Molecular Methods and Platforms for Infectious Diseases Testing

Molecular assays have considerably improved the field of infectious disease diagnostics, especially with the superior responsiveness and specificity linked by presenting clinical laboratories with accurate and rapidly obtained results. Recent advancements in Molecular Diagnostics and the availability of commercial Nucleic Acid Amplification Techniques (NAATs) based assays have transformed the way researchers perform laboratory diagnosis of infectious disease testing. 

Diagnostic tests play a significant role in the clinical care of patients with contagious diseases, including detecting specific pathogens, discovering new pathogens, determining appropriate therapy, monitoring response to treatment, assessing prognosis, and disease surveillance. Even though the first direct-specimen molecular testing for infectious diseases was approved by the US FDA 26 years ago, there has been a recent explosion of molecular technology in two primary directions: 

1.Next-generation sequencin

2.Direct-specimen rapid amplification and detection and platforms

The BioFire FilmArray pouch is a multiplex PCR assay that stores all the necessary reagents for sample preparation, reverse transcription PCR(rt pcr), PCR, and detection in a freeze-dried format. During a test run, the BioFire System extracts and purifies all nucleic acids from the unprocessed sample. 

These high-level techniques offer an opportunity to provide medical laboratory diagnoses at a speed and sensitivity that was never possible with standard microbiology. The demand for diagnostics that promote clinical care and public health has never been more prominent. There is a significant chance of securing new technologies to address evident unmet needs.

Principle of molecular testing

The starting principle behind molecular testing involves amplifying the amount of DNA or RNA within a specimen of thousands to millions of times before analyzing. The DNA sequence is amplified using cycling methods, such as polymerase chain reaction (PCR) or isothermal amplification. 

Molecular genetic testing is typically used to detect changes in a single gene and includes direct sequence analysis and large deletion/duplication detection. The amplification of DNA/nucleic acids is achieved using naturally occurring enzymes (proteins). DNA polymerase is one of the key enzymes used in this process, which is the basis for the qPCR assay methodology.

Other enzymes can be used for amplification, and these are typically grouped under the generic label of isothermal nucleic acid amplification tests (INAAT). The augmentation step makes molecular biology methodologies more receptive than immunological assays, and every amplification cycle doubles the amount of DNA in the sample. PCR detection involves amplification achieved through temperature cycling, while isothermal amplification assays use a uniform reaction temperature for amplification.

Infectious disease testing

Over 30 new infectious diseases have been identified during the last three decades, making it imperative to have a reliable method to determine the responsible pathogens. Access to sensitive and rapid infectious disease assays is essential for accurate diagnosis, effective treatment, and timely infection control. Let’s look at some of the infectious diseases

1.Hepatitis C virus

Hepatitis C virus (HCV) disease is the most prevalent cause of chronic viral hepatitis. Almost 20% of the 4 million carriers develop liver cirrhosis. HCV infections are usually diagnosed by detecting antibodies aimed against specific HCV antigens.

2. Cystic fibrosis

Cystic fibrosis (CF) is a common, lethal, autosomal recessive disorder among Caucasians. The disease frequency in the general population is 1:25; nevertheless, the frequency varies between diverse geographic forms. 

The condition is identified by growing lung disease, male infertility, pancreatic dysfunction and elevated electrolytes. In all clinical parameters, there is tremendous variability in disease diagnosis.

3. Group B Streptococcus

Streptococcus agalactiae is a foremost cause of sepsis, meningitis, and death amongst newborn infants. There are almost 10%, and GBS colonizes 40% of healthy adult women in the genital and gastrointestinal tracts. GBS can cause illness throughout pregnancy and labour

4. Respiratory Tract Viral Infections

Acute respiratory tract infections are the most prevalent viruses in humans, and respiratory tract viruses cause about 80% of these infections. Respiratory tract virus infections vary from moderate upper respiratory tract infections to acute lower respiratory tract infection

5. CNS Viral Infections

Viral CNS infections typically manifest as meningitis and encephalitis. Nucleic acid testing helps quickly diagnose viral meningitis and limits unnecessary antibiotic use and potential repeat spinal taps, notably in children.

6. CT and Neisseria gonorrhoeae (NG) 

CT and Neisseria gonorrhoeae (NG) are frequent causes of bacterial STDs, and both can produce urogenital tract infections varying from acute to asymptomatic infection. Significant underreporting conditions can occur due to silent disorders affecting the reproductive age group

7. HBV infection 

HBV is tagged with A–H genotypes, and its predominance varies significantly by geography and population subgroups. HBV infection is a universal public health problem, with 400 million worldwide long-term carriers and up to 25% mortality. It is necessary to detect and quantify all genotypes because there are significant variations in genotype-dependent hepatocellular carcinoma. There is no purpose for molecular testing in diagnosing acute HBV other than identifying asymptomatic patients throughout the pretransfusion screening of blood products.

Future of Infectious Diseases Diagnostics

The availability of well defined FDA-cleared or FDA-approved Molecular infectious disease tests reduces the strain on laboratories in producing or verifying laboratory-developed panel tests. It also helps implement molecular testing in laboratories and allows for uniformity and comparability of tests, thus significantly advancing health care outcomes. While there have been notable recent progressions in clinical microbiology and increased availability of molecular and other diagnostic tests, the tests are still not optimally integrated into clinical laboratory scientists’ care for the benefit of patients. 

• Is this because available trials do not address priority clinical needs? 
• Are healthcare providers inadequately informed about the availability and utility of many tests? 
• Are there barriers to the most needed diagnostics’ laboratory research, development, and regulatory approval? 
• What are the challenges to laboratory adoption and integration of diagnostics into diverse healthcare settings? 

Because of the influence on health care and the evolving technology, significant changes in molecular testing are expected in the next few years.

BioFire filmarray instruments run syndromic infectious disease tests. The FDA authorizes it for the point-of-care space under an emergency use authorization and SARS-CoV-2 in one multiplex PCR test. Visit the Helvetica Health Care website for more information on the product.