Test quantitation of (1-3) -β-D-glucan, as a method for diagnose of deep fungal infections


Information 03/01/2011.

The difficulties for diagnosis of deep fungal infections, by conventional microbiological methods (microscopic examination, culture of clinical specimens, detection of antibodies, ...), has promoted the development of alternative methods such as detection of fungal components, which are released during multiplication of the fungus in the tissues. Of those, which have prevailed in use, they are the components that detect the wall of Candida spp. (mannan) and Aspergillus spp. (galactomannan), by immunoenzymatic methods (ELISA capture or sandwich), and their nucleic acids that detect fungal genome by PCR (Polymerase Chain Reaction).

(1-3) - -D-glucan b is a wall component of most fungi species except Zygomycetos (Mucorales), and small amounts in Cryptococcus spp. From inside out, the cell wall of fungi outside of the cytoplasmic membrane, a layer of chitin [polysaccharide ? (1-4) -acetyl], then outward, layers ? is (1- 3) -glucan with galactomannans [? (1-4) linkages -manano with ? (1-6) galactose, in the case of Aspergillus and some other fungi], and in the cell wall of yeasts such as Candida albicans and other species, is a layer of mannose polysaccharides branched structure with a central axis formed by glycosidic linkages ? (1-6) branches and with ? (1-2) and ? (1-3). These polysaccharides mannose (mannans) are conjugated proeínas (mannoproteins), and are located in the outermost part of the cell wall. By exist the (1-3) - b -D-glucans, as pricipal component in the fungal cell wall, has been seen as a component to detect invasive fungal infections by several species of fungi, including opportunistic infections by Pneumocystis jirovecci, and excluding Mucorales (Rhizopus spp., Mucor spp.) that lack it, or Cryptococcus spp. who possesses in limited quantity.

      The existence of this component, important in the wall of most fungi, carried search antifungals recuse synthesis, trying to find an equivalent to the ?-lactam antibacterials block PBPs (penicillin binding proteins), holding synthesis of the cell wall. The synthesis of (1-3) - -D-glucans b, is performed by an enzyme [(1-3) - glucan synthetase b] which is, like the PBPs of bacteria in the cytoplasmic membrane . These antimicrobials block (1-3) - glucan synthetase b are echinocandins (caspofungin, anidulafungin and midecafungina). The first naturally occurring echinocandin papulacandina was obtained from a fungus (Papularia spp.), And subsequently cilafungina semisynthetic derivative was obtained. These antimicrobials, by its molecular structure of large, are not sufficiently absorbed when administered orally, so to be administered intravenously. These antifungals are not active against fungi which lack glycans on their wall (Cryptococcus spp., Trichosporon spp., Rhodotorula spp., Zygomycetos -Mucorales). They possess synergistic activity with amphotericin B and fluconazole, by acting on different targets. Generally they possess fungicidal activity, except for some fungi that have fungistatic activity (Aspergillus spp.).

      Tests designed to quantify (Fungitell; Glucatell, ...), are based on the activation of the coagulation system of some species of crustaceans (crabs), as Limulus polyphemus or Tachypleus tridentatus. The rationale is similar to that of the LAL test, the amebocyte lysate of Limulus to detect bacterial endotoxins. In the test lysate of Limulus amebitos to detect bacterial endotoxins, they interact with the factor C amebocyte lysate of generating an enzymatic activity that converts coagulogen into a clot (coagulin). In the case of (1-3) - -D-glucans b, these interact with the G present in the lysate factor of amebocytes, causing transformation coagulogen into coagulin (coagulation). Tests using this procedure use amebocyte lysate which have withdrawn factor C, so that, if bleeding provoked by exposing a clinical sample, coagulation has had to be induced by (1-3) - b -D-glucan, the G factor of the clotting cascade of these crabs activated. This activation, to reside in a serine protease enzyme activity type currently detected by a kinetic method with a chromogenic substrate for this enzyme activated.

      Throughout the experience with the application of this method, we found some situations of false positive results. This occurs in patients receiving antibiotics produced from cultures of fungi, such as amoxicillin-clavulanate, so in patients receiving parenteral treatment with this antibiotic, a careful evaluation of the results of the test should be performed with the clinical characteristics of the patient. Another similar situation has been described in patients with Pseudomonas aeruginosa infections, and to a lesser extent with Streptococcus pneumoniae infections Alcaligenes faecalis or. This is because these bacteria have in their periplasmic space components (1-3) - -D-glucan b. Moreover, it described elevation (1-3) - -D-glucan b in patients with mucosal colonization by Candida spp, without any invasive infection.. Also we found false positive results after ingestion of oatmeal ( "OAT"), barley ( "barley") or rye ( "rye").

The sensitivity of this test is considered to be between 65 and 70%, depending on the cut point for the quantification of (1-3) - -D-glucan b is 80 to 60 pg / ml. The specificity of 92 and 87%, for both cutoffs, respectively. The positive predictive value would be for both cutoffs 89 and 83%, respectively, and the negative predictive value of 73 and 75%. The absence of (1-3) - -D-glucan b has a negative predictive value of 100%.

Situation in IVAMI: in IVAMI, we have not yet introduced this test in the diagnosis because they do not consider that there is still a clear acceptance indications, probably because of doubts raised by its use, nor do we know that is collected by the recommendations of Guidelines on schemes therapeutic-dignósticos decision. In general, it is considered as an added test when assessing a patient. Moreover, it has other alternatives for more experience possessed. In this sense perform testing for infections mannan Candida spp., Galactomannan for Aspergillus infections spp., And PCR for detection of fungal infections by any species.