Ann. Medit. Burns Club - vol. 6 - n. 2 - June 1993


Di Lonardo A., Ferrante M., Maggio G., Bucaria V., Del Zotti M., Brienza E.

Istituto di Chirurgia Plastica Ricostruttiva ed Estetica, UniversitA clegli Studi, Bari, Italy

SUMMARY. In the context of recent developments reported in the literature regarding methods for the histological assessment of burn wound infection, in addition to traditional microbiological techniques, the results are presented of a study of 40 patients admitted to the Bari Bums Centre in the period 1990-91. The histological analysis of the biopsies provided useful information which made it possible to have a better understanding of the parameters supplied by traditional microbiological tests and therefore to adopt a more personalized therapeutic approach in individual patients.

The frequency and the gravity of septic complications in the seriously burned patient impose the need for an accurate diagnostic and therapeutic approach. The microbiological monitoring of surface swabs and biopsies from burn wounds provides precise information as to the type of infection present and the bacterial charge, but it gives no indication as to the depth of the infection. Microbiological investigation should therefore be complemented by histological examination in order to detect the possible presence of micro-organisms in the tissues beneath the eschar. This study combines the results obtained by microbiological monitoring and by histological analysis, and establishes the infection rate in burndamaged areas on a scale of values of increasing severity. Our experience has shown this method to be a very reliable criterion of assessment for the choice of therapeutic strategies, particularly as regards the timing and the site of surgical intervention.

Material and methods

The study included all burn patients admitted to the Bari Burns Centre Intensive Care Unit between January 1990 and December 1991 within 24 hours of the burn lesion and belonging to classes III, IV and V of the Roi index. Biopsies were taken from each patient, both from the burned areas and from apparently healthy areas in the vicinity of the burn, on the basis of a predetermined map. Two samples were taken from each biopsied area. One of these was fixed in 10% formalin and subjected to histological examination; the other was kept dry in a sterile container and examined microbiologically.
Biopsies were taken on the first, fourth, eighth and twelfth day.
Further biopsies were taken only from patients requiring more than one reconstructive surgical operation. The time interval was maintained at every fourth day. The number of biopsies performed in each patient was related to the extent of the burn area in order to obtain statistically significant evaluations (I biopsy every 10 CM2) (9). All biopsies were by disposable sterile biopsy punch (diameter 4 mm).

Histological technique

The biopsies fixed in 10% formalin were sent to the Bari University Pathological Histology Laboratory. After inclusion in paraffin, serial sections were prepared perpendicular to the skin surface, with a thickness of 5 ~t (6, 8). Twenty slides were prepared for each histological sample, with the following stains:

  • haematoxylin-eosin
  • Gomorj Grocott
  • gram-modified
  • methylene blue.
  • The histological specimens were examined by optical microscope (magnification 400 and 1000 times). The depth reached by the bacterial invasion was graded according to the following scale (9):

    GRADE  0: no micro-organisms

    GRADE  1: some micro-organisms

    • la: low bacterial charge
    • lb: high bacterial charge

    GRADE  2: invasion of surface dermis

    GRADE  3: invasion of all dermis

    GRADE  4: invasion of hypodermis and/or underlying tissues.

    Microbiological technique

    The biopsies were weighed, homogenized, diluted as necessary in physiological solution and inoculated in doses of I ml into sterile Petri dishes to which about 15 ml of ordinary broth were added for the bacterial count and 15 ml of Sabouraud's agar for the cultivation of fungi. After 48-72 hours of incubation at 37 'C a count was made of any colonies that had developed. At the same time, the 1/100 dilution in physiological solution was used for seeding the following cultivation media:

  • Sabouraud's agar
  • Murtz's lactose agar
  • mannitol salt agar (MSA)
  • Cryptococcus specific medium
  • blood agar.
  • A portion of the solution (about 0.1/0.2 ml) was seeded in ordinary broth and in Sabouraud's agar from which subcultures were prepared after 24 hours respectively on the Murtz medium and on MSA from ordinary broth and on Sabouraud's agar from Sabouraud's broth.

    Case histories and results

    Using the above methods we studied 40 patients admitted to the Bari Burns Centre Intensive Care Unit between January 1990 and December 1991. The patients belonged to classes 3, 4 or 5 on the Roi prognostic index and were aged between 26 and 45 years. The burns ranged between 30% and 70% BSA. The results were obtained using the double-blind technique and relate to a total of 352 biopsies (176 histological and 176 microbiological).

    Histological results

    Both the level of infection in the tissues and the search for possible mycotic contamination were considered. Haematoxylin-eosin staining enabled us to distinguish the various tissue planes and to detect the presence of fungi, and also to hypothesize the possibility of bacterial infection. Grarn-modified and methylene blue staining made it possibile to detect the presence of bacteria in the tissues and to assess the depth reached. Table 1 shows the histological results related to the grading of infection observed in the 176 biopsies examined.

    Microbiological results

    The microbiological assessment of the various biopsies supplied a number of parameters. Here we give only those relative to the tissue bacterial charge (expressed as the number of bacteria per gm of tissue) and to the presence of fungal colonies. The results of the 176 biopsies are given in Table 1.

    Biopsies Grading Fungi bact. 10 bact. 10 fungi
    72 0 72
    33 1a 33
    22 1b 3 18 4
    24 2 5 15 9 3
    14 3 5 6 8 2
    11 4 3 5 6


    Two main considerations can be made on the basis of these results.
    Firstly, there is a clear discrepancy between the septic risk expressed by the microbiological assessment and the actual extent of the tissue sepsis as demonstrated histologically (2, 4). Table I shows that the histological finding in 11 biopsies of deep tissue invasion -(4th grade) - a clear indication of considerable systemic septic risk (9) - corresponded to a microbiological finding of only 6 biopsies with a bacterial charge of more than 10 bacteria per gm of tissue. Also, the 22 grade lb biopsies (massive surface
    contamination) corresponded to the proportionately high number of 4 findings of a bacterial charge greater than 10, which would suggest generalized sepsis.
    Secondly, the histological study enabled us in some cases to detect significant mycotic contaminations in the lesions not revealed by the corresponding microbiological analysis.
    The reasons for this discrepancy are still being studied. It would appear likely that a phenomenon of bacteria/fungi competition may in vitro inhibit the development of mycotic colonies.
    On the basis of these findings the following clinical and prognostic considerations can be made:

    1. From the point of view of reconstructive surgery, the concept of early escharectomy in the severely burned patient (which in our experience means operations performed before day 10) must be associated with that of emergency necrectomy when, in the presence of documented massive bacterial invasion of the viable tissues underlying the eschar, it is necessary to operate very early (day 4 or 5) in the districts most exposed to risk.

    The level to which escharectomy should extend depends on the histological assessment of the depth of the infection. In areas where bacterial colonization of the subcutaneous tissue is demonstrated (Grade 4) necrectomy must be performed as far as the muscle fascia independently of the apparent viability of the tissues as observed intraoperatively.
    Aimed systemic antibiotic therapy may therefore be initiated immediately in order to sterilize the viable, well vascularized deep sublesional tissues which have been reached by the bacterial colonization.
    The histological monitoring of the extent of the infection in the tissues confirms the effectiveness of the topical antiseptic therapy practised or suggests alternative techniques (9). Progressive extension of the infection in the tissues, associated with a simultaneous increase in the bacterial charge, is a clear indication for more energetic topical therapy (more frequent daily medications, use of alternative topical antiseptics preselected on the basis of their microbiological effectiveness) (9).
    The histological visualization of mycotic contamination, even if not confirmed by the corresponding microbiological finding, makes it necessary in therapy to associate specific systemic antifungals selected on the basis of the antimycogram.
    The numerical value of 10 bacteria per gm of tissue loses its significance as a point of reference for the maximum limit for systemic sepsis if it is not associated with the histological demonstration of the level of bacterial and/or fungal invasion in the peri and sublesional viable tissues.
    In the light of the above considerations our therapeutic approach in the patients was conditioned by the specific histopathological and microbiological indications.


    The histological investigation conducted on burn lesion biopsies represents in our experience a further step towards a better understanding and definition of the intricate problem of infection in seriously burned patients. The method described here, followed according to standardized protocols described in the literature (9), allowed a more rational analysis of the parameters supplied by microbiological tests and thus enabled us to grade the tissue infection more accurately. This method made it possible to adopt a personalized therapeutic approach on the basis of precise laboratory indications, and not merely a standard therapy.
    The results obtained in these first years of application have been sufficient to allow us to make some surprising and interesting considerations that amply confinn the importance of the study.

    RESUME. Dans le contexte des plus récents développements décrits dans la littérature pour ce qui concerne les méthodes pour l'évaluation histologique de l'infection des brûlures, outre les techniques microbiologiques traditionnelles, les auteurs présentent les résultats d'une étude effectuée sur 40 patients hospitalisés chez le Centre des Brûlés de Bari, pendant la période 1990-91. L'analyse histologique des biopsies a fourni des informations utiles qui ont permis aux auteurs d'avoir une meilleure compréhension des paramètres fournis par les analyses mierobiologiques traditionnelles et d'adopter en conséquence une approche thérapeutique plus personnalisée chez les patients individuels.


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