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, but patients that receive this intervention are at an increased risk of developing a severe condition called ventilator-associated pneumonia (VAP). VAP is , and the most common cause of in critically ill patients. The serious consequences of this disease demand quick and accurate diagnostic testing, yet no gold standard exists. recommend 3 diagnostic methods for VAP: bronchoalveolar lavage (BAL), lung biopsy and tracheal aspirate. Of the 3, tracheal aspirate specimens are to collect, but they have and rarely distinguish between colonizing microbiota and microbiota causing infection. Diagnostic uncertainty leads providers to empirically treat for VAP, regardless of whether the patient is confirmed to have it or not. Ultimately, presumed VAP results in high resource utilization and is the in the ICU. This overuse has been in the ICUs of both adult and pediatric institutions. Even though the tracheal aspirate culture method lacks accuracy in diagnosing VAP, clinicians are left with little choice but to use it given that other diagnostic methods are limited and other specimen types are more difficult to obtain. 

It is especially important to manage mechanically ventilated patients as appropriately as possible in the time of SARS-CoV-2/COVID-19. that the United States only has 62,000 modern ventilators, far less than the anticipated number of severe COVID-19 cases that are expected to occur. Reducing the number of ventilator-days per patient could make ventilators available for the next patient in need sooner, and interpreting tracheal aspirate culture results with caution could prevent unnecessary side-effects from antimicrobial use.

A Brief History of Respiratory Specimen Rejection Criteria

While respiratory samples will always be a challenge, not all respiratory specimens are without guidance. Rejection criteria for sputum samples is still broad and not universal, but they have provided algorithms for laboratories to follow in order to prevent the culture of samples that are grossly contaminated with oropharyngeal flora. In general, the criteria suggest that a specimen with a high number of epithelial cells by Gram stain is contaminted and is likely unacceptable for culture. To support the use of these criteria, the requires the following when inspecting a clinical laboratory for compliance:

1. Gram stains should be routinely performed to determine specimen acceptability and used as a guide for culture workup. This must be documented in a laboratory procedure.
2. Laboratories must provide evidence that they are using some form of Gram stain rejection criteria and not setting up culture on specimens deemed inappropriate by these criteria.

Currently, there are no standards for laboratory workup of tracheal aspirate cultures. Two studies performed in the 1990’s proposed Gram stain rejection criteria for tracheal aspirate specimens with >10 squamous epithelial cells or no organisms detected on the Gram stain for and the absence of organisms on Gram stain for the. However, guidelines have never been established, and as a result, these cultures remain unreliable, variable and costly. 

Tracheal Aspirate Cultures: The Challenges

So, what is so challenging about these cultures? The truth is, the inconsistencies begin with specimen collection and continue all the way through to susceptibility reporting. 

1. The use of to collect a specimen. The in-line suction technique collects mucus from the closed tracheal tube system that has been connected to the patient since they were hooked up to the ventilator. when in-line suctioning is used compared to the use of a sterile catheter for suctioning. This is common practice for many hospitals, and the use of this method is more likely to collect biofilm-forming organisms that colonize plastics, resulting in misleading results that suggest the patient has pneumonia caused by one of these opportunistic pathogens. 
2. Specimen transport. Specimens should be delivered  to the microbiology laboratory as soon as possible after collection, preferably within 2 hours. If immediate transportation is not possible, specimens should be refrigerated in order to improve pathogen isolation. Many organisms are susceptible to temperature changes or delays in testing. Additionally, fastidious (but important) organisms may be overgrown by non-fastidious organisms within the specimen before it is ever cultured in the laboratory, resulting in misleading culture results. Some laboratories have specimen transport requirements for tracheal aspirate culture specimens and others do not. 
3. The use of saline during collection and/or specimen processing. The effect of saline on specimen quality will vary depending on transport time, processing and culture methods. , which could have a significant impact on what grows in culture. Additionally, the use of saline to collect tracheal aspirate specimens or during the processing of the specimen leads to dilution. If the amount of saline used to collect the specimen is unknown and quantitative culture is used in the laboratory, the quantities of growth reported from culture are
4. Gram stain rejection criteria. As mentioned above, Gram stain rejection guidelines exist for sputum samples, but not for tracheal aspirate specimens. Rejection criteria vary between laboratories, and this results in inconsistent culturing, reporting and overall management of tracheal aspirate cultures. The lack of a quality check through the use of Gram stain criteria results in the culturing of poor-quality specimens and culture results that mislead clinicians. 
5. Quantitative versus qualitative plating techniques. Several studies have examined the utility of both quantitative and qualitative plating methods, but the study designs and results are variable. As with many other components of the tracheal aspirate culture process, guidelines and universal recommendations for specimen plating have not been established.
6. Organism identification, reporting and antibiotic susceptibility testing. Understanding the in the development of VAP is incredibly challenging. While the presence of an organism in culture could simply suggest colonization, those colonizing organisms also tend to be the  most likely to cause pneumonia. The reporting of organisms varies between laboratories. Microbiologists must walk a fine line between over-reporting organisms (which can lead to unnecessary antibiotic use and increased length of stay), or under-reporting, which may fail to mention the presence of an organism causing serious disease. This is also true for susceptibility testing, which should only be offered when the reported organism is believed to be the cause of infection and the use of antibiotics is warranted. 

Real-life Examples of Challenging Tracheal Aspirate Cultures

Case 1

Gram stain results: 

• No polymorphonuclear cells.
• No squamous epithelial cells.
• No organisms detected.

This specimen was plated using the semi-quantitative plating method (four quadrants streaked with growth reported as rare (Q1), few (Q2), moderate (Q3) or heavy(Q4)). The specimen was plated on blood, chocolate and MacConkey agars. After 24 hours of incubation, the following growth was observed on the plates:

 

 

The Gram stain for this culture was completely insignificant, showing no inflammatory cells, epithelial cells or organisms. However, the plates tell a different story. The blood plate shows moderate growth of a white colony (Staphylococcus) that is the predominant organism growing in moderate quantities. The chocolate plate shows the same, except that the Staphylococcus is growing in a heavy quantity. The MacConkey agar has rare growth of both a lactose fermenting gram-negative rod and a non lactose-fermenting gram-negative rod. 

• Based on the original Gram stain, should this specimen have been set up for culture at all?
• None of the organisms were seen in the original Gram stain, but there is a predominant organism and it is growing in significant amounts. Due to this fact, it is likely that the organism will be fully identified and reported.
• If the organism is fully identified and reported, should susceptibility testing be performed?
• There are gram-negative rods growing in this culture. However, these are growing in insignificant amounts, are not predominant and were not seen in the Gram stain. Knowing that the reporting of gram-negative rods tends to prompt clinicians to treat, should these be mentioned at all? Or, should they be included in normal upper respiratory microbiota?
• If the gram-negative rods are reported, should susceptibility testing be performed?

For this case, if the patient has risk factors for VAP. Additionally, that treatment regimen will vary depending on the patient’s risk of developing an infection with an antimicrobial-resistant organism, recent infections or history of previous organisms, severity of illness and the distribution of local susceptibility patterns at the institution. Empiric therapy often covers methicillin resistant Staphylococcus aureus (MRSA), as well as Pseudomonas aeruginosa. When culture results are released, it is often up to the stewardship team to encourage de-escalation of antibiotics if the treatment is too broad or unnecessary. Although the gram-negative rods in this culture are likely insignificant, a clinician might be hard-pressed to remove Pseudomonas coverage once a non-lactose fermenting gram-negative rod is reported. Unnecessary treatment with these antibiotics can lead to complications, such as Clostridioides difficile infection, and can promote the spread of antimicrobial resistant organisms. 

Case 2

Gram stain results: 

• Heavy polymorphonuclear cells (quantitated using the 100X objective).
• Rare squamous epithelial cells.
• Rare gram-positive cocci in pairs.

This specimen was plated using the semi-quantitative plating method (four quadrants streaked with growth reported as rare, few, moderate or heavy). The specimen was plated on blood, chocolate and MacConkey agars. After 24 hours of incubation, the following growth was observed on the plates:

 

 

The original Gram stain of this specimen could be considered relatively significant by some labs. There is a significant amount of polymorphonuclear cells, which could suggest infection. However, the significance of these cells on Gram stain has been , as they can be seen as a result of other medical conditions or respiratory tract inflammation from the placement of the endotracheal tube. There are rare squamous epithelial cells which suggests that the specimen might be of acceptable quality, and there is a single organism type seen, albeit rare. From this Gram stain alone, we might expect to see significant growth of a single organism type on the plates. 

However, that's not the case. The blood and chocolate agar plates show few growth of 5 or more colony types, with no particular organism growing in heavier quantities than the others. This looks like normal respiratory microbiota without the presence of a predominant pathogen. Although a few of the 5 colony types have morphologies consistent with S. aureus or S. pneumoniae, they are not predominant and the only organism seen in the Gram stain was rare.

• S. pneumoniae and S. aureus are common causes of VAP, but they are also colonizers of the oropharynx. Is it necessary to mention their growth in culture even if they are not predominant?
• If these organisms are reported, should susceptibility testing be offered?
• Should the gram-positive cocci in the Gram stain be considered significant, regardless of the low quantity?

In this case, no predominant pathogen was identified in culture. If every potential pathogen was picked out and identified (such as S. aureus and S. pneumoniae) regardless of quantity, clinicians might be prompted to treat with drugs like vancomycin (in order to cover for MRSA) and meropenem, intense and broad-coverage drugs that can select for resistant organisms, induce side effects and lead to C. difficile infection if maintained over an extended period of time. If the culture results are reported as “mixed normal upper respiratory flora,” clinicians may be more likely to de-escalate empiric treatment and search for other causes of the patient’s symptoms.

Moving Forward

Chances are, the answers to these questions and interpretation of these cultures will vary between the microbiologists reading this article. While a low level of subjectivity is normal in the microbiology lab, it is exceedingly common with tracheal aspirate cultures. The lack of standardization or guidelines for specimen processing and workup present a challenge for both microbiologists and clinicians, and highlight the need for future research and the development of guidelines in this area. 

Developing standards and guidelines around specimen collection, transport, rejection criteria and culture reporting for trachael aspirate cultures will lead to more meaningful interpretation of results and decrease poor patient outcomes. Mechanical ventilation is a life-saving measure that is essential for critical care. It is important that measures are taken to prevent the development of VAP, but also that these measures do not lead to inappropriate treatment or extended time in the hospital. Complications related to mechanical ventilation (such as VAP) will likely increase in frequency across the world as SARS-CoV-2 continues to spread. Appropriately managing these patients clinically will be key to reducing their length of stay and preventing unnecessary side effects. In the long term, development of tracheal aspirate guidelines could help alleviate confusion and ensure that diagnostic results are meaningful and predictive.