Case Report

Abstract

A case of false-negative serum latex agglutination cryptococcal antigen (CRAG) test in a 45-year-old HIV-positive male with Cryptococcus-positive culture is described. The patient was presented to a hospital in Botswana, with breathlessness and a diffuse papular rash. His CD4 count was 25 cells/μL. Despite the suspicion for disseminated cryptococcal disease, an initial serum CRAG latex test was negative. Results of subsequent Indian ink staining, culture of cerebrospinal fluid and skin scrapings, and serum lateral flow immunoassay (LFA) were all positive for Cryptococcus neoformans. There are several possible explanations for the false-negative CRAG latex test. Given the positive LFA result, we speculate that disease may have been caused by Cryptococcus gattii, which is estimated to be responsible for between 15% and 30% of all cryptococcal diseases in Botswana. Reduced sensitivity of CRAG latex assays for detecting C gattii may lead to underdiagnosis of cryptococcal infection.

Introduction/Summary

Cryptococcal meningitis is extremely common in areas with high HIV prevalence. Reports suggest that it is the most common cause of meningitis in sub-Saharan Africa (SSA), with a 1-year mortality of approximately 40%. ¹ Early diagnosis and treatment are essential to ensure a favorable outcome. The usual symptoms include headache, fever, nausea, neck stiffness, abnormal behavior, and photophobia. However, cutaneous lesions are also recognized signs of disseminated disease. We present a case where prompt diagnosis of disseminated cryptococcosis was not made because of an initial false-negative serum cryptococcal antigen (CRAG) test result. The accuracy of immunoassays for diagnosis of cryptococcosis is important, given the increasing use of these assays in SSA to screen and diagnose cryptococcal disease in individuals with advanced HIV and subclinical infection. ^2,3

Case Report

A 45-year-old HIV-infected male presented to a district hospital in southern Botswana with a 1-week history of breathlessness. He also reported dry cough, diffuse rash, and generalized weakness that had worsened over the previous 7 days. He had been diagnosed with HIV 4 months prior when admitted to the same hospital with shortness of breath. During that hospitalization, he was treated for pneumocystis jiroveci pneumonia (PJP) and commenced on highly active antiretroviral therapy (HAART).

Initial physical examination revealed a temperature of 36.7°C, blood pressure of 145/87 mm Hg, and a pulse of 115 beats/min. His respiratory rate was 45 breaths/min, and oxygen saturation was 75% in room air. On respiratory examination, crackles were heard in the lower lung zones bilaterally. The patient was alert but disoriented to time and person. Neither meningeal signs nor focal neurological deficits were detected. He had a diffuse, umbilicated, papular rash involving his face, trunk, and arms. Many of the papules were ulcerated (Figure 1).

Figure 1.

 

Routine laboratory results on the day of admission included a hemoglobin level of 11.1 g/dL, platelet count 428 × 10³/μL, white blood cell count of 3800/μL (with neutrophils, 88.8%), urea 4.7 mg/dL, creatinine 0.77 mg/dL, aspartate aminotransferase 32 mU/mL, and alanine aminotransferase 22 mU/mL. CD4 count was 25 cells/μL. A serum blood sample sent for CRAG by latex agglutination (LA; Remel kit) was negative, following pronase pretreatment and performed according to manufacturer’s instructions. Arterial blood gases and chest X-rays were not available at the hospital.

On the presumption that his respiratory distress and delirium were due to either PJP or community-acquired pneumonia, he was started on empiric therapy with parenteral cefotaxime and cotrimoxazole. Supplemental oxygen, intravenous fluids, and oral prednisolone were also initiated. The rash was attributed to Molluscum contagiosum. Unfortunately, despite these therapies, his condition deteriorated over the next 6 days. On Day 7, he was transferred to the regional referral hospital.

On transfer, further investigations were performed, given the suspicion for disseminated Cryptococcus, in spite of the earlier negative serum CRAG latex result. Skin scrapings of the umbilicated papules were positive with India Ink staining and CRAG by LA. A lumbar puncture was also performed, wherein the opening pressure was 38 cm, India Ink staining of the cerebrospinal fluid (CSF) and the CRAG latex assay were positive. The CSF culture revealed heavy growth of Cryptococcus; the isolate was not speciated.

Based on these findings, the patient was initiated on parenteral amphotericin B and oral fluconazole, in accordance with the local guidelines. With confirmed disseminated Cryptococcus, the initial negative serum CRAG result was called into question. A repeat serum sample was sent to an external reference laboratory for repeat CRAG testing using the latex assay (Remel kit). This test was also negative, despite pronase pretreatment and serial dilutions. By contrast, when the serum sample was processed using lateral flow immunoassay (LFA; IMMY kit), the CRAG test was positive.

The patient completed 21 days of intravenous amphotericin and fluconazole (800 mg/d) at the referral hospital but remained in respiratory compromise. He died 30 days after his initial admission from respiratory failure.

Discussion

HIV-associated cryptococcosis is a spectrum of diseases caused by Cryptococcus neoformans and Cryptococcus Gattii, which results in 20% to 25% of AIDS-related deaths globally each year. ⁴ In SSA, cryptococcal infection accounts for 13% to 44% of deaths among HIV-infected adults. ^{5 –7} Due to the high morbidity and mortality associated with cryptoccoccal infection, the importance of rapid and accurate laboratory tests cannot be overstated. Immunoassays targeting the glucuronoxylomannan (GXM) antigen have become a mainstay in the diagnosis. ^8,9 The LA assays are considered the gold standard method, in part, because of their high sensitivity in detecting Cryptococcus both in CSF and in serum. ^10,11 Nevertheless, this case illustrates the potential problem of false-negative results with CRAG latex assays.

There are several possible explanations for why the serum CRAG latex result was negative in our case. Rarely false negatives are caused by the prozone phenomenon (also referred to as the “hook” effect), when there is failure of antibody–antigen precipitation due to antibody excess ¹² ; excess antibody prevents adequate antibody cross-linking and interferes with antibody–antigen precipitation. ¹³ The prozone effect seems unlikely in this case, since the serum sample was pretreated with a pronase enzyme before the LA immunoassay was run to mitigate the effect of antibodies preventing antibody–antigen precipitation. ¹⁴ Rarely, cryptococcal disease is caused by unencapsulated Cryptococcus. This can also result in a negative CRAG test. However, it is improbable that unencapsulated Cryptococcus was the reason for the aberrant result in our case, given the positive India Ink result, which is consistent with encapsulated Cryptococcus.

We speculate that this patient was infected with Cryptococcus serotype C, C gattii, for which CRAG latex assays are notoriously insensitive. ^2,15 That the CRAG latex assay was only positive with skin and CSF samples, presumably at very high antigen titers, would be consistent with this conclusion, since available data suggest that at high enough titers, even Cryptococcus serotype C will be positive with most CRAG latex tests. ¹⁵ A recent study by Percival et al ² demonstrated that LA assays show serotype bias in GXM detection. In this study, 3 commercially available CRAG latex immunoassays all had significantly diminished sensitivity for detecting serotype C compared to serotypes A, B, or D (Table 1). The assays were only positive for C gattii (serotype C) at very high antigen titers. By contrast, most LFAs have high sensitivity for all serotypes, including serotype C, even at very low C gattii titers. ^2,11

Table 1.

Sensitivities of 3 Commercially Available Latex Agglutination Assays for Detection of GXM of Different Serotypes.^a

Minimum Concentration of GXM (ng/mL) Producing a Positive Resultb
Immunoassay	Assay format	Ac		Bc		Cc			Dc
		CN6	MU-1	184	409	34	298	24 066	127	M0024
Immunomyologics	LA	24	32	27	68	432	260	460	62	63
Meridian CALAS	LA	18	20	52	22	>2000	470	360	44	65
Inverness	LA	38	NT	64	NT	>2000	940	>2000	50	NT
Meridian Premier	ELISA	35	22	25	21	>2000	>2000	>2000	900	630

Abbreviations: LA, latex agglutination; ELISA, antigen capture sandwich enzyme-linked immunosorbent assay; CALAS, cryptococcal antigen latex agglutination system; GXM, glucuronoxylomannan; NT, not tested.

^aTable reproduced with permission of Kozel et al. First published in Clinical and Vaccine Immunology, August 2011, pp. 1292-1296.

^bResults are shown for GXM isolated from different strains of each serotype. >2000, GXM of some strains produced negative results at all concentrations up to 2000 ng/mL.

^cFor each serotype, selected strains deemed representative of the serotype included in analysis.

While initial studies of cryptococcosis in patients with AIDS found a predominance of C neoformans var. grubi (serotype A) and C neoformans var. neoformans (serotype D) isolates, ^{16 –18} several studies suggests that the frequency of C gattii in southern Africa may be around 15%. ^19,20 A recent report from Botswana reported that the prevalence in our setting maybe as high as 30%. ²¹

Because neither culture serotyping nor quantitative antigen titers are routinely performed in Botswana, we were unable to confirm that serotype bias was the explanation for the initial false-negative CRAG latex result. Furthermore, we only used 1 CRAG latex test—the Remel assay—and cannot confirm that other latex assays would have resulted in the same test. However, given the lack of other plausible explanations, the limitation of the CRAG latex assay used, and the established prevalence of C gattii in HIV-infected adults with cryptococcal disease in Botswana, ²¹ this is a reasonable explanation for the false-negative serum results.

Summary

Although most available CRAG assays have excellent operating characteristics, false-negative results can delay diagnosis and therapy. Given that substantial minority of cryptococcal infections in parts of SSA are caused by C gattii, clinicians should be aware that latex assays have significantly reduced sensitivity for detecting serotype C Cryptococcus. The prevalence of different cryptoccal serotypes should impact the choice of CRAG tests used, especially given the increasing use of serum CRAG assays for screening HIV-infected adults.

Given the limitations of latex assays, we advocate for scale-up in the provision of CRAG LFA testing in high HIV prevalence settings. More fundamentally, clinicians should always be cautious about relying on a laboratory test if it does not fit with a strong clinical suspicion—especially if such a decision would have potentially grave clinical consequences, as in this case.