**6.2. Gram stain**

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

A decision strategy based on the results of Gram stain was proposed to assist the clinician in the empirical prescription of antibiotics [68]:


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 Gram stain [69].

#### **6.3. Molecular methods**

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 genes [70, 71].

or the resistance gene markers; possibility to search for multiple agents and resistance markers; direct detection in clinical specimens; and higher sensitivity. On the other hand, among important drawbacks are: most of them are qualitative, risk of contamination, high costs, and

AST: antimicrobial susceptibility testing; FISH: fluorescence *in-situ* hybridization; MALDI-TOF: matrix-assisted laser desorption/ionization time-of-flight; MRSA: methicillin resistant *S. aureus*; MSSA: methicillin sensitive *S. aureus*; MS: mass spectrometry; NGS: next-generation sequencing; PCR: polymerase chain reaction; PCR/ESI-MS: PCR-electrospray

**Systems No. of pathogens/markers Technology**

Accelerate PhenoTest™ BC kit 27 bacteria and 2 yeasts and AST FISH Amplidiag® CarbaR+VRE 5 carbapenemase and 2 vancomycin-resistance markers

Curetis Unyvero™ 16 bacteria, 1 fungus, 18 resistance markers PCR

resistance markers

15 resistance markers

FilmArray® Respiratory Panel 17 viruses and 3 bacteria Multiplex RT-PCR

NxTAG® Respiratory Pathogen Panel 18 viruses and 3 bacteria Multiplex RT-PCR R-Biopharm RIDA® GENE-kits *mecA*/*mecC*, SCCmec cassette, and *S. aureus* Multiplex RT-PCR Verigene® Respiratory Pathogens Flex Test Up to 13 viruses and 3 bacteria (customized) Multiplex RT-PCR

and *P. aeruginosa*

GeneXpertMRSA/SA Only MRSA and MSSA RT-PCR MALDI-TOFI Wide spectrum of bacteria and fungi MS

**Table 2.** Commercial molecular systems for detection of respiratory pathogens and resistance markers.

PCR/ESI-MS

Microbiology of Ventilator-Associated Pneumonia http://dx.doi.org/10.5772/intechopen.69430

NSG

Multiplex RT-PCR

117

Multiplex RT-PCR

Abbot IRIDICA System 780 bacteria, 200 fungi, 13 viruses, and 4

CE-IVD HAI BioDetection kit 12 most common nosocomial pathogens and

FTD Bacterial pneumoniae HAP Detection and quantification of *K. pneumoniae*

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].

lack of validation.

**6.4. Exhaled breath metabolomics**

ionization mass spectrometry; RT-PCR: real time-PCR.

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, including bacteria, viruses, and fungi.

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/


AST: antimicrobial susceptibility testing; FISH: fluorescence *in-situ* hybridization; MALDI-TOF: matrix-assisted laser desorption/ionization time-of-flight; MRSA: methicillin resistant *S. aureus*; MSSA: methicillin sensitive *S. aureus*; MS: mass spectrometry; NGS: next-generation sequencing; PCR: polymerase chain reaction; PCR/ESI-MS: PCR-electrospray ionization mass spectrometry; RT-PCR: real time-PCR.

**Table 2.** Commercial molecular systems for detection of respiratory pathogens and resistance markers.

or the resistance gene markers; possibility to search for multiple agents and resistance markers; direct detection in clinical specimens; and higher sensitivity. On the other hand, among important drawbacks are: most of them are qualitative, risk of contamination, high costs, and lack of validation.
