Recent Trends In Molecular Microbial Diagnosis
Real time polymerase chain reaction technology has revolutionized the diagnosis, monitoring, and treatment in many infections such as HIV, hepatitis B, and hepatitis C. Diseases of epidemiologic importance such as swine flu (H1N1) are rapidly being diagnosed and outbreaks controlled by using reverse transcriptase PCR methods. Commercial high throughput systems with complete automation right from extraction, PCR, to result are now available for laboratories with large workloads.
Tuberculosis (TB) is a disease of great public health importance in our country. Recently, cartridge based version of semi-nested PCR has been made widely available. Use of cartridge based nucleic acid amplification technology known as CBNAAT allows for a rapid diagnosis of TB and rifampicin susceptibility within two hours. Early TB treatment not only plays an important role in faster patient cure but also helps in preventing spread of infection and advent of multidrug/extended drug resistance (MDR/XDR).
Hybridization techniques such as Line probe assays have been used in diagnosis of TB. Major advantage of this technique is that along with TB diagnosis it also helps in the screening for MDR and XDR. The technology has been adapted for both samples and cultures.
Probe based assays such as solution-phase hybridization and chemiluminescence are used in detection of both Neisseria gonorrhoeae and Chlamydia trachomatis in a single specimen (one specimen, two separate probes). Hybrid capture systems are used for screening of high risk human papillomavirus (HPV) in cervical scrapings. Multiplex PCR technology allows for direct simultaneous identification of multiple pathogens from a single sample. Multiplexing methods although very convenient may have lower sensitivity.
Microarray techniques can simultaneously detect multiple DNA/RNA targets in a single run. The technology can be adapted to detect simultaneously bacteria, fungi, parasites, and viruses in a single sample. As each target is spotted multiple times on the microarray chip, the sensitivity and specificity of the technique is excellent. User friendly point-of-care systems employing microarray technique are a boon for hospitalized setups for the syndromic diagnosis and management of sepsis, meningitis, encephalitis, gastroenteritis, respiratory, and such other infections. The tests can be also used as Rule out tests to rule out bacterial infections, avoiding unnecessary antibiotic treatment. Microarrays can also be used for detecting antimicrobial resistance (genes), typing of microbial pathogens, and monitoring of microbial infections by investigating host genomic expression and polymorphism profiles.
DNA sequencing is considered as the gold standard method for microbial identification. Over time the speed of DNA sequencing has considerably reduced and applications include viral genotyping, antiviral drug resistance, and diagnosis of sepsis using 16s RNA based methods. Next generation sequencing potentially allows for identification of every microorganism and prediction of drug resistance directly from the specimen. Currently largely research based, scientific developments will definitely bring NGS to the diagnostic laboratory in the future.
Complex technology, high costs, good infrastructure, and requirement of highly skilled staff have been limitations of molecular technology. Annual maintenance costs and requirement of electricity and internet are some constraints that still exist. With improved techniques and detection of gene mutations, interpretive concerns with low range results and variants of unknown significance remain. Molecular technology has evolved from highly complex systems requiring elaborate infrastructure and highly skilled technical personnel to user friendly point-of-care systems. With excellent sensitivity, specificity, and speed, molecular diagnostics have a crucial role to play in the diagnosis, monitoring, treatment, and management of microbial diseases. Research and development is constantly underway to bring compact systems, improved sensitivity, and broader targets. It is only a matter of time that complex techniques such as NGS and microarrays will make their foray into the diagnostic laboratory.
