4. Discussions
Currently, culture-based techniques that routinely employed to isolate
pulmonary pathogens often using selective culture media designed for
specific groups of microorganisms. The culture conditions are biased
towards known, previously encountered microorganisms, and some novel and
rare microorganisms might be missed. So the detection rate of
microorganisms in conventional culture is low. Miao et al.’s study
reported the sensitivity of culture for diagnosing infectious disease
was 35.2% [22]. Jain et. al reported that no bacterial pathogen was
ever isolated by culture in up to 75% of pneumonia
cases [4], which was similar
to the culture-negative rate seen in another study
[3]. The present study found
that the culture-positive rate for the diagnosis of lung infections was
even lower, as low as 16.3%. The possible reasons for the lower
culture-positive rate are as follows, first of all, all of the patients
in the present study received empiric broad spectrum antibiotics prior
to sample collection. Detection of microorganisms from routine culture
is limited due to the early administration of broad-spectrum or
prophylactic antimicrobial drugs that could have been sufficient to
affect culture results but not to eradicate infection [23,
27]. Secondly, 74% patients
were immune-impaired in the present study, microorganisms infecting the
immune-impaired host can be fastidious to grow or non-cultivable
[3, 28, 29].
The mNGS allows for unbiased detection of virtually any pathogen present
in a given sample through direct sequencing of the sample’s extracted
DNA [30, 31]. We conducted a
literature review and found that there are currently several studies on
the use of mNGS for the diagnosis of pulmonary infections. Although mNGS
had different sensitivities for detecting pathogens in these studies,
and the gold standards used to calculate sensitivity vary, some were
based traditional methods [19 ~ 21] and some were
based on the final diagnosis [22, 23] as the gold standard. However,
a similar conclusion is reached that mNGS is more sensitive and more
advantageous than traditional methods in identifying pathogenic
microorganisms. Li et al. applied mNGS to detect the microbial pathogens
in CT-guided puncture lung biopsy tissues, they reported that the
sensitivity and specificity were 100.0% and 76.5% for bacteria, 57.1%
and 61.5% for fungi when compared to culture [19]. Langelier et
al.’s study applied mNGS of BALF to detect microbial pathogens in
hematopoietic cell transplant patients with acute respiratory illnesses,
which reported the sensitivity of mNGS for detecting respiratory
microbes (human metapneumovirus, respiratory syncytial virus,
Stenotrophomonas maltophilia, human herpesvirus 6 and cytomegalovirus)
was 100% when compared to standard testing [20]. Zhang et al.’s
study reported 13 cases of Pneumocystis pneumonia (PCP) identified
through mNGS of BALF or sputum or blood. Pneumocystis jirovecii was
detected by mNGS in all samples and by conventional methods in 5 out of
13 samples, respectively. They concluded that mNGS showed satisfying
Pneumocystis pneumonia detection rate compared to conventional methods
[21]. Pan et al.’s study explored the application of mNGS of BALF in
the microbiologies diagnosis of community acquired pneumonia in
immune-impaired patients. They reported standard procedures identified
pathogens in 6 out of 13 patients, while mNGS detected pathogens in 12
out of 13 patients [32]. Miao et al.’s study reported the
sensitivity of mNGS for diagnosing infectious disease was 50.7%.
However, in their study, the study was conducted on patients with
various types of infectious diseases, not just lung infections and
specimens were not limited to respiratory specimens [22]. In the
present study, the gold standard used to calculate sensitivity and
specificity was the final diagnosis. We found that the sensitivity of
mNGS of BALF to detect pathogenic microorganisms was significantly
higher than that of traditional culture of BALF, regardless of bacterial
pneumonia (85.7% versus 42.9%), fungal pneumonia (71.4 % versus
4.8%), or generalized pneumonia (81.4% versus 16.3%).
It is worth noting that this study demonstrates that the advantages of
mNGS in the field of fungus testing are more prominent. Culture
indentified pathogens in only one fungal pneumonia patients (1/21),
while mNGS detected pathogens in 19 fungal pneumonia patients (19/21).
The two patients missed by mNGS were a patient with cryptococcal
pneumonia and a patient with Aspergillus pneumonia. The culture and GM
test of the patients with Aspergillus pneumonia missed by mNGS happen to
be positive. Although the culture-positive rate is low, it is very
necessary to combine culture, GM test and mNGS for the diagnosis of
fungal pneumonia, so as to avoid missed diagnosis to the greatest
extent. It should be emphasized that mNGS is not omnipotent. For
cryptococcal pneumonia, mNGS does not have an advantage. As shown in
this study, the capsular polysaccharide antigen was positive in all four
cases of cryptococcal pneumonia, but one case was missed by mNGS. ROSE
also played an important role in the diagnosis of cryptococcal
pneumonia. We saw granulomas in TBLB specimens of all 4 patients by ROSE
for the first time, and Cryptococcus was found in TBLB specimens in 3 of
them by ROSE. Therefore, our recommendation for the diagnosis of
cryptococcal pneumonia is that further detection of capsular
polysaccharide antigen is of great significance, while mNGS is not
necessary, if cryptococcal pneumonia is highly suspected based on the
patient’s exposure history, clinical manifestations, imaging findings
and ROSE results.
In terms of detecting bacteria, mNGS still has advantages over culture,
although this advantage is not as prominent in fungal detection.
Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli are
easily detected by culture. However, some bacterial pathogens detected
by mNGS, including Nocardia, Mycobacterium tuberculosis, Pneumocystis
jirovecii, Haemophilus parainfluenzae, Pyramidobacter piscolens and
Prevotella, were not detected by culture. These bacteria are either
fastidious microbes (such as anaerobe) or require long incubation times
(such as Mycobacterium tuberculosis). In P4, two obligate anaerobic
bacteria (Pyramidobacter piscolens and Prevotella) were identified, in
P25, Pyramidobacter piscolens were identified. Pyramidobacter piscolens
and Prevotella were usually isolated from the oral cavity of patients
with dental pulp disease, periodontal infection or alveolar abscess and
healthy people. They may be a potential pathogen of pulp disease and
periodontal disease [33, 34]. Study also reported that Prevotella
induces severe bacteremic pneumococcal pneumonia in mice
[35].
In the present study, 74% patients were immune-impaired and 23% of
patients with pulmonary infection were confirmed to be mixed infections
by mNGS. Conventional culture is powerless in identifying mixed
infections, while mNGS exhibited its remarkable advantages in detecting
pathogens of mixed pulmonary infections in immune-impaired patients.
Therefore, mNGS might be more likely to benefit immune-impaired patients
who are susceptible population of various pathogens.
Using mNGS, we further explored the characteristics of flora composition
in the three respiratory specimens (including the relative abundance of
pathogens and the relative abundance and richness of common
oropharyngeal microbiota). Surprisingly,
we found that TBLB and BB samples
were similar in flora composition except for the richness of common
oropharyngeal microbiota, while the relative abundance and richness of
common oropharyngeal microbiota in BALF were higher than TBLB.
In our study, in comparison to BALB and BALF, BB gave a higher number of
true positive, in comparison to BALF, BB gave much lesser number of
false positive and false negative cases, showing its superiority in
diagnosing infective PPLs. So regardless of the diagnostic efficacy, or
the relative abundance of the pathogenic microorganisms and the
contamination of common oropharyngeal microbiota, BB is no worse than
TBLB. In the present study, BB yielded a better diagnostic performance
most likely because it allowed cells and microorganisms to be collected
from a larger area. This suggests that in future,
high throughput sequencing of the
BB samples might be an alternative choice for patients with infectious
PPLs but couldn’t tolerate more invasive TBLB procedures, such as
patients with hematological diseases who cannot tolerate TBLB because of
thrombocytopenia or poor platelet function.
The current study has several limitations: the first limitation is a
small sample size, the number of patients who had mNGS in all three
samples is limited due to the relatively expensive cost of mNGS
sequencing. The second limitation is using a retrospective design. The
third limitation is that this study did not make a statistical
comparison of specificity differences. Because ROSE was performed before
the mNGS specimens were taken, if the clinical information, CT results
and ROSE performance were all indicative of infectious lesions, then the
sample was further sampled for mNGS, so the number of negative cases was
small. In this case, the calculated specificity is not reliable, so the
specificity is not compared between groups.