Molecular epidemiological investigation of Cryptococcus spp. isolated from cats in Japan using multi-locus sequence typing.

Cryptococcosis is an important fungal infection for both humans and cats, but molecular epidemiological studies on strains isolated from cats are limited. We conducted multi-locus sequence typing analysis and antifungal susceptibility testing of 14 Cryptococcus spp. strains from domestic cats in Japan and one strain isolated from a cat in Singapore. All 14 strains from domestic cats in Japan were identified as Cryptococcus neoformans molecular type VNI. The sequence types (STs) included eight cases of ST5, five cases of ST31, and one novel ST. VNI ST5 is the most frequently isolated strain in Japanese patients as well, while there are no records of VNI ST31 being isolated from Japanese patients. The Singaporean cat strain was identified as C. gattii VGIIb (C. deuterogattii), ST7. We compared these results with strains previously reported to have been isolated from cats. This comparison suggested that molecular types of Cryptococcus spp. isolated from cats may differ depending on the country. In the antifungal susceptibility testing of C. neoformans, one strain each exceeded the epidemiological cutoff value (ECV) for amphotericin B and 5-fluorocytosine, while two strains exceeded the ECV for fluconazole. This study reveals the molecular epidemiology of Cryptococcus spp. isolated from cats with cryptococcosis in Japan. It suggests that investigating Cryptococcus spp. carried by cats, which share close living environments with humans, may contribute to the health of both cats and human populations.

Cryptococcosis is an important fungal disease in both humans and cats. We genotyped strains isolated from cats with cryptococcosis in Japan. Our findings revealed that the most common genotype infecting both cats and humans in Japan is identical.

Clinical features and prognostic factors of cryptococcal infections in HIV-infected patients: a 10-year study from an infectious disease specialist hospital.

Background: Cryptococcosis is an invasive infection that commonly affects immunosuppressed individuals, especially patients with HIV infection. Cryptococcal infection in HIV-infected patients should be considered a major health concern because it is associated with high morbidity and mortality rates. In this study, we aimed to evaluate the clinical characteristics and prognostic factors of cryptococcal infections in human immunodeficiency virus (HIV)-infected patients to facilitate effective clinical management and improve patient outcomes. Methods: We reviewed and analyzed the clinical data and relevant laboratory test results of HIV-infected patients with positive cryptococcal cultures and reserved strains between 2013 and 2023 from Beijing Youan Hospital affiliated to Capital Medical University. The clinical characteristics and laboratory test results of the patients were compared, and the correlation between parameters and the prognoses of the patients at different observation timepoints (3, 6, 9, and 12 months) was analyzed. Results: A total of 76 patients (70 males and six females; median age, 37 years) were included in this study. The results indicated that the later the initiation of antiretroviral therapy (ART) after the diagnosis of HIV infection (> 6 months), the higher the probability of death. Analysis of the correlation between the time of ART initiation and the timing of treatment for cryptococcal infections showed that the time of ART initiation was strongly related to survival at different timepoints. Initiation of ART time within 0-4 weeks, 4-6 weeks and more than 6weeks of starting treatment for Cryptococcus infection was associated with a lower mortality rate at 12-month, the 3-month, 6- and 9-month follow-up timepoint separately. Conclusions: Although cryptococcal infection in HIV-infected patients continues to be a challenging and intricate issue, ART is a key factor that affects its prognosis. The later ART is started, the worse the prognosis of the infection. The time of ART initiation and the timing of treatment for cryptococcal infections should be further refined and balanced based on different clinical courses. Thus, clinicians should pay closer attention to cryptococcal infections in patients with HIV infection and initiate ART based on the patient's clinical condition.

Bilateral adrenal cryptococcosis causing adrenal insufficiency in an immunocompetent patient.

ABSTRACT: Cryptococcosis usually occurs in immunocompromised patients and presents as meningitis and lung disease. Adrenal gland involvement may be observed, yet primary adrenal insufficiency by cryptococcal infection is infrequent. We present a case of a middle-aged immunocompetent man with primary adrenal insufficiency and bilateral adrenal lesions, splenomegaly, and miliary mottling in the lungs on imaging. No evidence of meningitis was witnessed. The clinico-radiological findings led toward the differential diagnosis of disseminated tuberculosis or fungal infection. Detection of cryptococcus organism was done on fine-needle aspiration cytology and biopsy on periodic acid-Schiff stain and Gomori`s methenamine silver stain. Thus, it is recommended to keep the possibility of cryptococcosis in mind while dealing with instances that have a tuberculosis-like clinico-radiological presentation. The detection of the causal organism on Fine needle aspiration (FNA)/biopsy examination may be useful in confirming the diagnosis and determining the appropriate medical treatment.

Recomendaciones para el diagnóstico y tratamiento de la infección por Cryptococcus spp / Recommendations for the diagnosis and treatment of infection by Cryptococcus spp

Las infecciones por levaduras del género Cryptococcus pueden causar un abanico amplio de manifestaciones clínicas, dependiendo de si se trata de una infección invasora o no. Los pacientes susceptibles, especialmente de las formas invasoras, comparten el compromiso de la inmunidad celular, ya sea por afecciones primarias o secundarias. El grupo más estudiado es el de personas que viven con VIH. La mortalidad es alta, especialmente en entornos de recursos reducidos. El esquema de tratamiento es en fases, inicialmente combinado, para luego continuar con monoterapia por un periodo prolongado, dependiendo de la duración del factor de riesgo subyacente. Hacemos una revisión de la evidencia y recomendaciones actualizadas.

Infection by yeast of the Cryptococcus genus can cause a wide range of clinical manifestations, depending on whether it is an invasive infection or not. Susceptible patients, especially those with invasive forms, share the compromise of cellular immunity, either due to primary or secondary conditions. The most studied group is that of people living with HIV. Mortality is high, especially in resource-poor settings. The treatment scheme is in phases, initially combined, to then continue with monotherapy for a prolonged period, depending on the duration of the underlying risk factor. We review the evidence and update recommendations.

Recurrent pulmonary cryptococcosis during chronic HBV infection: A case report.

RATIONALE: Pulmonary cryptococcosis is one of the important opportunistic infections and has a wide range of symptoms depending on the underlying conditions. Here, we reported a case living with chronic hepatitis B virus infection who had a recurrent pulmonary cryptococcosis. PATIENT CONCERNS: A 51-year-old male patient was admitted to our center because of cough, fatigue, and shortness of breath for 2 weeks. DIAGNOSIS: Pulmonary infection was suggested by chest computed tomography. Most lab examinations for infection were negative and only cryptococcal antigen testing was positive. Therefore, a clinical diagnosis of pulmonary cryptococcosis was made. INTERVENTIONS: Fluconazole (200 mg/day) and bicyclol (50 mg/day) was given orally. OUTCOMES: During the follow-up of 3 and 6 months, his conditions improved, and he recovered fully. Moreover, cryptococcal antigen level was 12.57 ng/mL. During the 2-year follow-up, no recurrence occurred. LESSONS: This case highlights the importance of the awareness of opportunistic infections during chronic hepatitis B virus infection, especially the potential of recurrence.

fuPCR as diagnostic method for the detection of rare fungal pathogens, such as Trichosporon, Cryptococcus and Fusarium.

Fungal respiratory tract colonization is a common finding in patients with hematologic neoplasms due to immunosuppression inherent in the diseases and exacerbated by therapy. This greatly increases the risk of fungal infections of the lungs, which is associated with significant mortality. Therefore, reliable diagnostic methods with rapidly available results are needed to administer adequate antifungal therapy. We have established an improved method for fungal DNA extraction and amplification that allows simultaneous detection of fungal families based on a set of multiplexed real-time PCR reactions (fuPCR). We analyzed respiratory rinses and blood of 94 patients with hematological systemic diseases by fuPCR and compared it with the results of culture and serological diagnostic methods. 40 healthy subjects served as controls. Regarding Candida species, the highest prevalence resulted from microbiological culture of respiratory rinses and from detection of antibodies in blood serum in patients (61 and 47%, respectively) and in the control group (29 and 51%, respectively). Detection of other pathogenic yeasts, such as Cryptococcus and Trichosporon, and molds, such as Fusarium, was only possible in patients by fuPCR from both respiratory rinses and whole blood and serum. These fungal species were found statistically significantly more frequent in respiratory rinses collected from patients after myeloablative therapy for stem cell transplantation compared to samples collected before treatment (P < 0.05i). The results show that fuPCR is a valuable complement to culturing and its inclusion in routine mycological diagnostics might be helpful for early detection of pathophysiologically relevant respiratory colonization for patients with hematologic neoplasms.

We validated a set of PCR reactions (fuPCR) for use in routine diagnostic. In contrast to culture and serological methods, only by fuPCR pathogenic yeasts (Cryptococcus and Trichosporon) and molds (Aspergillus and Fusarium) were detected in respiratory rinses and blood of hematological patients.

Analysis of Cryptococcal Extracellular Vesicles: Experimental Approaches for Studying Their Diversity Among Multiple Isolates, Kinetics of Production, Methods of Separation, and Detection in Cultures of Titan Cells.

Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. IMPORTANCE Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.

Cryptococcus depauperatus, a close relative of the human-pathogen C. neoformans, associated with coffee leaf rust (Hemileia vastatrix) in Cameroon.

The genus Cryptococcus is well known for its two species -Cryptococcus neoformans and C. gatii- that are etiological agents of cryptococcosis, an important fungal disease of mammals, including humans, and which is particularly common in immunocompromised patients. Nevertheless, Cryptococcus is a large and widely distributed genus of basidiomycetes occupying a broad range of niches, including mycoparasitism. One such mycoparasitic species is Cryptococcus depauperatus, which was firstly mistakenly described as a pathogen of scale insects under the name Aspergillus depauperatus. The "Aspergillus" conidiophores were later shown to be basidia of a Cryptococcus and the new combination C. depauperatus was proposed. Additionally, instead of an entomopathogen, the fungus was found to be a mycoparasite growing on the entomopathogen Akanthomyces (Lecanicillium) lecanii. Recently, during surveys for mycoparasites of coffee leaf rust (Hemileia vastatrix) in the context of a biocontrol project, white colonies covering rust pustules were observed in Cameroon. Upon close examination, instead of a member of the "white colony forming complex" of Ascomycetes, commonly collected growing on H. vastatrix, such colonies were found to represent a basidiomycete fungus with basidia-bearing chains of basidiospores, typical of the genus Cryptococcus. Morphological and molecular evidence was generated supporting the identification of the fungus on rust pustules as C. depauperatus. This is the first record of C. depauperatus from Africa and of its association with coffee leaf rust.

Prevalence of Advanced HIV Disease, Cryptococcal Antigenemia, and Suboptimal Clinical Outcomes Among Those Enrolled in Care in Vietnam.

BACKGROUND: People living with advanced HIV disease are at high risk of morbidity and mortality. We assessed the prevalence of cryptococcal antigenemia (CrAg) and clinical outcomes among patients newly presenting with CD4 ≤100 cells/µL in Vietnam. SETTING: Twenty-two public HIV clinics in Vietnam. METHODS: During August 2015-March 2017, antiretroviral therapy (ART)-naïve adults presenting for care with CD4 ≤100 cells/µL were screened for CrAg. Those who consented to study enrollment were followed up for up to 12 months and assessed for clinical outcomes. RESULTS: Of 3504 patients with CD4 results, 1354 (38.6%) had CD4 ≤100 cells/µL, of whom 1177 (86.9%) enrolled in the study. The median age was 35 years (interquartile range 30-40); 872 (74.1%) of them were men, and 892 (75.8%) had CD4 <50 cells/µL. Thirty-six patients (3.1%) were CrAg-positive. Overall, 1151 (97.8%) including all who were CrAg-positive initiated ART. Of 881 patients (76.5%) followed up for ≥12 months, 623 (70.7%) were still alive and on ART at 12 months, 54 (6.1%) had transferred to nonstudy clinics, 86 (9.8%) were lost to follow-up, and 104 (11.8%) had died. Among all 1177 study participants, 143 (12.1%) died, most of them (123, 86.0%) before or within 6 months of enrollment. Twenty-seven patients (18.9%) died of pulmonary tuberculosis, 23 (16.1%) died of extrapulmonary tuberculosis, 8 (5.6%) died of Talaromyces marneffei infection, and 6 (4.2%) died of opioid overdose. Eight deaths (5.8%) occurred among the 36 CrAg-positive individuals. CONCLUSIONS: Late presentation for HIV care was common. The high mortality after entry in care calls for strengthening of the management of advanced HIV disease.

Cryptococcal antigen carriage among HIV infected children aged 6 months to 15 years at Laquintinie Hospital in Douala.

BACKGROUND: Up to 15% of deaths of people living with HIV is attributable to meningeal cryptococcosis, with nearly 75% occuring in sub-Saharan Africa. Although rare in children, it is a major cause of morbidity and mortality in people living with HIV. A strong association between cryptococcal antigenemia and the development of meningeal cryptococcosis has been shown in adults. Thus, in 2018, the World Health Organization published an updated version of its guidelines for the diagnosis, prevention and management of cryptococcal infection in adults, adolescents and the HIV-infected child. GOAL: To determine the prevalence of cryptococcal antigenemia and to identify its determinants in children infected with HIV. METHODS: An analytical cross-sectional study was carried out at the approved treatment center of Laquintinie hospital in Douala over a period of 4 months. Children were recruited consecutively after informed parental consent. Cryptococcal antigenemia and CD4 assay were performed using a Cryptops® immunochromatographic rapid diagnostic test and flow cytometry, respectively. The data collected included the socio-demographic, clinical and paraclinical variables of the children, as well as their antecedents. Data analysis was performed using Epiinfo software version 3.1 and SPSS 21.0. The significance threshold was set at 5%. RESULTS: A total of 147 children were enrolled. The mean age was 9.8 ± 4.09 years. The majority were on antiretroviral therapy (142, 96.60%). Only 13 (8.80%) were in severe immunosuppression. No child showed signs of meningeal cryptococcosis. The prevalence of cryptococcal antigenemia was 6.12%. Severe immunosuppression [OR: 10.03 (1.52-65.91), p = 0.016] and contact with pigeons [OR: 9.76 (1.14-83.65), p = 0.037] were independent factors significantly associated with the carriage of the cryptococcal antigen. CONCLUSION: We recommend screening for cryptococcal antigenemia and routine treatment with fluconazole of all HIV positive children with cryptococcal antigen whether symptomatic or not.

CROI 2021: Neurologic Complications of HIV-1 Infection or COVID-19.

The 2021 Conference on Retroviruses and Opportunistic Infections (CROI) featured a timely review of the neurologic complications of COVID-19 as well as new research findings on mechanisms by which SARS-CoV-2 may affect the brain. CROI included new and important findings about the neurologic complications of HIV-1, human polyomavirus 2 (also known as JC Virus), and cryptococcus. New long-term analyses of cognition in people with HIV-1 identified that cognitive decline over time is associated with multimorbidity, particularly diabetes, chronic lung disease, and vascular disease risk conditions. These conditions are associated with aging, and the question of whether people with HIV are at risk for premature aging was addressed by several reports. New findings from large analyses of resting state networks also provided valuable information on the structural and functional networks that are affected by HIV-1 infection and cognitive impairment. Several reports addressed changes after initiating or switching antiretroviral therapy (ART). Findings that will improve understanding of the biologic mechanisms of brain injury in people with HIV were also presented and included evidence that host (eg, myeloid activation, inflammation, and endothelial activation) and viral (eg, transcriptional activity and compartmentalization) factors adversely affect brain health. Other research focused on adjunctive therapies to treat HIV-1 and its complications in the central nervous system. This summary will review these and other findings in greater detail and identify key gaps and opportunities for researchers and clinicians.

A Spuriously Negative Cryptococcal Antigen Assay Result in Disseminated Cryptococcosis: the Deception of the Postzone Phenomenon.

Cryptococcus is a basidiomycetous yeast responsible for considerable HIV-related morbidity and mortality. A cachectic 26-year-old HIV-positive man with a CD4 count of 103 cells/µl presented with fever, breathlessness, and bilateral lower limb weakness. A brain computed tomography scan could not elucidate the neurological deficit. His blood was sent for culture and serum cryptococcal antigen detection, with the latter testing as negative. By the fourth day of admission, the patient's condition had deteriorated drastically. A lumbar puncture was performed, and like his serum sample, the cerebrospinal fluid also tested negative for cryptococcal antigens. By this time, Cryptococcus neoformans was isolated from the admission blood culture. The laboratory diluted both the serum and cerebrospinal fluid specimens to retest for cryptococcal antigens, and finally, an antigen titer of ≥1:2560 was recorded.

[A prospective multi-center clinical investigation of HIV-negative pulmonary cryptococcosis in China].

Objective: To investigate the current status of the diagnosis and treatment of pulmonary cryptococcosis in respiratory medicine and improve the understanding of the clinical characteristics of HIV-negative pulmonary cryptococcosis in China. Methods: A prospective multi-center open cohort study was designed to screen for pulmonary cryptococcosis in the general wards and intensive care units of the Department of Respiratory Diseases in 22 hospitals. The HIV-negative patients with positive cryptococcal etiological diagnosis based on smear culture, antigen detection and histopathology were enrolled in the study. The clinical data of enrolled patients were collected and analyzed. Results: A total of 457 cases of pulmonary cryptococcosis were enrolled, among which 3.28% (15/457) were disseminated infections. The case fatality rate was 0.88% (4/457). The majority of the cases were diagnosed by histopathological examinations (74.40%, 340/457) and cryptococcus antigen detection (37.64%, 172/457). Patients with pulmonary cryptococcosis accounted for 2.04‰ (457/223 748) of the total hospitalized patients in the Department of Respiratory Diseases during the same period, and the ratio was the highest in south and east China. Meanwhile, 70.24% (321/457) of the patients had no underlying diseases, while 87.75% (401/457) were found to have immunocompetent status. Cough and expectoration were the most common clinical symptoms in patients with pulmonary cryptococcosis. However, 25.16% (115/457) of the patients had no clinical symptom or physical signs. In terms of imaging features on pulmonary CT, multiple pulmonary lesions were more common than isolated lesions, and there were more subpleural lesions than perihilar or medial lesions. Morphologically, most of the lesions were middle-sized nodules (1-5 cm) or small-sized nodules (3 mm to 1 cm). The sensitivity of serum cryptococcus antigen test was 71.99% (203/282). Moreover, antigen-positive patients differed from antigen-negative patients in terms of basic immune status, clinical symptoms, imaging features and infection types. Meanwhile, immunocompromised patients differed from immunocompetent patients in terms of clinical symptoms, physical signs, infection-related inflammation indicator levels, imaging features, serum cryptococcus antigen positive rate and prognosis. Conclusions: The majority of cases of HIV-negative pulmonary cryptococcosis in China had no underlying disease or immunocompromised status, and the overrall prognosis was favorable. However, early diagnosis of HIV-negative pulmonary cryptococcosis remains challenging due to the complicated manifestations of the disease.

FastFung: A novel medium for the culture and isolation of fastidious fungal species from clinical samples.

We developed a novel culture medium, referred to FastFung medium as suitable for the culture of clinical fungi, including fastidious ones, for both research and diagnostic studies. It is based on Schædler agar supplemented with many essential components for the growth of fastidious fungi. It also contains selective antibacterial agents for the inhibition of contaminant bacteria growth. In this preliminary study, the FastFung medium was compared to the gold standard Sabouraud medium for 98 fungal and 20 bacterial strains. The fungal strain positive culture rate was 100% vs. 95% and the bacterial strain inhibition was 100% vs. 20%, for the FastFung and Sabouraud media, respectively. When compared to the Sabouraud medium on 120 clinical samples, the FastFung medium displayed both a higher fungal colonies count, and a lower culture contamination rate. Storage at 4 °C for 4 weeks did not alter the FastFung culture medium performances for the six isolates of Candida, Cryptococcus, and Penicillium tested. These encouraging results suggest future development of using the FastFung medium in clinical mycology and in mycobiome characterization. Further prospective evaluation aiming at assessing whether implementing the FastFung medium in the routine workflow simplifies and strengthen fungal isolation capacities in the clinical laboratory is warranted.

Burden of Pediatric Central Nervous System Infection and Cost-Benefit Simulation of Multiplex Polymerase Chain Reaction in Japan.

To investigate the clinical use of multiplex polymerase chain reaction (mPCR) in Japan, epidemiological and clinical data for central nervous infections are needed. Here, we report on the epidemiology and economic burden of central nervous system infections and a simulation of the cost-benefit analysis of the Filmarray® Meningitis/Encephalitis (FAME) test for possible clinical use in Japan. We performed FAME tests on samples from 27 patients with pleocytosis aged between 0 and 20 years seen in six community hospitals in Nara and Osaka prefectures. All clinical management procedures were performed without knowledge of the mPCR test results. We analyzed the clinical data and calculated the required reduction in average length of stay for the FAME test to be cost-beneficial. Among the 27 cases, the FAME test revealed causal pathogens in 13 cases (48.1%). The average medical and social costs per case were ¥299,118 ($2,719.2) and ¥171,768 ($1,561.5), respectively. The minimal needed reduction in average length of stay for the FAME test to be cost-beneficial was 0.32- 0.86 days per meningitis case. The result can be informative for evaluating the cost-effectiveness of the clinical use of the FAME test in Japan.

Cryptococcal empyema treated with tube thoracostomy and intrapleural fibrinolysis.

A 55-year old woman with a history of relapsed T-cell ALL presented with right pleuritic chest pain and decreased breath sounds over the right hemithorax. Imaging of the chest showed loculated effusions. Tube thoracostomy was performed with intrapleural application of alteplase and dornase alpha over a 3-day period. Repeat imaging demonstrated a marked decrease in the volume of the effusion. In most prior published cases of pleural cryptococcosis, surgical drainage was required in addition to prolonged antifungal agents. More than 50% of patients with cryptococcal infection have severe underlying disease or immunodeficiency state making them high risk for surgery. This is the first case to our knowledge of cryptococcal empyema successfully treated with tube thoracostomy and intrapleural fibrinolysis.

Attempted Isolation of Cryptococcus Species and Incidental Isolation of Exophiala dermatitidis from Human Oral Cavities.

Recent molecular studies suggest that Cryptococcus may inhabit the normal human mouth. We attempted to isolate Cryptococcus from 21 adult non-acutely ill patients and 40 volunteer medical and non-medical staff in Southeastern Wisconsin, USA. An upper lip sulcus culture and an oral rinse specimen were inoculated separately onto Staib (birdseed) agar containing chloramphenicol and incubated in gas impermeable zip lock bags at 35 °C. No cryptococci were grown from any of the 122 samples from the 61 subjects. Both specimens from a woman with no risk factors for fungal disease yielded a black yeast at 4 days on Staib agar. This isolate was shown to be Exophiala dermatitidis by colony and microscopic morphology, analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and sequencing through the internal transcribed spacer ribosomal RNA gene. This appears to be a novel isolation of E. dermatitidis from the oral cavity of a generally healthy human.

First case report of Cryptococcus laurentii knee infection in a previously healthy patient.

BACKGROUND: The purpose of this case report was to report a case of Cryptococcus laurentii infection in the left knee of a previously healthy 29 year old male patient. CASE PRESENTATION: After an initial misdiagnosis and 7 months of failed treatment, the patient received nearly a month of treatment with voriconazole (200 mg IV q12 h) and knee irrigation with amphotericin B until the infection was controlled. The treatment continued with fluconazole for nearly 7 months and approximately 5 weeks of antibiotic treatment for a skin bacterial coinfection. In the end, the patient's symptoms disappeared completely, the left knee recovered well, and there was no recurrence of infection. CONCLUSION: The key points of successful treatment in this case were the thorough debridement, the adequate course of knee irrigation with antifungal drugs and more than 6 months of oral antifungal drugs that were able to eradicate the infection.

Cryptococcosis in non-human immunodeficiency virus-infected patients: A clinical dilemma and diagnostic enigma.

Cryptococcosis is a fungal disease with worldwide distribution and wide array of clinical manifestations, caused by encapsulated basidiomycetous yeasts called Cryptococcus spp. It has traditionally been considered an opportunistic infection known to occur in immunocompromised hosts, particularly those who are infected with human immunodeficiency virus. However, this infection has also been reported in phenotypically 'normal' or otherwise clinically non-immunocompromised patients. The seemingly mysterious nature of this potentially fatal illness has always kept clinicians and diagnosticians in a dilemma. This case series reiterates this perspective.

A comparative evaluation of three methods for the rapid diagnosis of cryptococcal meningitis (CM) among HIV-infected patients in Northern Malawi.

Introduction: Cryptococcal meningitis (CM) is the most common systemic fungal infection in patients with HIV infection. Rapid diagnosis and timely initiation of antifungal therapy are key to reducing mortality rate associated with CM. This study aims to evaluate the ability of four different diagnostic tests (Gram stain, India ink, and two types of commercial lateral flow assay [LFA]) to identify CM-positive patients and to compare the sensitivity and specificity of these tests. Methods: This was a prospective cross-sectional study on diagnostic tests accuracy conducted in Northern Malawi. The target population was HIV-infected adult patients presenting with features of meningitis. Four types of diagnostic tests were conducted: India ink, Gram stain, and two types of commercial lateral flow assay (LFA) (Immy, Inc., OK, USA and Dynamiker Biotechnology (Tianjin) Co., Ltd), Singapore). Culture was conducted as the reference standard. Results: A total of 265 samples were collected. The rate of positive CM detection ranged from 6.4% (using India ink) to 14.3% (using LFA). India ink exhibited the lowest sensitivity of 54.8% (95% confidence interval [CI]: 36.0%-72.7%), followed by Gram stain (61.3%; 95% CI: 42.2%-78.2%). The Dynamiker LFA exhibited the highest sensitivity of 100.0% (95% CI: 90.0%-100.0%) but a lower specificity (97.0%; 93.9%-98.8%) compared to the Immy LFA (98.3%; 95% CI: 95.7%-99.5%). Conclusion: LFA diagnostic methods have the potential to double the detection rate of CM-positive patients in resource-limited countries such as Malawi. As such, LFAs should be considered to become the main diagnostic tests used for CM diagnostics in these countries. Our data indicate that LFAs may be the best method for diagnosing CM and exhibits the highest diagnostic accuracy as it has shown that it outperforms cell culture, the current gold standard.