Recently mNGS testing and analyses have become commercially available. For example, Charles Chiu and colleagues from the University of California, San Francisco (UCSF) are pioneers in the development of mNGS testing for the diagnosis of central nervous system (CNS) infections. In 2014, the first use of mNGS was reported for the diagnosis of neuro-leptospirosis on CSF from a 14-year-old boy presenting the signs of meningoencephalitis. This was the first report demonstrating the use of mNGS with medically actionable information and successful clinical diagnosis that led to the appropriate treatment of the patient. Since then, UCSF provides validated mNGS DNA and RNA testing for patients with meningitis and/or encephalitis. The UCSF diagnostic lab also offers mNGS DNA testing for patients with sepsis and disseminated infections. UCSF software analyzes sequence reads, identifies those reads which align to pathogens in the GenBank database, and issues a report showing the presence of pathogens in a clinical sample, along with clinical interpretation. At least 66.7% of pathogens detected from CSF were true positives, and only 5.6% were found to be false positives. The mNGS test at USCF for pathogen detection from CSF specimens showed a sensitivity of 86.1% and a specificity of 97.9%. The mNGS test for pathogen detection from plasma samples showed a sensitivity of 77% and specificity of 86%. The turnaround time from shipping samples to delivery of a report is generally 1-2 weeks.
The Karius test (Karius, California, United States) is another example of how mNGS is useful for the diagnosis of bloodstream infections (BSIs) and sepsis. The Karius test involves extraction of cell-free DNA (cfDNA) from plasma, then a sequencing library is created and sequenced using Illumina technology. The sequence data is compared to an internal reference database encompassing a number of microbial genomes. A published study by Thair et al. confirmed that the Karius test detected approximately three times more positive cases than culture-based detection. However, the limitation is that the test can give false positive results. The Arizona-based Fry Laboratories also provides DNA sequencing diagnostic services for cutaneous, gastrointestinal, hematologic, musculoskeletal, and pulmonary infections.
The Beijing Genomic Institute (BGI Genomics), a China-based company, is one of the largest companies that provide clinical mNGS services for the detection of pathogens causing respiratory infections such as Coronavirus and other pathogenic microorganisms. The sequencing services by the Zhejiang, China-based IngeniGen XunMinKang Biotechnology company also provide the detection of undiagnosed pathogens in patients with respiratory diseases.
mNGS is an unbiased culture-independent and hypothesis-free sequencing technology that has shown tremendous clinical application particularly in the diagnosis of CNS infections, bloodstream infections, and respiratory tract infections. Examples of recent applications of mNGS in the diagnosis of these infections are provided in Table 2. Below is a brief overview of the areas where mNGS has made considerable impact and the implications.
Pneumonia is considered among the top 10 causes of death in the United States, especially among immunocompromised patients such as those with hematologic malignancy or undergoing hematopoietic stem cell transplant. The identification of the causative agent of pneumonia is difficult and often inaccurate due to the pathogen diversity, heterogeneity of sampling, and limited detection methods. Traditional molecular diagnosis for pneumonia is pathogen-specific but unreliable for novel or unexpected pathogens. The ability of mNGS to provide a comprehensive view of pathogens makes it useful in the diagnosis of unexplained pneumonia and disease of unknown etiology. Recently, mNGS has improved the diagnosis of pulmonary infections over traditional methods by detecting a broad range of organisms including bacteria, viruses and fungi in a number of recent investigations. Remarkably, the causative agent was identified only by mNGS in two recent studies. Importantly, mNGS led to the treatment modifications and guided treatment decisions for 127 patients with pulmonary infections. Moreover, mNGS detected 50% of cases coinfected with bacteria of different respiratory origin in another study.
A mNGS approach can be superior to traditional methods for pathogen detection and confirmation of respiratory infections, particularly for Mycobacterium tuberculosis. Mycobacterium tuberculosis, can be quite challenging to detect, however, it has been shown in the last few years that mNGS could potentially be used as the first-line diagnostic test for tuberculosis. Karius-based mNGS testing of plasma samples detected suspected tuberculosis in 60% of adults and 50% of pediatric patients.
Lastly, RNA viruses are also considered one of the primary causes of respiratory infections. mNGS can detect a number of viruses that are usually not screened for in respiratory infections using routine diagnostic assays. It has shown good sensitivity and specificity compared to conventional testing and can identify viruses such as Influenza, Rhinovirus, and HIV. An additional advantage to mNGS is the potential to document and describe emerging, and re-emerging viral infections associated with outbreaks. For example, RNA-based viral metagenomics has detected the presence of novel human coronavirus variants from patients with respiratory symptoms.
In 2017, it was estimated that 48.9 million cases and 11 million deaths were related to sepsis globally. Thus, the early and accurate diagnosis of BSI is critical to initiate appropriate antibiotic therapy and for patient survival. Recent findings indicate sequencing microbial cfDNA using mNGS is a valuable approach for the detection of BSI pathogens when the conventional diagnostics fail to detect the etiological agent. A retrospective multi-center study utilizing the cfDNA and RNA showed that mNGS had a positive impact in 7.3% of cases, a negative impact in 3.7% of cases, and no impact in 86.6% of cases in patients with suspicion of multiple infections. Another study applied mNGS on cfDNA in septic and non-septic intensive care unit (ICU) patients and was able to diagnose sepsis and predicted mortality as soon as the first day. Similarly, cfDNA of relevant pathogens was detected in the blood plasma of cystic fibrosis patients. mNGS testing improved the detection rate of BSI in patients having fever of unknown origin or patients with suspected BSI from 38% to 87.1% when compared to conventional methods. However, no difference was observed in specificity between two methods for patients with clinical suspicion of infections. In one report, mNGS pathogen detection rate was comparable with routine diagnostics in 37% of cases. In some scenarios, the pathogen detection rate for mNGS varied by organism. For instance, mNGS was 100% sensitive for the detection of Staphylococcus aureus and Escherichia coli. However, mNGS test missed the presence of Streptococcus pyogenes. In contrast, 37% of BSI cases were found to be positive by only mNGS test in patients with clinical suspicion of sepsis in another study.
Neuroinflammatory diseases such as meningitis and encephalitis can be diagnostically challenging due to the requirement of invasive procedures for CSF collection, limited availability/low volume of CNS samples, and difficulty of detection by traditional culture. Furthermore, meningoencephalitis is related with increased risk of morbidity and mortality and thus needs prompt diagnosis and disease management. CSF culture is considered the gold-standard method for the diagnosis of meningitis. However, prior antibiotic therapy may reduce the sensitivity of CSF cultures, increasing the possibility of false-negatives. mNGS has potential to detect pathogens of unknown etiology as evidenced by clinical series demonstrating the success of mNGS in the detection of hard-to-diagnose CNS infection cases. Recently published studies have confirmed the diagnostic sensitivity of mNGS ranging 22% to 95% in patients with CNS infections. mNGS testing guided treatment decisions and clinical actionable management for 34.1%-53% of patients in these reports. However, contaminants from skin flora can lead to false positive bacterial sequences in CSF specimens obtained by lumbar puncture.