A retrospective study on the frequency of cryptococcosis in University Malaya Medical Centre from year 2013 to September 2019
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Background: Cryptococcosis is an infectious disease with worldwide distribution caused by Cryptococcus species. Cryptococcus neoformans and Cryptococcus gattii are the common species in human cryptococcal infection. Cryptococcus neoformans is a major cause of illness in people diagnosed with Human Immunodeficiency Virus (HIV), with an estimated 220,000 cases of cryptococcal meningitis occurring worldwide each year. This is a retrospective study on the frequency of cryptococcosis in different age groups and gender; as well as to identify the types of Cryptococcus species isolated in this population in University Malaya Medical Center from year 2013 to September 2019. Methods and materials: Data on cryptococcosis diagnosed in University Malaya Medical Center from year 2013 to September 2019 was extracted from the laboratory information system at the center. 59 newly diagnosed cryptococcosis cases were included in this study. Cryptococcus species were isolated from blood, cerebrospinal fluid, pus, body fluid, tissue and skin. Methods of identification of Cryptococcus species were via API 20C AUX, Vitek 2 and MALDI-TOF. Results: In this population of study, the frequency of cryptococcosis is 83.06% and 16.94% in males and females respectively. The frequency of Cryptococcus neoformans is 81.36%, with 83.33% comprising males (age 10–87) and 16.67% comprising females (age 33–81). The frequency of Cryptococcus neoformans var. grubii is 3.39% (2 males aged 33 and 53), Cryptococcus albidus is 3.39% (2 females aged 65 and 70), Cryptococcus uniguttulatus is 3.39% (two males aged 35 and 87) and Cryptococcus sp. is 3.39% (2 males aged 25 and 36). Cryptococcus gattii (34 year old male), Cryptococcus laurentii (71 year old male) and Cryptococcus humicola (59 years old male) are 1.70% in frequency each. Conclusion: In conclusion, the frequency of cryptococosis in this period of study is high in the male population with Cryptococcus neoformans as the commonest isolated species. Early diagnosis followed by treatment of cryptococcosis offers good to excellent prognosis for the patients. However, the prognosis varies in the immunocompromised. Targeted screening programmes should be carried out to detect early cryptococcal disease, especially in HIV-infected persons to reduce morbidity and mortality, and improve the overall quality of life.Keywords:
Cryptococcus gattii
Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATα-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii.
Cryptococcus gattii
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Cryptococcus spp is a major cause of opportunistic infections in immunocompromised patients, primarily due to Cryptococcus neoformans and Cryptococcus gattii. There are occasional reports of other Cryptococcus species causing invasive human disease. However, their epidemiology and clinical significance are not fully defined. We sought to describe cases with cultures positive for Cryptococcus species other than C neoformans and C gattii.A retrospective descriptive analysis of clinical and laboratory data of patients with cultures growing Cryptococcus species other than C neoformans and C gattii from November 2011 to February 2019 was performed. Three Mayo Clinic sites in Arizona, Florida, and Minnesota were included.From 176 cases with a culture growing Cryptococcus spp, 54 patients (30%) had a culture for Cryptococcus other than C neoformans and C gattii in the study time frame. The most common species were Cryptococcus magnus, Cryptococcus laurentii, and Cryptococcus ater. The organisms were isolated and identified in culture of bronchoalveolar lavage (11), skin (11), urine (7), oral (4), sinus (3), intraoperative soft tissue (3), sputum (2), synovial fluid (2), cerebrospinal fluid (2), and intravenous catheter (2), among others (7).Only 8 (15%) cases were considered to be potentially pathogenic, with 1 case of invasive disease. Antifungal treatment was fluconazole, itraconazole, and griseofulvin, for a mean systemic antifungal duration of 42 days.This large series of patients with Cryptococcus spp other than C neoformans and C gattii suggests that these species rarely cause clinically significant infection in humans. Only 1 case of invasive disease was found.
Cryptococcus gattii
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Objective To establish a multiplex PCR targeting the intergenic spacer regions (IGS) for the identification of Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii.Methods Primers were designed by using the software ClustalW2 and Oligo 6 based on the sequence of IGS1 region,which shows high sequence variability in the genome of Cryptococcus neoformans and Cryptococcus gattii.,for the multiplex PCR.Then,the developed multiplex PCR was performed to identify 51 Cryptococcus neoformans strains representing genotypes VNI-VNIV and VNB as well as 41 Cryptococcus gattii strains representing genotypes VGI-VGIV.The identification results were compared with those from common PCR by using primers GPA1A,CLA4D and SODlgattii specific to Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii,respectively,as well as with those from the canavanine-glycine-bromothymol blue (CGB) medium-based culture.Results The developed multiplex PCR successfully identified the 92 Cryptococcus neoformans and Cryptococcus gattii.strains,and yielded negative results from the other tested pathogenic yeasts,which revealed a high specificity of the designed primers.False positive results were observed in the identification of two Cryptococcus gattii strains with GPA1A primer-based PCR,one Cryptococcus gattii strain with CLA4D primer-based PCR,one var.grubii strain and one var.neoformans strain with CGB culture,while no false negative results were observed in the detection of these Cryptococcus strains by any of these methods.Conclusions The developed multiplex PCR in this study can rapidly and accurately identify Cryptococcus neoformans var.grubii,var.neoformans,AD hybrid,and Cryptococcus gattii,with superior performance in comparison with common PCR and CGB medium-based culture.
Key words:
Cryptococcosis; Polymerase chain reaction; Genotype
Cryptococcus gattii
Multiplex
Primer (cosmetics)
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This study compared the enzymatic activity of clinical isolates of Cryptococcus neoformans, Cryptococcus gattii, environmental isolates of C. neoformans and non-neoformans Cryptococcus. Most of the cryptococcal isolates investigated in this study exhibited proteinase and phospholipase activities. Laccase activity was detected from all the C. neoformans and C. gattii isolates, but not from the non-neoformans Cryptococcus isolates. There was no significant difference in the proteinase, phospholipase and laccase activities of C. neoformans and C. gattii. However, significant difference in the enzymatic activities of β-glucuronidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase between C. neoformans and C. gattii isolates was observed in this study. Environmental isolates of C. neoformans exhibited similar enzymatic profiles as the clinical isolates of C. neoformans, except for lower proteinase and laccase activities.
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Emerging environmental pathogenic fungal infections, including cryptococcosis, continue to pose a significant threat to humans with compromised immunity and, to some extent, healthy ones. Cryptococcus neoformans was originally identified as the main etiological agent of human cryptococcosis, but recent studies have also identified the occurrence of opportunistic infections caused by Cryptococcus gattii . These two saprophytic facultative yeasts present a paradox as they can infect humans without requiring a host for replication or survival, a phenomenon termed readymade virulence. Many cryptococcal virulence traits appear to have dual effects that provide survival advantages in both animal hosts and the environment. Several molecular techniques have been developed to provide in-depth knowledge of these species complexes. This review will focus on the description of the Cryptococcus neoformans and Cryptococcus gattii ( CnCg ) species complexes and associated cryptococcal pathogenesis, ecological niches, and virulence factors employed by the pathogens to cause disease.
Cryptococcus gattii
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Cryptococcus gattii
Phenotypic switching
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Cryptococcus neoformans and Cryptococcus gattii are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target Cryptococcus do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome b (cyt b) gene to detect C. neoformans and C. gattii in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The cyt b-directed assay accurately detected and identified all eight C. neoformans/gattii genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The Cryptococcus-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.
Cryptococcus gattii
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ABSTRACT We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex ( Cryptococcus neoformans var. grubii , Cryptococcus neoformans var. neoformans , and Cryptococcus gattii ). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.
Cryptococcus gattii
High Resolution Melt
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Background: Cryptococcosis is an infectious disease with worldwide distribution caused by Cryptococcus species. Cryptococcus neoformans and Cryptococcus gattii are the common species in human cryptococcal infection. Cryptococcus neoformans is a major cause of illness in people diagnosed with Human Immunodeficiency Virus (HIV), with an estimated 220,000 cases of cryptococcal meningitis occurring worldwide each year. This is a retrospective study on the frequency of cryptococcosis in different age groups and gender; as well as to identify the types of Cryptococcus species isolated in this population in University Malaya Medical Center from year 2013 to September 2019. Methods and materials: Data on cryptococcosis diagnosed in University Malaya Medical Center from year 2013 to September 2019 was extracted from the laboratory information system at the center. 59 newly diagnosed cryptococcosis cases were included in this study. Cryptococcus species were isolated from blood, cerebrospinal fluid, pus, body fluid, tissue and skin. Methods of identification of Cryptococcus species were via API 20C AUX, Vitek 2 and MALDI-TOF. Results: In this population of study, the frequency of cryptococcosis is 83.06% and 16.94% in males and females respectively. The frequency of Cryptococcus neoformans is 81.36%, with 83.33% comprising males (age 10–87) and 16.67% comprising females (age 33–81). The frequency of Cryptococcus neoformans var. grubii is 3.39% (2 males aged 33 and 53), Cryptococcus albidus is 3.39% (2 females aged 65 and 70), Cryptococcus uniguttulatus is 3.39% (two males aged 35 and 87) and Cryptococcus sp. is 3.39% (2 males aged 25 and 36). Cryptococcus gattii (34 year old male), Cryptococcus laurentii (71 year old male) and Cryptococcus humicola (59 years old male) are 1.70% in frequency each. Conclusion: In conclusion, the frequency of cryptococosis in this period of study is high in the male population with Cryptococcus neoformans as the commonest isolated species. Early diagnosis followed by treatment of cryptococcosis offers good to excellent prognosis for the patients. However, the prognosis varies in the immunocompromised. Targeted screening programmes should be carried out to detect early cryptococcal disease, especially in HIV-infected persons to reduce morbidity and mortality, and improve the overall quality of life.
Cryptococcus gattii
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Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.
Cryptococcus gattii
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