Fungal infections in bone marrow transplants - Etiology: Conventional microbiological tests; Molecular diagnosis (PCR); Species identification (sequencing).

Information 02-12-2019.

Hematopoietic cell transplantation is the treatment of choice in some malignant and non-malignant conditions. This procedure can cause complications with vital repercussions, especially in the early post-transplant phase. Several of its complications are accompanied by high morbidity and mortality despite improvements in supportive therapies.

Among the causes of morbidity and mortality are infections by the Herpes group viruses (Cytomegalovirus, Herpes simplex virus and Varicella-Zoster virus) and by many fungi (Candida spp., filamentous fungi and Pneumocystis jiroveci).

Some fungi such as yeasts of the genus Candida and Pneumocystis carinii can be found as commensals in our body and others such as filamentous fungi are very ubiquitous in the environment. These fungi, despite our daily contact with them, do not cause disease. However, in immunosuppression states, as in the post-transplant phase of hematopoietic cells, they can cause invasive infections.

The incidence of invasive fungal infections in recipients of allogeneic bone marrow transplants is usually around 15%, of which 3% would correspond to Candida infections and 12% to other invasive fungal infections. Most filamentous fungal infections (molds) are due to invasive aspergillosis (86%).

These infections are among the most dangerous complications in allogeneic or autologous bone marrow transplants, as they are accompanied by a very high mortality (> 95%) in patients transplanted with respiratory failure requiring endotracheal intubation and mechanical ventilation, especially when they are accompanied by liver and kidney dysfunction. The survival rate is 2 to 40% and among the predictors of mortality is mechanical ventilation equal to or greater than 4 days.

Yeasts are the fungi that most often colonize and infect people. Of the many existing species of yeast, only some, such as the genus Candida, cause human infections. Of the approximately 200 species of Candida, only some are part of the normal flora of the gastrointestinal tract of 80% of healthy individuals. Of these, the Candida albicans species represents between 50 and 70% of human yeast isolates (this species is found in 50 to 60% of invasive pulmonary infections after a bone marrow transplant). The rest is distributed among about 20 different species found occasionally (C. krusei, C. glabrata, C. tropicalis, C. lusitaniae and C. parapsilosis,…). The highest prevalence of the Candida albicans species is due to the virulence factors associated with its pathogenicity: rapid germination capacity, protease production, possession of adhesion factors, possession of receptors for proteins of the serum complement system, ability to change phenotypes and hydrophobicity.

Filamentous fungi are not usually found as commensals in the human organism. The most frequent infection entrance is the inspiration from the nostrils or respiratory tract or penetration through a solution of epidermal continuity (incision). Of the many existing filamentous fungi, Aspergillus spp. is the most prevalent human pathogen. The incidence of invasive infections by Aspergillus spp. is 0.5% of infections after an autologous bone marrow transplant; 2.3% after allogeneic transplantation with a compatible donor; or 3.9% after a transplant with an unrelated donor. Mortality caused by its invasive infections 3 months after its diagnosis ranges from 53.8% in autologous transplants to 84.6% in transplants with an unrelated donor.

Among its different species, Aspergillus fumigatus is the most prevalent cause of 56% of infections. Other species are: Aspergillus flavus (18.7%), Aspergillus terreus (16%), Aspergillus niger (8%), Aspergillus versicolor (1.3%), Aspergillus ustus and Aspergillus lentulus.

Other fungi that cause opportunistic invasive fungal infections are: Cryptococcus spp., Pneumocysts jiroveci, Fusarium spp., Trichosporon spp., Penicillium spp. Alternaria spp. Pseudallescheria spp., Scedosporium spp. and Mucorales such as Zygomycetes spp.

In some countries, due to their environmental geographical presence, the Histoplasma capsulatum and Coccidioides immitis species are added to the above.

The finding of all the fungi mentioned lacks clinical significance in immunocompetent patients. However, in immunosuppressed patients, they act as opportunists, crossing the host´s natural barriers, penetrating normally into sterile tissues and causing tissue damage.

Invasive infection occurs thanks to the interaction of several factors: acquisition by the host of the potential pathogen, effective competition of the potential pathogen with the microorganisms that are part of our mucosal commensal flora to grow in sufficient numbers to exceed the phagocytes, properties of inherent virulence of the potential pathogen to invade and cause tissue damage, and impairment of the host´s natural defense mechanisms.

In recent years the incidence of invasive fungal infections has decreased, which may be related to changes in transplants, including the use of non-myeloablative conditions, increased use of antifungal prophylaxis and supportive care.

Clinical manifestations of Candida spp.

Candida infections, especially those caused by Candida albicans, were recognized as especially frequent at the beginning of the 1980s at the beginning of bone marrow transplants. During this period of time, before the extension of prophylaxis with fluconazole, the incidence of invasive candidiasis was estimated at 11%, with an associated mortality of 39%. Azole prophylaxis decreased the overall rate of invasive candidiasis and changed prominent organisms, increasing the incidence of infections caused by Candida glabrata resistant to fluconazole, and Candida krusei.

More recently, the incidence of invasive candidiasis has decreased in allogeneic bone marrow transplant recipients after non-ablative preparation.

Invasive candidiasis is described as acute when it comes with blood infection, or chronic when it is treated with hepatosplenic involvement. Patients are usually febrile and may have signs of sepsis; peripheral manifestations such as chorioretinitis; skin lesions or abscesses of organs (eg. liver, kidneys). The most recent statistics indicate a low incidence of candidemia in bone marrow transplant recipients with a high representation of azole resistant species.

Hepatosplenic candidiasis usually develops during periods of neutropenia and mucositis, when organisms that colonize the gastrointestinal tract invade portal vascularization and spread to the liver and spleen. The infection usually manifests itself only after neutrophil transfer, with an increase in inflammation that causes the classic presentation of fever, elevation of liver enzymes and pain in the side. Inflammation in liver lesions can be necrotic or granulomatous; the organisms may or may not be observed in microscopic exams. Candida albicans is considered to be the most common cause of disease, probably associated with its ability to transition from yeast to hypha for invasion of the gastrointestinal tract.

The most common manifestation of Candida infection is unexplained fever. endophthalmitis, rare before grafting, is the most common sign of candidemia in the post-transplant period. Retinal lesions can be subtle and should be investigated by indirect ophthalmoscopy. Macronodular skin lesions, polyarthralgia or polymyalgia (due to invasion of joint and muscle tissues) and azotemia (due to an invasion of the renal tubules) are occasional manifestations of Candida fungemia. Persistent fever after transplantation, together with an elevation of serum alkaline phosphatase, may indicate the presence of hepatosplenic candidiasis.

Clinical manifestations of filamentous fungal infections

Filamentous fungal infections, especially those caused by Aspergillus fumigatus, were recognized after allogeneic transplantation during the 1990s. Incidence estimates vary from approximately 5 to 12% in allogeneic bone marrow transplant recipients after myeloablative therapies. During this period of time, it was reported that the mortality associated with the infection was as high as 80 to 90%. More recent studies have shown that the incidence of invasive aspergillosis has probably stabilized. Extensive epidemiological surveillance studies have found that the incidence continues to be between 5 and 10% although the rates reported by the centers are very variable, probably due to differences in diagnostic aggressiveness and the different long-term monitoring methods of the patient after bone marrow transplant.

Members of this genus usually cause disease after inhalation in the sinuses or lungs. Inhalation through these pathways allows possible hematogenous spread to other organs (brain, skin). Classic lung radiographic lesion is described as a nodular infiltrate, with or without a sign of "halo" and progression to cavitation; however, the radiographic presentation of invasive aspergillosis can also include lobular infiltrates in a unilateral or bilateral focal distribution. It has also been found that bronchopneumonia can occur more frequently in patients during the post-transplant period in allogeneic bone marrow transplant recipients.

Tracheobronchitis, a manifestation that has been reported most frequently in lung transplant recipients also occurs in bone marrow transplant recipients.

Some information indicates that some component of the clinical manifestations and radiological findings may be due to immunological reconstitution and inflammation caused by it, rather than to the fungal invasion itself, especially in patients who have neutropenia or are recovering from she.

Due to the difficulties of making the diagnosis through radiological findings, microbiological diagnostic methods are very important. However, conventional microbiological methods such as culture from tissue biopsies or bronchoalveolar lavage fluid (BAL) can be difficult and have a sensitivity of about 50%. For this reason, methods based on the detection of antigen (Aspergillus spp. galactomannan) or molecular methods (PCR) for the generic or specific detection of fungi are being applied.

Other filamentous fungi, such as Fusarium species, Scedosporium and Zygomycetes species, have increased in frequency as a cause of invasive disease in bone marrow transplant recipients since the late 1990s. Clinical manifestations can be very similar to infections caused by Aspergillus species.

Lung disease and, less frequently, sinusitis are manifestations of aspergillosis. The propensity of Aspergillus hyphae to invade blood vessels and cause pulmonary infarction produces signs of pleuritic chest pain, hemoptysis, localized wheezing, pleural friction, sinus tenderness, nasal discharge, epistaxis or nasal eschar. Any of these signs or symptoms in the context of persistent fever should lead to the suspicion of aspergillosis. Pulmonary infiltrates tend to be nodular and peripherally located.

CT scans of the chest and paranasal sinuses can detect the disease before simple x-rays. Primary cutaneous aspergillosis can occur at the exit site or in the tunnel of an intravenous catheter.

There are descriptions of outbreaks of Aspergillus catheter infections during a period of renovation (works) of the hospital caused by the introduction of aerosol spores into the surgical wound. The organisms recovered from air samples in operating rooms where infected catheters were placed, were not recovered from other operating rooms.

Brain abscesses in the patient with BMT are commonly due to fungi. In one center, 92% of all cases of brain abscesses were caused by fungi. Aspergillus accounted for 58% of cases and Candida 33%. Bacteria were involved in less than 10% of cases. Aspergillus brain abscesses generally occurred concomitantly with lung disease (87% of cases). Candida brain abscesses were associated with fungemia or neutropenia.

Microbiological diagnosis

Due to the non-specificity of clinical signs and symptoms and the occasional low usefulness of blood cultures, various techniques have been used to improve the early diagnosis of the infection.

The antigen detection tests of Candida spp. (mannan), Aspergillus spp. (galactomannan) are currently available and can be quite useful for detecting infections by both organisms, respectively. In countries where Histoplasma capsulatum is prevalent (eg. USA) the detection of its antigen is used.

Molecular diagnostics (PCRs) are useful for detecting different types of fungi, if they are applied to samples where there is usually no fungal colonizing flora (intestine) or respiratory tract (environmental fungi).

Tests performed in IVAMI:

  • Conventional microbiological diagnosis by sample culture.
  • Detection of mannan (Candida spp.) or galactomannan (Aspergillus spp.) in peripheral blood.
  • Antifungal sensitivity tests in case of availability of isolation in culture.
  • Molecular diagnosis of fungi by generic PCR tests when the samples are usually sterile (blood, lung aspirate, liver biopsy, spleen, etc.).
  • Molecular diagnosis of specific species of fungi (Candida spp., Aspergillus spp., Pneumocystis jiroveci, etc.), using specific PCR tests of each group.
  • Molecular identification of species in case of positive results by sequencing.

Sample recommended:

  • Complete blood drawn in tubes with EDTA (1.0 mL) in case of suspected systemic infection.
  • Aspirated sinuses in case of suspicion of sinus infection.
  • Deep respiratory samples in case of suspected bronchopulmonary infection: bronchopulmonary aspirate; bronchoalveolar lavage; aspiration or biopsy of transthoracic pulmonary puncture, …..
  • Punctures or tissue biopsies (depending on the location of the invasive infection).

Storage and sending of the sample:

  • Refrigerated (preferred) for less than 2 days.
  • Frozen: more than 2 days. If frozen sample is available, send in this state without defrosting.

Interference with the test:

  • Complete blood hemolysis.
  • Repeated freeze-thaw.
  • Conservation at temperature above refrigeration.
  • Sample extraction during convalescence phase.
  • Extraction of blood in tubes with heparin.

Cost of the test:

  • Conventional microbiological diagnosis by sample culture: Consult to
  • Tests of mannan (Candida spp.) or galactomannan (Aspergillus spp.): Consult to
  • Molecular diagnosis of fungi by generic PCR tests: Consult to
  • Molecular diagnosis of specific species of fungi (Candida spp., Aspergillus spp., Pneumocystis jiroveci, etc.,) by specific PCR tests (each group): Consult to
  • Molecular identification of species in case of positive results by sequencing: Consult to