​Complete summary of fungi inspection pictures and texts, experts come to draw the key points!

*It is only for medical professionals to read for reference.
It is no longer difficult to understand the fungal test report! Some fungi can invade the human body, grow and multiply in tissues, organs, and blood, and cause inflammatory reactions and infectious diseases of tissue damage
.

At present, common clinical fungal infections such as Candida, Aspergillus and so on
.

Laboratory techniques for diagnosing fungal infections include smears, culture, (1,3)-β-D glucan (G test), etc.
With the advancement of medical biotechnology, new diagnostic techniques have gradually entered the clinic
.

On December 11, 2021, the Respiratory Medicine Annual Meeting of the Chinese Medical Association was held in Xiamen, Fujian
.

At the meeting, Professor Ye Feng from the First Affiliated Hospital of Guangzhou Medical University brought us a special report on "Progress in Fungal Laboratory Testing", let's take a look
.

Traditional fungal laboratory detection technology Traditional fungal laboratory diagnostic technology: smear, culture, G test, antigen antibody, pathology
.

▌ Direct microscopic examination of fungal morphological characteristics Candida: often singular budding, with pseudohyphae at the end narrowed and attached to the fungus, like a "sausage" connection, and most of the hyphae are separated; Aspergillus flavus: there are overnight filaments, visible "double The 45-degree branch of the "sense type", with larger hyphae; Aspergillus niger: long conidiophore and rough surface of small conidia; Cryptococcus neoformans: large variation in size, spherical or rugby-like, appearing clear on the black background of ink Halo; bacterial cells can be seen in the halo
.

▌ Colony characteristics of fungi Candida: There are 5 types of clinically common Candida, as shown in Figure 1
.

Figure 1 Source: Screenshot of Professor Ye Feng’s "Fungus Laboratory Detection Progress" report.
Candida color identification culture: Candida albicans colonies are emerald green, Candida tropicalis is blue gray or iron blue; Candida glabrata is purple, Kerou Candida is light powder; other types of Candida are white
.

Aspergillus: Figure 2 Source: Screenshot of Professor Ye Feng's "Fungus Laboratory Testing Progress" report.
Aspergillus under the microscope (Figure 2) is "beautiful and enchanting", but it brings great danger to human health
.

Among them, infectious diseases caused by Aspergillus fumigatus are more common
.

Cryptococcus: Figure 3 Source: Screenshot of Professor Ye Feng's "Progress in Fungal Laboratory Detection" Report Cryptococcus can grow on grape peptone agar, Sabouraud, and caffeic acid media
.

Talaromyces marneffei: A temperature-sensitive biphasic clump bacteria, the colony is pilose, and it secretes a wine-like red pigment in the medium
.

The positive rate of culture in bone marrow and lymph node is the highest (both 100%), followed by skin (90%) and blood (76%)
.

▌ Tissue section and pathology Figure 4 Source: Professor Ye Feng’s "Fungus Laboratory Detection Progress" report screenshot is shown in Figure 4.
It is difficult to distinguish Aspergillus and Mucor on histopathological section
.

Professor Ye Feng pointed out: “Aspergillus is relatively thin, with sharp-angled branches that have more divisions; while Mucor is relatively stubby, with a right-angled or obtuse-angled branch
.

" Optical chromogenic methods (such as calcium fluorescence or cape blue stain) for direct identification and rapid staining of specimens; positive sterile body fluid culture and deep tissue pathological identification are still the gold standard for the diagnosis of invasive infections
.

▌ Fungus G (BDG, (1,3)-β-D glucan) test [1] (1,3)-β-D glucan is a component of the cell wall of fungi.
When phagocytes engulf the fungus, it can make ( 1,3)-β-D glucan was released in the blood in large quantities and was detected
.

Table 1 Note: ICU: Intensive Care Unit; SOT: Solid Organ Transplantation; SoR: Strength of Recommendation; QoE: Quality of Evidence; HSCT, Hematopoietic Stem Cell Transplantation; IFD: Invasive Mycosis; HIV: Human Immunodeficiency Virus; IA: Invasive Aspergillosis; PPV: positive predictive value; NPV: negative predictive value; GM: galactomannan Professor Feng Ye emphasized: “The sensitivity of the G test is about 50% to 70%, and dynamic observation of the changes in the G test value is more clinically meaningful
.

”▌ GM test[1] GM test is a common method for clinical diagnosis of fungal infections, and is mostly used to identify infections caused by Aspergillus
.

Table 2 Note: HRCT: high-resolution computed tomography; ODI: optical density index ▌ different methods for detecting cryptococcal antigens Table 3 ▌ polymerase chain reaction (PCR) detection of fungi [2] PCR refers to the use of a piece of DNA as a template , With the participation of DNA polymerase and nucleotide substrate, the DNA is amplified to a sufficient amount for structural and functional analysis
.

As shown in Figure 5, extraction of Aspergillus DNA from whole blood requires higher technical requirements than extraction from serum
.

Professor Ye Feng pointed out: “The sensitivities of whole blood PCR and serum PCR are 85.
1% and 78.
7%, respectively, and the sensitivity of whole blood PCR is higher; but extracting DNA from serum samples is faster and easier than whole blood
.

” Note: ELISA: Enzyme-linked immunosorbent assay; WB: whole blood; Serum: serum Figure 5▌ Molecular biological diagnosis of tissue biopsy specimens [1] Table 4▌ Combined detection improves the sensitivity of diagnosis Yes, combining multiple detection methods can eliminate more false positives and increase the sensitivity rate of diagnosis
.

"Table 5 New fungal laboratory diagnostic technology ▌ T2 Candida [3] Diagnostic performance: In addition to G test and PCR technology, T2 magnetic resonance (T2 magnetic resonance, T2MR) can also detect Candida, namely T2 Candida; T2 Candida It is the best diagnostic tool for the diagnosis of candidaemia
.

Limitations: T2 Candida, an automatic molecular detection method based on magnetic resonance, can detect the 5 most common Candida species within 5 hours, such as Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei
.

T2 Candida failed to detect all Candida, and there is still a lack of large-scale randomized controlled studies in the diagnosis of deep Candida infection and candidemia in children
.

▌ Matrix-assisted laser Analytical ionization-time-of-flight mass spectrometry (MALDI-TOF MS) [4] Figure 6 Description of MALDI-TOF mass spectrometry technology.
Source: Professor Ye Feng's "Progress in Fungal Laboratory Detection" report screenshot MALDI-TOF MS is a simple, fast and high-pass High-quantity, low-cost and high-efficiency microbial identification technology, suitable for the detection of pure culture strains and blood culture bottles of Baoyang
.
It
can be detected quickly (usually within 20 minutes), and the sensitivity and specificity of the culture bottles for identification of Baoyang are high and close to 100%
.

Insufficient: This method is mainly used for the identification of bacteria, yeasts, and mycobacteria, and a standard database system needs to be established for comparative analysis
.

▌ The application of next-generation sequencing (NGS) in the diagnosis and treatment of infectious diseases [5] Figure 7 Laboratory diagnostic procedures include an overview of the general workflow of NGS ▌ VOCs (volatile organic compounds) for the diagnosis of pulmonary aspergillosis [6] For patients with suspected fungal pneumonia, the characteristics of secondary metabolites of Aspergillus in their exhaled breath can be used to identify IA
.

Figure 8: Aspergillus respiratory metabolic difference reference standard and test parameters Aspergillus secondary metabolite characteristics of any element detection can distinguish patients with IA, IFD or other pneumonia, the sensitivity is 94% (95% CI, 81%-98 %), the specificity is 93% (95%CI, 79%-98%)
.

▌ Comparison of multiple detection methods in the diagnosis of aspergillosis [7] Table 6 Note: BAL: bronchoalveolar lavage fluid; BG: β-glucan fungal resistance detection of Aspergillus fumigatus resistance to triazole drugs The problem is getting more and more serious.
The resistance mechanism originating from the environment is mainly TR34/L98H, and TR34/L98H has been reported in many countries [8]
.

To explore the resistance mechanism of TR34/L98H, it mainly includes: ①The geographical (air) migration of resistant TR34/L98H spores; ②The transfer of TR34/L98H in unrelated strains; ③ Both
.

A new type of TR46/Y121F/T289A was discovered in the Netherlands, which can cause high resistance to voriconazole and lead to the failure of voriconazole treatment
.

Summary: After class, Professor Ye Feng summarized as follows: 1.
Routine laboratory testing methods for fungi include smear microscopy (fluorescence staining), culture, pathology, G test, antigen, and antibody; 2.
Focus on new fungal detection technology: PCR , T2 Candida, MALDI-TOF, next-generation sequencing, breath characteristic VOCs; 3.
Combined detection is often used in clinical practice to improve the sensitivity and specificity of diagnosis; 4.
Focus on the susceptibility test and drug resistance of fungi
.

References: [1]Ullmann AJ,Aguado JM,Arikan-Akdagli S,et al.
Diagnosis and management of Aspergillus diseases:executive summary of the 2017 ESCMID-ECMM-ERS guideline.
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[2]Springer J,Morton CO,Perry M,et al.
Multicenter comparison of serum and whole-blood specimens for detection of Aspergillus DNA in high-risk hematological patients.
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[3]Zacharioudakis IM,Zervou FN,Mylonakis E.
Use of T2MR in invasive candidiasis with and without candidemia.
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[4]Croxatto A,Prod'hom G,Greub G.
Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.
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[5]Deurenberg RH,Bathoorn E,Chlebowicz MA,Couto N, Ferdous M, García-Cobos S, Kooistra-Smid AM, Raangs EC, Rosema S, Veloo AC, Zhou K, Friedrich AW, Rossen JW.
Application of next generation sequencing in clinical microbiology and infection prevention.
J Biotechnol.
2017 Feb 10;243:16-24.
[6]Koo S,Thomas HR,Daniels SD,Lynch RC,Fortier SM,Shea MM,Rearden P,Comolli JC,Baden LR,Marty FM.
A breath fungal secondary metabolite signature to diagnose Invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40.
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis:recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45( 6):646-52.
doi:10.
1111/eci.
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Epub 2015 Apr 24.
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus:a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
Rosema S,Veloo AC,Zhou K,Friedrich AW,Rossen JW.
Application of next generation sequencing in clinical microbiology and infection prevention.
J Biotechnol.
2017 Feb 10;243:16-24.
[6]Koo S,Thomas HR,Daniels SD,Lynch RC,Fortier SM,Shea MM,Rearden P,Comolli JC,Baden LR,Marty FM.
A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40 .
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis:recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45(6):646-52.
doi:10.
1111/eci.
12448.
Epub 2015 Apr 24 .
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus: a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
Rosema S,Veloo AC,Zhou K,Friedrich AW,Rossen JW.
Application of next generation sequencing in clinical microbiology and infection prevention.
J Biotechnol.
2017 Feb 10;243:16-24.
[6]Koo S,Thomas HR,Daniels SD,Lynch RC,Fortier SM,Shea MM,Rearden P,Comolli JC,Baden LR,Marty FM.
A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40 .
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis:recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45(6):646-52.
doi:10.
1111/eci.
12448.
Epub 2015 Apr 24 .
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus: a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
Application of next generation sequencing in clinical microbiology and infection prevention.
J Biotechnol.
2017 Feb 10;243:16-24.
[6]Koo S,Thomas HR,Daniels SD,Lynch RC,Fortier SM,Shea MM,Rearden P,Comolli JC,Baden LR,Marty FM.
A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40.
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis: recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45(6):646-52.
doi:10.
1111/eci.
12448.
Epub 2015 Apr 24.
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus: a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
Application of next generation sequencing in clinical microbiology and infection prevention.
J Biotechnol.
2017 Feb 10;243:16-24.
[6]Koo S,Thomas HR,Daniels SD,Lynch RC,Fortier SM,Shea MM,Rearden P,Comolli JC,Baden LR,Marty FM.
A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40.
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis: recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45(6):646-52.
doi:10.
1111/eci.
12448.
Epub 2015 Apr 24.
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus: a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40.
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis:recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45(6):646-52.
doi:10.
1111/eci.
12448.
Epub 2015 Apr 24.
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus:a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.
Clin Infect Dis.
2014 Dec 15;59(12):1733-40.
[7]Arvanitis M,Mylonakis E.
Diagnosis of invasive aspergillosis:recent developments and ongoing challenges.
Eur J Clin Invest.
2015 Jun;45(6):646-52.
doi:10.
1111/eci.
12448.
Epub 2015 Apr 24.
[8]Vermeulen E,Lagrou K,Verweij PE.
Azole resistance in Aspergillus fumigatus:a growing public health concern.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.
Curr Opin Infect Dis.
2013 Dec;26(6):493-500.