Talaromycosis (Penicilliosis) in a Cynomolgus Macaque

Abstract

A sexually mature Chinese-origin female Macaca fascicularis assigned to the high-dose group in a 26-week toxicology study with an experimental immunomodulatory therapeutic antibody (a CD40 L antagonist fusion protein) was euthanized at the scheduled terminal sacrifice on study day 192. The animal was healthy at study initiation and remained clinically normal throughout the study. On study day 141, abnormal clinical pathology changes were found during a scheduled evaluation; splenomegaly was detected on study day 149 and supported by ultrasound examination. At the scheduled necropsy, there was marked splenomegaly with a nodular and discolored appearance. Cytologic examination of a splenic impression smear revealed yeast-like organisms within macrophages. Histologically, there was disseminated systemic granulomatous inflammation with 2- to 3-μm oval, intracytoplasmic yeast-like organisms in multiple organs identified as Talaromyces (Penicillium) marneffei. This organism, not previously reported as a pathogen in macaques, causes an important opportunistic infection in immunosuppressed humans in specific global geographic locations.

Opportunistic infections in nonhuman primates have been well documented and can occur as a result of immunosuppression from viral infections such as simian immunodeficiency virus, stress due to environmental conditions, or during safety assessment studies when animals are exposed to potential therapeutic agents that may be purposefully or unintentionally immunomodulatory.¹ A wide range of agents including bacteria, viruses, fungi, and parasites can be opportunistic pathogens in humans and nonhuman primates.⁹ The resulting infection may be subclinical or progress and lead to death or early euthanasia. We report a dimorphic fungal infection not previously described in macaques, which was asymptomatic but suspected based on periodic, scheduled laboratory evaluations as part of a 26-week toxicology study with an immunomodulatory molecule.

A 3.1-kg sexually mature female Macaca fascicularis from Yunnan province in Southern China was in the high-dose, intravenous administration group of a 26-week toxicology study with an experimental CD40 L antagonist fusion protein. The testing facility (Charles River Laboratories, Reno, NV) was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and was in full compliance with all applicable guidelines of the US Department of Agriculture. All research protocols were approved by the Testing Facility Institutional Animal Care and Use Committees prior to dose administration. Cynomolgus monkeys were obtained from a US Department of Agriculture–approved facility and passed veterinary inspection prior to assignment to study and were negative for simian retrovirus and tuberculosis. Certified Primate Diet (No. 5048, PMI, Richmond, MI) was provided daily in amounts appropriate for the age and size of the animals. The animals had ad libitum access to water (reverse osmosis purified and passed through ultraviolet light treatment) via an automatic watering system/water bottle. Animals also had access to enrichment opportunities (including some or all of the following: treats, fresh fruit, toys, grooming bars, and social interaction). The study animals were pair-housed by sex in climate-controlled rooms at 64–84°F with a relative humidity of 30–70%. The rooms had greater than 10 air changes per hour with 100% fresh air (no air recirculation) and a photoperiod cycle of 12 hours light/12 hours dark.

The test article was administered by weekly 30-minute intravenous infusion. Between study days 113 and 141, several clinical pathology parameters changed in this animal, including decreased erythrocyte mass and leucocyte counts, increased alkaline phosphatase, and decreased A/G ratios with a reversal of albumin and globulin concentrations (Supplemental Materials).

A previously scheduled physical examination on study day 149 detected splenomegaly which was confirmed by an ultrasound evaluation. Because of these findings, additional blood samples were collected every 7 to 14 days until the end of the study on day 191. The affected animal continued to maintain body weight and had normal activity and appetite.

The terminal necropsy occurred as scheduled on study day 192. Observations at necropsy were a skin abrasion with several scabs on the lower jaw, an adhesion between the parietal peritoneum and viscera, and marked splenomegaly. The spleen weighed 61 g versus 3.1 g for the mean of the female control animals, and was >2% of the body weight for this animal. The spleen was nodular with multifocal tan firm foci on the splenic capsule and was a homogeneous deep red on the cut surface with a meaty consistency (Supplemental Materials).

Impression smears of spleen and bone marrow were made and stained with modified Wright’s-Giemsa. The smears contained moderate numbers of intact nucleated cells and were markedly hemodiluted with scattered platelets. Macrophages were markedly increased in size and frequently contained 3- to 5-μm, oval to round organisms that were occasionally elongated up to 10-μm long with an eccentrically to centrally located round nucleus (Fig. 1). The organisms were also often seen extracellularly. There were mild increases in nondegenerate neutrophils with lower numbers of eosinophils and basophils. Occasional macrophages displayed erythrophagocytosis and contained one to numerous erythrocytes. The lymphocyte population was predominantly composed of small and intermediate cells. There were small numbers of erythroid precursors (predominantly metarubricytes and rubricytes) and rare megakaryocytes, indicating extramedullary hematopoiesis.

Figs. 1–4.

Talaromycosis, cynomolgus macaque. Fig. 1. Splenic impression smear. Yeast forms of Talaromyces marneffei are present in the cytoplasm of a macrophage. Modified Wright’s-Giemsa. Fig. 2. The splenic red pulp contains multiple coalescing foci of granulomatous inflammation. Hematoxylin and eosin (HE). Fig. 3. Yeast forms of T. marneffei in splenic red pulp macrophages. Gömöri methenamine silver. Fig. 4. Erosion and granulomatous inflammation of the tongue. Inset: Macrophages containing numerous yeast forms of T. marneffei. HE.Open in viewer

Histologically, all but one section of skin, all sections of lymph nodes, spleen, liver, lung, femoral bone marrow, and many other tissues had multiple minimal to marked coalescing foci of mononuclear cells, predominately macrophages, containing large numbers of yeast organisms in their cytoplasm (Fig. 2). In these cells, the organisms were oval, 2 to 3 μm, and surrounded by a clear space (Figs. 3, 4). Other tissues with minimal mononuclear cell infiltrates were brain, tongue, thymus, omentum, rectum, cervix, and vagina. Splenic extramedullary hematopoiesis correlated with the regenerative anemia and leukopenia due to bone marrow involvement and inflammatory infiltrates in numerous organs. Increased globulins were indicative of a humoral immune response, although infected macrophages contained intact organisms. No microscopic correlate was found for the increased alkaline phosphatase.

Fungal cultures at ambient temperature on Sabouraud dextrose agar performed by a Charles River Laboratory of tissue collected at necropsy showed velvety greenish yellow colonies excreting a red pigment. Microscopically, biverticilliate conidiophores with tapering phialides in clusters and round conidia in chains were observed. Growth at 37°C produced small elongate fission yeast. Polymerase chain reaction evaluation of frozen liver and spleen was performed at the Centers for Disease Control and Prevention using the QIAamp DNA formalin-fixed paraffin-embedded tissue kit (Qiagen, Hilden, Germany) and the primer set ITS3/ITS4 as previously described.⁶ The amplified DNA sequence was a 100% match to multiple isolates of Talaromyces (Penicillium) marneffei based on a BLAST search of the National Center for Biotechnology Information database. In addition, DNA amplified using the primer set Bt2A and Bt2B (which amplifies the tubulin gene and is more specific for species identification of Penicillium/Talaromyces) from both the DNA extracted from the tissue and the isolate was a 100% match to multiple isolates of T. marneffei but no better than a 95% match to any other species. As a thermophilic dimorphic fungus, the organism grows as a mold on Sabouraud medium at room temperature and as yeast at 37°C under physiologic conditions.

Penicillium sp. has been identified as part of normal flora on the skin of Cebus monkeys, and the abrasion on the skin of the face may have been the portal of entry.⁵ In humans, the organism may lie dormant for years, presumably in the lungs or lymph nodes, without clinical manifestation.¹⁰ Widespread dissemination may occur following immune modulation, as seen in humans. Recent work has shown that this organism can secrete laccase and Mp1p as factors that reduce the innate immune response and may play a role in the persistence of the yeast in macrophages.^8,12

Talaromyces (Penicillium) marneffei was first isolated as a commensal organism from a bamboo rat (Rhizomys sinensis) at the Pasteur Institute of Indochina, Dalat, South Vietnam, in 1956.² It was named Penicillium marneffei in the subgenus Biverticillium, and the infection by this organism was designated “penicilliosis” and appeared so-named in publications for nearly 60 years. In 2015, the Penicillium subgenus Biverticillium was reclassified into the genus Talaromyces, and the infection is now known as talaromycosis.⁷ It has been recognized as the cause of systemic mycosis in AIDS patients and was the third most common opportunistic infection in Southeast Asia in this population prior to the advent of highly active antiretroviral therapy.¹¹ Human disease is characterized by fungal invasion of multiple organs, especially blood, bone marrow, skin, lungs, and reticuloendothelial tissues, with high fatality rates notably with delayed diagnosis and treatment.¹³ It now is found in AIDS patients and those with cell-mediated immunodeficiencies involving the interleukin-12/interferon-γ signaling pathway.³ Infection is a risk factor for patients receiving anti-CD20 therapeutics, chronic administration of T-lymphocyte–depleting drugs, or kinase inhibitors. The infection has rarely been reported among hematologic cancer patients, including those from disease-endemic regions.⁴ It is not known to be a pathogen of otherwise healthy patients, and cases in the United States are not required to be reported at the state or national level. The mechanism enabling infection in this specific animal was believed to be directly linked to the immunosuppressive pharmacology of the anti-CD40 L fusion protein.