of Molecular and Immunohistochemical Adjunct Modalities in the Diagnosis of Soft Tissue Neoplasms.

The accurate diagnosis of soft tissue neoplasms has crucial therapeutic and prognostic importance. There is frequent morphologic overlap between entities, and ancillary modalities are used in the vast majority of diagnoses. Immunohistochemistry is rapid and inexpensive, and in addition to the older markers that mainly detected cytoplasmic proteins, antibodies can indirectly detect tumor-specific genetic and molecular abnormalities. The use of molecular diagnostic techniques is now widespread, with molecular services often integrated into routine histopathology laboratories; as their cost and turnaround times begin to parallel those for immunohistochemistry, we compared the usefulness of ancillary immunohistochemistry, molecular genetic, and molecular cytogenetic techniques in the diagnosis of soft tissue lesions. We evaluated the number and contribution of immunohistochemical tests and panels and of ancillary molecular techniques in the primary histopathologic diagnosis of 150 soft tissue lesions. Ninety of 150 cases required either only one immunohistochemical panel or minimal immunohistochemistry for diagnosis, while 39/150 required 2 to 4 panels. In 5/150, ancillary molecular tests alone (without immunohistochemistry) were diagnostically sufficient. The majority of cases required one immunohistochemical panel for diagnosis, with a smaller proportion requiring a second, and a minority requiring a third or fourth (which mainly comprised neoplasms for which the final diagnosis was uncertain). Certain neoplasms required both extensive immunohistochemistry and ancillary molecular testing, despite which the final diagnosis was inconclusive. Ancillary molecular techniques now make a significant contribution to soft tissue tumor diagnosis, being required in over one third (52/150) of cases, and were useful in confirming or excluding tumors that were not possible to conclusively diagnose with histology and immunohistochemistry. Only a small proportion of soft tissue neoplasms (16/150; all benign) did not require immunohistochemistry or ancillary molecular methods, with morphology alone being sufficient for diagnosis.

Symposium on viral respiratory disease surveillance

Recognition of the need for respiratory disease surveillance for informing prevention strategies dates back to the late 1940’s. Sir Christopher Andrews realized that the development of a successful influenza vaccination programme would be hindered by the unpredictable nature of the influenza virus and proposed the formation of a world influenza centre and a network of linked national laboratories which tracked influenza epidemics, to the World Health Organization (WHO). This network, now the WHO Global Influenza Surveillance Network (GISN) remains in place today and, although it’s primary goal is still to inform seasonal influenza vaccine strain selection, it has had to widen its activities to address emerging public health needs. There has been a renewed focus on understanding the overall burden of disease caused by influenza, especially in countries with no seasonal influenza prevention programmes, as a strategy to increase influenza awareness. While the zoonotic and pandemic threats from the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the realization that pandemic influenza arises from avian influenza viruses have stimulated global and national pandemic preparedness activities. Nevertheless, improved surveillance systems for both animal (avian and pig) and human influenza viruses are needed for informing and refining prevention strategies. Although the public health focus globally has been on influenza, the burden of disease caused by other respiratory viruses is also substantial and is largely under-recognised. A range of respiratory viruses are implicated in pneumonia, a disease recognised as a major killer of young children in developing countries and of the elderly in developed countries. There is also evidence that some respiratory viruses may be evolving allowing virulent variants to cause outbreaks in certain at-risk populations presenting another possible pandemic risk, highlighting the need to monitor unusual outbreaks of respiratory disease and the importance of the surveillance of respiratory viruses in general. Overall, prevention strategies for respiratory viruses other than influenza are less well defined, undoubtedly contributing to their perceived lower public health priority. Major advances in the diagnosis of respiratory virus infections have been made through the wider use of molecular diagnostic techniques which are now enabling rapid and sensitive respiratory pathogen surveillance. It is now possible to identify a wide range of respiratory pathogens in a higher proportion of respiratory infections, along with multiple pathogen infections. The application of these molecular techniques has also lead to the discovery of a number of new respiratory viruses; the clinical importance of the human metapneumovirus (hMPV) is now well established, while with others such as the human bocavirus (HBoV) and human rhinovirus type C (HRV-C), their clinical correlates are only just being elucidated. The knowledge base on these viruses urgently needs expanding through well designed controlled studies and other activities, including widened surveillance. The future challenge is to build on what has been achieved for the global surveillance of influenza by establishing standardized surveillance protocols which incorporate a widening range of respiratory viruses. The purpose being to inform and refine respiratory disease prevention strategies. These strategies must then be communicated to governments and public health messages developed for the education of the general public. DOI:10.1111/j.1750-2659.2010.00140.x www.influenzajournal.com

Update on human infections caused by intestinal protozoa

To update the reader on the recent literature discussing human infections due to intestinal protozoa. Diarrheal diseases due to intestinal protozoa cause significant morbidity and mortality worldwide in a variety of patient populations. New literature on key pathogens Cryptosporidium, Cyclospora, Isospora, Giardia, and Entamoeba histolytica are discussed. The recent medical literature on these pathogens has focused on new advances in understanding human genetic markers associated with susceptibility to infection and the use of molecular diagnostic techniques for both outbreak investigations (molecular epidemiology) and to understand the variability of clinical disease within species and subspecies.

Panorama actual de la epidemiología, diagnóstico y tratamiento de las infecciones de transmisión sexual

In the last decade, cases of sexually-transmitted infections (STIs) have progressively increased in Europe. The reasons for this increase are unclear, but may involve changes in social behavior, migration and international travel, coupled with the emergence of risk groups that have not been taken into sufficient consideration to date. The routine use of molecular diagnostic techniques for many of these infections has solved many problems of sensitivity and the suitability of samples for microbiological diagnosis: non-invasive samples can be used, which has undoubtedly contributed to the increase in the number of cases. Moreover, molecular methods have also been introduced for antibiotic and antiviral susceptibility testing, as well as for molecular characterization of clinical isolates. All of these factors, together with the approval of the vaccine against the human papillomavirus, have changed the landscape of STIs across Europe.

Genetic and molecular genetic studies in the diagnosis of atypical lymphoid hyperplasias versus lymphoma

The major emphasis in this section of the Workshop was the use of molecular diagnostic techniques in the differential diagnosis of benign versus malignant lymphoid proliferations at nodal and extranodal sites. In 1 case, these techniques were used to help make a very specific benign diagnosis. In the remaining cases, their main intention was to help prove that a lymphoid proliferation was benign or malignant. Review of the cases described herein highlighted both their utility in this regard as well as how they can be problematic and misleading. They also highlighted how important it is for the diagnostic pathologist to know the precise studies performed and to correlate the results with the morphological, phenotypical, and other findings before reaching a conclusion. Some of these issues are discussed in greater detail herein. J. Melnick, S. Shenoy, and M. Kraus of Washington University in St. Louis discussed a classic case (90) of autoimmune lymphoproliferative syndrome (ALPS) (or Canale-Smith syndrome).1 A 16-month-old male presented with anemia, adenopathy, and splenomegaly and a lymph node biopsy showed a very worrisome and very typical interfollicular lymphoid proliferation (Fig 1). Flow cytometric analysis of the “large cell” gate and immunohistochemical evaluation of the interfollicular cells demonstrated the following classic “double-negative” T-cell phenotype: CD2 , CD3 , CD5 , and CD43 with diminished CD7 but absent CD45RO, CD4, CD8, gamma-delta, CD20, CD30, CD56, myeloperoxidase, cytomegalovirus, and Epstein-Barr virus.1 Southern blot analysis did not demonstrate evidence of either a clonal B-cell or T-cell population. Fas (CD95) gene sequencing showed a heterozygous mutation in the death domain. These findings supported the benign nature of the lymph node biopsy and confirmed the diagnosis of ALPS. It is of interest that the patient’s mother is also heterozygous for the mutation so that, as is well recognized, other factors must play a role in the phenotypic expression of this disorder.2,3 The child was reported to be doing well at 6.5 years on low-dose steroid therapy. Criteria for ALPS include (1) chronic nonmalignant lymphoproliferation, (2) defective lymphocyte apoptosis in vitro and (3) 1% T-cell receptor (TCR) alpha/beta , CD4 , CD8 (double-negative) T cells in peripheral blood and/or the presence of doublenegative cells in lymphoid tissue.2 In addition to the marked paracortical expansion as described earlier, the lymphoproliferation includes florid follicular hyperplasia often with progressively transformed follicular centers and a polyclonal plasmacytosis.1 The double-negative T-cell population also expresses cytotoxic proteins, such as TIA-1, and many are CD57 .1 A CD5 polyclonal B-lymphocytosis is also characteristic.1 Supporting criteria include autoimmunity/autoantibodies and mutations in TNFRSF6 (Fas gene), FasL (Fas ligand), or caspase 10 gene. Fas is a cell surface receptor that binds Fas ligand and then initiates apoptosis initially by binding FADD, which in turn leads to activation of caspase 8 or 10. Patients with ALPS usually present as infants or young children with lymphadenopathy, splenomegaly, and variable autoimmune phenomena, particularly hemolytic anemia and thrombocytopenia. Hepatomegaly is seen in about 50% of patients. There are also many other less common clinical manifestations.2 Although the lymphoproliferative component is said to regress over time, the autoimmune phenomena are more likely to occur. Molecular studies have been used to identify 4 different types of ALPS.4,5 These include cases with generally heterozygous mutations in TNFRSF6 (type Ia), in the Fas ligand gene (Ib) or in the gene for the “downstream” caspase 10 (II), and those without any recognized molecular abnormalities (III). The Fas mutations are found throughout the gene, but most are in the exon that contains the death domain. Mutations in the extracellular portion of Fas are less likely to have ALPS and, if they do, to have less severe ALPS.4 It should be recognized that Fas gene mutations are not restricted to patients with ALPS and their relatives. Fas mutations occur normally in germinal centers,6 have been reported in a patient fulfilling the criteria for systemic lupus erythematosus,7 and occur in mucosaassociated lymphoid tissue and other lymphomas8 as well as in other malignancies.2 Molecular studies are also important in these cases, because they can be used (as in the case presented here) to support a benign diagnosis in the face of worrisome morphology by failing to demonstrate a From the Division of Hematopathology, University of Pittsburgh Health System, Pittsburgh, PA. Accepted for publication December 1, 2002. Address correspondence and reprint requests to Steven H. Swerdlow, MD, UPMC-Presbyterian, UPMC Health System, Division of Hematopathology, 200 Lothrop St., Room PUH C606, Pittsburgh, PA 15213-2582. © 2003 Elsevier Inc. All rights reserved. 0046-8177/03/3404-0008$30.00/0 doi:10.1053/hupa.2003.95

Viral myocarditis: Changing concepts in pathogenesis and implications in diagnosis and treatment

Viral myocarditis is an important potential precursor to idiopathic dilated cardiomyopathy. An understanding of its pathogenesis has evolved with the recent clarification of the role of immune mechanisms, which appears to have both protective and autodestructive effects. In animal models immune modulation has led to paradoxical worsening of the disease, and clinical trials of immunosuppression have not shown any beneficial effects. Through the use of molecular diagnostic techniques, a possible direct role for the virus itself is now increasingly recognized in human disease. This is supported by animal models demonstrating viral cytopathic effects and chronic persistence of virus within the myocardium. A novel contribution from microvascular ischemia has also recently been demonstrated; this may play a particularly important role in the evaluation and treatment of dilated cardiomyopathy. These changing concepts in pathogenesis will have an impact on diagnosis; myocardial biopsy will be used for molecular diagnosis of viral infection, in addition to providing important histological information. Therapy for viral myocarditis will also evolve in parallel to include trials of antiviral agents, targeted immune modulators, and anti-ischemic or vasodilator therapy. With new tools of molecular diagnosis in myocarditis complementing the traditional morphological and immunological assessments, we now have avenues of opportunity to explore in depth the pathogenesis, epidemiology, and prognosis of this challenging disease.