March 2020

Cryptococcal necrotizing soft tissue infections in solid organ transplants

Dr Elizabeth Prentice
Dr Chad Centner

Case Presentation
An HIV-negative 45-year-old female* with end stage kidney disease secondary to polycystic kidneys received a renal transplant and was on maintenance immune-suppression with tacrolimus and prednisolone. Her course was unremarkable over the following 2 years. She then presented to hospital in extremis with a hot, swollen left arm, extremely tender to touch. A diagnosis of necrotizing fasciitis was made.

Management
The clinical team commenced antibiotic therapy with cloxacillin, ciprofloxacin and clindamycin, and the patient proceeded to surgical management. A debrided tissue sample was submitted to the laboratory for microbiological testing.

Differential diagnosis
Necrotizing fasciitis is caused by a variety of organisms, depending on the various predisposing factors. Common Gram-positive organisms include Streptococcus pyogenes and Staphylococcus aureus. Clostridium species are also common causes especially in relation to contaminated trauma wounds. Polymicrobial infections of facultative Gram-negative bacilli and anaerobic organisms are common in the elderly or those with underlying illnesses. Vibrio vulnificus and Aeromonas hydrophila are uncommon causative organisms associated with water exposure.

Final diagnosis
The initial Gram stain demonstrated abundant irregularly stained round-to-oval yeast cells with no bacteria observed (Figure). A diagnosis of cryptococcal necrotizing fasciitis was considered and the patient commenced treatment with amphotericin B and flucytosine. The patient did not report any risk factors for cryptococcal disease such as exposure to birds or bird droppings. She did not have any symptoms or signs of meningitis and a lumbar puncture was normal, including a negative CSF cryptococcal antigen test. However, her serum cryptococal antigen test was positive. The laboratory later isolated a pure growth of Cryptococcus neoformans identified by Vitek 2 (Biomerieux, France). The patient responded rapidly to surgical debridement and antifungal therapy. After completion of induction and consolidation phases of the cryptococcal management regimen, she commenced long-term maintenance with fluconazole.

Discussion
Necrotising soft tissue infections include those infections of the skin, subcutaneous tissue and/or the superficial fascia in which necrosis is present1. This includes necrotising fasciitis, which is classically divided into type I and type II on the basis of causative organisms: type I is a polymicrobial infection caused by facultative organisms, usually enteric Gram-negatives and anaerobes while type II is monomicrobial, most commonly caused by Group A streptococci and clostridial species2 . Less common causes of type II, associated with water exposure, are Aeromonas hydrophila and Vibrio vulnificus. Some authorities propose that these should be classified as Type III3. The same authors propose that fungal necrotizing faciiitis be classified as Type IV3. However, this includes trauma associated zygomycete infections in immunocompetent hosts and not opportunistic fungi, like Cryptococcus species, in immunocompromised people3.

There are more than 30 species of cryptococcus4. The taxonomy of cryptococcus is currently in flux with molecular methods currently being used for classification. The old nomenclature of Cryptococcus neoformans var grubii (Serotype A), Cryptococcus neoformans var neoformans (Serotype D) and Cryptococcus gattii (Serotypes B and C), are now referred to as C. neoformans species-complex. This encompasses the aforementioned species as well as other molecular types and hybrids (personal communication Prof. N Govender). Globally, most disease is due to those molecular types falling into C. neoformans var. grubii group (molecular types VNI and VNII)4. In South Africa the most common molecular type causing meningo-encephalitic disease in HIV positive people is VNI5. There are few data regarding molecular types commonly causing skin and soft tissue infections.

C. neoformans is an encapsulated yeast in the genus of basidiomycetous fungi. They are saprophytic, ubiquitous in nature and are commonly associated with rotting wood, soil contaminated with bird guano and certain types of trees. Virulence factors include the polysaccharide capsule, the formation of which is promoted by conditions present in the human host6. The capsule affects immune function in a variety of ways including its antiphagocytic and complement depleting properties6. Other virulence factors are the production of antioxidant melanin, and growth at high temperatures, which other species of cryptococci are unable to do, thus inhibiting growth in the human host6. The lifecycle involves both sexual and asexual stages. The asexual yeast (about 3µm) as well as the sexual basidiospores (1-2 µm) are infectious to the host7, which, once inhaled, reach the terminal alveoli in the lungs. The respiratory tract is the main portal of entry for infection, although primary infection through inoculation of the skin has been reported6. The latter generally does not lead to disseminated disease6.

Once in the lungs the yeasts come into contact with alveolar macrophages where they elicit a TH1 response and depending on host T-cell function one of three outcomes may occur: the pathogen may be eliminated by an effective immune response; an asymptomatic pulmonary lymph node complex may be established in which the yeasts are dormant and may later re-activate; or proliferation and dissemination of the yeast occurs6. The latter two scenarios lead to symptomatic disease in immunocompromised patients, where disease may occur immediately following exposure or during later periods of immune suppression.

The two major sites of clinical disease in cryptococcal infections are the lungs and the central nervous system4,6. These are followed by the skin, prostate and eye4. In an immunocompetent host pulmonary cryptococcosis is commonly asymptomatic, identified only by an abnormal chest X-ray4. In immunocompromised patients, however, infection is usually symptomatic ranging from pneumonia to life-threatening acute respiratory distress syndrome4.

The clinical manifestations of central nervous system disease are many and varied, from altered mental status to cranial neuropathies and signs and symptoms of meningitis6. CNS disease is a meningo-encephalitis, where brain parenchymal involvement may include cryptococcomas4. Clinical manifestations are a result of host immunity as although commonly a chronic presentation with severe immunocompromise, a more acute presentation with high fungal burden is observed4.

The skin is the third most common site of cryptococcal infection4. The classic lesion is a molluscum-type lesion of a raised papule with an umbilicated centre6. However, any type of lesion can be observed, including cellulitis, abscesses and rarely necrotizing fasciitis6. These more severe manifestations are common in severely immunosuppressed patients4. Mostly skin involvement is a result of disseminated disease, however, primary cutaneous cryptococcosis following inoculation has been reported8.

Aside from HIV infection, the most common underlying disorders associated with cryptococcal disease are haematological malignancies, solid organ transplantation, corticosteroid treatment and other immunosuppressant use, sarcoidosis and autoimmune diseases9. It has been suggested that cryptococcal disease in haematological malignancies especially, is the result of increased use of lymphocyte-depleting chemotherapeutic regimens9, and corticosteroids and Alemtuzumab therapy used in solid organ transplants have also been associated with an increased risk for cryptococcosis7. Interestingly, tacrolimus is negatively associated with CNS cryptococcosis but positively associated with skin, soft tissue and bone disease, as this drug has anti-cryptococcal properties at 37 °C to 39°C6,7.

In solid organ transplant recipients, cryptococcosis is the third most common invasive fungal infection after candidiasis and aspergillosis7. A multicenter surveillance network in the United states reported cryptococcosis in 8% of cases overall, with renal and heart transplants being the most common10. In solid organ transplant patients, the disease may be a result of primary infection after transplantation, or due to reactivation secondary to post transplant immunosuppression7. Other possibilities include donor-derived, or un-recognized pre-transplant infection7. Time to presentation of disease after transplant is longer than for candidiasis and aspergillus, about 20-36 months7,10. Clinically, CNS and pulmonary involvement are the most common, although clinical features differ between different solid organ transplants7. In renal transplant recipients, necrotizing soft tissue infection8,11–13, prostatitis, parathyroid gland infection, pyelonephritis and skull osteomyelitis have been reported along with various neurological abnormalities. Cellulitis and necrotizing soft tissue infection have also been reported with heart, heart and lung transplants as well as liver transplants7.

Diagnosis of cryptococcosis may be made by routine laboratory methods. The encapsulated yeast cells (5-10 µm) may be visualized by gram stain as gram positive organisms (Figure 1)6. The classic use of India Ink to negatively stain the encapsulated yeasts, is best used on CSF. This methodology is difficult to interpret when used on other body fluids such as urine or respiratory specimens6. In histopathological specimens the yeast may be identified by capsular stains such as mucicarmine or alcian blue. Gomori methenamine silver stains the yeast itself, which may be identified by narrow based budding in tissue specimens6.

Cryptococcus species grow well on routine media such as blood and chocolate agar (Figure 2) as well as on fungal media such a Sabouraud agar. The yeasts grow as pale, creamy colonies, usually after 48-72 hours of incubation4 and may be presumptively identified as cryptococcus species by the production of urease. On birdseed agar (Nigerseed agar), melanin production by cryptococcus distinguishes it from many other yeasts. However, many laboratories use automated systems for identification such as the VITEK 2 (Biomerieux, France), system or by MALDI-TOF mass spectrometry.

Serological tests such as enzyme immunoassays, latex agglutination or newer lateral flow assays for the detection of cryptococcal polysaccharide are used in serum and CSF. These have good sensitivity and specificity, >90%4,6. The lateral flow assay itself, which is used at point of care, has sensitivity and specificity of >98% for both serum and CSF, and can also be used in urine with a sensitivity of 85%4. In isolated pulmonary disease in immunocompetent hosts, antigen tests may be negative4, however approximately 83% of solid organ transplant patients with pulmonary disease will test positive by antigen detection7. Similar to cryptococcosis in HIV disease, lumbar puncture is also recommended for patients with cryptococcosis and solid organ transplants7.

The definitive treatment of necrotizing fasciitis is surgical debridement. Empirical antimicrobial therapy would typically include cover for anaerobic organisms, streptococci, staphylococci and Gram negative organisms, including Pseudomonas aeruginosa, depending on the site of infection and other risk factors. Fungal necrotizing fasciitis is rare and in the context of a compatible medical history, the laboratory should be informed that this diagnosis is suspected and close communication between the clinical and laboratory team should be maintained. The treatment for disseminated cryptococcosis in HIV+ and HIV- patients is similar to that for cryptococcal meningitis15 and has been outlined in recent South African national guidelines informed by WHO recommendations14. The 2-week induction phase comprises 1 week with amphotericin B (1 mg/kg/day) plus flucytosine, and followed by 1 week of high-dose oral fluconazole (1200 mg/day). If flucytosine is not available, amphotericin B and fluconazole are extended from 1 week each to both cover the full 2-week induction phase. The consolidation phase is 8 weeks of fluconazole 800 mg/day. It has been suggested that the maintenance phase (fluconazole 200 mg/day) be lifelong in cases of disseminated cryptococcosis presenting in chronically immunosuppressed patients12 such as the one presented in this report.

* Fictionalized case

Recommended reading

• Stevens, D. L. & Bryant, A. E. Necrotizing soft-tissue infections. New England Journal of Medicine 377, 2253–2265 (2017).
  A comprehensive review article on necrotising soft tissue infections, which includes necrotising fasciitis. Good explanations of classification as well as excellent discussion on pathogenesis and diagnosis
• Maziarz, E. K. & Perfect, J. R. Cryptococcosis. Infectious Disease Clinics of North America 30, 179–206 (2016).
  A review of crytococcal disease in many types of patient including HIV/AIDS and solid organ transplant. Written by the foremost expert on cryptococcosis, it provides concise, yet comprehensive overview.
• Henao-Martínez, A. F. & Beckham, J. D. Cryptococcosis in solid organ transplant recipients. Current Opinion in Infectious Diseases 28, 300–307 (2015).
  This article reviews the epidemiology, pathogenesis and various unique clinical presentations of Cryptococcosis in solid organ transplant patients. It is a comprehensive and extensive review of recent findings.