Talaromyces amestolkiae Infection in an AIDS Patient with Cryptococcal Meningitis

Concurrent infections caused by multiple fungal pathogens in immunocompromised patients can pose diagnostic and treatment challenges. Here, we presented the first reported case in Taiwan of an AIDS patient who had concurrent infection with Cryptococcus neoformans meningitis and Talaromyces amestolkiae lymphadenopathy. The patient presented with an enlarged inguinal lymph node and was diagnosed with T. amestolkiae lymphadenitis. The species T. amestolkiae was identified using DNA sequencing, which had the capability of differentiating it from other Talaromyces species. The patient was discharged from the hospital following treatment with amphotericin B and subsequent administration of voriconazole. This case highlights the importance of maintaining a suspicion of co-infections and utilizing appropriate diagnostic tools, such as DNA sequencing, to identify possible pathogens. Further studies are needed to determine the optimal treatment for T. amestolkiae and other co-infecting fungal pathogens.


Introduction
The genus Talaromyces was first described by Benjamin in 1955 based on phylogenetic information, which comprised several perfect stages of Penicillium species [1].It was later reclassified by Yimaz et al. based on morphological, molecular, and physiological characters, and consisted of over 80 species, including T. marneffei, which was frequently found in immunocompromised patients [2].Other Talaromyces species, including Talaromyces amestolkiae, Talaromyces purpurogenus, and Talaromyces piceus, have also been reported to cause invasive fungal infections [3].
Yilmaz et al. first described T. amestolkiae in 2012.T. amestolkiae was mostly isolated from the environment, including indoor dust, indoor air, soil, and wheat.It was also found in the sputum and lungs of cystic fibrosis patients [4].Although T. amestolkiae has been shown to be a potential pathogen in humans, the current diagnostic tools are insufficient.
While there are several diagnostics tools available, including microbial cultures, immunological techniques, microscopy, and PCR assays, certain fungal infections remain misdiagnosed or underdiagnosed.Differentiating among various Talaromyces species using conventional clinical microbiology tools is difficult, as most species are red pigmentproducing and exhibit similar features under the microscope.Therefore, the development of more specific and sensitive diagnostic techniques for Talaromyces species, including T. amestolkiae, is essential for accurate identification and timely treatment [3].However, currently, there is limited information in the literature regarding the Talaromyces species, other than T. marneffei.
This report discussed the first AIDS case with cryptococcal meningitis and lymphadenopathy due to a T. amestolkiae infection.This report covered details regarding clinical characteristics and treatments of T. amestolkiae infection.Molecular and morphological comparisons between T. amestolkiae and T. marneffei were carried out to facilitate better differentiation between the two species.

Case Report
A 31-year-old male patient with AIDS was admitted to the hospital with headache, diplopia, bilateral inguinal lymphadenopathy, and several erythematous papules with central crusted ulcers.This patient was diagnosed HIV since 2017 and he received ART with Bictegravir/Emtricitabine/Tenofovir Alafenamide with poor adherence.The patient had a low CD4 count of 19 cells/mm 3 and a high HIV viral load of 81,500 copies/mL upon admission to the hospital.The patient had been in his usual state of health until one week prior to admission, when he began to experience pain in the occipital area and the neck.A brain CT was arranged to rule out mass lesion, herniation, and other structural abnormalities.Following admission, Cryptococcus neoformans meningitis with fungemia was diagnosed with CSF analysis and serum laboratory survey.In CSF, it revealed a positive India ink result, latex of Cryptococcus test (+, 1:512) (IMMY CrAg ® LFA), and Cryptococcus neoformans in CSF culture.In serum analysis, positive latex of Cryptococcus test (1:4096) (IMMY CrAg ® LFA) and Cryptococcus neoformans in blood culture were found.
We started amphotericin B and flucytosine upon admission for the treatment of cryptococcal meningitis.However, we discontinued flucytosine on day 19 due to severe neutropenia (serum WBC decreased from 6460/µL to 1320/µL) and anemia (Serum Hb from 11.8 g/dL to 6.8 g/dL).Amphotericin B was switched to liposomal amphotericin B due to worsening renal function (serum creatinine increased from 1.04 mg/dL to 1.53 mg/dL).Although symptoms presented upon admission had been alleviated, bilateral inguinal lymphadenopathy persisted following antifungal therapy.These lymph nodes were painless, fixed, and 1 cm in diameter.An excisional biopsy was performed on the inguinal lymph nodes, which revealed a fungal infection caused by Talaromyces amestolkiae, instead of Cryptococcus neoformans (Figure 1).
To further verify the presence of T. amestolkiae, we sent the lymph node specimen to Tri-ibiotech, Taiwan.This company performed amplification and sequencing of the internal transcribed spacer rDNA area (ITS) and β-tubulin (BenA) with primer sets ITS5/ITS4 and Bt2a/Bt2b [5].ITS works as a DNA sequencing marker for fungi, but there is insufficient variation to distinguish all the closely related species of Penicillium and many other genera of ascomycetes.Because the ITS reference sequence has its limitations, a second DNA sequencing marker is needed to identify isolates to species level.BenA is the best option for a secondary identification marker for Penicillium.The ITS sequence of our strain showed 98.8% identity to T. amestolkiae strain M26 (MN086355.1),while the BenA sequence was identical (99.29%) to that of the other strain T. amestolkiae isolate AC5806 (KJ413360.1).As suggested, we further analyzed the ITS and BenA sequences of M26 and AC5806.The length of these two sequences was different as the M26 ITS (MN086355.1)was 868 bp and the AC5806 ITS (KJ413385.1)sequence was 536 bp in length.The overlapping region was from 115 to 650 bp of M26 ITS and was 100% identical.The BLAST of our ITS showed the overlapping region was from 119 to 701 of M26 ITS.Regarding to the BenA sequence of these two referenced strains, the M26 BenA sequence (MN097073.1)was 428 bp and the AC5806 BenA sequence was 422 bp in length, and the identity of these two sequences was 99% (400/403).In conclusion, the ITS and BenA sequences of our sample showed that there was a T. amestolkiae in the patient.To further verify the presence of T. amestolkiae, we sent the lymph node specimen to Tri-ibiotech, Taiwan.This company performed amplification and sequencing of the internal transcribed spacer rDNA area (ITS) and β-tubulin (BenA) with primer sets ITS5/ITS4 and Bt2a/Bt2b [5].ITS works as a DNA sequencing marker for fungi, but there is insufficient variation to distinguish all the closely related species of Penicillium and many other genera of ascomycetes.Because the ITS reference sequence has its limitations, a second DNA sequencing marker is needed to identify isolates to species level.BenA is the best option for a secondary identification marker for Penicillium.The ITS sequence of our strain showed 98.8% identity to T. amestolkiae strain M26 (MN086355.1),while the BenA sequence was identical (99.29%) to that of the other strain T. amestolkiae isolate AC5806 (KJ413360.1).As suggested, we further analyzed the ITS and BenA sequences of M26 and AC5806.The length of these two sequences was different as the M26 ITS (MN086355.1)was 868 bp and the AC5806 ITS (KJ413385.1)sequence was 536 bp in length.The overlapping region was from 115 to 650 bp of M26 ITS and was 100% identical.The BLAST of our ITS showed the overlapping region was from 119 to 701 of M26 ITS.Regarding to the BenA sequence of these two referenced strains, the M26 BenA sequence (MN097073.1)was 428 bp and the AC5806 BenA sequence was 422 bp in length, and the identity of these two sequences was 99% (400/403).In conclusion, the ITS and BenA sequences of our sample showed that there was a T. amestolkiae in the patient.
Antiretroviral therapy with abacavir, lamivudine, and dolutegravir was initiated after 6 weeks of anti-fungal medications.India ink staining of the cerebrospinal fluid was negative after 4 weeks of amphotericin B liposome during this admission.We changed amphotericin B to voriconazole as maintenance therapy for cryptococcal meningitis and the T. amestolkiae infection.The patient was discharged under stable condition.

Results and Discussion
This report described an AIDS patient with cryptococcal meningitis who also had a Talaromyces amestolkiae infection, which presented with lymphadenopathy.It was difficult Antiretroviral therapy with abacavir, lamivudine, and dolutegravir was initiated after 6 weeks of anti-fungal medications.India ink staining of the cerebrospinal fluid was negative after 4 weeks of amphotericin B liposome during this admission.We changed amphotericin B to voriconazole as maintenance therapy for cryptococcal meningitis and the T. amestolkiae infection.The patient was discharged under stable condition.

Results and Discussion
This report described an AIDS patient with cryptococcal meningitis who also had a Talaromyces amestolkiae infection, which presented with lymphadenopathy.It was difficult to diagnose this fungal infection due to the limitation of clinical diagnostic tools and challenges in distinguishing T. amestolkiae from other Talaromyces species without performing PCR or genome sequencing.A limited number of T. amestolkiae infections have been reported to date, and thus, this species is still relatively unknown.Nevertheless, the advent of PCR assays and DNA sequencing technology has improved its identification and raised awareness of its existence.The use of PCR combined with microscopic examination, fungal culture, and clinical symptoms allows for more accurate diagnosis of T. amestolkiae.When dealing with uncommon pathogenic fungi, misdiagnosis or failure to identify the correct pathogen is common.Therefore, we reviewed the reported pathogenic species of Talaromyces and the differences between T. marneffei and T. amestolkiae.
In 2011, Samson et al. proposed that Penicillium subgenus Biverticillium should be taxonomically unified with the Talaromyces species.Using sequencing data from the ITS and RPB loci, they determined that it was a phylogenetically distinct species characterized by conidiogenous cells with acerose and/or loosely woven ascocarp walls and placed them in the Talaromyces genus [6].Talaromyces species may easily confuse the clinician due to ambiguous microscopic findings.Hence, early diagnosis with DNA sequencing markers is necessary due to the high mortality rate of untreated T. marneffei patients.With this goal in mind, sequencing data from the ITS, BenA, and RPB2 gene regions were used to define relationships within Talaromyces [2].There are other possible secondary markers, including calmodulin (CaM) and the second largest subunit of RNA polymerase II (RPB2) genes [7].The ITS reference sequence of T. marneffei is JN899344 (alternative markers: BenA = JX091389; CaM = KF741958; RPB2 = KM023283).The ITS reference sequence of T. amestolkiae is JX315660 (alternative markers: BenA = JX315623; CaM = KF741937; RPB2 = JX315698) [2].With these DNA sequencing markers, we can accurately identify fungal pathogens and prescribe corresponding treatment precisely.
Several clinically significant Talaromyces species listed in Table 1 were previously reported.Among these species, T. marneffei is a fatal fungal pathogen that poses a particular risk to immunocompromised patients.This species was previously known as Penicillium marneffei; cases infected with this pathogen were mainly distributed in East Asian countries, such as China, Taiwan, Thailand, and Vietnam [8][9][10][11].Talaromyces indigoticus was isolated from skin and nail lesions from a man who was affected by onychomycosis in Western Panama [12].Talaromyces piceus was found in an X-linked chronic granulomatous disease (X-CGD) patient who suffered from rib osteomyelitis [13].T. piceus fungemia was diagnosed in a woman with alcohol-abuse disorder who was diagnosed cholangiocarcinoma after autopsy [14].T. amestolkiae and Talaromyces stollii were isolated from the lungs and sputum of immunocompromised patients [4].In this report, T. amestolkiae was isolated from the lymph node of our AIDS patient, suggesting a new clinical presentation.As T. amestolkiae has not been encountered as frequently compared to T. marneffei, we searched for relevant articles in medical databases and compared the two species with respect to risk factors, clinical manifestations, microbiological diagnosis, and treatment regimen to aid in future clinical diagnosis and treatment.A summary of the information is presented in Table 2.
Both T. amestolkiae and T. marneffei were predominantly isolated from immunocompromised patients.T. amestolkiae was reported in sputum samples from cystic fibrosis patients, bronchoalveolar lavage fluids from patients with acute lymphoblastic leukemia who recently underwent chemotherapy, and a lymph node biopsy from our AIDS patient with a CD4 count of 19 cells/mm 3 .Furthermore, T. marneffei was found in patients with a range of conditions, including AIDS, autoimmune diseases, cancers, and diabetes mellitus.

Risk factors
Immunocompromised patient.Case reported in an acute lymphoblastic leukemia patient [15] and we found this pathogen in an AIDS patient.

Respiratory symptoms
Productive cough, mild dyspnea, and occasional low grade fever [15] Other symptoms Lymphadenopathy Respiratory symptoms Nonproductive cough, fever, dyspnea, and chest pain Gastrointestinal symptoms Diarrhea and abdominal pain Skin lesions Papules on the face, chest, and extremities.Subsequently, the center of the papule becomes necrotic, giving the appearance of an umbilicated papule, which can resemble molluscum contagiosum [22].Mucosal lesions Mucosal lesions appear similar to skin lesions.

Definitive diagnosis
Culture 7 days in CYA at 25 Morphological transition from a mold to a yeast, producing colonies without a red diffusible pigment [22] Molecular diagnostics PCR amplification Sequence identification of specific regions PCR amplification Sequence identification of specific regions

Special consideration
For patients who cannot tolerate any form of amphotericin induction therapy with IV Voriconazole 6 mg/kg every 12 h on day 1 (loading dose), then 4 mg/kg every 12 h or with oral Voriconazole 600 mg every 12 h on day 1 (loading dose), then 400 mg every 12 h for 2 weeks is recommended [28,29] The patient with T. amestolkiae pneumonia exhibited symptoms of productive cough, dyspnea, and low-grade fever, while our patient presented with painless inguinal lymphadenopathy.In addition, T. marneffei infection typically resulted in subacute non-specific symptoms, such as fever, weight loss, hepatosplenomegaly, lymphadenopathy, and respiratory and gastrointestinal abnormalities.Furthermore, T. marneffei induced central necrotic papules over the face, chest, and extremities; the appearance of the papules resembles molluscum contagiosum.A definite diagnosis of Talaromyces relies on fungal culture and DNA sequencing.The culture of T. amestolkiae has center-raising and low margin colonies and grows rapidly on Czapek yeast agar (CYA) and malt extract agar (MEA).The culture has red soluble pigment and dark to violet-brown reverses.However, these features were absent in some strains [2].Thus, it can easily be confused with other Talaromyces species such as T. purpurogenus.On the other hand, T. marneffei is the only dimorphic species in the Talaromyces genus.It produces filamentous growth at 25 degrees Celsius and a yeast phase at 37 degrees Celsius.Red soluble pigment was found on general media and conidiophores have flask-shaped to acerose phialides [2].Because both fungi share similar growth characteristics on culture media, they can be difficult to distinguish by culture alone.
The antifungal therapy for T. amestolkiae was uncertain and there was a patient who had T. amestolkiae pulmonary infection.In the case report, the administration of voriconazole was initiated for the patient based on the suspicion of aspergillosis.Because the clinical improvement of the patient, they continued the voriconazole and it showed resolved symptoms and radiographic improvement after two months [15].As for our patient who presented T. amestolkiae lymphadenopathy and cryptococcal meningitis, he received amphotericin B for 6 weeks then oral voriconazole.
For T. marneffei infection, the treatment includes three phases: induction, consolidation, and maintenance.The recommended induction therapy for all patients is intravenous amphotericin B for 2 weeks [22].The induction phase is followed by consolidation therapy with oral itraconazole 200 mg every 12 h for 10 weeks [26].After receiving the treatment of induction and consolidation, the patient was suggested to continue the maintenance therapy (secondary prophylaxis) with oral itraconazole 200 mg/day until the CD4 count rises above 100 cells/mm 3 for more than 6 months [27].In the event that the patient experiences intolerance to amphotericin B therapy, voriconazole could be considered as an alternative treatment option.In the subsequent consolidation phase, voriconazole can be continued or itraconazole may be considered as suitable therapeutic alternatives [28,29].
When HIV-infected patients are diagnosed with cryptococcal meningitis, clinicians tend to attribute all of the clinical manifestations to a single pathogen.However, in our case, we discovered through further investigations that the patient was simultaneously infected with both C. neoformans and T. amestolkiae.In addition to the two species found in our case, previous studies disclosed other co-infectants were Mycobacterium, Pneumocystis jiroveci, Histoplasma, T. marneffei, and Aspergillus.Details related to these events are presented in Table 3.These pathogens mainly caused lung infections, lymphadenopathy, and skin lesions.Each patient received treatments for Cryptococcal infection, except for one with non-tuberculous mycobacteria infection, since the report was issued after the patient had been discharged.Most patients completed treatments and were discharged with oral medications.Diagnosing co-infections with fungal pathogens can be challenging.Hence, clinicians should maintain a heightened level of attentiveness and employ optimal diagnostics to identify possible co-pathogens.

Conclusions
To our knowledge, this is the first documented instance of concurrent infection with both C. neoformans and T. amestolkiae in a patient with AIDS.These findings highlight the possibility of co-infections with fungal pathogens in immunocompromised patients and underscore the importance of maintaining a high level of clinical suspicion in these cases.Immunocompromised patients have a high risk of getting fungal infections.Broadspectrum antifungals are frequently prescribed empirically without definite microbiological diagnosis, which may result in the development of drug resistance.Clinicians should be vigilant in utilizing appropriate diagnostic tools to identify the causative agents, which can guide therapeutic strategies.This report further illustrates that T. amestolkiae is not only an environmental fungus but also a human pathogen which morphologically resembles Penicillium.In cases where conventional diagnostic methods fail to differentiate closely related fungi, DNA sequencing can be utilized for further verification.The administration of amphotericin B followed by voriconazole successfully treated our patient.However, due to the limited number of cases reported, further studies are required to determine the optimal treatment for T. amestolkiae.The continued surveillance of fungal infections in immunocompromised patients is essential for identifying and managing co-infections and developing effective treatment strategies.

Figure 1 .
Figure 1.Specimen of the inguinal lymph node: (A) Left inguinal lymph node excisional biopsy; (B) Hematoxylin and eosin stain showing lymph node tissue with numerous fungal elements; (C) Periodic-Acid Schiff stain highlighting the fungal elements; (D) Gomori's Methenamine Silver stain highlight the fungal elements.

Figure 1 .
Figure 1.Specimen of the inguinal lymph node: (A) Left inguinal lymph node excisional biopsy; (B) Hematoxylin and eosin stain showing lymph node tissue with numerous fungal elements; (C) Periodic-Acid Schiff stain highlighting the fungal elements; (D) Gomori's Methenamine Silver stain highlight the fungal elements.