NEW YORK (GenomeWeb) — Molecular testing methods such as nucleic acid sequence-based amplification (NASBA) and qPCR, administered either in combination or separately, are superior to routinely used ELISA-based assays for diagnosing invasive aspergillosis, according to a newly published retrospective study.
The findings support the idea of routine molecular testing for early diagnosis of invasive aspergillosis, which is crucial since the infection can often be fatal in severely immunocompromised patients. In addition, NASBA in particular appears to be promising as a routine diagnostic method because of its advantages in sensitivity, simplicity, and speed, according to the study's authors.
The study, conducted by researchers from the First Affiliated Hospital of Chongqing Medical University and Chongqing Zhongshan Hospital in China, was published online last week in The Journal of Molecular Diagnostics.
In it, the investigators retrospectively examined blood samples taken from 80 patients at high risk for invasive aspergillosis, including patients with high beta-glucan levels (often a warning sign of fungal infection), immunocompromised patients, patients with hematological disease, and recent recipients of allogeneic stem cell transplants.
A total of 52 samples were identified as positive by one of three tests: NASBA, qPCR, or galactomannan enzyme-linked immunosorbent assay (GM-ELISA), the latter of which is routinely performed along with histopathology and culture to diagnose invasive aspergillosis.
Both the NASBA and qPCR assays were developed by the Chinese researchers. The NASBA assay used a total RNA extraction kit from BioTeke and previously published primers for a highly conserved 18s RNA region specific for the Aspergillus genus. Meantime, the qPCR assay comprised total DNA extraction using a Qiagen kit followed by SYBR Green PCR using previously published primers targeting the 28S rRNA gene.
The GM-ELISA assay, which detects galactomannan —a polysaccharide component of fungal cell walls that is often released into serum during fungal infections — was a commercial kit from Bio-Rad, and was cleared by the US Food and Drug Administration in 2009.
Comparison of the three assays revealed that NASBA had the highest sensitivity, at 76.47 percent (compared to 67.65 percent for qPCR and 52.94 percent for GM-ELISA); while qPCR had the highest specificity, at 89.13 percent (compared to 80.43 percent for both NASBA and GM-ELISA).
In addition, the researchers evaluated the tests in various combinations in order to determine an optimal diagnostic strategy, and here the molecular methods shined: serial testing (in which any one negative yields an overall negative result) using both NASBA and qPCR demonstrated perfect specificity and perfect positive predictive value. Meantime, parallel testing (in which any one positive yields an overall positive result) using both molecular assays resulted in a sensitivity of 94.12 percent.
The combination of molecular methods in a serial fashion, the researchers wrote in their paper, "should be useful in excluding invasive aspergillosis in suspect cases, thus reducing both suffering and expense for immunocompromised patients. On the other hand, the combination of NASBA and qPCR in parallel testing could be more suitable for screening patients suspected of invasive aspergillosis because this assay [combination] had the highest sensitivity."
In an email to PCR Insider, Yun Xia, a researcher at the First Affiliated Hospital of Chongqing Medical University and corresponding author on the paper, noted that GM-ELISA is currently routinely performed to diagnose invasive aspergillosis, but is not considered the gold standard for testing in China because "it only provides … evidence for Aspergillus infection as it has low sensitivity."
He also noted that he was not aware of any commercial molecular assays for Aspergillus detection, and said that his group has an interest in producing commercial NASBA or qPCR assays and is "preparing for kit development."
Perhaps the most notable example of a molecular Aspergillus assay under development is through a collaboration struck between Becton Dickinson's BD Diagnostics and Lab21 in 2011. The goal of this partnership was to develop a qPCR-based assay for use on the BD Max molecular testing platform using certain fungal DNA extraction and assay technologies originally developed by Myconostica, which Lab21 acquired in 2011. However, the current status of this assay-development partnership is in limbo, as Irish clinical diagnostics firm Trinity Biotech acquired certain molecular assets from Lab21 last October.
In their paper, Xia and colleagues underscored NASBA as a "promising tool for routine diagnosis" of invasive aspergillosis because it is sensitive, simple to administer, and fast. NASBA has been around since the early 1990s, and is an RNA-directed isothermal transcription-based amplification process that specifically amplifies RNA even in the presence of genomic DNA. The study's authors pointed out that it also has greater amplification efficiency than PCR, yielding more than 1012 amplicons in as little as 30 minutes.
Brian Wickes, a professor of microbiology and immunology and director of the Advanced Nucleic Acids Core Facility at the University of Texas Health Science Center at San Antonio, told PCR Insider in an email this week that the Chinese researchers may be on the right track in terms of considering molecular methods for at least surveillance, if not routine diagnosis, of aspergillosis.
"I think an assay that can be made cheap and easy to perform can be used for surveillance of at-risk patients, which would be its real value for treating patients that become infected with Aspergillus sp.," said Wickes, who was not involved in the JMD study, but earlier this year co-authored a study demonstrating a new method of extracting and PCR amplifying fungal DNA.
"Catching these infections early is crucial to successful treatment outcomes," Wickes said. "PCR assays are inexpensive and can be prepared as ready-to-go master mixes, which further enhances their diagnostic utility. In contrast to qPCR, NASBA is particularly cost-effective because temperature cycling is not needed, thereby eliminating the need for specific and expensive instrumentation."
Wickes also noted that molecular methods are broadly applicable because, in contrast to serological assays, "targets can be developed for nucleic acid-based assays relatively easily and can be adapted depending on specificity needs, which are often dictated by treatment strategies."
However, he warned that sample prep may be the most important component of any nucleic acid-based diagnostic assay, and noted that although it might be feasible to implement both NASBA and qPCR in a clinical setting, "qPCR uses DNA as the template nucleic acid while NASBA uses RNA … [and] it is not cost efficient or a good use of labor to use two different extractions primarily because in many cases sample specimens can be limited."
Xia said that the Chinese researchers believe that there would be little cost difference between performing GM-ELISA and the molecular tests, but he did not elaborate. The authors did concede in their paper that combination testing, whether parallel or serial, generally costs more than single individual testing. "However, for serial testing, it is an option to perform the second test only if the result of the first test is positive; in this case, there would be no increase of cost compared with single individual testing if invasive aspergillosis is not diagnosed."