Diagnostic Molecular Pathology

A Guide to Applied Molecular Testing

William B. Coleman, Gregory J. Tsongalis, William B. Coleman, Gregory J. Tsongalis

• Publisher: Academic Press
• Year: 2016
• ISBN:
  9780128011577
• Topic: Biological Sciences
• Subtopic: Genetics & Genomics
• Index: Biological Sciences

Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing is organized around disease types (genetic disease, infectious disease, neoplastic disease, among others). In each section, the authors provide background on disease mechanisms and describe how laboratory testing is built on knowledge of these mechanisms. Sections are dedicated to general methodologies employed in testing (to convey the concepts reflected in the methods), and specific description of how these methods can be applied and are applied to specific diseases are described.

The book does not present molecular methods in isolation, but considers how other evidence (symptoms, radiology or other imaging, or other clinical tests) is used to guide the selection of molecular tests or how these other data are used in conjunction with molecular tests to make diagnoses (or otherwise contribute to clinical workup). In addition, final chapters look to the future (new technologies, new approaches) of applied molecular pathology and how discovery-based research will yield new and useful biomarkers and tests.

Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing contains exercises to test readers on their understanding of how molecular diagnostic tests are utilized and the value of the information that can be obtained in the context of the patient workup. Readers are directed to an ancillary website that contains supplementary materials in the form of exercises where decision trees can be employed to simulate actual clinical decisions.

• Focuses on the menu of molecular diagnostic tests available in modern molecular pathology or clinical laboratories that can be applied to disease detection, diagnosis, and classification in the clinical workup of a patient
• Explains how molecular tests are utilized to guide the treatment of patients in personalized medicine (guided therapies) and for prognostication of disease
• Features an ancillary website with self-testing exercises where decision trees can be employed to simulate actual clinical decisions
• Highlights new technologies and approaches of applied molecular pathology and how discovery-based research will yield new and useful biomarkers and tests

Section II

Molecular Testing in Infectious Disease

Outline

Chapter 5 Molecular Testing for Human Imunodeficiency Virus

Chapter 6 Molecular Testing in Hepatitis Virus Related Disease

Chapter 7 Molecular Testing for Human Papillomaviruses

Chapter 8 Molecular Testing for Herpes Viruses

Chapter 9 Molecular Testing for Parvoviruses

Chapter 10 Molecular Testing for Polyomaviruses

Chapter 11 Molecular Testing for Respiratory Viruses

Chapter 12 Molecular Testing for Diseases Associated with Bacterial Infections

Chapter 13 Agents Associated with Sexually Transmitted Infections

Chapter 14 Molecular Methods for Healthcare-Acquired Infections

Chapter 15 Molecular Testing in Emerging Infectious Diseases

Chapter 5

Molecular Testing for Human Imunodeficiency Virus

M. Memmi^1,2, T. Bourlet^1,2 and B. Pozzetto^1,2, ¹GIMAP EA3064, University of Lyon, Saint-Etienne, France, ²Laboratory of Infectious Agents and Hygiene, University Hospital of Saint-Etienne, Saint-Etienne, France

Abstract

Molecular tests occupy a key role in the diagnosis and follow-up of human immunodeficiency virus (HIV) infection and notably in those due to HIV type 1 (HIV-1) that has already concerned 78 million people worldwide since the beginning of the pandemic. The plasmatic detection of HIV RNA has been introduced recently in the algorithm used for diagnosing HIV infection in those individuals with indeterminate serological results. Molecular HIV-1 screening is essential for identifying HIV-1-infected subjects who are donors of products of human origin, including blood, organs, tissues, and semen; it also plays a key role for the detection and follow-up of infants born to HIV-1-infected mother. The measure of HIV-1 RNA load in plasma is the key element for the follow-up of HIV-1-infected people and for starting an antiretroviral treatment, even in low-resource areas where point-of-care tests are developed to overcome the lack of trained laboratories. More recently, quantitative tests have been proposed for measuring the DNA viral load in blood cells and different tissues with the objective of managing the cure of HIV-1 reservoirs. Finally, molecular tests are currently used for monitoring HIV-resistant strains in subjects receiving antiretroviral therapy.

Keywords

Human immunodeficiency virus; plasma RNA viral load; molecular HIV screening; blood safety; mother-to-child HIV infection; point-of-care tests; low-resource areas; antiretroviral resistance determination

Introduction

The emergence of human immunodeficiency virus (HIV) infection in the 1980s [1] was contemporary of the discovery of polymerase chain reaction (PCR) [2], the most popular method used for the molecular diagnosis of viral diseases. Consequently, molecular testing occupies a large place in the screening and follow-up of HIV-infected subjects. The HIV species, a member of the Retroviridae family and of the Lentivirus genus, includes two serotypes named HIV-1 and HIV-2—both of them are responsible for severe immunodeficiency in humans (AIDS) but the first one is distributed worldwide whereas the other is rather limited to West Africa. Given this major difference in terms of Public Health, most of the attention will be dedicated to HIV-1 in this review.

Background on HIV Infection and AIDS

Overall Epidemiology

Since the beginning of the HIV pandemic, almost 78 million people have been infected with the HIV-1 virus and about half of them died. At the end of 2013, according to the World Health Organization (WHO), 35 million (33.1–37.2 million) people were living with HIV [3]. That same year, some 2.1 million people became newly infected, and 1.5 million died of AIDS-related causes. Approximately 0.8% of individuals aged 15–49 years worldwide are living with HIV. The burden of the pandemic varies dramatically between countries and regions, Sub-Saharan Africa remaining the most severely affected, with nearly 1 in every 20 adults living with HIV and accounting for nearly 71% of the people living with HIV worldwide (women comprised 59% of infected people in this area).

Life Cycle of HIV

HIV is a single-stranded, positive-sense RNA enveloped virus of about 120 nm in diameter. Due to the glycolipids constituting its envelope, the virion is relatively fragile in the environment. The entry of the virus through the envelope glycoproteins into the competent cells (mainly immune cells and notably T cells, monocytes–macrophages, and dendritic cells) requires the presence of receptors (mainly CD4 molecule) and chemokine co-receptors (CCR5 or CXCR4) at the surface of the cell. Once the viral RNA is delivered into the cell, it is reverse transcribed into DNA by an RNA-dependent DNA polymerase encoded by the viral genome. The newly generated double-stranded DNA is exported to the nucleus where it is integrated within the cellular DNA by a viral integrase. This phase, which is mandatory for the continuation of the viral cycle, is also crucial for the constitution of viral reservoirs that will persist definitively, despite the further use of antiviral or immunomodulating treatments (at least at this stage of our knowledge). The viral DNA is then transcribed into different RNAs that are used for generating both genomic RNA and messengers coding for viral proteins that are further processed by viral proteases. The virion is then released from the cell by budding through the cytoplasmic membrane.

Transmission of HIV

The virus is mostly transmitted via sexual route, mainly by vaginal and anal intercourse, although oral sex can also be incriminated. The transmission via contaminated products of human origin is another common way of infection, notably in intravenous drug users sharing syringes and in patients receiving unsafe blood products. The latter mode of transmission explains the need for screening donors of human products (blood, semen, other tissues, organs) with sensitive techniques. The third way of HIV transmission is from mother to child (MTC) during pregnancy, delivery, and breastfeeding. The detection of infected babies is also a major goal of HIV diagnosis.

Natural History of HIV Infection

After a stage of primary infection that can be clinically symptomatic or asymptomatic, the infected subject experiences a long phase of clinical latency during which the viral replication is ongoing in most cases, but at variable levels. A minority of individuals, called long-term nonprogressors, remain asymptomatic for years without developing immunodeficiency. By contrast, most of the infected subjects, if not diagnosed and treated, develop in less than 10 years a progressive loss of their immune functions, affecting principally the T-cell repertoire. AIDS is characterized by an acquired immunodeficiency that results in the occurrence of opportunistic infections and/or cancers that are responsible for the death of patients.

HIV Evolution During Treatment

There is presently no preventive vaccine against HIV infection and also no definite cure. However, lifelong effective treatment with antiretroviral (ARV) drugs can control the virus so that HIV infection can now be considered a chronic disease. Current typical treatments include a combination of three drugs in order to avoid the emergence of resistant strains. More than 25 approved molecules belonging to different classes of ARV are available, some of them being combined in the same pill for facilitating the daily observance of treatment. In most cases, this regimen is able to maintain the plasmatic viral load at an undetectable level and to preserve the immune defenses. In 2013, 12.9 million people living with HIV were receiving ART, of which 11.7 million from low-income or middle-income countries [4]. In the latter areas and during the same year, one-third of the total number of infected adults had access to ART whereas this proportion was only of one in four for children.

Molecular Tools in the Diagnosis and Follow-Up of HIV Infection

The first role of the clinical laboratory in the management of HIV infection is to identify the subjects who are not already recognized as infected by HIV, whatever the stage of...