A comparison of efficacy of parenteral and perineural dexmedetomidine with 0.25% ropivacaine for post-thyroidectomy analgesia using bilateral superficial cervical plexus block.

Background and Aims: Opioids are conventionally used for post-thyroidectomy pain, regional anesthesia is becoming popular due to its feasibility and efficacy in minimizing use of opioids and hence its side effects. This study compared analgesic efficacy of bilateral superficial cervical plexus block (BSCPB) using perineural and parenteral dexmedetomidine with 0.25% ropivacaine in thyroidectomy patients. Material and Methods: In this double-blind study, 60 American Society of Anesthesiologists (ASA) physical status I and II thyroidectomy patients, aged 18-65 years were randomized into two groups. Group A (n = 30) received BSCPB with 0.25% ropivacaine, 10 mL on each side with dexmedetomidine 0.5 µg/kg IV infusion. Group B (n = 30) received 0.25% ropivacaine plus dexmedetomidine 0.5 µg/kg, ten mL on each side. Duration of analgesia by measuring pain visual analog scores (VAS), total dose of analgesic requirement, Haemodynamics parameters and adverse events were recorded for 24 h. Categorical variables were analyzed using Chi-square test and continuous variables were computed as mean with standard deviation and analyzed using independent sample t-test. Mann-Whitney U test was used for analysis of ordinal variables. Results: Time to rescue analgesia was longer in Group B (18.6 ± 3.27 h) as compared to Group A (10.2 ± 2.11 h) (P < 0.001). Total analgesic dose required was also found to be lesser in Group B (50.83 ± 20.37 mg) as compared to Group A (73.33 ± 18.27 mg) (P < 0.001). No significant hemodynamic changes or side effects were observed in both groups; (P > 0.05). Conclusion: Perineural dexmedetomidine with ropivacaine in BSCPB significantly prolonged the duration of analgesia with reduced rescue analgesic requirement.

Post-operative analgesic efficacy of 0.25% ropivacaine with dexmedetomidine versus dexamethasone as an adjuvant in bilateral superficial cervical plexus block for thyroidectomy under general anaesthesia - A comparative randomized clinical study.

Background and Aims: The bilateral superficial cervical plexus block (BSCPB) is efficacious for post-operative analgesia in thyroid surgeries. We assessed the analgesic efficacy of dexmedetomidine and dexamethasone administered as adjuvants with 0.25 percent ropivacaine in BSCPB for thyroidectomy under general anaesthesia with regard to the duration of analgesia, total amount of rescue analgesic requirement, changes in intra- and post-operative haemodynamic parameters, VAS scores, and adverse events, if any. Methods: A prospective double-blind trial was planned with 80 adults undergoing thyroidectomy, randomized into two equal groups and given BSCPB with 20 ml 0.25% ropivacaine with adjuvants as either dexmedetomidine 50mg (group A) or dexamethasone 4mg (group B), 10 ml on each side, after the induction of general anaesthesia. Post-operative pain was monitored using the visual analog scale and the duration of analgesia was measured by time to first rescue analgesia. Post-operative haemodynamics and any adverse events were recorded. Results: The mean duration of analgesia was slightly prolonged in group A but statistically non-significant as compared to group B (1037 ± 97 vs. 1004 ± 122 minutes; P0.18). The post-operative median VAS scores and vital parameters were relatively comparable for both groups (P > 0.05) for the first 24h. There was a significant reduction in the incidence of PONV (P < 0.05) in group B. Conclusion: Although dexamethasone offers a slight advantage of decreased incidence of PONV, BSCPB using ropivacaine with either dexmedetomidine or dexamethasone as an adjuvant imparted adequate analgesia with stable haemodynamics and may be used as a pre-emptive analgesic technique in thyroid surgeries.

Comparison of Effects of Oral Melatonin with Oral Alprazolam used as a Premedicant in Adult Patients Undergoing Various Surgical Procedures under General Anesthesia: A Prospective Randomized Placebo-Controlled Study.

BACKGROUND AND AIMS: Preoperative anxiety is a major concern in patients undergoing any surgical procedure. Various drugs have been used to alleviate it. This study aims to compare the effects of oral melatonin versus oral alprazolam on preoperative anxiety, sedation, orientation, and cognitive function. MATERIALS AND METHODS: A total of 90 adult patients were randomly allocated into three groups. Group M (n = 30) received oral melatonin 3 mg (2 tablets); Group A (n = 30) received oral alprazolam 0.25 mg (2 tablets), and Group P (n = 30) received placebo, multivitamin tablet (2 tablets) as premedication. The objectives of our study were to compare anxiety, sedation, orientation, and cognitive function using visual analog scale (VAS) anxiety score, Ramsay Sedation Score (RSS), orientation score, and Digit symbol substitution test (DSST) score before and after 120 min of giving premedication. For statistical analysis, paired t-test, Chi-square test, analysis of variance, and post hoc bonferroni test were used. RESULTS: Mean VAS scores for anxiety (after 120 min) were significantly reduced in Group M (3.9 ± 1.53) and Group A (4.43 ± 1.67) when compared to Group P (4.73 ± 0.82) (P < 0.05). RSS was higher in Group A as compared to Group M and Group P (P < 0.05). No significant difference in orientation score was observed in all the three groups before and after premedication (P > 0.05). DSST score (cognitive function) was better in Group M when compared to both Group A and Group P (P < 0.05). CONCLUSION: Oral melatonin (6 mg) when used as a premedicant is an effective alternative to alprazolam regarding providing better anxiolysis, lesser sedation along with maintenance of cognitive, and psychomotor function.

Comparison between lignocaine nebulization and airway nerve block for awake fiberoptic bronchoscopy-guided nasotracheal intubation: a single-blind randomized prospective study.

BACKGROUND: The preferred management strategy for difficult airways is awake fiberoptic bronchoscopy-guided intubation, which requires effective airway anesthesia to ensure patient comfort and acceptance. This randomized single-blind prospective study was conducted to compare lignocaine nebulization and airway nerve block for airway anesthesia prior to awake fiberoptic bronchoscopy-guided intubation. METHODS: Sixty adult patients scheduled for surgical procedures under general anesthesia were randomly allocated to two groups. Group N received jet nebulization (10 ml of 4% lignocaine) and Group B received bilateral superior laryngeal and transtracheal recurrent laryngeal nerve blocks (each with 2 ml of 2% lignocaine) followed by fiberoptic bronchoscopy-guided nasotracheal intubation. All patients received procedural sedation with dexmedetomidine. The intubation time, intubating conditions, vocal cord position, cough severity, and degree of patient satisfaction were recorded. Student's t test was used to analyze parametric data, while the Mann-Whitney U test was applied to non-parametric data and Fisher's test to categorical data. P values < 0.05 were considered statistically significant. RESULTS: The time taken for intubation was significantly shorter in Group B [115.2 (14.7) s compared with Group N [214.0 (22.2) s] (P = 0.029). The intubating conditions and degree of patient comfort were better in Group B compared with Group N. Although all patients were successfully intubated, patient satisfaction was higher in Group B. CONCLUSIONS: Airway nerve blocks are preferable to lignocaine nebulization as they provide superior-quality airway anesthesia. However, nebulization may be a suitable alternative when a nerve block is not feasible.

Effects of Inhaled Nitrous Oxide on the Induction Dose and Time Requirements of Propofol: A Prospective, Randomized, Double-blind Study.

CONTEXT: Propofol is a commonly used induction agent during general anesthesia. As a sole agent, it does not provide any strong analgesic effect. The nitrous oxide (N2O) used along with propofol for induction of anesthesia augments the induction characteristics and reduces the dose of propofol. AIMS: To study the effects of inhaled N2O on the induction dose and time of propofol during general anesthesia and also its hemodynamic response and adverse effects. SETTINGS AND DESIGN: The present research is a prospective, randomized, double-blind comparative study. SUBJECTS AND METHODS: The study population consisted of eighty patients aged 18-60 years from either sex, American Society of Anesthesiologists physical status 1 and 2 which were scheduled for various elective surgical procedures under general anesthesia. The patients were randomly allocated into two groups comprising forty patients in each group. All patients were premedicated with glycopyrrolate 0.2 mg, ondansetron 4 mg, and fentanyl 1 µg/kg intravenously. Group FN received breathing mixture of gases (67% N2O @ 4 L/min and 33% O2 @ 2 L/min), and propofol and Group FO received 100% O2 @ 6 L/min and propofol. The different hemodynamic parameters (heart rate, mean arterial pressure, systolic blood pressure, diastolic blood pressure, and SpO2) were measured. STATISTICAL ANALYSIS: All observations were analyzed using Chi-square test, Student's t-test, and analysis of variance. RESULTS: The mean induction time and dose were significantly less in Group FN as compared to Group FO (P < 0.05). The mean induction time was 172 ± 32 s in Group FN as compared to 242 ± 43 s in Group FO (P < 001), whereas the mean induction dose was 56.10 ± 13.92 mg in Group FN as compared to 81.67 ± 17.64 mg in Group FO (P < 0.05). The hemodynamic parameters remained stable with no complications. CONCLUSION: The coadministration of N2O during induction of anesthesia with propofol not only reduced the induction dose of propofol but also reduced induction time significantly. Furthermore, it provided stable hemodynamics without any complications.

Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence.

The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans' replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2Δ) was generated, and confirmed a predicted shortened RLS in sir2Δ cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2Δ cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal's effect. Importantly, no difference in virulence was observed with drug treatment when sir2Δ cells were used for infection, which confirmed target specificity and ruled out non-specific effects of the drugs on the Galleria host. Thus, this study suggests that RLS modulating drugs, such as Sir2p agonists, shift lifespan and vulnerability of the fungal population, and should be further investigated as a potential class of novel antifungal drug targets that can enhance antifungal efficacy.

Effect of bispectral index versus end-tidal anesthetic gas concentration-guided protocol on time to tracheal extubation for halothane-based general anesthesia.

BACKGROUND AND AIMS: Early extubation is a desirable goal after general anesthesia. Very few studies have compared the effect of bispectral index (BIS) monitoring versus standard end-tidal anesthetic gas (ETAG) concentration monitoring on tracheal extubation time for halothane-based anesthesia. The aim of this study was to compare the effect of BIS versus ETAG-guided anesthesia on time to tracheal extubation for halothane-based anesthesia in general surgical setting. METHODS: This was a randomized, controlled double-blind study. Sixty patients with the American Society of Anesthesiologists physical status Class 1 or 2, receiving halothane-based general anesthesia were randomized to BIS-guided (n = 30) and ETAG-guided anesthesia (n = 30). Time to tracheal extubation was measured. In BIS group, BIS value was kept between 40 and 60 while in ETAG group; ETAG value was kept between 0.7 and 1.3 minimum alveolar concentration. The two groups were compared using Student's t-test, and P < 0.05 was considered statistically significant. Data were processed and analyzed using SPSS version 17 software. RESULTS: Mean time to tracheal extubation was significantly longer in BIS group (9.63 ± 3.02 min) as compared to ETAG group (5.29 ± 1.51 min), mean difference 4.34 min with 95% confidence interval (3.106, 5.982) (P < 0.05). CONCLUSION: In our study, the extubation time was significantly longer in BIS-guided anesthesia as compared to ETAG-guided anesthesia. ETAG monitoring promotes earlier extubation of patients as compared to BIS monitoring during halothane anesthesia.

ALL2, a Homologue of ALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 (ALL1) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 (ALL2). ALL2's function was characterized by generating null mutants in C. neoformans. In contrast to ALL1, loss of ALL2 attenuated virulence in the pulmonary infection model. The all2Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, the all2Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. Unlike ALL1's involvement in iron homeostasis, we now present data on ALL2's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight that C. neoformans, a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche.

Anesthetic considerations in a child with unrepaired D-transposition of great arteries undergoing noncardiac surgery.

D-transposition of great arteries (D-TGA) is the most common cyanotic congenital heart disease diagnosed at birth. There is ventriculoarterial discordance leading to parallel circulation. The postnatal survival depends on intercirculatory mixing of oxygenated and deoxygenated blood at various levels through atrial septal defect, ventricular septal defect or patent ductus arteriosus. The anesthesiologist must have an understanding of concepts of shunting and other long-term consequences of transposition of great arteries (TGA) in order to tailor the anesthetic technique to optimize the hemodynamic variables and oxygenation in the perioperative period. The preoperative evaluation includes echocardiography to delineate the type of TGA, associated lesions and extent and direction of shunts. Oxygen saturation is influenced by the ratio of pulmonary vascular resistance (PVR) to systemic vascular resistance. Thus, care should be taken to avoid an increase in PVR which can lead to decreased pulmonary blood flow leading to hypoxia. We report a case of an 8-year-old child with unrepaired D-TGA, who presented to us for craniotomy for drainage of brain abscess.

Microrheology with optical tweezers: measuring the relative viscosity of solutions 'at a glance'.

We present a straightforward method for measuring the relative viscosity of fluids via a simple graphical analysis of the normalised position autocorrelation function of an optically trapped bead, without the need of embarking on laborious calculations. The advantages of the proposed microrheology method are evident when it is adopted for measurements of materials whose availability is limited, such as those involved in biological studies. The method has been validated by direct comparison with conventional bulk rheology methods, and has been applied both to characterise synthetic linear polyelectrolytes solutions and to study biomedical samples.

Estimating the attack rate of pregnancy-associated listeriosis during a large outbreak.

BACKGROUND: In 2011, a multistate outbreak of listeriosis linked to contaminated cantaloupes raised concerns that many pregnant women might have been exposed to Listeria monocytogenes. Listeriosis during pregnancy can cause fetal death, premature delivery, and neonatal sepsis and meningitis. Little information is available to guide healthcare providers who care for asymptomatic pregnant women with suspected L. monocytogenes exposure. METHODS: We tracked pregnancy-associated listeriosis cases using reportable diseases surveillance and enhanced surveillance for fetal death using vital records and inpatient fetal deaths data in Colorado. We surveyed 1,060 pregnant women about symptoms and exposures. We developed three methods to estimate how many pregnant women in Colorado ate the implicated cantaloupes, and we calculated attack rates. RESULTS: One laboratory-confirmed case of listeriosis was associated with pregnancy. The fetal death rate did not increase significantly compared to preoutbreak periods. Approximately 6,500-12,000 pregnant women in Colorado might have eaten the contaminated cantaloupes, an attack rate of ~1 per 10,000 exposed pregnant women. CONCLUSIONS: Despite many exposures, the risk of pregnancy-associated listeriosis was low. Our methods for estimating attack rates may help during future outbreaks and product recalls. Our findings offer relevant considerations for management of asymptomatic pregnant women with possible L. monocytogenes exposure.

Cryptococcus neoformans ex vivo capsule size is associated with intracranial pressure and host immune response in HIV-associated cryptococcal meningitis.

BACKGROUND: The Cryptococcus neoformans polysaccharide capsule is a well-characterized virulence factor with immunomodulatory properties. The organism and/or shed capsule is postulated to raise intracranial pressure (ICP) in cryptococcal meningitis (CM) by mechanical obstruction of cerebrospinal fluid (CSF) outflow. Little is known regarding capsule phenotype in human cryptococcosis. We investigated the relationship of ex vivo CSF capsular phenotype with ICP and CSF immune response, as well as in vitro phenotype. METHODS: In total, 134 human immunodeficiency virus (HIV)-infected Ugandan adults with CM had serial lumbar punctures with measurement of CSF opening pressures, quantitative cultures, ex vivo capsule size and shedding, viscosity, and CSF cytokines; 108 had complete data. Induced capsular size and shedding were measured in vitro for 48 C. neoformans isolates. RESULTS: Cryptococcal strains producing larger ex vivo capsules in the baseline (pretreatment) CSF correlated with higher ICP (P = .02), slower rate of fungal clearance (P = .02), and paucity of CSF inflammation, including decreased CSF white blood cell (WBC) count (P < .001), interleukin (IL)-4 (P = .02), IL-6 (P = .01), IL-7 (P = .04), IL-8 (P = .03), and interferon γ (P = .03). CSF capsule shedding did not correlate with ICP. On multivariable analysis, capsule size remained independently associated with ICP. Ex vivo capsular size and shedding did not correlate with that of the same isolates grown in vitro. CONCLUSIONS: Cryptococcal capsule size ex vivo is an important contributor to virulence in human cryptococcal meningitis.

Multistate outbreak of listeriosis associated with cantaloupe.

BACKGROUND: Although new pathogen-vehicle combinations are increasingly being identified in produce-related disease outbreaks, fresh produce is a rarely recognized vehicle for listeriosis. We investigated a nationwide listeriosis outbreak that occurred in the United States during 2011. METHODS: We defined an outbreak-related case as a laboratory-confirmed infection with any of five outbreak-related subtypes of Listeria monocytogenes isolated during the period from August 1 through October 31, 2011. Multistate epidemiologic, trace-back, and environmental investigations were conducted, and outbreak-related cases were compared with sporadic cases reported previously to the Listeria Initiative, an enhanced surveillance system that routinely collects detailed information about U.S. cases of listeriosis. RESULTS: We identified 147 outbreak-related cases in 28 states. The majority of patients (127 of 147, 86%) were 60 years of age or older. Seven infections among pregnant women and newborns and one related miscarriage were reported. Of 145 patients for whom information about hospitalization was available, 143 (99%) were hospitalized. Thirty-three of the 147 patients (22%) died. Patients with outbreak-related illness were significantly more likely to have eaten cantaloupe than were patients 60 years of age or older with sporadic illness (odds ratio, 8.5; 95% confidence interval, 1.3 to ∞). Cantaloupe and environmental samples collected during the investigation yielded isolates matching all five outbreak-related subtypes, confirming that whole cantaloupe produced by a single Colorado farm was the outbreak source. Unsanitary conditions identified in the processing facility operated by the farm probably resulted in contamination of cantaloupes with L. monocytogenes. CONCLUSIONS: Raw produce, including cantaloupe, can serve as a vehicle for listeriosis. This outbreak highlights the importance of preventing produce contamination within farm and processing environments.

Allergen1 regulates polysaccharide structure in Cryptococcus neoformans.

Cryptococcus neoformans is an important human, fungal pathogen that sheds polysaccharide (exo-PS) into host tissues. While shed exo-PS mediates numerous untoward effects (including promoting increased intracranial pressure), little is known about the regulation of this phenomenon. Since downregulation of the Allergen 1 (ALL1) gene is associated with high ICP, we investigated the relationship between ALL1 expression and exo-PS structure using a variety of biophysical techniques. The Δall1 mutants of two serotypes produced a shorter exo-PS with less branching and structural complexity than the parental strains. Consistent with lower branching, these exo-PSs manifested higher intrinsic viscosity than the parental strains. The Δall1 mutant strains manifested differences in epitope expression and significant resistance to phagocytosis. Exo-PS of Δall1 mutant exhibited anti-phagocytic properties. Comparative transcriptome analysis of mutant and parental strain under iron-deprived conditions indicated a role of ALL1 in iron homeostasis, characterized by differential regulation of genes that mediate iron reduction and transport. Together, our results demonstrate a role of ALL1 in regulating conformational aspects of PS structure and iron homeostasis. These findings provide a mechanism to explain how changes in ALL1 expression influence virulence of switch variants and suggest that structural changes and polymer length are epigenetically regulated.

Pneumothorax complicating pulmonary embolism after combined spinal epidural anesthesia in a chronic smoker with open femur fracture.

Pulmonary embolism during or after regional anaesthesia is although very rare, it has been reported in cases undergoing lower limb orthopedic procedures. We presenting a 48 years old male, a known smoker since 25 years, with history of road traffic accident and open fracture right femur for external fixation. Combined spinal epidural anaesthesia was given. After 35 minutes patient complained dyspnea and chest pain. SpO2 decreased to 82% from 100%. Continuous positive airway pressure with 100% oxygen was given. SpO2 increased from 82% to 96%. Suddenly he had bouts of cough and SpO2 became 79-80% with unstable haemodynamics. On chest auscultation there was decreased breath sounds on right side with limited expansion. Trachea was intubated after inducing anaesthesia with fentanyl 70 µg and thiopental 300 mg. Chest radiograph showed right sided pneumothorax. Intercostal drain with a water seal was put. After 5 minutes HR was 80/min, BP was 110/69 mmHg and SpO2 was 97%. Pulmonary thromboembolism secondary to deep vein thrombosis was suspected and was confirmed by D-dimer Elisa and color Doppler of lower limbs. Patient was shifted to intensive care unit after completion of surgery. Anticoagulant therapy was started. He was weaned from the ventilator on 3rd day and trachea was extubated. Chest drain was removed after 9 days and he was discharged from hospital on 15th post operative day.

Antigenic and phenotypic variations in fungi.

Mechanisms to vary the phenotypic characteristics of fungi are diverse and can be important for their life cycle. This review summarizes phenotypic variability in fungi and divides this phenomenon into three topics: (i) morphological transitions, which are environmentally induced and involve the entire fungal population, (ii) reversible phenotypic switching between different colony morphologies, which is restricted to a small fraction of the population, and (iii) antigenic variation of surface antigens, which can be immuno-dominant epitopes happens in individual fungal cells.

Isolation and characterization of senescent Cryptococcus neoformans and implications for phenotypic switching and pathogenesis in chronic cryptococcosis.

Although several virulence factors and associated genes have been identified, the mechanisms that allow Cryptococcus neoformans to adapt during chronic infection and to persist in immunocompromised hosts remain poorly understood. Characterization of senescent cells of C. neoformans demonstrated that these cells exhibit a significantly enlarged cell body and capsule but still cross the blood-brain barrier. C. neoformans cells with advanced generational age are also more resistant to phagocytosis and killing by antifungals, which could promote their selection during chronic disease in humans. Senescent cells of RC-2, a C. neoformans strain that undergoes phenotypic switching, manifest switching rates up to 11-fold higher than those of younger cells. Infection experiments with labeled cells suggest that senescent yeast cells can potentially accumulate in vivo. Mathematical modeling incorporating different switching rates demonstrates how increased switching rates promote the emergence of hypervirulent mucoid variants during chronic infection. Our findings introduce the intriguing concept that senescence in eukaryotic pathogens could be a mechanism of microevolution that may promote pathoadaptation and facilitate evasion of an evolving immune response.

Biofilm formation in clinical Candida isolates and its association with virulence.

Biofilm formation, an important virulence trait of Candida species was measured in 107 Candida isolates from 32 candidemic patients by XTT [2,3-bis (2-methoxy-4nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] activity and compared to biofilm formation of Candida isolates from oropharyngeal lesions of 19 AIDS patients. Biofilm formation by XTT varied among species and C. albicans; C. lusitaniae and C. krusei produced more biofilm than the other Candida species. C. tropicalis was the most dominant species isolated from blood followed by C. albicans, and other non-albicans species whereas only C. albicans was recovered from oral lesions. Importantly, though Biofilm formation was variable within a species it was stable in sequential isolates during chronic infection. Sequential isolates exhibited identical Karyotype pattern or RAPD patterns unless patients were co-infected with more than one strain. High biofilm formation was associated with slow growth rate but not with adherence. Murine infection studies demonstrated that, degree of in-vitro biofilm formation was associated with virulence in mice, as mice infected both with no and low biofilm formers survived longer than mice infected with high biofilm former C. albicans (p< or =0.001). We conclude that biofilm formation is a stable but strain specific characteristic that can greatly vary among C. albicans and non-albicans strains, and plays an important role in persistence of infection.

Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans.

Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Delta gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Delta cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Delta mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.