Despite the great progress in prevention and treatment with prophylactic antibacterial and colony-stimulating factors, FN is still one of the most common and serious complications in Hematological and malignant diseases, which may lead to treatment delay, poor consequence and even death[16]. 20%-30% of patients need hospitalization, and the overall hospitalization mortality is about 10%. For 24-72 hours without relief in fever, reevaluation is needed to adjust the treatment plan according to European guidelines[1,17]. Bacteremia can be detected in 72 hours by traditional blood culture, but its sensitivity is low, often affected by the technician's technology, physical and chemical environment and contamination[18]. The instability and incompleteness drive us to find a better detection method. In recent years, new detection methods, such as polymerase chain reaction (PCR), not relying on culture, was adopted by some microbial technician. However it can only detect common pathogens qualitatively[19]. Since 2008, a large number of studies from more than twenty countries have shown that mNGS, directly detecting plasma free cells, has high practicability in the diagnosis of undiagnosed infections, and it performs well in the identification of rare, novel, occult and mixed infection [20].This makes the new technology suitable for detection of fever pathogens in children with hematological and malignant tumors.
According to the analysis of 37 samples in mNGS and TT group, we eventually detected 39 pathogens in 32 cases. The positive rate of mNGS for bacteria was 35.1% higher than the TT group(Mainly through blood culture)which was only 8.1%. That’s not surprisingly, as blood culture was always known with low sensitivity. In the past ten years, the common bacteria reported by different research centers are different. Gram-negative bacteria is slightly higher than Gram-positive bacteria[21]. In our study, Gram-positive and negative bacteria were found equal in 14 cases, different from Yan Chenhua's multicenter and prospective epidemiological study on FN in China in 2016[22]. Considering the regular use of carbapenem antibiotics treatment, many cases of Gram-negative bacteria infection might cure within 3 days, and not incorporated in the group. With further analysis, despite of the high positivity for bacteria, the re-detection did not promote much adjustment of antibacterials. That may because of the sufficient experience in the use of antibacterial drugs in Hematological and malignant diseases, which the clinicians had.
15 fungal infections were detected in 37 cases, accounting for a large proportion. Histopathological diagnosis is the gold standard for IFD, but it is time consuming and invasive. We make clinical diagnosis by G test, galactomannan test and chest CT.[25,26,27]. G test and galactomannan test are somehow lack of classification of different phylum of fungi. Early during the collection phase in our study,we found a case,of which the early G test was highly positive. Antifungal (Voriconazole) was added to combine with the initial empiric antibacterial therapy, meanwhile mNGS were given. However the disease still worsened and could not be reversed. Two days later, the patient died, with the blood samples of mNGS reported as tropical candidiasis. The precise selection of antifungal drugs is directly related to the therapeutic effect in the treatment of fungal bacteremia,which is more dangerous. mNGS, which provided more specific species of fungal, could have helped if given earlier. In our study, totally 4 cases got more active and effective antifungal treatment due to mNGS. Here we suggest that mNGS accurately did provide positive improvements to treatment.
In addition, it is more sensitive to virus with the unbiased detection, and is also simpler operating with multiple viruses. However, the presence of contamination in DNA extraction is obvious in virus detection. We received the reports of multiple viruses in some cases with various levels of sequence expression although the technicians had already performed the comparison and selection according to the most advanced approach[28,29]. Although with higher sequence expressions of virus in some cases, they were still confirmed as previous infection in combination with subsequent pathogen antibody detection. For the confirmed virus infection, we reduced the use of antibacterial drugs and adopted targeted treatment. In addition, for rare infections like parasitic, without giving a clear indication in the previous clinical examination, it is difficult to diagnose through routine laboratory tests. At this moment, mNGS absolutely provided help.
In summary, a total of 9 cases (24.3%) got a positive effect on the treatment strategy, especially for fungal infection. In the remaining cases, with most of the pathogens ware found, although there was no change in treatment, the diagnosis was clear and it was still helpful for the clinician to keep track of the diseases.
To learn more about the effect, we also compared fever duration time in children who received mNGS and TT with those only had traditional ones before. The results showed that the duration of fever in mNGS and TT group was shorter than that of the TT group. For children of FN with hourly mortality, the fever duration is closely related to the survival rate. With the high positive rate, mNGS contributed to precise anti-infective treatment, and this finally resulted in earlier control of infection and for some less experience burden. In addition, we want to clarify a situation in this retrospective study. Children with more severe symptoms (like higher temperature, worse cough, poor mental state, et) are more likely to agree with the new detection. For those relatively mild ones, parents balanced interests, and disagree with this check finally. Therefore, in fact, the condition of children in mNGS and TT group is somehow more serious than that in the TT group. We believe If given more random and equal trial conditions, duration of fever of mNGS and TT group could be shorter than the actual value.
Theoretically, given enough sequencing length, almost all microorganisms can be uniquely identified through mNGS by multiple matching with microbial genome and reference database [30]. With the comprehensive and multi-pathogen coverage, positivity of pathogens in FN which with complex pathogens and atypical manifestations is high. However, the specificity of this method is opposite. This is also a challenge for technician at present. The accurate interpretation of pollution and pathogens not only requires a more scientific sequence analysis method, but also emphasizes the clinical thinking of clinicians. Diagnosis requires clinicians to combine with the patient's clinical manifestations. In our study, pathogens was selected by discussion with multiple clinicians and sometimes should be verified by other experimental methods, so as to minimize false positive rates. Imitation of our study was the small sample size. The price was an important factor affecting its large-scale promotion. However, with the maturity of the test technology and the standardization of the process, the turnaround time can be constantly shortened, and definitely the cost will constantly be reduced. Then mNGS will become a widely accepted method, not just a last resort[20].
mNGS was found to have practical clinical value for FN in our research. However, to ensure the correct use of the new model of microbiology test, clinician and microbiologists should fully understand the function and limitations of this method.