**6.4. Exhaled breath metabolomics**

**6.2. Gram stain**

116 Contemporary Topics of Pneumonia

Gram stain [69].

genes [70, 71].

**6.3. Molecular methods**

including bacteria, viruses, and fungi.

The Gram stain of respiratory specimens can provide rapid information regarding morphological aspects of the bacterial pathogen and whether it is Gram-positive or Gram-negative. Additionally, microscopic examination may reveal whether the smear is suggestive of infection. It is generally accepted as active infection when the biological sample has more than 25

A decision strategy based on the results of Gram stain was proposed to assist the clinician in

• **If the Gram stain of the ETA is negative:** Antibiotic prescription can wait until the microbiological culture result is available, since it is very unlikely that the patient have VAP. • **If the Gram stain of PSB is positive**: The antibiotic therapy can be initiated and based on the result of Gram stain, since it is very likely that the patient has VAP. Later, it can be ad-

• **If the Gram stain of PSB is negative and the Gram stain of the ETA is positive:** The antibiotic therapy may only be initiated depending on the severity of the patient's clinical

Nevertheless, the utility of Gram staining in the diagnosis of VAP and as a guide for the antibiotic empirical therapy of VAP is a very controversial subject. It is usually accepted that this old diagnostic tool has a high negative predictive value, i.e., VAP is unlikely with a negative

Several molecular-based methods have been proposed for the detection of respiratory pathogens that offer a reliable diagnosis, with high sensitivity and specificity. Most of them are nucleic acid-based amplification methods that identify, simultaneously, multiple and specific target gene sequences (multiplex assays) of a wide range of bacterial species and resistance

Considering that the etiology of the VAP is very different from the community-acquired pneumonia, some main potential gene targets are *mecA* gene in *S. aureus*; *blaVIM* and *blaIMP* genes in *P. aeruginosa*; *blaOXA* genes in *Acinetobacter* spp.; and *blaKPC* gene in members of the Enterobacteriaceae family, in addition to the detection of *Stenotrophomonas maltophilia* [72].

Currently, a variety of platforms or systems are available to identify respiratory pathogens using distinct technologies. Some molecular diagnostic systems detect a small number of microorganisms, such as GeneXpertMRSA/SA that detects MRSA and MSSA. On the other hand, IRIDICA and MALDI-TOFI can detect a wide range of pathogens and resistance markers. **Table 2** shows the major commercial systems available to detect respiratory pathogens,

Depending on the methods, the advantages of molecular methods include rapid results; detection of very low amounts of gene sequences; target sequences to identify the agent and/

neutrophils and less than 10 epithelial cells per 10× low-power field.

condition or when the VAP is confirmed by the culture.

the empirical prescription of antibiotics [68]:

justed according to the culture result.

Recent advances in diagnostic technologies have pointed to metabolomics as an emerging and faster method to aid in the diagnosis of various diseases, such as cancer, asthma, among others. The procedure can be performed with samples such as plasma and also with noninvasive samples, such as exhaled air and saliva. Results can return within a matter of hours, compared with days of conventional culture. In the case of exhaled air, the method consists in determining the profile of volatile organic compounds (VOCs) emitted by the patient through respiration [73]. These metabolic degradation products present in the expired air are derived from the patient and the pathogen. The VOC profile is detected through sensitive procedures such as nuclear magnetic resonance spectroscopy [74] and gas chromatography-mass spectrometry [75]. Studies in patients with VAP have allowed the determination of distinct VOC patterns in clinical cases associated to different pathogens, showing good correlation with the microbiological culture and offer great potential as biomarkers [76, 77].

Major drawbacks of this method include: (i) the sampling methodology, which should enable to sample from beyond the endotracheal tube and hence to exclude air from the upper respiratory tract; (ii) discovery of more pathogen-specific metabolites; and (iii) the need of trained personnel to operate the analytical methodology by gas chromatographymass spectrometry apparatus.

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