Patients with pulmonary fungal infections have diverse clinical manifestations and atypical imaging features, laboratory pathogenic examination becomes an important basis for diagnosis, among which smear microscopy and fungal culture of clinical fluid specimens are the most widely used, although it is difficult to identify whether it is colonization or infection in the diagnosis of certain fungal infections, but the results as “microbiological evidence “, undoubtedly for the diagnosis and treatment of invasive fungal diseases have important reference value. At present, the value of microscopic examination and culture results of different body fluid specimens is still controversial in clinical practice, and this issue is now discussed in order to provide reference for clinical diagnosis and treatment.
1. Clinical specimen acquisition and processing
1.1 Acquisition and processing of qualified sputum specimens
Sputum is the most important and commonly used clinical specimen for the pathogenic diagnosis of respiratory tract infection, and the correct method of sputum specimen collection and whether the sputum specimen is qualified or not are also the keys to whether the fungal culture can detect pathogenic bacteria. Like bacterial cultures, fungal sputum specimens should be collected before clinical suspicion of pulmonary fungal disease and the use of antifungal drugs, collected several times if necessary, and sent for examination within 2 hours, and if not sent in time should be stored at 4°C and processed within 24 hours. The current pass rate of sputum specimens sent for clinical examination is only 40% to 60%. The correct method of collection is to ask the patient to gargle first and cough deeply, and then retain purulent sputum or secretions for examination. In patients without sputum who need to be examined for Pneumocystis carinii, sputum can be induced by nebulized inhalation with hypertonic saline.
Sputum specimen processing should follow the principle of pathogenic detection of lower respiratory tract specimens by smear examination followed by culture, but in clinical practice people usually focus only on sputum culture, while sputum smear microscopy is often neglected. It is important to emphasize that sputum smear results have a very important indirect and direct diagnostic value for clinical purposes. First of all, smear microscopy can screen out qualified sputum specimens. Screening can be done by picking the purulent part of the specimen for Gram staining, when squamous epithelial cells <10/low magnification field, multinucleated leukocytes >25/low magnification field, or a ratio of both <1:2.5 is considered a qualified sputum specimen. Second, sputum smears can be stained with hematoxylin-eosin (HE) or related special stains for direct microscopic examination to find pathogens. HE staining is a non-specific fungal staining method that can improve the identification of mycelium to some extent. Fungal-specific stains such as hexosamine silver stain (GMS) and periodate-Schiff stain (PAS stain) can be applied to stain fungal pathogens such as Aspergillus, Trichoderma and Candida, and the combination of both stains can further improve the detection rate of fungal pathogens [1]. Respiratory specimens such as Aspergillus or Trichoderma mycelium, Candida pseudomycorrhizae and outgrowth spores are found to be of greater clinical significance.
1.2 Intra-airway suction acquisition and specimen handling
In patients with tracheal intubation and tracheotomy, microscopic examination and culture of airway secretions drawn through the artificial airway is an important basis for the pathogenic diagnosis of respiratory tract infection, and is more reliable than sputum specimens. The literature shows a high sensitivity and specificity of its pathogenic detection [2]. Tracheal suctioning is mostly performed by blind aspiration. Before the operation, the patient’s general condition should be improved as much as possible and adequate oxygen should be given, and in the case of ventilator-supported patients, pure oxygen inhalation can be given briefly to prevent hypoxemia caused during the operation. All operations need to follow the principle of asepsis, use sterile suction tubes and sputum collectors, adjust the negative pressure to 150-200 mmHg (adults) before suction, and the operation should be gentle, agile, and intermittent suction. Specimen processing is basically the same as sputum specimens.
There is no large-scale clinical report on the accuracy of the results of the “12-stage method” and “7-stage method” for semi-quantitative culture of fungi in respiratory secretions, but the graded report results can provide more reference value for the clinic. The purulent part of the respiratory secretion specimen was selected and inoculated with one blood plate, one chocolate plate and one Sabo plate, and one direct smear. The chocolate plate was placed in a candle jar, and the other plates were placed in an ordinary incubator, incubated at 35℃ for 48 hours to observe the morphology of the colonies, pick out various colonies, and mixed smear in the first area,
Gram staining, oil microscopy. Record and report the results of bacteria (including fungi) culture according to the following “12-level method”: no growth of the bacteria (including fungi) in all three plates as (-); only in Sabo or chocolate or blood agar growth, or not in the counting area and not visible in the counting area, or in the counting of various colonies 100 In the count of 100 bacteria (including fungi) colonies have 1 to 4, is judged as a small amount (<0. 1); 5 to 14, for 0. 1; 15 to 24, for 0. 2; 25 to 34, for 0. 3; and so on; pure culture for 1. 0. Direct smear Gram staining oil microscopy, first determine the quality of the specimen, according to the following "7-level method "Record and report the smear test results: fungal mycelium or spores are not seen in the whole film as (-); < 3 / 100 field of view, as a very small amount; 3 to 9 / 100 field of view, as a small amount; 1 to 9 / 10 field of view, as (+); 1 to 9 / each field of view, as (++); 10 to 99 / each field of view, as ( +++); ≥100 per field of view, for (++++) [3].
1.3 Protective brush sampling and sample processing
Relative to sputum culture examination, either transbronchoscopically guided protective brush (PSB) technique or blind pick-up method protective brush (BPSB) can reduce specimen contamination and have better accuracy and reproducibility [4,5]. Transoral or nasal insertion is performed according to the routine bronchoscopic examination, and no suctioning of secretions is performed during bronchoscopy insertion when possible. Prior to insertion of the bronchoscope via the artificial airway for sampling, adequate oxygen needs to be administered in advance, and ventilator-supported patients can also use a three-way tube to maintain ventilator use during the sampling process. After guiding the bronchoscope to the imaging-located lesion site (it is advisable to select the area with the most obvious infiltrative lesions or purulent secretions), push out the cannula and brush in turn, brush the secretions from the lesion site, withdraw the cannula after retreating the brush into the cannula. After disinfecting the outer wall and tip of the cannula with 75% medical ethanol, the brush is extended and dipped into a tube containing 1 ml of sterile saline, shaken thoroughly, and sent to culture in a sterile sealed tube.
BPSB can be used in patients with an established artificial airway and is currently mostly used in the pathogenic diagnosis of ventilator-associated pneumonia, with some literature suggesting no significant difference in diagnostic efficacy between bronchoscopically guided PSB and BPSB [4]. Operationally, a slow insertion via tracheal intubation or cannula is required to master the appropriate insertion depth (about 30-50 cm for nasal and oral tracheal intubation and about 20-40 cm for tracheotomy), and in case of resistance to deeper penetration, the brush is extended to brush the secretions and withdrawn after retraction of the brush. Subsequent operations and specimen processing were the same as before.
1.4 Bronchoalveolar lavage fluid acquisition and processing
Bronchoalveolar lavage fluid (BALF) microscopy and culture are the most reliable methods for diagnosing infectious lung diseases, and the literature shows a direct microscopic positivity rate of 30.9% for BALF and 27.4% for culture in patients with confirmed and clinically diagnosed invasive pulmonary fungal disease [6]. Our Technical Specification for Cytological Detection of Bronchoalveolar Lavage Fluid (Draft) requires that: ① lavage site: the middle lobe of the right lung (B1 or B5) or the lingual segment of the left lung is selected for diffuse interstitial lung disease, while limited lung lesions are lavaged in the corresponding bronchopulmonary segment. ② Procedure: In the lung segment to be lavaged, inject 1~2 ml of local anesthesia with 2% lidocaine through the silicone tube via the biopsy hole, wedge the tip of the fibrinoscope closely into the segment or sub-segment bronchial opening, rapidly inject sterile saline at 37°C, 25~50 ml each time, 100~250 ml in total, and immediately recover the lavage fluid by negative pressure suction at 50~100 mmHg. 60 %. The recovered fluid is immediately filtered with double-layer sterile gauze to remove mucus, and the total amount is recorded and loaded into a sterile container and sent for testing at low temperature on ice. ③Qualification criteria: no airway secretions mixed in BALF; recovery rate > 40%, surviving cells accounted for more than 95%; erythrocytes < 10% (except trauma/hemorrhage factors), epithelial cells < 3-5%, smear cell morphology intact, no deformation, uniform distribution.
Blinded mini-bronchial lavage (BMBL) is recommended for the establishment of an artificial airway, especially in critically ill patients treated with ventilatory support, and is a less invasive procedure. The operation is performed by pre-giving the patient pure oxygen inhalation for 1 minute, taking a sterile suction tube, placing it through the artificial airway, and slowly delivering it to the bronchial ton and then injecting 20-30 ml of sterile saline, ensuring that the recovery volume is above 3 ml [7]. The specimens were centrifuged and the precipitated smears were taken for observation or culture.
1.5 Retention and processing of urine specimens
Urine microscopy and culture are of limited value in invasive fungal infections, but have some diagnostic value if Candida spp. or Penicillium manefaciens are found [8]. Specimen collection should strive to precede the administration of antibiotics, and clean mid-stream urine is preferably retained in the morning and collected using a wide-mouthed sterile container made of inert material and stored with a closed lid. For urine collection with an indwelling catheter, the exterior of the catheter should be disinfected first and urine should be aspirated using a syringe to puncture the catheter according to the aseptic method, avoiding collection from the urine collection bag [9]. Urine specimens should be stored at room temperature for no more than 2 hours and at 4°C for no more than 8 hours. For microscopic examination of urine sediment smear, 5-10 ml of specimen can be taken after centrifugation at 3000-4000 r/min for 30 minutes, and the sediment smear is taken for direct microscopic examination or microscopic examination after performing relevant staining. Urine culture can be used to inoculate the ring coated on the corresponding medium, take the centrifuged urine sediment culture can improve the positive rate, culture 18 ~ 24 hours of sterile growth, should continue to culture 24 hours before observation.
1.6 Blood
Blood culture detection of pathogenic bacteria is one of the important bases in the diagnosis of invasive fungal disease. Its operation is the same as routine bacterial culture blood specimen collection, to pay attention to the principle of asepsis, better blood collection before the application of antifungal drugs, should take different parts of the double blood for culture; if there is an indwelling intravascular catheter, at least the blood specimen collected by the catheter should be included; while removing the catheter, the catheter tip should be sent to culture simultaneously.
1.7 Pleural fluid
Pleural effusion may be present in 36% of patients with invasive fungal disease [6], but positive pleural fluid cultures are not common in patients, and as a sterile cavity fluid, its positivity has confirmatory value. Pleural fluid specimens need to be centrifuged and the precipitate taken for direct smear microscopy or inoculated for culture.
2. Interpretation of positive clinical specimens
2.1 Candida
The isolation rate of Candida clinical specimens is high, and Candida can be isolated from the sputum of 20% to 55% of normal people, and colonization in the airway is common, and it is difficult to distinguish colonization or infection by positive sputum Candida culture, so the clinical significance is limited, and the positive results must be interpreted with caution, even if the culture of the specimens examined by protective brush under bronchoscopy is positive, it cannot be used as a basis for diagnosing invasive Candida infection. Clinical studies have shown that patients with positive sputum and BALF Candida cultures did not develop systemic infections and had no increased mortality in conditions where no antifungal treatment was taken [10,11]. Although it has been suggested that multiple positive sputum cultures can help to exclude the possibility of Candida contamination and colonization, current national and international guidelines do not consider positive Candida culture of airway specimens as a microbiological basis for diagnosis and do not recommend antifungal therapy on this basis [12].
Direct microscopic examination of airway specimens is of greater value compared to culture. Candida is a yeast-like fungus that grows slowly, in small numbers and mostly in spore form when in a colonized state. When microscopic examination finds Candida forming pseudomycorrhizae (budding spores) and transforming into fungal filaments under certain conditions, it may become the state of infection. Therefore, in qualified sputum specimens, induced sputum or BALF, direct microscopic examination reveals a large number of outgrowing bacteriophages and Candida hyphae, indicating that they multiply rapidly may be in a pathogenic state.
Candida colonization is a prerequisite for the development of invasive infections, and multisite Candida colonization in critically ill patients is an independent risk factor for the development of invasive Candida infections [13]. The colonization index (CI) and corrected colonization index (CCI) are used to help determine the colonization load of Candida in patients by using quantitative culture techniques of clinical specimens, which can guide the early detection and preemptive treatment of high-risk groups in the clinic. The criteria are as follows: five specimens from the patient’s airway aspirate (sputum), pharyngeal swab, gastric fluid, urine and rectal swab (stool) were collected for semi-quantitative count of Candida. Candida counts of ≥1×102 colony forming unit (CFU)/ml in pharyngeal/rectal swabs and ≥1×105 CFU/ml in gastric fluid, airway aspirates and urine were defined as positive. CCI = total number of positive sites/total number of specimens. It is now considered that in high-risk groups, anti-Candida therapy can be given early in sepsis patients with CCI ≥ 0.4 [13]. It was found that the colonization index reached the threshold an average of 6 days earlier than the onset of Candida infection, which has a high clinical predictive value, and clinical preemptive treatment of Candida based on this can result in a significant decrease in the incidence of invasive Candida infection without increasing the rate of drug resistance [14].
Therefore, clinical anti-Candida treatment should not be based on sputum culture results alone, but needs to refer to the clinical risk factors of the patient at that time (especially disruption of the mucosal barrier, colonization of pre-existing Candida, immune function suppression, etc.), corresponding clinical symptoms and imaging changes, sputum or airway secretion smear findings (such as the presence or absence of pseudomycelia or hyphae, emergent spores, etc.), colonization index (especially corrected colonization index) The clinical value of the test is determined by combining the results of serum markers (e.g. G test) with the results of the test, and then deciding whether empirical treatment is needed. If a patient with a positive blood culture for Candida also presents with evidence of respiratory infection and new lesions on chest imaging that cannot be explained by other causes such as bacterial infection, sputum culture that is repeatedly positive and consistent with the results of blood culture can be used as a microbiological basis for secondary pulmonary Candida infection.
Positive culture of Candida in sterile cavity fluid (blood and pleural fluid) is an important microbiological basis to confirm the diagnosis of invasive Candida infection.
Urine sediment smear microscopy is feasible for suspected urinary system Candida infection. In patients without catheterization, if the urine specimen culture is positive twice or more, it can be used as a microbiological basis for diagnosis; in patients with catheterization, a positive urine specimen culture cannot be used as a microbiological basis for diagnosis. Interpretation of a positive urine specimen culture must be interpreted in the clinical context. In the absence of any clinical symptoms, treatment for candiduria is generally not recommended unless the patient is in a high-risk environment for Candida transmission; removal of predisposing factors usually resolves candiduria. However, in symptomatic candiduria, or in patients with candiduria where disseminated candidiasis is suspected, treatment should be administered with a regimen equivalent to that of candidemia.
In recent years, the rate of non-Candida albicans infections has been increasing [15]. The development of Candida chromogenic media by CHROMagar in France at the end of the last century enabled the direct identification of common Candida spp. pathogens such as Candida albicans (blue-green), Candida tropicalis (blue), Candida klebsiella (pink), Candida smoothis (pink) and Candida subsmoothis (light white) in one step culture. This method provides the conditions for early clinical species judgment and provides a reference basis for the selection of drugs for early treatment.
2.2 Aspergillus
Aspergillus spores 2-5μm in diameter, easily suspended in the air, aspirated spores can cause Aspergillosis, mostly seen in lung and sinus infections, can often be detected in airway specimens, due to the presence of contamination and colonization may, the interpretation of the results need to be cautious.
Clinical specimen smear microscopy, the specimen can be pretreated with 10% potassium hydroxide to remove the protein component while ensuring the integrity of the mycelium, and easier to observe. In order to further improve the sensitivity of the microscopic examination, a variety of staining methods can be selected, staining after the microscopic observation of slender, acute bifurcation of the separated mycelium can be confirmed as Aspergillus mycelium. will be significantly reduced. In specific staining, GMS staining is a combination of stain and polysaccharide in the fungal cell wall, through the aldehyde group contained in the fungal cell wall to reduce the hexamine silver to black metallic silver, so that the mycelium appears black. PAS staining mycelium is red, while the cell composition and structure as the background also shows in purple, clinically can be combined with GMS staining and PAS staining to improve the detection rate of Aspergillus mycelium [1]. .
Aspergillus spp. are suitable for growth in standard cultures and most laboratories are able to identify the species. The group is divided by colony morphology and the color of the conidial head, and then the species is identified by the morphology and color of the conidia, the number of spore-producing structures, the morphology of the acrosome, and the morphology of the sexual spores.
The isolation rate of Aspergillus from clinical airway specimens is not high, the sensitivity and specificity of Aspergillus culture is limited [12], and the positive rate of Aspergillus culture in BALF is less than 15% [16]. 2008 American College of Infectious Diseases clinical practice guidelines for the diagnosis and management of Aspergillosis proposed that: specimens in the airway such as qualified sputum specimens, endotracheal suction, BALF or brush test specimens microscopically found mycelium, sputum specimens cultured for 2 consecutive isolations of A single positive culture of Aspergillus species and BALF can be used as a microbiological basis for the diagnosis of pulmonary aspergillosis [17]. However, clinicians should still be aware that the results must be combined with host factors and clinical features to determine their clinical significance.
Positive blood cultures in patients with Aspergillosis are rare, and even if systemic infection occurs, the rate of positive Aspergillus blood cultures is still low, so positive blood cultures for Aspergillus must exclude whether it is due to exogenous contamination, and the results should be interpreted with caution. Positive results of pleural fluid culture can be used as a basis for confirming the diagnosis, but it is not common in the clinic. It is worth noting that if the patient had received systemic antifungal therapy before specimen collection, a negative microbiological examination cannot exclude the possibility of invasive Aspergillus infection.
2.3 Cryptococcus
Cryptococcal infection is most common with Cryptococcus neoformans, and common staining methods for detection include HE staining, ink staining, PAS staining, Alcian blue staining, and GMS staining, etc. HE and GMS staining are easy to detect Cryptococcus, but may be confused with Candida or Histoplasma. Ink staining is usually used to examine cryptococci in cerebrospinal fluid or secretion smear, with a positive rate of about 60%. It has the advantages of convenience, rapidity and cost saving, and is the preferred method to examine cryptococcal infection in smear, which is not suitable for application in mucous respiratory secretions. Alcine blue method is often used to identify Cryptococcus neoformans, because Cryptococcus neoformans entrapment membrane belongs to mucus material, can be Alcine blue and carmine coloring, while other fungi and podoconiosis no coloring, so Alcine blue staining method is relatively specific for Cryptococcus neoformans, can identify Cryptococcus neoformans and its morphology, size similar yeast. The diagnosis can be made after staining with a round or oval-shaped body with a broad pod membrane outside.
Cryptococcal cerebrospinal fluid culture should be collected from 3 to 5 ml or more specimens for examination [18]. Cryptococcal isolation culture with Sabo culture based on about 30°C is the most appropriate, 2 to 5 days to form a typical cryptococcal colony, take the bacteria microscopic examination, can see round or oval body, no pseudofilament formation.
In clinical airway specimens from patients with cryptococcosis, the sputum culture and smear positivity rate is generally less than 25%. Because Cryptococcus neoformans can reside in normal populations, the presence of Cryptococcus neoformans in sputum or even in tracheal suction cultures should be judged on a clinical basis to determine whether there is a pulmonary cryptococcal infection. Usually, no cryptococci are present in the healthy airways, while patients with chronic structural lung disease are likely to have colonization [19]. Of concern is that patients with abnormal immune function with pulmonary cryptococcal infection are prone to systemic dissemination, especially in the central nervous system, and extra-pulmonary dissemination is more likely if such patients are clearly diagnosed by surgical pathology or treated surgically. Therefore, cerebrospinal fluid examination should be performed as early as possible for suspected meningitis, and the positive rate of cerebrospinal fluid smear in early meningitis can be more than 85%, and the positive rate of culture is also higher. It is inconclusive whether patients with confirmed pulmonary cryptococcosis need routine cerebrospinal fluid examination, but it is recommended for patients with abnormal immune function [20].
2.4 Pneumocystis
Pneumocystis pneumonia (PCP) is a pulmonary infection caused by the opportunistic pathogen Pneumocystis epidermidis. Pneumocystis has two pathogenic forms, encysted and trophozoite, and is one of the leading causes of mortality in patients with AIDS. Although PCP has a high morbidity and mortality rate, early diagnosis and prompt treatment is curable in 70% of patients.
Unlike other fungi, Pneumocystis cannot be detected by reliable culture methods, so the main way to confirm its infection is by direct observation of the pathogen through multiple staining techniques with a high degree of specificity. The staining methods commonly used for Pneumocystis envelope and trophozoite detection are GMS staining, Richter-Giemsa staining, Pap staining, and fluorescent whitening dye (CW) staining. Studies have shown that GMS selectively stains the cyst wall of Pneumocystis with a diagnostic sensitivity of 76.9% but a specificity of 99.2% [21]; Richter-Gimza stain stains all stages of the pathogen and is suitable for smear microscopy; Pap stain stains the eosinophilic material around the pathogen called “bubble body” and is Pap stain stains the eosinophilic material around the pathogen called “foamy bodies” and is a reliable diagnostic method with sensitivity and accuracy [19]. The most reliable basis for PCP diagnosis is the direct microscopic detection of pathogens in BALF or induced sputum [22].
3 Summary
In the diagnosis and treatment of invasive fungal disease, on the one hand, there is neglect and inadequate interpretation of microscopy and culture of clinical specimens, especially airway specimens, such as ignoring the significance of positive sputum culture results and one-sidedly identifying contamination and colonization, which delays the best time for treatment. On the other hand, there is also a tendency to overestimate the clinical value of the results, using positive culture of airway specimens as “microbiological evidence” for the clinical diagnosis of invasive fungal disease, which leads to overtreatment.
Clinical reliance on new microbial antigen detection or molecular biology tests at the expense of microscopy and culture of clinical specimens should be avoided. In many cases, microscopy may be a faster, more cost-effective, and more reliable method of detection. At the same time, specimen fungal microscopy and culture should be based on qualified specimen retention methods and standardized specimen handling procedures, so that the results may become strong evidence in the clinician’s diagnosis. And in clinical interpretation, a comprehensive judgment needs to be made by combining regional microbiological characteristics, high-risk factors, clinical manifestations and other laboratory test results.
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