False-Negative Serum Cryptococcal Antigen Lateral Flow Immunoassay Result for a Patient with Disseminated Cryptococcal Disease

Introduction

Cryptococcosis is a fungal infection caused primarily by Cryptococcus neoformans and C. gattii.¹ C. neoformans is an opportunistic fungal pathogen, with its critical virulence factor being a polysaccharide capsule located externally to the cell wall. Polysaccharide capsules can be released into the environment, where they are termed exopolysaccharides.

Cryptococcosis is definitively diagnosed from pathogen cultures in Sabouraud dextrose agar. However, the culture results typically take several days because of its slow growth.² Thus, rapid and reliable diagnostic methods are necessary because early diagnosis can decrease the mortality from cryptococcosis. Serological methods for detecting CrAg, including the latex agglutination (LA) test, enzyme immunoassay (EIA), and lateral flow immunoassay (LFA), have been used in clinical practice to diagnose cryptococcosis. The LFA is useful because it provides stable results at room temperature with minimal required laboratory equipment. Compared with the EIA and LA test, the LFA shows equivalent or superior overall sensitivity. In 2011, the World Health Organization recommended the CrAg LFA test as an effective method for diagnosing cryptococcosis;³ however, it occasionally yields false-negative results.

This case report describes a false-negative result from a serum CrAg LFA in a patient with disseminated cryptococcal disease, whose blood culture tested positive for C. neoformans. This case highlights the need for better knowledge of this situation and discussion of its possible causes.

Case Presentation

A 79-year-old woman presented to our infection division with hyperpyrexia. She had nephrotic syndrome and diabetes and had been treated with traditional Chinese medicine, hormones and insulin for a long time. Two weeks prior (day 0), she was referred to a local hospital for fever with shivering. A chest computed tomography (CT) scan revealed inflammatory lesions in both lungs. She was treated with cefazolin and levofloxacin, which were changed to imipenem combined with moxifloxacin on day 7 after her blood culture results indicated an Escherichia coli infection; however, her symptoms persisted after 1 week of treatment.

The patient was subsequently admitted to our infection division for further treatment. At the time of admission (day 15), her physical examination results were temperature, 38.2°C; blood pressure, 124/56 mmHg; heart rate, 99 beats/minute; and respiratory rate, 18 breaths/minute. The patient was lethargic and dispirited. Her laboratory examination results were white blood cells, 36,300/µL with 92.2% neutrophils; C-reactive protein, 105 µg/mL; procalcitonin, 0.5205 ng/mL; and interleukin, 60.042 ng/mL. During her hospitalization, her temperature continued to increase to 40°C (day 21). Her blood, sputum and cerebral spinal fluid (CSF) specimens were sent to our department for microbiological examinations. The CrAg LFA yielded a negative result for the serum (IMMY Inc., Norman, OK, USA) and a positive result for the CSF. India ink staining and CSF culture results were both negative. Parenteral voriconazole was initiated as empiric treatment pending diagnostic testing (day 21).

After 4 days and 16 hours (day 24) of blood culturing using an automated blood culture system (BACTEC FX,BD Becton, Dickinson and Company), pathogens were detected microscopically from two aerobic blood culture bottles. Microscopically, the pathogens were gram-positive, budding, round conidia, suggesting Cryptococcus spp. India ink staining of the fluid in the positive blood culture bottles revealed the pathogenic capsules. Microscopically, the pathogens appeared as rounded cells with thick halos (Figure 1A). The strains were isolated from Sabouraud dextrose agar after 24 hours of aerobic incubation and appeared as beige mucoid yeast colonies (Figure 1B). Identification using the VITEK 2 compact automated system (BioMerieux, France) yielded a positive result for C. neoformans. With confirmation of disseminated cryptococcosis, the initial negative serum LFA test result was questioned. Per the manufacturer’s instructions, we serially diluted the serum and processed it using the CrAg LFA test kit. Increasing dilutions showed that the 1:5 dilution was negative, 1:10 and 1:20 were weakly positive, and 1:40 was positive.

Figure 1 (A) Morphology of C. neoformans in blood culture bottle by Indian ink staining (×400). (B) Colony morphology of C. neoformans on Sabouraud dextrose agar.

Antimicrobial susceptibility tests were performed via the microdilution broth method using ATB FUNGUS 3 (BioMerieux). In vitro antimicrobial susceptibility testing showed that the minimal inhibitory concentrations for the C. neoformans were 5-fluorouracil (5-FC) 4 µg/mL, amphotericin B (AMB) 0.5 µg/mL, fluconazole (FLU) 1 µg/mL, voriconazole (VOR) 0.125 µg/mL, and itraconazole (ITR) 0.06 µg/mL. As per previous studies, the epidemiological cut-off values for C. neoformans were 8 µg/mL for 5-FC and FLU, 0.25 µg/mL for ITR and VOR, and 1 µg/mL for AMB.^4–6 Thus, the patient’s antibiotics were changed to AMB plus 5-FC on day 26. After 1 week of treatment, her infection was controlled, and she was transferred to a local hospital for rehabilitation.

Discussion

Cryptococcosis is definitively diagnosed from pathogen cultures in Sabouraud dextrose agar. However, C. neoformans may take several days to culture because of its slow growth. Additionally, the number of pathogens as well as previous use of antifungal agents may influence the culture results.³ Cryptococcal meningitis (CM) can be diagnosed by microscopy of fresh CSF using India ink staining, which has 100% specificity but only 50% sensitivity depending on cell titers and capsule production.⁷ In the early stages, India ink staining results may appear normal, as with the patient in this case. The CrAg, a component of the glucuronoxylomannan polysaccharide capsule, which is soluble and detectable in serum or body fluids as a marker of infection, is used for timely and sensitive diagnoses of cryptococcosis.^8,9 Several serological methods for CrAg detection, such as LA, EIA and LFA tests, have been developed. In the LA test, particles are coated with specific antibodies and agglutinated CrAg in the sera of infected patients.¹⁰ LA sensitivity in serum ranges from 83%–97%, and its specificity ranges from 93%–100%,¹¹ but the test is manual, subjective and time-consuming. The EIA is a direct, microplate-based, immunoenzymatic sandwich assay, with 100% sensitivity and 98% specificity.¹¹ The LFA is widely used as a rapid and cheap method for diagnosing cryptococcosis, with both 100% sensitivity and specificity.¹² The LFA test requires minimal laboratory equipment; thus, it can be easily performed in many laboratories.

In our patient, the C. neoformans blood culture and the positive LFA result for the CSF revealed that the patient had disseminated cryptococcosis. C. neoformans first infects the lungs, then spreads to the central nervous system (CNS) through the blood. The patient had not yet shown obvious symptoms of CNS infection, indicating that the CM was in the early stage of infection. The culture and India ink staining of the CSF were negative; the CrAg detection in CSF is significant for the diagnosis of CM.

Although the patient was diagnosed with disseminated cryptococcosis, the serum LFA test result was negative. Mahajan et al reported a case of unencapsulated C. neoformans with a negative CrAg.¹³ India ink staining of the fluid in the positive blood culture bottles showed that the C. neoformans appeared as rounded cells with thick halos, indicating that the patient was not infected by an unencapsulated or weakly capsulated strain. Another case report described a false-negative serum CrAg result via LFA in an HIV-positive patient, as demonstrated by a blood culture from a matched sample that was positive for Cryptococcus spp. (Table 1).¹⁴ The serum CrAg test may have been negative owing to the postzone effect. Unfortunately, the culture specimen was not stored, so the CrAg LFA could not be titrated to evaluate this hypothesis. Therefore, the serum was diluted, and LFA tests were repeated to resolve the discrepancy. After diluting the serum ten times, the LFA result was weakly positive. The false-negative LFA result occurred due to the postzone phenomenon, also known as the “hook” effect. When the antigen concentration is too high, excess antibodies will prevent cross-linking and interfere with antibody-antigen precipitation, leading to false-negative results.

Reports of false-negative results due to the postzone phenomenon are uncommon. Our review included 8 cases of false-negative CrAg LFAs described in five reports (Table 1). Six were in the CSF and in HIV-infected patients.^15–18 However, the only specimens tested were CSF. A retrospective study of patients with culture-confirmed CM showed that the CrAg LFA had 100% sensitivity in the serum.¹⁹ Testing serum for CrAg is recommended, especially for patients who cannot undergo an immediate lumbar puncture or when lumbar puncture is contraindicated.

Table 1 Reports of False-Negative Cryptococcal Antigen Lateral Flow Assay in Recent Years

Cryptococcosis treatment is complex. “Practice Guidelines for the Management of Cryptococcal Disease” was first published by the Infectious Diseases Society of America (IDSA) in 2000.²⁰ According to these guidelines, a three-part regimen of “induction-consolidation-maintenance” is recommended for immunocompromised patients. This includes AMB plus 5-FC for 2 weeks, FLU for 8–10 weeks, then a lower dose of FLU for 6–12 months. In this case, the patient was treated according to these guidelines, and the infection was controlled well.

Conclusion

This is the first reported case of the postzone phenomenon occurring on an LFA from an HIV-negative patient who had disseminated cryptococcosis caused by C. neoformans. When disseminated cryptococcosis is highly suspected, but the CrAg LFA is negative, a serial dilution must be performed to rule out a false-negative test due to the postzone phenomenon. Clinicians should always be cautious when laboratory test results do not fit the strong clinical suspicion.