Using force or EMG envelope as feedback signal for motor control system.

PURPOSE: This work studied muscle neuro-mechanics during symmetrical up-going ramp (UGR) and down-going ramp (DGR). AIM: to evaluate during the modulation of muscular action the outcome of force feedback (FF) or neural feedback (NF) on the behavior of the trailing signals - i.e. the EMG envelope (eEMG) for FF or force signal for NF. METHOD: Subjects: 20. Investigated muscles: dorsal interosseous (FDI) and tibialis anterior (TA). Detected signals: force and EMG. Visual feedback: force (FF), eEMG (NF). Effort triangles: ramps duration 7.5 s, vertex at 50 and 100 % of the maximal voluntary action. Eventually, each subject performed FF50%, FF100%, NF50% and NF100% per each muscle. In each condition the areas beneath the force and eEMG signals were computed to calculate the ratios between the DGR and UGR values during the different tasks (force area DGR / force area UGR; eEMG area DGR / eEMG area UGR). Electro-mechanical coupling efficiency (EMCE) was estimated through the eEMG area / force area ratio for both UGR and DGR in each condition. RESULTS: a) FF. FDI: eEMG area ratio was 0.84 ± 0.15 and 0.73 ± 0.17 for FF50% and FF100%, respectively. TA: eEMG area ratio was 0.88 ± 0.11 and 0.91 ± 0.17 for FF50% and FF100%, respectively. b) NF: FDI: force area ratio was 1.18 ± 0.13 and 1.17 ± 0.13 for NF50% and NF100%, respectively. TA: force area ratio was 1.17 ± 0.21 and 1.07 ± 0.19 for NF50% and NF100%, respectively. c) DGR EMCE was greater than UGR EMCE in all four tasks. CONCLUSION: The influence of UGR on deployed EMCE in the following force decrement phase underpins the changes of trailing signals area during DGR. This underlines the necessity of a careful evaluation of the features of FF or NF for experimental studies or rehabilitation purposes involving the motor control system.

Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Influence of age on force and re-lengthening dynamics after tetanic stimulation withdrawal in the tibialis anterior muscle.

PURPOSE: During alternate movements across a joint, the changeover from one direction of rotation to the opposite may be influenced by the delay and rate of tension reduction and the compliance to re-lengthening of the previously active muscle group. Given the aging process may affect the above-mentioned factors, this work aimed to compare the dynamics of both the ankle torque decline and muscle re-lengthening, mirrored by mechanomyogram (MMG), in the tibialis anterior because of its important role in gait. METHODS: During the relaxation phase, after a supramaximal 35 Hz stimulation applied at the superficial motor point, in 20 young (Y) and 20 old (O) subjects, the torque (T) and MMG dynamics characteristics were measured. RESULTS: The T and MMG analysis provided: (I) the beginning of the decay after cessation of stimulation (T: 22.51 ± 5.92 ms [Y] and 51.35 ± 15.21 ms [O]; MMG: 27.38 ± 6.93 ms [Y] and 61.41 ± 18.42 ms [O]); (II) the maximum rate of reduction (T: - 110.4 ± 45.56 Nm/s [Y] and - 52.72 ± 32.12 Nm/s [O]; MMG: - 24.47 ± 10.95 mm/s [Y] and - 13.76 ± 6.54 mm/s [O]); (III) the muscle compliance, measuring the MMG reduction of every 10% reduction of torque (bin 20-10%: 15.69 ± 7.5[Y] and 10.8 ± 3.3 [O]; bin 10-0%: 22.12 ± 10.3 [Y] and 17.58 ± 5.6 [O]). CONCLUSION: Muscle relaxation results are different in Y and O and can be monitored by a non-invasive method measuring physiological variables of torque and re-lengthening dynamics at the end of the electromechanical coupling previously induced by the neuromuscular stimulation.

Environmental and bioclimatic factors influencing yeasts and molds distribution along European shores.

The present study employed data collected during the Mycosands survey to investigate the environmental factors influencing yeasts and molds distribution along European shores applying a species distribution modelling approach. Occurrence data were compared to climatic datasets (temperature, precipitation, and solar radiation), soil datasets (chemical and physical properties), and water datasets (temperature, salinity, and chlorophyll-a concentration) downloaded from web databases. Analyses were performed by MaxEnt software. Results suggested a different probability of distribution of yeasts and molds along European shores. Yeasts seem to tolerate low temperatures better during winter than molds and this reflects a higher suitability for the Northern European coasts. This difference is more evident considering suitability in waters. Both distributions of molds and yeasts are influenced by basic soil pH, probably because acidic soils are more favorable to bacterial growth. Soils with high nitrogen concentrations are not suitable for fungal growth, which, in contrast, are optimal for plant growth, favored by this environment. Finally, molds show affinity with soil rich in nickel and yeasts with soils rich in cadmium resulting in a distribution mainly at the mouths of European rivers or lagoons, where these metals accumulate in river sediments.

ICU environmental surfaces are a reservoir of fungi: species distribution in northern Italy.

BACKGROUND: Preventing and reducing nosocomial infections is a public health goal. Concern about healthcare-associated fungal infections has increased in recent years due to the emergence and spread of new pathogens, increasing antifungal resistance and outbreaks in hospital settings. AIM: To investigate the presence of medically relevant fungal species on environmental surfaces in 12 intensive care units of eight hospitals in Milan, Italy. METHODS: Environmental samplings, using contact plates on surfaces near bed stations and medical workstations, were conducted between November 2019 and January 2020. Fungi isolated were identified, and some were tested in vitro for antifungal susceptibility. FINDINGS: In total, 401 environmental samples were collected from 61 bed stations and 17 medical workstations. Positive samples were found in all hospitals except one, with positivity rates ranging from 4% to 24.2%. Filamentous fungi were found mainly on infusion pumps (23.2%) and patient tables (21.2%), whereas yeasts were found mainly on computers (25%) and floors (10.9%). Fungi were isolated from 12% of total samples. Filamentous fungi, mainly Aspergillus fumigatus, grew in 70.8% of positive samples, and yeasts grew in 27.1%, mainly Candida parapsilosis (42.8%) and Candida glabrata (28.6%). Fungi were detected near patients' beds and on surfaces at workstations, indicating potential for environment-to-patient, patient-to-patient and healthcare worker-to-patient transmission. CONCLUSIONS: Surveillance in hospital settings through environmental sampling may be an important component of fungal infection prevention.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Tobacco and e-cigarette smoking habits among Italian healthcare students.

BACKGROUND: Healthcare professionals might play a significant role in tobacco control. The aims of this study were to investigate tobacco cigarette and electronic cigarette (e-cigarette) consumption among university students enrolled in courses of the healthcare area, and to understand whether training in healthcare could induce to smoking cessation. STUDY DESIGN: Cross-sectional survey using a self-administered, structured questionnaire. METHODS: Tobacco smoking habits of 560 students of four different medical area courses at the University of Milan, enrolled both in the first and in the last course year during the 2017-2018 academic year, were collected. RESULTS: The prevalence of smokers was 34.8%, almost the same for males and females, and higher in Italian students compared to foreigners. Smoking prevalence was higher among employed (46.9%) than unemployed (35.1%) students, without differences between healthcare and non-healthcare workers. The influence of family, and particularly friends, is confirmed to be relevant. About 25% of respondents tried e-cigarettes, mainly smokers (44.6%) and former smokers (38.6%) with the goal of quitting smoking. More than 44% were dual users of both tobacco cigarettes and e-cigarettes. Comparing smoking habits between first-year and final-year students, only students of healthcare assistance course showed a significant drop in smoking during the university studies. CONCLUSIONS: The implementation of specific educational curricula on smoking dangers and on smoking cessation techniques might have the double effect of supporting students in quitting smoking and of properly preparing them for their future task of helping people to quit smoking.

Normalized maximal rate of torque development during voluntary and stimulated static contraction in human tibialis anterior: Influence of age.

The risk of falling in older adults has been related, among other factors, to the reduction of the rate of torque development (RTD) with age. It is well known that both structural/peripheral and neural factors can influence the RTD. The purpose of this study was to compare the normalized RTD in young and older participants obtained during a) rapid voluntary tension production and b) neuromuscular electrical stimulation. The tibialis anterior of 19 young subjects (10 males and 9 females; age 21-33 years old) and 19 older participants (10 males and 9 females; age 65-80 years old) was studied. The subjects performed a series of maximal isometric explosive dorsiflexions and underwent trains of supra-maximal electrical stimulations (35 Hz) on the tibialis anterior motor point. Muscle shortening was indirectly measured using a laser (surface mechanomyogram, MMG). Both torque and MMG were normalized to their maximum value. Using a 20 ms sliding window on the normalized torque signal, the normalized maximum RTD was calculated for both voluntary and stimulated contractions. Active stiffness of the muscle- tendon unit was calculated as the area of the normalized torque with respect to the normalized MMG. Normalized maximum RTD was found significantly lower in older adults during voluntary activity (young: 751.9 ± 216.3%/s and old: 513.9 ± 173.9%/s; P < .001), and higher during stimulated contractions (young: 753.1 ± 225.9%/s and old: 890.1 ± 221.3%/s; P = .009). Interestingly, active stiffness was also higher in older adults (young: 3524.6 ± 984.6‰ and old 4144.6 ± 816.6‰; P = .041) and significantly correlated to the normalized maximum RTD during stimulated contractions. This dichotomy suggests that modifications in the structural/peripheral muscle properties are not sufficient to counteract the age-related decrease in neural drive to the muscle during voluntary isometric contractions in aged participants.

Prospective multicentre study on azole resistance in Aspergillus isolates from surveillance cultures in haematological patients in Italy.

OBJECTIVES: This study was conducted to assess the prevalence of azole resistance in Aspergillus isolates from patients with haematological malignancies or who were undergoing haematopoietic stem cell transplantation and to identify the molecular mechanism of resistance. METHODS: In this 28-month prospective study involving 18 Italian centres, Aspergillus isolates from surveillance cultures were collected and screened for azole resistance, and mutations in the cyp51A gene were identified. Resistant isolates were genotyped by microsatellite analysis, and the allelic profiles were compared with those of resistant environmental and clinical isolates from the same geographical area that had been previously genotyped. RESULTS: There were 292 Aspergillus isolates collected from 228 patients. The isolates belonged mainly to the section Fumigati (45.9%), Nigri (20.9%), Flavi (16.8%) and Terrei (4.8%). Three isolates showed itraconazole resistance: Aspergillus fumigatus sensu stricto, Aspergillus lentulus (section Fumigati) and Aspergillus awamori (section Nigri). The itraconazole resistance rates were 1% and 1.48% considering all Aspergillus spp. isolates and the Aspergillus section Fumigati, respectively. The prevalence of azole resistance among all the patients was 1.3%. Among patients harbouring A. fumigatus sensu stricto isolates, the resistance rate was 0.79%. The A. fumigatus isolate, with the TR34/L98H mutation, was genotypically distant from the environmental and clinical strains previously genotyped. CONCLUSIONS: In this study, the Aspergillus azole resistance rate was 1% (3/292). In addition to A. fumigatus sensu stricto, A. lentulus and A. awamori azole-resistant isolates were identified. Therefore, it is important have a correct identification at the species level to address a rapid therapy better, quickly understand the shift towards cryptic species and have an updated knowledge of the local epidemiology.

Evolution of fungemia in an Italian region.

BACKGROUND: Fungemia represents a public health concern. Knowing aetiology and activity of the antifungals is critical for the management of bloodstream infections. Therefore, surveillance on local/international levels is desirable for a prompt administration of appropriate therapy. METHODS: Data on fungi responsible for fungemia and antifungal susceptibility profiles were collected from a laboratory-based surveillance over 2016-2017 in 12 hospitals located in Lombardia, Italy. The trend of this infection in twenty years was analysed. RESULTS: A total of 1024 episodes were evaluated. Rate of candiaemia progressively increased up to 1.46/1000 admissions. C.albicans was the most common species (52%), followed by C. parapsilosis (15%) and C glabrata (13%). As in the previous surveys the antifungal resistance is rare (echinocandins<2%, fluconazole 6%, amphotericin B 0.6%). Fungi other than Candida were responsible for 18 episodes: Cryptococcus neoformans (5 cases), Fusarium spp. (4), Magnusiomyces clavatus (3), Saccharomyces cerevisiae (3), Rhodotorula spp. (2), Exophiala dermatitidis (1). All fungi, except S.cerevisiae, were intrinsically resistant to echinocandins. Some isolates showed also elevated azole MIC. CONCLUSIONS: No particular changes in terms of species distribution and antifungal susceptibility patterns was noted. However, surveillance programs are needed to monitor trends in antifungal resistance, steer stewardship activities, orient empirical treatment.

Influence of age on motor control accuracy during static ramp contractions.

We investigated the influence of the ageing process on the performance of the motor control system accuracy during a challenging motor task throughout the analysis of force output oscillations. The force signal of the first dorsal interosseous during linearly varying static contraction, 0-100-0% of the maximal volitional abduction in 15 s, was studied in 11 young and older adults. The relative error between the target and the actual force as well as several parameters of the force oscillations (corrections) were estimated. To understand the experimental results, we analyzed the force output generated by a set of computational simulations of a pool of motor units controlled by a proportional-integral-derivative system. Compared to young adults the older subjects presented larger errors and a lower number of corrections with longer duration and larger relative amplitude. The motor control system modelling varied the error update frequency (UF) of the controller (from 1 to 2.5 Hz) as well as the range of contraction time (CT) of the recruited motor unit (30-90 ms and 60-120 ms reflecting young and old ranges, respectively). The simulation generated force profiles with parameters similar to experimental recordings in young (UF = 1.5; CT 30-90 ms) and older (UF = 1; CT 60-120 ms) adults. Interestingly, the results of the simulations suggested that the improvement in the error update frequency of the controller was not able to compensate for the contractile changes in the motor unit twitches. In conclusion, the peripheral contractile changes with age can influence motor unit control strategies and represent a crucial phenomenon in the generation of larger force oscillations in older adults.

First report of two cases of cryptococcosis in Tripoli, Libya, infected with Cryptococcus neoformans isolates present in the urban area.

Cryptococcosis is a potentially fatal fungal disease caused by the basidiomycetes yeasts Cryptococcus neoformans and C. gattii with high predilection to invade the central nervous system mainly in immunocompromised hosts. Skin can be secondarily involved in disseminated infection or be exceptionally involved as primary cutaneous infection by inoculation with contaminated materials. We report the first two Libyan cases of cryptococcal meningitis in HIV patients, in which one of them presented a secondary cutaneous involvement due to systemic dissemination. The first patient was a 17-year-old female, had fever, cough, headache and intractable vomiting as well as itchy water bumps on her skin and upper limbs. The cutaneous eruption prompted the accurate diagnosis. Cultures were positive for C. neoformans in both cerebrospinal fluid and skin specimens, as well as cryptococcal antigen was detected in serum. The isolate was identified, by molecular analysis, as C. neoformans AD-hybrid belonging to molecular type VNIII and mating type αAAα, the same genotype found for some environmental isolates recovered from olive trees in Tripoli. The second patient was a 36-years-old male with a long history of HIV on irregular treatment. Cryptococcal antigen in serum was positive and cultures yielded the growth of C. neoformans var. grubii, molecular type VNI and mating type αA. Both patients did not respond adequately to treatment and died of impaired central nervous system function and respiratory failure, respectively.

Molecular characterization of environmental Cryptococcus neoformans VNII isolates in Jos, Plateau State, Nigeria.

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeasts able to cause fatal neurological infections in both human and other mammals. Cryptococcosis is the most common fungal infection of the central nervous system and has a huge burden in sub-Saharan Africa and South East Asia. Bird excreta are considered an environmental reservoir for C. neoformans in urban areas, therefore a study aimed at isolating and characterizing this yeast is important in disease management. In this study, one hundred samples of pigeon droppings were collected in Jos, Plateau State, Nigeria. C. neoformans was isolated from three samples and initially identified using standard phenotypic and biochemical tests. Molecular analysis revealed that all three isolates belonged to C. neoformans genotype VNII, mating type α and were assigned to the sequence type ST43 by multilocus sequence typing analysis. This study reports, for the first time, the molecular characterization of C. neoformans in Nigeria, where little is still known about the environmental distribution of the genotypes, serotypes and mating types of this important human pathogen.

Cryptococcus gattii sero-mating type allelic pattern determined by multiplex PCR.

Molecular methods to differentiate serotypes, mating types and molecular types of Cryptococcus neoformans and C. gattii are important tools to understand epidemiology and pathogenesis of these pathogens. In this study, a multiplex polymerase chain reaction (PCR) approach was applied to sero-mating typing of C. gattii strains. Four pairs of primers were designed to target 4 allele-specific genes located in the mating-type locus. Twenty-three C. gattii strains, presenting different mating types and serotypes, were tested to validate the method. The method was able to identify all sero-mating allelic patterns including hybrid combinations, and therefore, it represents a simple one-step PCR for sero-mating typing of C. gattii strains.

Azole-resistant Aspergillus fumigatus in the environment of northern Italy, May 2011 to June 2012.

In recent years acquired azole resistance in Aspergillus fumigatus has been increasingly reported and a dominant mechanism of resistance (TR34/L98H) was found in clinical and environmental isolates. The aim of the present study was to investigate the prevalence of azole resistance in environmental A. fumigatus isolates collected in northern Italy. A. fumigatus grew from 29 of 47 soil samples analysed. Azole-resistant isolates were detected in 13% (6/47) of the soil samples and in 21% (6/29) of the soil samples containing A. fumigatus. High minimal inhibitory concentrations (MIC) of itraconazole (≥16 mg/L) and posaconazole (≥0.5 mg/L) were displayed by nine isolates from six different soil samples, namely apple orchard (1 sample), rose pot compost (2 samples), and cucurbit yields (3 samples). Seven isolates had a MIC=2 mg/L of voriconazole. Seven of nine itraconazole and posaconazole resistant isolates harboured the same TR34/L98H mutation of cyp51A. These findings, together with the occurrence of resistant clinical isolates, suggest that azole resistance should be considered in primary patient care.

Heterozygosis and pathogenicity of Cryptococcus neoformans AD-hybrid isolates.

Nineteen Cryptococcus neoformans AD-hybrid isolates were investigated to assess whether hybrid genomic background could affect virulence in a mouse model. The level of heterozygosity of each strain was analyzed using primers specific for allele A and D of 15 polymorphic genes. Virulence was tested in a mouse model of systemic infection by measuring time of survival. In addition, the putative virulence attributes, melanin, phospholipase, and capsule production, as well as growth at 39°C and UV sensitivity were investigated. Eight strains showed to be heterozygous in up to 70% of loci, other eight strains were heterozygous in less than 60% of loci, while the remaining three strains were homozygous at all tested loci. Mice infected with hybrids with a high percentage of heterozygosis showed significantly (P < 0.01) shorter survival than mice infected with the other hybrids. Mortality was not correlated with the mating-type locus pattern, as well as it was not correlated with the level of expression of the different virulence attributes investigated. The present study confirms that hybridization in C. neoformans could represent an important evolutionary driving force in increasing the fitness of this yeast in the environment and in the host.

Increased mortality in young candidemia patients associated with presence of a Candida albicans general-purpose genotype.

The yeast Candida albicans causes life-threatening candidemia. A general-purpose genotype (GPG; corresponds to clade 1) causes more infections than other C. albicans genotypes. To investigate if GPG strains also cause higher mortality, we developed a duplex PCR assay which was 98% accurate in identifying GPG strains in an international collection of strains typed with probe Ca3. We applied the assay to 635 European C. albicans candidemia isolates. Of these, 18% conformed to the GPG genotype, 4% were of a borderline genotype, and 78% were of a non-GPG genotype, broadly consistent with genotype distributions in earlier studies. The prevalence of GPG strains was increased in females and in younger patients, exceeding 40% in infants aged ≤1 year. Logistic regression confirmed sex and age as significant determinants of GPG prevalence. Across the entire patient cohort, there was no difference in mortality for patients infected with GPG strains or other strains (36% versus 37%). However, mortality in patients aged ≤48 years was 33% for infection with GPG strains but only 15% for infection with other strains (z test; P < 0.01). Mortality rates associated with GPG and non-GPG strains were comparable in older patients (39% versus 46%). A logistic regression analysis confirmed the age-dependent impact of genotype on mortality. Thus, GPG strains may be more virulent than other strains in younger patients. Because candidemia is usually caused by endogenous strains, our PCR assay could potentially be used as a risk assessment tool for identifying younger patients most at risk of death from candidemia.

Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis.

OBJECTIVES: To characterize the pathogenicity of 15 strains of Cryptococcus neoformans belonging to several serotype/mating type allele patterns (Dalpha, Da, A(alpha), A(a), A(alpha)/D(a) and D(alpha)/A(a)) in experimental models of murine cryptococcosis. METHODS: CD1-infected mice were examined for survival and fungal loads in either brain or lung during the course of infection. RESULTS: All strains, with the exception of one Da strain, produced melanin in vitro. Similarly, all strains were encapsulated and produced phospholipase. When CD1 mice were challenged intravenously (i.v.) with 5x10(5)CFU/mouse and observed for 60 days post-infection, a significant variation of mortality rate was observed among mice infected with different strains. A(alpha) and A(alpha)/D(a) strains all produced 100% mortality within the study period with mean survivals significantly shorter than those of mice infected with strains belonging to any other allele type (P<0.0001). A wide range of pathogenicity was shown by haploid and diploid strains presenting D(alpha) allele. This finding was confirmed by an intranasal model of challenge. To investigate the progression of infection, the mice were challenged i.v. with 5x10(4)CFU/mouse and tissue burden experiments (brain and lung) were performed on days 6 and 12 post-infection. Only the mice infected with A(alpha) and A(alpha)/D(a) strains showed a >1 log(10) increase of CFU/g in both tissues throughout the study period. CONCLUSIONS: Our results suggest that the presence of the A(alpha) mating type allele in either haploid or diploid strains is correlated with virulence, while the presence of the A(a) or D(a) allele in haploid strains is associated with moderate or no virulence. Finally, either haploid or diploid strains presenting D(alpha) allele vary in virulence.

Determination of Cryptococcus neoformans var. neoformans mating type by multiplex PCR.

Mating type plays an important role in the epidemiology and virulence of Cryptococcus neoformans. The present study designed a multiplex PCR method to distinguish the six mating type patterns (Aa, Da, Aalpha, Dalpha, Aa/Dalpha, and Aalpha/Da) of C. neoformans var. neoformans. PCR amplification identified one fragment for Aa (860 bp), Dalpha (413 bp) and Da (645 bp) strains, two fragments for Aalpha (320 and 400 bp) and Aa/Dalpha (860 and 413 bp) strains, and three fragments (645, 400, 320 bp) for an Aalpha/Da strain. The method appears to be a valid, simple and relatively inexpensive tool for epidemiological and virulence studies.