Patients Characteristics Twenty-two patients were finally included, including 21 males (94.54%) with a median age was 36 years (IQR: 27-46 years). With 17(77.27%) CT/MRI positive and only 1 patient (4.55%) on Antiroviral Therapy (Table 1). The cohort consisted mainly of isolated meningitis (13 patients [59.09%]) or encephalitis (7 patients [31.81%]), with 9.09% presenting with myelitis (2 patients). 31.81% (7 patients) study patients were severely ill, all of whom were admitted to intensive care unit (ICU). The median CD3+4+ T cell count 74cells/ul (IQR: 13–214 cells/ul). The median time from admission to CSF sampling for metagenomic NGS was 3 days (IQR:0–14 days). The median white blood cell count in CSF was 9/mm3 (IQR: 4–90/mm3).
Characteristic
|
Value
|
Age years median(range)
|
36(27-46%)
|
Male sex - no. (%)
|
21(95.45%)
|
Syndrome - no. (%)
|
Meningitis only
|
13(59.09%)
|
Encephalitis with or without meningitis
|
7(31.81%)
|
Myelitis with or without meningitis
|
2(9.09%)
|
Department
|
Infectious disease department
|
15(68.19%)
|
Intensive care unit
|
7(31.81%)
|
MRI/CT(+)
|
17(77.27%)
|
Antiroviral Therapy (%)
|
1(9%)
|
CD3+4+ T cell count (cells/ul) median(range)
|
74 (13-214)
|
White blood cell count in CSF (10E6/L)
|
9(4-90)
|
Median time after hospital admission that CSF sampling for metagenomic NGS (range) - days
|
3(0-14)
|
All cases
|
22
|
Performance of mNGS for diagnosing CNS infections
Diagnostic performance of mNGS Relative to Conventional Methods
Twenty-two CSF samples were tested by mNGS and conventional microbiological studies. 19 infections were identified by mNGS, while 13 infections were detected by traditional direct-detection tests alone (defined as culture, PCR, x-pert /RIF or CSF antigen tests, excluding serological tests or samples other than CSF). As expected, mNGS increased the sensitivity rate by approximately 40% in comparison with that of conventional methods (86.36% vs. 45.21%) (Table 2).
In total, NGS detected 5 bacterial species, of which M. tuberculosis (2cases) was the most common, 20 viral species, of which (EBV)(9) and Human betaherpes virus 5(CMV)(4) were the most common, 6 fungal species, both of which were Cryptococcus (2)and Talaromyces marneffei (2), and 1 parasitic species, of which was the Toxoplasma gondii. (Figure 1-A) Nine co-infections were detected, of which Human gammaherpesvirus 4 (EBV) (7cases) and cytomegalovirus(3cases) was the most common (Figure 1-B). The percentage of co-infection in mNGS positive specimens was notably higher than that of conventional methods positive specimens. (40.91% vs. 14.39%).
Table 2 Diagnostic performance of mNGS Relative to Conventional Methods
|
mNGS
|
Conventional methods
|
Positive percent (n/N) (%)
|
86.36(19/22)
|
59.09(13/22)
|
Pathogens
|
Bacteria(n)
|
5
|
2
|
DNA Viruses(n)
|
20
|
12
|
Fungi(n)
|
6
|
3
|
Parasites(n)
|
1
|
0
|
Co-infection(n/N) (%)
|
40.91(9/22)
|
17.39(4/22)
|
EBV+ Acinetobacter bau-mannii
|
1
|
0
|
EBV+Talaromyces marneffei
|
1
|
1
|
EBV+Mycobacterium tuberculosis
|
1
|
1
|
EBV+CMV+JC polyomavirus
|
1
|
0
|
EBV+Human alphaherpesvirus 3(VZV)
|
1
|
0
|
CEB+CMV+Mycobacterium tuberculosis
|
1
|
1
|
CMV+Talaromyces marneffei
|
1
|
0
|
Cryptococcus neoformans+JC polyomavirus
|
1
|
0
|
EB+CMV+Aspergillus fumigatus
|
1
|
0
|
EB+CMV+ Talaromyces marneffei
|
0
|
1
|
Concordance Between mNGS and conventional methods in Detection of Pathogens
In our results, 13 of the 22 cases (59.9%) with mNGS and traditional methods were both positive, and 2 of the 22 cases (0.09%) were both negative. Only mNGS positive was found in 5 cases (22.73%), but no positive was found in conventional methods only.For the double positive samples, 6 of the 13 cases were a perfect match, and 2 cases were a complete mismatch. “Partly matched” were found in 5 cases, indicating that at least 1 overlap of pathogens when multiple microbial results were observed (Figure 2).
The clinical implications of mNGStesting in real-world settings.
Clinician Feedback (Pathogens identified by mNGS in 19 cases)
A total of 27 pathogens were identified in 19 patients (86.36%). Based on mNGS diagnosis, 4(21%)) modified the initial diagnosis, while up to 10(53%) identified microorganisms corroborated the clinical diagnosis. 2 (10%) pathogens were not well related with clinical diseases, while 3 (16%) were inconsistent with clinical diagnosis (Figure 3).
For mNGS identified pathogens, the initial empirical diagnosis was often confirmed by mNGS (10.53%) or modified (21%), while only 16% were considered unreliable (unsupported) and 10% uncertain.
Clinical Effect
The mNGS detected pathogens but not detected by conventional methods included: 3 bacterial species,4 viral species, 3 fungal species, and 1 parasitic species. Among the pathogens identified by the metagenomic NGS alone, 6 pathogens the clinician declared that the results of mNGS approvingly influenced their clinical reasoning and 7 pathogens guided antimicrobial therapy (Table 3).
Table 3 Clinical Effect (16 Extra Pathogens in 14 cases identified by mNGS only)
|
All
|
Active
antimicrobial
therapy
|
Non-active
antimicrobial
therapy
|
No
antimicrobial
therapy
|
Extra Pathogens identified by
mNGS (n)
|
|
|
|
|
Bacteria
|
|
|
|
|
Acinetobacter baumanniic
|
1
|
|
|
1
|
Haemophilus parahaemolyticus
|
1
|
|
1
|
|
Pseudomonas aeruginosa
|
1
|
|
1
|
|
DNA Virus(n)
|
|
|
|
|
Human alphaherpes virus 3(VZV)
|
2
|
1
|
|
1
|
Human gammaherpes virus 4(EBV)
|
2
|
1
|
|
1
|
Human betaherpes virus 5(CMV)
|
2
|
1
|
|
1
|
JC polyomavirus
|
2
|
|
|
2
|
Fungi(n)
|
|
|
|
|
Cryptococcus neoformans
|
2
|
|
|
1
|
Aspergillus sydowii
|
1
|
1
|
|
|
Aspergillus fumigatus
|
1
|
1
|
|
|
Parasites(n)
|
|
|
|
|
Toxoplasma gondii
|
1
|
1
|
|
|
total
|
16
|
6
|
2
|
7
|