Comparative performance of cryptococcosis diagnostic tests.
Cryptococcosis is an important systemic mycosis that threatens the lives of humans and animals. The disease is caused by two species of the genus Cryptococcus: Cryptococcus neoformans and Cryptococcus gattii. The diagnosis of cryptococcosis is made through microscopy, fungal culture followed by biochemical tests, and detection of the cryptococcal capsular antigen (CrAg). Despite the existence of an established diagnostic protocol, the search for new diagnostic tests is necessary due to the high incidence of the disease, with estimates of approximately 1 million cases of cryptococcal meningitis per year and more than 600,000 deaths in patients infected with human immunodeficiency virus (HIV), the potential for C. gattii to cause the disease in immunocompetent individuals, and the disease’s rapid worldwide dissemination. With the development of biotechnology, synthetic peptides have opened up new possibilities as a source of pure epitopes and molecules for the diagnosis of various diseases, based on the detection of circulating antibodies. Synthetic peptides can also be used for the development of vaccines. Studies on Leishmaniasis, Chagas disease, paracoccidioidomycosis, tuberculosis, and, more recently, on cryptococcosis, among others, have shown that this approach shows potential for the early diagnosis of the disease, thus reducing the morbi-lethality of individuals affected by this infection and ultimately changing their prognosis.
- synthetic peptides
- B cell
Cryptococcosis is an important systemic mycosis that threatens the lives of humans and animals. It manifests primarily through respiratory system diseases and meningoencephalitis. Cryptococcosis is among the emergent fungal infections with significant morbi-lethality, and it is the fourth most frequent cause of opportunistic infection in human immunodeficiency virus (HIV)-positive patients. The disease is caused by two species of the genus
Annually, AIDS-related cryptococcal meningitis is responsible for approximately 15% of the mortality in these individuals . Sub-Saharan Africa has the largest rate of coinfection with
2. Etiological agents
However, this classification becomes difficult, as significant divergences between serotypes are frequently observed at the molecular level . Serotype limits do not entirely coincide with genetic groupings; therefore, serotyping is not regarded as a reliable technique for differentiating strands of
A series of molecular studies were conducted, including polymerase chain reaction (PCR) fingerprinting and amplified fragment length polymorphism (AFLP) analysis of the orotidine monophosphate pyrophosphorylase (
Genotype VGI is endemic in Australia and has also been described in Papua New Guinea, Asia, and southern California. The VGIII and VGIV genotypes are found less frequently, with the VGIII type isolated in the Ibero-American regions and in India and type VGIV recorded in South Africa and in the U.S. [19, 25–29].
3. Natural history of the disease
Cryptococcosis is a systemic mycosis with a pulmonary gateway, which is caused by infection with either
Once in the lung,
4. Virulence factors
The pathogenic species of the genus
The polysaccharide capsule is composed of 90 to 95% glucuronoxylomannan (GXM), 5% galactoxylomannan (GalXM), and approximately 1% mannoproteins (MPs) [40, 41]. It is regarded as one of the most important virulence factors for
5. Diagnosis of cryptococcosis
The laboratory diagnosis of cryptococcosis is based upon a number of principles: the demonstration of the yeast in the clinical material, the isolation of the yeast in the culture followed by biochemical tests for the final identification, anatomic-pathological examination, and research into circulating antigens. Several biological materials may be used for the identification of fungal infection, e.g., serum, plasma, blood, tissue, and CSF, which is the major biological material used for the diagnosis of cryptococcal infection in the CNS .
The direct research of the fungus can be accomplished using CSF, sputum, bronchial washing, cutaneous-mucosal lesion pus, urine, macerates of biopsy tissue, prostatic secretion, blood, and bone marrow biopsy specimens. Clinical samples analyzed with India ink indicate the presence of the capsulated yeasts (Figure 1). This method is fast and low-cost but is not very sensitive and cannot distinguish between species. Due to the high yeasts load found in samples from AIDS patients, the sensitivity of this method may reach 80% for cryptococcal meningitis, whereas in immunocompetent individuals, this sensitivity may be as low as 30–72% [46–48]. In addition, the success of this technique is dependent upon the expertise of the microbiologist, and there are reports in the literature of false negatives in 20–30% of the results from infections with
The culturing of
After obtaining the isolate, it is necessary to differentiate the species type for clinical and epidemiological purposes [50–52]. Only
The production of urease is a biochemical test used to identify only the genus as both
During infection, the capsular polysaccharides of
The detection of the capsular antigen by agglutination of sensitized particles of latex (LA), which until sometime ago was the immunological method with the most widespread clinical use, may be accomplished in samples from the serum, urine, bronchoalveolar lavage, and CSF. The serological reaction to latex agglutination (LA) is sensitive and specific, emphasizing titers equal to or higher than 1/8 and being able to present cross reaction with the serum of patients with rheumatoid arthritis [62, 64]. The enzyme-linked immunosorbent assay (ELISA) may detect antigens from a cryptococcal infection earlier and at lower titers; however, it is time consuming, expensive, and is laborious. Although CrAg-latex performs as well as EIA and culture, its major limitations are that latex is a manual test and that the resulting interpretation of it is subjective. CrAg-latex and EIA also require laboratory equipment and refrigeration of reagents, making them inadequate for use in environments with minimal infrastructure . The need for refrigeration drastically increases the cost of the test in places with limited resources. Studies report that serological tests with CrAg-latex and EIA may show lower sensitivity when used with strands of some genotypes of
Recently, a new sensitive, low-cost, fast, and non-laborious immunochromatographic assay known as the lateral flow immunoassay (LFA) was made available for purchase for use in serum, CSF, and urine . This method has demonstrated good sensitivity for the detection of cryptococcal antigen (CrAg), primarily in HIV-positive patients . The World Health Organization (WHO) has recommended the use of antigen detection using LFA for patients infected with HIV who show low CD4 cells and are asymptomatic from a neurological viewpoint . This strategy enables the early identification of patients with a cryptococcal disease in the subclinical stage . It has been used in various studies as a form of screening and diagnosis, thus easing its application to clinical practice. Nevertheless, reasonably good results have been accomplished in multiple types of biological specimens, e.g., blood, CSF, and urine .
6. Synthetic peptides
The concept of synthetic peptides and protocols for their artificial synthesis was introduced in the early 20th century . Since then, peptides have become increasingly important for biochemistry, medicine, and biotechnology. In 1963, Bruce Merrifield described the development of solid-phase peptide synthesis, a technique that made the large-scale production of synthetic peptides a reality. Since then, various studies with different sizes of synthetic peptides have been reported .
In the early 1990s, with the development of biotechnology, recombinant antigens were widely used in clinical diagnosis to detect specific antibodies. However, their use in diagnostic tests presented some problems, such as low immunoreactivity compared with the corresponding purified human antigens, laborious and expensive production, and variation in inter-assay reactivity [72–76].
In this regard, synthetic peptides have opened up a new field and perspective as a source of pure epitopes and molecules for the diagnosis of various infectious and noninfectious diseases based on the detection of circulating antibodies and antigens and can also be used for the development of vaccines . Bioinformatics tools are widely used to predict antigenic and immunogenic regions. These programs are capable of predicting B and T cells epitopes, primarily by building on the known properties of amino acids, e.g., their hydrophilicity, charge, flexibility, exposed surface area, and secondary structure [78–80].
Some factors must be taken into account when dealing with synthetic peptides. The first factor to observe is whether the epitopic area is continuous or discontinuous because the amino acids belonging to the epitope are often separated in the linear sequence and become juxtaposed only when the antigen is in its native conformation. The second factor for observation is the size of the epitope. When this field of study began, researchers worked with only small epitopes as prior to the development of solid-phase peptide synthesis, one could not synthesize very large peptides. The very large peptides (>25–30 amino acids) are more expensive and difficult to produce and also have lower yields. For these reasons, peptides of 10–15 amino acid residues are usually recommended for the production and detection of antibodies [81–83].
The use of synthetic peptides for diagnostic tests confers several advantages, e.g., they are innocuous, easy to store and transport, have a high level of reproducibility with low levels of nonspecific reactions, and retain the possibility of changing the chemistry of the peptide by inserting cysteine residues, fatty acids, or carrier proteins or even by incorporating post-translational modifications, such as phosphorylation [84–86].
Over the past 20 years, several peptide sequences have been used to improve the sensitivity and specificity of tests that use recombinant or native protein as antigens [87–93]. However, the use of synthetic peptides as antigens has grown, with many diagnostic systems that are based on synthetic peptides in production, with some being commercially available at the present time. Some diagnostic tests that use synthetic peptides may already be part of the routine clinical diagnosis of certain diseases that involve viruses, parasites, or autoimmune diseases.
Some of the tests that are already available on the market include tests for Epstein–Barr virus, which examines various epitopes on the capsid protein; hepatitis C virus, which includes synthetic peptides that mimic its structural and nonstructural regions (NS4 and NS5); coronavirus, which is composed of synthetic peptides derived from epitopes of the nucleocapsid and spike proteins and can detect the presence of antibodies from human serum and plasma specimens;
Recent advances have been made in the search for more easily available immunodiagnostic tests for fungal infections. Various methods with high specificity and sensitivity are still under development, with a particular emphasis on the search for markers that are able to detect infections at an early stage. In this regard, Caldini et al.  used synthetic peptides from the gp75
With regards to cryptococcosis, the search for new diagnostic tests is necessary due to the high incidence of the disease, with estimates of approximately 1 million cases of cryptococcal meningitis per year and more than 600,000 deaths in HIV-infected patients, the potential for
As previously mentioned, diagnostic methods based upon the detection of antibodies have been developed and successfully applied to various other infectious diseases. The efficacy of these methods is not impacted by the antigenic charge of the microorganism, which is particularly relevant for the diagnosis of cryptococcosis, whose major diagnostic tests, LA and LFA, are dependent on the charge of the antigen.
The early diagnosis of cryptococcosis is a challenge that science and the health system must face as in most cases, the disease is diagnosed late, which results in significant morbidity and mortality. Thus, efforts should be made toward finding a rapid, sensitive, and specific diagnosis. In this sense, the identification of multiple immunogenic targets and the possibility of synthesizing these artificial targets appear to be a promising alternative for the development of more accurate tests for the diagnosis of systemic mycosis.
In this area, Martins et al.  have adopted an innovative strategy that combines the technology of proteomics and bioinformatics, with the aim of identifying multiple immunogenic targets for a diagnostic test for cryptococcosis. Linear B-cell epitopes of immunoreactive proteins for
In the search for a faster and more specific test, Brandão et al.  tested various synthetic peptides derived from immunoreactive proteins of
Hsp70 is a conserved protein that has been increasingly studied worldwide for its role in various biological processes, including the interaction of
Higher diagnostic performance can be achieved with multi-epitope chimeric proteins. This type of antigen becomes more attractive because it has more than one antigen-binding site, thus multiplying the possibilities for increasing antigenicity. Brandão et al. demonstrated in a theoretical model (
The use of this technology for the development of a diagnostic test capable of the early identification of cryptococcosis and the possibility of building an effective vaccine for this disease are essential for significant reduction in the morbidity and mortality of individuals affected by this infection and may ultimately change their prognosis.
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