Resource-limited countries, especially those in sub-Saharan Africa and Southeast Asia, continue to experience a high incidence of cryptococcosis; managing this disease is a persistent public health challenge [2
]. Although several CrAg tests are currently available for cryptoccoccal diagnosis, these tests are not readily accessible in resource-limited settings, resulting in no diagnosis or underdiagnosis of these often fatal infections. In the present study, a recently developed cryptococcal assay, the LFA, was evaluated for test sensitivity and agreement with other available diagnostic methods using archived serum and urine specimens from HIV-infected patients in Thailand. Results showed that, with serum specimens, the LFA was 100% sensitive when compared with the gold standard blood culture. Additionally, the LFA had a high level of agreement with the cryptococcal EIA. When urine was evaluated, the LFA was found to be very sensitive (92%) when compared with blood culture, and moderately sensitive (70.7%) when compared with EIA-positive samples.
One of the limitations of this study was that the specimens were collected from patients hospitalized with acute respiratory illness for whom complete clinical details were not available, including whether they had meningitis. Patients with more invasive infections (ie, meningitis) may have a higher burden of circulating organisms and therefore antigen, and this may impact the performance of tests that measure CrAg. Accordingly, discrepant results between serum EIA and LFA were more often observed in serum with lower EIA optical density values, possibly reflecting the presence of low levels of circulating antigen.
This study was performed in a reference laboratory and therefore the performance of the LFA in a field or hospital setting is unclear at this time. However, the LFA was simple to perform and did not require any additional laboratory equipment. Incubation could be performed at room temperature and the assay itself could be accomplished in 3 easy steps since this method does not require any pretreatment of specimen (to remove rheumatoid factor). Finally, the LFA yielded results that were unambiguous. Thus, the LFA has many characteristics that may make it a valuable POCT in resource-limited areas. In addition, this study demonstrates that the CrAg LFA satisfies most of the WHO ASSURED criteria [16
]: specifically, the assay is sensitive, user-friendly (small specimen volume, simple to use), rapid (10–15 minutes to perform), and equipment-free (including no requirement for refrigeration). The rapid turnaround time will allow diagnoses to be potentially provided during patient visits, allowing treatment to begin immediately if warranted. Recently, Jarvis et al [19
] strongly recommended the integration of CrAg screening into national antiretroviral treatment programs in sub-Saharan Africa to reduce the human and economic costs due to the disease [19
]. Although not tested in this study, the LFA may also have utility as a screening tool for early diagnosis of cryptococcosis.
Extending the incubation time for sera from 5 to 15 minutes improved the sensitivity of the LFA when compared with EIA, thereby increasing agreement between the EIA and the LFA. Since the time of this study, the manufacturer of the LFA has modified the testing protocol, now recommending testing twice the volume of patient specimen, reducing the recommended specimen:diluent ratio from 1:5 to 1:2, increasing the amount of conjugate in the chromatographic strip, and increasing the recommended incubation time of the LFA to 10 minutes. Currently the LFA has CE marking (a mandatory conformance marking for the European Economic Area) for use in the European Union and in countries that use CE approval and has been submitted to the United States Food and Drug Administration for approval (personal communication, Sean Bauman, IMMY).
For POCTs to have maximum value in remote settings, the use of minimally invasive, easily obtained, processing-free specimens is optimal. This study was performed with sera and urine as test specimens in a controlled laboratory setting (reference laboratory); however, in remote areas with insufficient technical expertise or specimen processing capabilities, sera may not be the ideal specimen type. The lower sensitivity of the LFA for urine compared with serum could be due to reduced excretion of CrAg into the urine, compared with the blood. HIV infection [20
] and treatment of HIV with the antiretroviral drugs tenofovir and indinavir [21
] have been previously demonstrated to reduce the glomerular filtration rate and, potentially, reduce the amount of CrAg excreted in the urine. Urine has been used for antigen detection in other fungal [23
] and nonfungal diseases [26
]. The kinetics of excretion of CrAg antigen in urine is unclear, and additional studies testing CrAg in urine need to be performed, including studies where urine is collected under controlled conditions.
Another minimally invasive specimen type is whole blood from a finger stick. Point-of-care assays for whole blood have been developed for viral and parasitic diseases [8
], all of which have been useful in resource-limited countries. Further studies evaluating the LFA using finger-stick blood would enhance the accessibility of this assay. Additionally the test’s cost, ranging from $1.25 to $2.50 per test (depending on the country and volume of purchase) is based on the World Bank’s list of economies, thereby ensuring affordability to the countries most in need.
In summary, this study demonstrates that the LFA is a sensitive test for Cryptococcus spp compared with the gold standard culture, and has a high level of agreement with EIA. Given the ease of use, temperature stability, minimal requirements for laboratory infrastructure, and potential low cost of this test, the LFA shows great promise as a POCT for diagnosis of cryptococcosis. The availability of this assay as a POCT for use in remote locations could have a meaningful impact on cryptococcal diagnosis.