MICROMYCETES INFECTION IN PATIENTS WITH THERMAL TRAUMA

INTRODUCTION

Severely and critically burned patients are exposed every day to a wide range of potentially pathogenic microorganisms (PPM).¹ Infectious complications represent a predominant cause of mortality in these patients. This fact is mainly due to improved quality of care for these patients, when patients who would certainly die many years ago can survive an extensive thermal trauma today. Another reason is also increasing resistance of potentially pathogenic microorganisms.² Recently we also noticed change in the occurrence of each PPM in the patients with thermal trauma. Bacteria are still the predominant PPM in these patients, however especially micromycetes infections play an important role today as well.³

This is mainly due to extensive loss of skin cover, or compromised function of skin as a barrier and local immunologic function. An important factor is also the fact that severe and critical burns are associated with a decline of immunological performance with a character of immune paralysis to immunosuppression. Antibacterial strategies and also antibiotics lead to selection of resistant forms of bacteria and they also facilitate propagation of yeasts and fungi.⁴

MATERIAL AND METHODS

This is a retrospective monocentre study, which enrolled all burn patients over the age of 18 with a thermal trauma who were hospitalized at the Department for Burns and Reconstructive Surgery of the University Hospital Brno within the period from 01/01/2007 to 31/12/2015. Yeasts or fibrous fungi were demonstrated in cultures in all of the patients in the group during hospitalization. Basic epidemiological markers were evaluated in these patients. ABSI score (Abbreviated Burn Severity Index) was used to determine the degree of the thermal trauma.

Culture of yeasts and fungi

Biological material sent for culture examination is processed according to the sample character. Culture on blood agar and MacConkey agar is a standard examination for all types of biological material; they represent the basic media for culture examination. Other selective culture media with a higher content of sodium chloride are subsequently added according to the sample character to demonstrate Staphylococci, VL agar is added to demonstrate anaerobic bacteria, etc. Microscopic preparation belongs to a standard for liquid materials; some of them are processed quantitatively (bronchoalveolar lavage, sputum, urine).

In case of a suspicious micromycetes infection, cultivation on standard media is supplemented with inoculation of material on mycological media: the most frequently used medium is Sabouraud agar. Biological material is inoculated on Sabouraud agar on Petri dishes and on several inclined Sabouraud agars in a tube intended for growth of fibrous fungi. Cultivation takes place for 7 days and under a temperature of 28–30°C for yeasts. Fibrous fungi are cultured in room temperature and simultaneously under thermostat temperature of 35–37°C. Growth on a chromogenic medium is used to determine the type of grown yeasts (colour change), production of chlamydospores during a growth on rice agar and weight spectrometry-MALDI-TOF. Fibrous fungi are classified into a genus and species based on their macroscopic appearance and microscopic preparation from the culture.

Statistical analysis

Continuous and ordinal parameters are described in tables with mean, median, minimum and maximum. Categorical variables are described as a number of patients and their relative count in given groups. The results of statistical evaluation are presented as p-values of statistical tests. Mann-Whitney test is used, when continuous or ordinal parameters are compared between two groups of patients. Fisher’s exact test is used to examine the association between two categorical variables. Statistically significant results at significance level 0.05 (p-value <0.05) are provided in bold. For all statistical analysis IBM SPSS Statistics ver. 23 (IBM Corporation, 2015) was used.

RESULTS

There were 61 patients with thermal trauma in whom yeast or fibrous fungi were isolated during the period of observation. There were 37 males and 24 females (M:F ratio – 1.5:1) in the group. Average age of the patients was 57.3 years (29 patients below 60 years of age, 32 patients were over 60 years, inclusive). 6 patients died (mortality was 9.8%). The average extent of the burn area was 21.6% TBSA (median 14.0%). There were 21 patients with severe thermal trauma, i.e. with an extent of the burn over 20% TBSA; there were 12 patients with a critical burn (extent of the burn over 40% TBSA). Average value of ABSI was 7.9. The highest number of the patients in the group was 5-8 within the ABSI range (39 patients, 63.9%). The basic epidemiological data of the patients in the monitored group are provided in Table 1.

There were 90 strains of micromycetes obtained from cultures in total in these patients (79 yeasts, 11 fibrous fungi). Micromycetes were isolated from burn area in 30 patients, from the lower airways in 19 patients, from the urogenital area in 15 patients and from blood culture in 7 patients. Non-albicans Candida strains were predominant among yeasts (60 species), Candida albicans was isolated totally 16 times. Aspergillus fumigatus (4 isolations) and Fusarium species (2 isolations) were predominant species among fibrous fungi. All of the isolated strains of yeasts and fibrous fungi are shown in the Table 2.

Micromycetes were obtained from culture in 7 patients in total within the first 5 days of hospitalization (11.5%). The number of patients with micromycetes isolation increased during further course. Micromycetes were cultured in 15 patients during the 6^th - 10^th day of hospitalization (25.0%), in 8 patients during the 11^th–5^th day (14.0%) and in 33 patients (61.1%) after the 16^th day of hospitalization. The evaluation of the extent of thermal trauma expressed by the ABSI value is also interesting including its influence on the risk of micromycetes infection development. Within the 10^th day of hospitalization the patients with positive culture for yeasts or fungi have a lower ABSI value than patients without positive culture. Higher ABSI value contributes on the development of infection caused by micromycetes until the 11^th day of hospitalization. The effect of ABSI on the risk of yeast or fungi infection development is shown in Table 3.

During the monitoring of the effect of particular risk factors on the development of multipathogenic micromycetes infection in the patients from our group, we could not identify any of the factors with a statistically significant effect. In spite of that is interesting the effect of gender, while infectious complications in females were more frequent (p=0.093) or the effect of burn area extent (p=0.244). The most important risk factors for multipathogenic micromycetes infection are shown in Table 4.

Other risk factors for the development of infectious complications in the specific localisation are shown in Tables 5 and 6.

A strong effect of mechanical ventilation or tracheostoma (p<0.001, and p<0.001 respectively) was demonstrated from the infection development point of view in the area of the lower airways, when micromycetes were also isolated from the material. However, it is also interesting that tracheostoma and artificial pulmonary ventilation represent a risk factor for the development of micromycetes infection only in patients over the age of 60. This risk is not statistically significant in younger patients. Verification of inhalation trauma itself is close above the margin of statistical significance (p=0.053). On the contrary, the effect of mechanical ventilation duration was not shown as important for isolation of yeasts or fungi from the area of the lower airways (p=0.581).

Micromycetes was most frequently isolated from burn area. In spite of that, we could not demonstrate any significant effect on the development of infection in this localisation in any of the monitored parameters, even if the values were close to statistical significance in case of absence of a deep burn or extent of the burn over 40% TBSA (p=0.053, and 0.059, respectively).

DISCUSSION

Even today, infectious complications represent a predominant cause of mortality and morbidity of burn patients. Burns represent such type of trauma, in which development of opportune infections may be very common. Paradoxically, the increase of infectious complications caused by micromycetes is due to extensive therapeutic approach in local and also systemic control of bacterial infection. Especially topical antibacterial dressing started the rising incidence of mycotic infections, which continues until today.⁶ Candida infections are predominant in burn patients. The incidence of non-Candida infectious complications is much lower, however this fact is observed also in other groups of critically ill patients. ^7,8

Infection of the burn area is the most common location with regards to the occurrence of infectious complications in burn patients, no matter whether this is a bacterial or non-bacterial aetiological agent. Fungi are not predominant pathogens in the development of burn area infection, however their contribution gradually rises. Burn area infected with Candida albicans is shown on Figure 1.

According to a retrospective study of the American Burn Association (ABA), micromycetes were demonstrated as an aetiological agent in 6.3% of all infectious complications in the burn area.⁹ Contribution of micromycetes in the aetiology of burn area infection reaches much higher values (20–40%) in most studies.^10,11 Questionable remains the real incidence. With regards to a common absence of specific clinical symptoms, omission of micromycetes role in burn patients or difficult laboratory diagnostics, the infectious complication may be missed in several patients. In relation to the incidence of infectious complications caused by fungi in burn patients, it is not possible to demonstrate whether there is a clear geographic prerequisite. This fact is due to worldwide presence of micromycetes.¹²

Isolation of Candida albicans within monitoring of each micromycetes is currently predominant in most of studies. Very surprising finding in our group of patients is that occurrence of non-albicans Candidas is much higher than the count of isolated Candida albicans strains. Count of the isolated Candida krusei and Candida glabrata strains is also alarming with regards to the sensitivity to the most commonly available antimycotic drug at present. Both these yeasts are virtually resistant to fluconazole (Candida krusei is naturally resistant, Candida glabrata acquires resistance very quickly). Another option for therapy of infection caused by these micromycetes is administration of echinocandins. Therefore occurrence of these yeasts means always a dramatic increase of economic costs of therapy. Most studies identify the basic risk factors for the development of candidemia, and possibly candidiasis. Greater extent of the burn area, prolongation of the period from trauma to admission, presence and extent of deep burns, number of surgical procedures, total parenteral nutrition or therapy with antibiotics (cotrimoxazole, amikacin, vancomycin and other) are such risk factors.¹² We focused on the two most common locations in our group with regards to the development of mycotic infection, i.e. on the burn area and lower airways. In case of burn area, no risk factor that would significantly contribute on the development of infection was demonstrated, although the extent of burn area and absence of a deep burn are on the margin of statistical significance. In case of infectious complication development in the area of the lower airways, the presence of a tracheostoma and requirement of mechanical ventilation were important risk factors, similarly to the presence of inhalation trauma.

The diagnostics and therapy of mycotic infections represent a great challenge. Culture of mycotic infections is quite lengthy. Since prompt diagnostics is currently greatly emphasized, the methods that demonstrate mycotic antigens are preferred more often, not only in burn patients. Mycotic antigens are actually parts of the body of the fungi, which can be demonstrated in various materials (blood, bronchoalveolar lavage and other). The most frequently used antigens today, which we encounter in common practice, include galactomannan for detection of aspergillum infection and also 1,3-β-D glucan as a so-called panfungal antigen in several fungi.¹³

Early diagnostics of zygomycetes infections is still problematic. Rising incidence has been observed recently also in these fibrous fungi. In relation with zygomycetes, Absidia species, Mucor species, Rhizomucor species and Rhizopus species occur in burn patients.¹⁴ Area after necrectomy in left lower limb with isolation of Absidia species is shown on Figure 2.

CONCLUSION

Immune paralysis or immunosuppression can develop in patients with thermal trauma, especially in case the extent of the burn is over 20% TBSA. This fact together with compromised local skin barrier and presence of other risk factors (inhalation trauma, mechanical ventilation and other) cause that the burn patients are susceptible for development of infectious complications caused by micromycetes.

Corresponding author:

Břetislav Lipový, M.D., Ph.D.

Department of Burns and Reconstructive Surgery,

University Hospital Brno

Jihlavská 20,

625 00 Brno

Czech Republic

E-mail: bretalipovy@gmail.com