Human immunodeficiency virus (HIV) is a retrovirus that causes acquired immune deficiency syndrome (AIDS) (1). HIV destroys and disables the immune system, often leading patients to die from secondary infections and cancer (2). According to the Joint United Nations Program on HIV and AIDS (UNAIDS), 38 million people were living with HIV worldwide in 2019 (3). The Chinese Center for Disease Control and Prevention (CCDC) estimated that, in China, there were more than 0.86 million people living with HIV at the end of 2018, with 83.4% of them receiving anti-retroviral therapy (ART) (4, 5).

Upon acquiring the infection, HIV specifically attacks CD4⁺T lymphocytes in humans, resulting in the decrease or dysfunction of CD4⁺T lymphocytes. This leads to secondary infection or secondary tumors due to immune deficiency syndrome (6). Among secondary infections, fungal infection causes at least a quarter of deaths in people living with HIV (PLWH) (7). Talaromyces marneffei (Penicillium marneffei) is one of the major causes of opportunistic fungal infection in HIV-related secondary infections prevalent in Southeast and East Asia, with the highest incidence in China, Thailand, and Vietnam (8, 9). Diagnosis can be inferred from fungal culture and microscopic examination (10). Patients with lower CD4⁺T lymphocytes are more likely to experience severe clinical manifestations (11, 12). When infected, patients usually develop papulonecrotic skin lesions, fever, lymphadenectasis, and gastrointestinal abnormalities, often accompanied by other opportunistic infections such as tuberculosis and salmonella infections (13, 14). Even with ART treatment, the mortality rate of the T.marneffei infection can still reach 33% in China (15). Given the high fatality rate, timely diagnosis and treatment are crucial for HIV patients (16). In the current times, the most accurate diagnosis is the microscopic identification of morphological and dimorphic fungi separated and cultured from clinical specimens (17). However, the fungal culture needs to be incubated for at most 2 weeks, resulting in an elevated case fatality rate (18).

Ultrasound examination uses ultrasound microwaves to image the organs and tissues inside patients. Unlike computed tomography (CT), which produces ionizing radiation, ultrasound examinations do not harm the human body. For T.marneffei-infected patients, ultrasound examination can be used to investigate changes in lymph node (LN) lesions. While there are several case reports and clinical studies on the manifestation and CT results of HIV patients with T.marneffei (19–22), studies on ultrasound imaging of the T.marneffei infection are rarely reported. Based on the studies cited above, we speculate that there are some distinguishable sonographic features and a potential relationship between these features and the immune status of these patients. Therefore, the objectives of this study were to retrospectively characterize the ultrasound features of superficial LNs and explore the associations between lymphocyte subsets and sonographic features in HIV patients infected with T.marneffei to provide assistance and understanding for diagnosis and therapy.

In this retrospective study, a small cohort of 26 HIV patients with enlargement of LNs and T.marneffei coinfection were included. All patients underwent ultrasound-guided needle puncture biopsy of superficial LNs, confirming infection with T.marneffei. Clinical information, including patient details, sonographic data, and lymphocyte population, and ultrasound images were collected. Through observation and statistical analysis, we characterized the ultrasound features of superficial LNs in HIV patients infected with T.marneffei and showed the possible associations between sonographic data and lymphocyte population.

It is worth noting that diagnoses of AIDS-related superficial LN tuberculosis (ASLTB) and AIDS-related diffuse large B cell lymphoma (ADLBL) should be differentiated in cases of T.marneffei-related LN infection. Ultrasound findings for ASLTB often show superficial LN enlargement in multiple locations, with the neck being the most common site. The images are complex and variable, often showing hypoechoic signs, thinned or absent hilum structure, and internal liquefaction, which makes it difficult to differentiate from T.marneffei-infected LNs (Supplementary Figure S4). However, ASLTB has a specific chronic inflammatory response, and the lesion grows slowly, with an increased probability of edema in the surrounding tissue. There is usually no obvious tenderness upon local compression, and LNs often fuse with visible calcification. Some cases may involve the skin and form sinus tracts. In contrast, T.marneffei infection of LNs has a more acute onset and rapid progression, often accompanied by tenderness upon local compression. Adjacent LNs are rarely fused, and skin lesion is rare, with no formation of sinus tracts. When liquefaction occurs, the solid part still shows diffuse echogenicity. Lymphoma is a common AIDS-related malignancy. The internal part of the affected LN shows extremely low echogenicity, which is often lower than that of the surrounding muscle tissue, and typical small mesh-like changes can be noticed. The hilum is basically absent, and calcification is rare. Color Doppler flow imaging (CDFI) often shows a rich portal-type blood flow, with many branches and a few cases showing mixed or marginal-type blood flow (Supplementary Figure S5). The progression is usually slow, and the affected LNs are usually large, with an L/S ratio of less than 2 and a mean diameter of over 5 cm. There is usually no tenderness upon local compression. In CDFI scans of patients with the T.marneffei infection, the mean diameter of the involved superficial LNs is rarely over 3 cm. The internal echogenicity is higher than that of ADLBL, and liquefaction is rare. The hilum of LNs may exist or become thinner, and tenderness is often present upon local compression. The type of T.marneffei-infected LNs characterized by the absence of diffuse echogenicity increase in the LN and the usually present or thinner hilum is prone to misdiagnosis and relies more on clinical diagnosis. Therefore, sometimes, it is difficult to discriminate by ultrasound alone, and ultrasound-guided puncture biopsy of LNs is often needed for pathological diagnosis.

Talaromyces marneffei is a thermally dimorphic fungus primarily prevalent in Southeast Asia and East Asia (24). It usually infects people who have low immunity, especially immunocompromised HIV patients (25). In HIV patients, the quantity of CD4⁺T cell can reflect the body’s immunity because it is the major target cell of HIV. Clinical researchers found that the majority of T.marneffei-infected HIV patients had less than 50 CD4⁺T cell counts (26), and it had certain diagnostic efficacy for early AIDS combined with the T.marneffei infection (27). The same result was found in this study, with CD4⁺T cell counts less than 200, 100, and 50 cells/μL in 23 (92%), 21 (84%), and 20 (80%) patients, respectively. The correlation analysis between CD4⁺T cell counts, CD4⁺T cell ratios, CD4⁺T/CD8⁺T cell ratios, and axillary LN L/S ratios showed that the quantity of CD4⁺T cell had a significantly positive correlation with axillary LN L/S ratios. Decreased CD4⁺T cells indicated that the immune system was damaged. The LN L/S ratio less than 2 was known as malignant enlargement of LNs (28), so this relationship between CD4⁺T cell and LN L/S ratios may be explained as the worse immune status resulting in a heavier infection. Another subset of blood lymphocytes, NK cells, was reported to be decreased in T.marneffei-infected HIV-negative patients (29, 30). Apart from their decrease in HIV patients, lower NK cell counts and NK cell ratios were observed in patients who had retroperitoneal lymphadenectasis. To summarize, similar to lymphocyte subsets such as CD4⁺T cells, ultrasound findings of LNs could serve as indicators for the T.marneffei infection in HIV patients.

This study had several limitations, with the major one being the small sample size. The reason only 26 patients were chosen for the study was the inclusion criteria set by us. The present study aimed at exploring the ultrasound characteristics of superficial LNs; only patients confirmed to be infected with T.marneffei through ultrasound-guided needle puncture biopsy of superficial LNs, followed by the tissue culture, met the inclusion criteria. This led to a small sample size. Additionally, affected by the limited sample size, the credibility of the results of statistical analysis in this study was reduced. In future research, we might expand the sample size of T.marneffei-infected HIV patients with enlarged LNs and collect patients with HIV and other pathogen coinfection, except T.marneffei, who also have enlarged LNs into another group. Following this, we might attempt to characterize the differences among patients who have LN enlargement due to infections of different causes.

In summary, T.marneffei infection of LNs is common in HIV patients with a rapid onset and often present with superficial LN enlargement, mostly involving the cervical LNs. The T.marneffei-infected LNs have several characteristics in echogenicity, hilum, and blood flow signal, which is different from ALSTB or ADLBL. Furthermore, there might be associations between lymphocyte subsets and enlarged superficial LNs. Ultrasound examination should be paid attention to if patients have superficial LN enlargement and if the diagnosis of T.marneffei infection is considered.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Clinical Research Ethics Committee of the Hangzhou Xixi Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because This research used data or specimens obtained in previous clinical trials, disease surveillance or clinical studies.

LP conceived and designed this study and collected the data and images. HS designed this study, analyzed the data and wrote the manuscript. All authors reviewed and approved the final manuscript.