Current German Recommendations and International Research on the Use of COVID-19 Boosters among Health Care Providers in 2024: A Narrative Review.

While the World Health Organization (WHO) has de-escalated coronavirus disease 2019 (COVID-19) from a global health emergency, ongoing discussions persist as new viral variants. This article aimed to consolidate German recommendations and international research to offer health care providers (HCPs) a comprehensive guide on COVID-19 boosters in 2024. The review outlines key recommendations from the German Robert Koch Institute. HCPs should receive COVID-19 boosters at least 12 months after their last vaccination or COVID-19 infection, contingent on the prevalent viral variant(s) in the region. However, excessive doses and/or frequent boosters, especially with mRNA vaccines, may lead to immune imprinting, T-cell exhaustion, and immunoglobulin (Ig) switching. Notably, this review highlights the significance of Ig, particularly IgA and IgG subclasses, in influencing infection risk and disease progression. Furthermore, it explores the implications of mRNA vaccine technology and potential adverse effects related to excessive dosing. In conclusion, this article provides a comprehensive analysis of COVID-19 vaccine boosters for HCPs, synthesising current recommendations, scientific debates, and considerations for optimising protection against SARS-CoV-2 in the evolving landscape of the post-pandemic era.

BactoBattle: a game-based learning companion for medical bacteriology.

A card game called BactoBattle has been developed to help medical students who have just started learning medical bacteriology to improve their learning efficacy and satisfaction, especially on the topic of antimicrobial resistance. Copies of the game were placed in the students' study room (approximately 1 set per 12 students) and made available to the students throughout the study period so that they could choose to play the game during their free time if desired. After the study period had ended, the students were asked to complete a questionnaire and a post-test. In total, 33 students completed the questionnaire, and were split into 2 groups: the player group, comprising 12 (36.4 %) students who had played the game, and the non-player group. The player group perceived that they could memorize more knowledge compared to the non-player group and indeed recorded higher post-test scores than the non-player group (10.4 vs 8.3 out of 15 points, P=0.031). However, there was no difference in learning motivation (P=0.441) or enjoyment (P=0.562) between the two groups. A majority of the players said they would continue playing the game after the study period and would recommend the game to other students. In short, the BactoBattle game can be a useful tool to improve the learning efficacy of students, but its effect on learning satisfaction remains unclear.

In vitro modeling of isoniazid resistance mechanisms in Mycobacterium tuberculosis H37Rv.

Introduction: Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, has been a global threat to human beings for several decades. Treating tuberculosis has become more difficult as the prevalence of drug-resistant tuberculosis has increased globally. Evidence suggests that the comprehensive landscape of resistance mechanisms in MTB is ambiguous. More importantly, little is known regarding the series of events connected to resistance mechanisms in MTB before exposure to anti-TB drugs, during exposure to the drugs, and finally, when the MTB becomes resistant after exposure, upon analyses of its genome. Methods: We used the wild-type strain of MTB (H37Rv) in an in vitro model for generating induced resistance using a sub-inhibitory concentration of isoniazid, and the generated resistance-associated variants (RAVs) were identified using the whole genome sequencing method. Results: The detection of an inhA promoter mutation (fabG1-15C>T), which results in increased production of InhA protein, was found to be a major mechanism for developing resistance to isoniazid in the first place. We observed adaptation of MTB resistance mechanisms in high isoniazid stress by alteration and abolishment of KatG due to the detection of katG S315N, the common region of mutation that confers isoniazid resistance, along with katG K414N, katG N138S, and katG A162E. Furthermore, we detected the ahpC-72C>T and ahpC 21C>A mutations, but further investigation is needed to determine their role in compensating for the loss of KatG activity. Discussion: This suggests that increased InhA production is the main mechanism where there are low levels of isoniazid, whereas the alteration of KatG was found to be utilized in mycobacterium with a high concentration of isoniazid. Our work demonstrates that this in vitro approach of generating induced resistance could provide clinically relevant information after the fabG1-15C>T mutation, which is the common mutation found in clinical isolates. Moreover, other mutations detected in this work can also be found in clinical isolates. These findings may shed light on the impact of isoniazid in generating RAV and the resistance mechanism scenario that mycobacterium used under various isoniazid-pressuring conditions. More research is needed to understand better the role of RAV and mechanical resistance events within the mycobacterium genome in promoting a promising drug prediction platform that could lead to the right treatment for patients with MDR-TB and XDR-TB.

Effect of fenestrated sterile drape and face mask on bacterial dispersion toward the periocular area during intravitreal injection.

This experimental crossover study was performed to investigate whether fenestrated surgical drapes (covering the nose and mouth but with an opening over the periorbital area) with or without patients' surgical face masks increase periorbital bacterial dispersion during simulated intravitreal injection conditions. Each of the 16 healthy volunteers performed 14 scenarios involving different mask and drape conditions in both silent and speaking situations. In each scenario, the subject lay down flat on the back with a blood agar plate being held at the inferior orbital rim perpendicular to the face to capture airflow from breathing/speaking. Another blood agar plate placed 50 cm away from the subject served as an experimental control. A total of 224 experiments were performed. Speaking situations significantly showed more colony forming units (CFUs) compared with their controls (P = 0.014). There were no significant differences in CFUs between wearing vs not wearing the masks (P = 0.887 for speaking and P = 0.219 for silent) and using vs not using the drapes (P = 0.941 for speaking and P = 0.687 for silent). Reusable and disposable drapes were also not significantly different (P = 1.00 for speaking and P = 0.625 for silent). Streptococcus spp., the oropharyngeal microbiota, were only cultivated from speaking scenarios. While refraining from speaking (for both practitioners and patients) is the mainstay of reducing bacterial dispersion and risks of post-injection endophthalmitis, the use of fenestrated surgical drapes or patients' face masks did not significantly affect the amount of bacterial dispersion toward the periorbital area.

Clinical characteristics and diagnosis of intestinal tuberculosis in clinical practice at Thailand's largest national tertiary referral center: An 11-year retrospective review.

BACKGROUND: Diagnosing intestinal tuberculosis (ITB) is challenging due to the low diagnostic sensitivity of current methods. This study aimed to assess the clinical characteristics and diagnosis of ITB at our tertiary referral center, and to explore improved methods of ITB diagnosis. METHODS: This retrospective study included 177 patients diagnosed with ITB at Siriraj Hospital (Bangkok, Thailand) during 2009-2020. RESULTS: The mean age was 49 years, 55.4% were male, and 42.9% were immunocompromised. Most diagnoses (108/177) were made via colonoscopy; 12 patients required more than one colonoscopy. Among those, the sensitivity of tissue acid-fast bacilli (AFB), presence of caseous necrosis, polymerase chain reaction (PCR), and culture was 40.7%, 13.9%, 25.7%, and 53.4%, respectively. Among patients with negative tissue histopathology, 4 (3.7%) and 13 (12.0%) were ITB positive on tissue PCR and culture, respectively. The overall sensitivity when all diagnostic methods were used was 63%. Seventy-six patients had stool tests for mycobacteria. The overall sensitivity of stool tests was 75.0%. However, when analyzing the 31 patients who underwent both endoscopy and stool testing, the sensitivity of stool testing when using tissue biopsy as a reference was 45.8%. Combining stool testing and tissue biopsy did not significantly increase the sensitivity compared to tissue biopsy alone (83.9% vs. 77.4%, respectively). CONCLUSION: Despite the availability of PCR and culture for TB, the overall diagnostic sensitivity was found to be low. The sensitivity increased when the tests were used in combination. Repeated colonoscopy may be beneficial. Adding stool mycobacteria tests did not significantly increase the diagnostic yield if endoscopy was performed, but it could be beneficial if endoscopy is unfeasible.

The Risk Factors and Mechanisms of Azole Resistance of Candida tropicalis Blood Isolates in Thailand: A Retrospective Cohort Study.

In recent decades, an epidemiological shift has been observed from Candida infections to non-albicans species and resistance to azoles. We investigated the associated factors and molecular mechanisms of azole-resistant blood isolates of C. tropicalis. Full-length sequencing of the ERG11 gene and quantitative real-time RT-PCR for the ERG11, MDR1, and CDR1 genes were performed. Male sex (odds ratio, 0.38), leukemia (odds ratio 3.15), and recent administration of azole (odds ratio 10.56) were associated with isolates resistant to azole. ERG11 mutations were found in 83% of resistant isolates, with A395T as the most common mutation (53%). There were no statistically significant differences in the expression of the ERG11, MDR1, and CDR1 genes between the groups resistant and susceptible to azole. The prevalence of azole-resistant isolates was higher than the usage of antifungal drugs, suggesting the possibility of environmental transmission in the healthcare setting. The unknown mechanism of the other 17% of the resistant isolates remains to be further investigated.

Assessment of Biofilm Formation by Candida albicans Strains Isolated from Hemocultures and Their Role in Pathogenesis in the Zebrafish Model.

Candida albicans, an opportunistic pathogen, has the ability to form biofilms in the host or within medical devices in the body. Biofilms have been associated with disseminated/invasive disease with increased severity of infection by disrupting the host immune response and prolonging antifungal treatment. In this study, the in vivo virulence of three strains with different biofilm formation strengths, that is, non-, weak-, and strong biofilm formers, was evaluated using the zebrafish model. The survival assay and fungal tissue burden were measured. Biofilm-related gene expressions were also investigated. The survival of zebrafish, inoculated with strong biofilms forming C. albicans,, was significantly shorter than strains without biofilms forming C. albicans. However, there were no statistical differences in the burden of viable colonogenic cell number between the groups of the three strains tested. We observed that the stronger the biofilm formation, the higher up-regulation of biofilm-associated genes. The biofilm-forming strain (140 and 57), injected into zebrafish larvae, possessed a higher level of expression of genes associated with adhesion, attachment, filamentation, and cell proliferation, including eap1, als3, hwp1, bcr1, and mkc1 at 8 h. The results suggested that, despite the difference in genetic background, biofilm formation is an important virulence factor for the pathogenesis of C. albicans. However, the association between biofilm formation strength and in vivo virulence is controversial and needs to be further studied.

EQUAL Candida score, an effective tool for predicting the outcomes of Candida tropicalis candidaemia: A retrospective cohort study.

BACKGROUND: Candida tropicalis is the most common non-albicans Candida species found in Asia-Pacific countries, including Thailand. The pathogen is known for its great virulence, which causes a high case-fatality rate. Associations between case fatality and patient characteristics, infectious disease unit consultation and EQUAL Candida score were investigated. METHODS: This retrospective cohort study was conducted with 160 cases of C. tropicalis bloodstream infection between 2015 and 2019 at a single, large, tertiary centre in Thailand. Clinical characteristics, clinical presentations, patient outcomes (30-day case-fatality rate) and independent predictive factors were analysed. RESULTS: The 30-day case-fatality rate was 68.1%. The median of the EQUAL Candida score was 8. Independent factors for the prediction of case fatality were septic shock (hazard ratio, 1.84), the use of mechanical ventilation (hazard ratio, 2.03) and the EQUAL Candida score (hazard ratio, 0.75). CONCLUSIONS: The predictive factors for 30-day case fatality were septic shock, mechanical ventilation use and the EQUAL Candida score. It is recommended that the EQUAL score be considered for patients infected with C. tropicalis candidaemia to reduce the case-fatality rate.

Immunopathogenesis of Emerging Candida auris and Candida haemulonii Strains.

The emergence of a multidrug-resistant Candida species, C. auris and C. haemulonii, has been reported worldwide. In Thailand, information on them is limited. We collected clinical isolates from Thai patients with invasive candidiasis. Both species were compared with a laboratory C. albicans strain. In vitro antifungal susceptibility and thermotolerance, and pathogenesis in the zebrafish model of infection were investigated. Both species demonstrated high minimal inhibitory concentrations to fluconazole and amphotericin B. Only C. auris tolerated high temperatures, like C. albicans. In a zebrafish swim-bladder-inoculation model, the C. auris-infected group had the highest mortality rate and infectivity, suggesting the highest virulence. The case fatality rates of C. auris, C. haemulonii, and C. albicans were 100%, 83.33%, and 51.52%, respectively. Further immunological studies revealed that both emerging Candida species stimulated genes involved in the proinflammatory cytokine group. Interestingly, the genes relating to leukocyte recruitment were downregulated only for C. auris infections. Almost all immune response genes to C. auris had a peak response at an early infection time, which contrasted with C. haemulonii. In conclusion, both emerging species were virulent in a zebrafish model of infection and could activate the inflammatory pathway. This study serves as a stepping stone for further pathogenesis studies of these important emerging species.

Emergence of Multi-Drug Resistance and Its Association With Uncommon Serotypes of Streptococcus agalactiae Isolated From Non-neonatal Patients in Thailand.

Group B streptococcus (GBS) or Streptococcus agalactiae is an opportunistic pathogen that causes serious illness in newborns, pregnant women, and adults. However, insufficient detection methods and disease prevention programs have contributed to an increase in the incidence and fatality rates associated with this pathogen in non-neonatal patients. This study aimed to investigate factors of the observed increased incidence by investigation of serotype distribution, virulence factors, and antimicrobial susceptibility patterns from invasive GBS disease among non-neonatal patients in Thailand. During 2017-2018, a total of 109 S. agalactiae isolates were collected from non-pregnant patients. There were 62 males and 47 females, with an average age of 63.5 years (range: 20 - 96). Serotypes were determined by latex agglutination assay and multiplex polymerase chain reaction (PCR)-based assay. Among those isolates, seven virulence genes (rib, bca, pavA, lmb, scpB, cylE, and cfb) were detected by PCR amplification, and were determined for their susceptibility to 20 antimicrobial agents using a SensititreTM Streptococcus species STP6F AST plate. Among the study isolates, serotype III was predominant (52.3%), followed by serotype V and serotype VI (13.8% for each), serotype Ib (11.9%), and other serotypes (8.2%). Of the seven virulence genes, pavA was found in 67.0%. Except for one, there were no significant differences in virulence genes between serotype III and non-serotype III. Study isolates showed an overall rate of non-susceptibility to penicillin, the first-line antibiotic, of only 0.9%, whereas the resistance rates measured in tetracycline, clindamycin, azithromycin, and erythromycin were 41.3, 22.0, 22.0, and 22.0%, respectively. Strains that were resistant to all four of those drugs were significantly associated with non-serotype III (p < 0.001). Using multi-locus sequence typing (MLST), 40.0% of the four-drug-resistant isolates belonged to serotype VI/ST1, followed by serotype Ib/ST1 (35.0%). Cluster analysis with global GBS isolates suggested that the multiple drug-resistant isolates to be strongly associated with the clonal complex (CC) 1 (p < 0.001). Compared to the 2014 study of 210 invasive GBS isolates conducted in 12 tertiary hospitals in Thailand, the proportion of serotype III has dramatically dropped from nearly 90% to about 50%. This suggests that resistances to the second-line antibiotics for GBS might be the selective pressure causing the high prevalence of non-serotype III isolates.

Invasive Fungal Diseases in Children with Acute Leukemia and Severe Aplastic Anemia.

Although the outcomes of childhood leukemia and severe aplastic anemia (SAA) have improved, infectious complications are still the major concern. Particularly worrisome are invasive fungal diseases (IFDs), one of the most common causes of infectious-related deaths in patients with prolonged neutropenia. A retrospective study was conducted of IFDs in pediatric patients with newly diagnosed or relapsed acute leukemia, or with SAA, at Siriraj Hospital, Mahidol University, Thailand. There were 241 patients: 150 with acute lymphoblastic leukemia (ALL), 35 with acute myeloid leukemia (AML), 31 with relapsed leukemia, and 25 with SAA. Their median age was 5.4 years (range, 0.3-16.0 years). The overall IFD prevalence was 10.7%, with a breakdown in the ALL, AML, relapsed leukemia, and SAA patients of 8%, 11.4%, 19.3%, and 16%, respectively. Pulmonary IFD caused by invasive aspergillosis was the most common, accounting for 38.5% of all infection sites. Candidemia was present in 34.6% of the IFD patients; Candida tropicalis was the most common organism. The overall case-fatality rate was 38.5%, with the highest rate found in relapsed leukemia (75%). The incidences of IFDs in patients with relapsed leukemia and SAA who received fungal prophylaxis were significantly lower than in those who did not (P = N/A and 0.04, respectively). IFDs in Thai children with hematological diseases appeared to be prevalent, with a high fatality rate. The usage of antifungal prophylaxes should be considered for patients with SAA to prevent IFDs.

Gastrointestinal mucormycosis due to Rhizopus microsporus following Streptococcus pyogenes toxic shock syndrome in an HIV patient: a case report.

BACKGROUND: Gastrointestinal (GI) mucormycosis is a rare and often deadly form of mucormycosis. Delayed diagnosis can lead to an increased risk of death. Here, we report a case of GI mucormycosis following streptococcal toxic shock syndrome in a virologically suppressed HIV-infected patient. CASE PRESENTATION: A 25-year-old Thai woman with a well-controlled HIV infection and Grave's disease was admitted to a private hospital with a high-grade fever, vomiting, abdominal pain, and multiple episodes of mucous diarrhea for 3 days. On day 3 of that admission, the patient developed multiorgan failure and multiple hemorrhagic blebs were observed on all extremities. A diagnosis of streptococcal toxic shock was made before referral to Siriraj Hospital - Thailand's largest national tertiary referral center. On day 10 of her admission at our center, she developed feeding intolerance and bloody diarrhea due to bowel ischemia and perforation. Bowel resection was performed, and histopathologic analysis of the resected bowel revealed acute suppurative transmural necrosis and vascular invasion with numerous broad irregular branching non-septate hyphae, both of which are consistent with GI mucormycosis. Peritoneal fluid fungal culture grew a grayish cottony colony of large non-septate hyphae and spherical sporangia containing ovoidal sporangiospores. A complete ITS1-5.8S-ITS2 region DNA sequence analysis revealed 100% homology with Rhizopus microsporus strains in GenBank (GenBank accession numbers KU729104 and AY803934). As a result, she was treated with liposomal amphotericin B. However and in spite of receiving appropriate treatment, our patient developed recurrent massive upper GI bleeding from Dieulafoy's lesion and succumbed to her disease on day 33 of her admission. CONCLUSION: Diagnosis of gastrointestinal mucormycosis can be delayed due to a lack of well-established predisposing factors and non-specific presenting symptoms. Further studies in risk factors for abdominal mucormycosis are needed.

Population diversity and virulence characteristics of Cryptococcus neoformans/C. gattii species complexes isolated during the pre-HIV-pandemic era.

Cryptococcosis has become a major global health problem since the advent of the HIV pandemic in 1980s. Although its molecular epidemiology is well-defined, using isolates recovered since then, no pre-HIV-pandemic era epidemiological data exist. We conducted a molecular epidemiological study using 228 isolates of the C. neoformans/C. gattii species complexes isolated before 1975. Genotypes were determined by URA5 restriction fragment length polymorphism analysis and multi-locus sequence typing. Population genetics were defined by nucleotide diversity measurements, neutrality tests, and recombination analysis. Growth at 37°C, melanin synthesis, capsule production, and urease activity as virulence factors were quantified. The pre-HIV-pandemic isolates consisted of 186 (81.5%) clinical, 35 (15.4%) environmental, and 7 (3.1%) veterinary isolates. Of those, 204 (89.5%) belonged to C. neoformans VNI (64.0%), VNII (14.9%) and VNIV (10.5%) while 24 (10.5%) belonged to C. gattii VGIII (7.5%), VGI (2.6%) and VGII (0.5%). Among the 47 sequence types (STs) identified, one of VNII and 8 of VNIV were novel. ST5/VNI (23.0%) in C. neoformans and ST75/VGIII (25.0%) in C. gattii were the most common STs in both species complexes. Among C. neoformans, VNIV had the highest genetic diversity (Hd = 0.926) and the minimum recombination events (Rm = 10), and clinical isolates had less genetic diversity (Hd = 0.866) than environmental (Hd = 0.889) and veterinary isolates (Hd = 0.900). Among C. gattii, VGI had a higher nucleotide diversity (π = 0.01436) than in VGIII (π = 0.00328). The high-virulence genotypes (ST5/VNI and VGIIIa/serotype B) did not produce higher virulence factors levels than other genotypes. Overall, high genetic variability and recombination rates were found for the pre-HIV-pandemic era among strains of the C. neoformans/C. gattii species complexes. Whole genome analysis and in vivo virulence studies would clarify the evolution of the genetic diversity and/or virulence of isolates of the C. neoformans/C. gattii species complexes during the pre- and post-HIV-pandemic eras.

An Association of an eBURST Group With Triazole Resistance of Candida tropicalis Blood Isolates.

Candidemia, a bloodstream infection caused by genus Candida, has a high mortality rate. Candida albicans was previously reported to be the most common causative species among candidemia patients. However, during the past 10 years in Thailand, Candida tropicalis has been recovered from blood more frequently than C. albicans. The cause of this shift in the prevalence of Candida spp. remains unexplored. We conducted in vitro virulence studies and antifungal susceptibility profiles of 48 C. tropicalis blood isolates collected during 2015-2017. To compare to global isolates of C. tropicalis, multilocus sequence typing (MLST), a minimum spanning tree, and an eBURST analysis were also conducted. C. tropicalis and C. albicans were the most (47-48.7%) and second-most (21.5-33.9%) common species to be isolated from candidemia patients, respectively. Of the C. tropicalis blood isolates, 29.2, 0, 100, and 93.8% exhibited proteinase activity, phospholipase activity, hemolytic activity, and biofilm formation, respectively. Moreover, 20.8% (10/48) of the isolates were resistant to voriconazole and fluconazole, and also showed high minimum inhibitory concentrations (MICs) to posaconazole and itraconazole. In contrast, most of the isolates were susceptible to anidulafungin (97.9%), micafungin (97.9%), and caspofungin (97.9%), and showed low MICs to amphotericin B (100%) and 5-flucytosine (100%). The MLST identified 22 diploid sequence types. Based on the eBURST analysis and minimum spanning tree, 9 out of 13 members (69.2%) of an eBURST group 3 were resistant to voriconazole and fluconazole, and also showed high MICs to posaconazole and itraconazole. Association analysis revealed the eBURST group 3 was significantly associated with the four triazole resistance (p < 0.001). In conclusion, the eBURST group 3 was associated with the triazole resistance and resistance to many antifungal drugs might be collectively responsible for the prevalence shift.

Development of a New Scoring System To Accurately Estimate Learning Outcome Achievements via Single, Best-Answer, Multiple-Choice Questions for Preclinical Students in a Medical Microbiology Course.

During the preclinical years, single-best-answer multiple-choice questions (SBA-MCQs) are often used to test the higher-order cognitive processes of medical students (such as application and analysis) while simultaneously assessing lower-order processes (like knowledge and comprehension). Consequently, it can be difficult to pinpoint which learning outcome has been achieved or needs improvement. We developed a new scoring system for SBA-MCQs using a step-by-step methodology to evaluate each learning outcome independently. Enrolled in this study were third-year medical students (n = 316) who had registered in the basic microbiology course at the Faculty of Medicine, Siriraj Hospital, Mahidol University during the academic year 2017. A step-by-step SBA-MCQ with a new scoring system was created and used as a tool to evaluate the validity of the traditional SBA-MCQs that assess two separate outcomes simultaneously. The scores for the two methods, in percentages, were compared using two different questions (SBA-MCQ1 and SBA-MCQ2). SBA-MCQ1 tested the students' knowledge of the causative agent of a specific infectious disease and the basic characteristics of the microorganism, while SBA-MCQ2 tested their knowledge of the causative agent of a specific infectious disease and the pathogenic mechanism of the microorganism. The mean score obtained with the traditional SBA-MCQs was significantly lower than that obtained with the step-by-step SBA-MCQs (85.9% for the traditional approach versus 90.9% for step-by-step SBA-MCQ1; p < 0.001; and 81.5% for the traditional system versus 87.4% for step-by-step SBA-MCQ2; p < 0.001). Moreover, 65.8% and 87.8% of the students scored lower with the traditional SBA-MCQ1 and the traditional SBA-MCQ2, respectively, than with the corresponding sets of step-by-step SBA-MCQ questions. These results suggest that traditional SBA-MCQ scores need to be interpreted with caution because they have the potential to underestimate the learning achievement of students. Therefore, the step-by-step SBA-MCQ is preferable to the traditional SBA-MCQs and is recommended for use in examinations during the preclinical years.

Cryptococcus neoformans/gattii Species Complexes from Pre-HIV Pandemic Era Contain Unusually High Rate of Non-Wild-Type Isolates for Amphotericin B.

INTRODUCTION: The Cryptococcus neoformans/gattii species complexes are a leading cause of fatality among HIV-infected patients. Despite the unavailability of clinical breakpoints (CBPs) for antifungal agents, epidemiological cutoff values (ECVs) were recently proposed, and non-wild-type isolates for polyenes and azoles are being increasingly reported. However, the distributions of the susceptibility patterns for pre-HIV-era isolates have not been studied. METHODS: We determined the in vitro antifungal susceptibility patterns of 233 Cryptococcus isolates, collected at the National Institutes of Health, USA, in pre-HIV pandemic era, to study minimum inhibitory concentrations (MICs) to the important drugs for cryptococcosis and to compare the results with strain genotypes. Amphotericin B susceptibility was compared to published ECV of C. neoformans. RESULTS: The 233 Cryptococcus strains consisted of 89.7% C. neoformans species complex and 10.3% C. gattii species complex. Most were from clinical sources (189, 81.1%), and the major molecular type was VNI (146, 62.7%). The highest geometric mean (GM) was observed for fluconazole (GM = 0.96 µg/mL) while the lowest was for itraconazole (GM = 0.10 µg/mL). MICs to fluconazole in C. gattii species complex were significantly higher than C. neoformans species complex (p < 0.001). Moreover, C. neoformans/VNI strains showed significantly higher MICs than others such as C. neoformans/VNII to fluconazole (p < 0.0001) and C. deneoformans/VNIV to amphotericin B (p = 0.022) and fluconazole (p = 0.008). In our collection of 167 clinical C. neoformans species complex strains, 85 (50.9%), 24 (14.4%), and 3 (1.8%) strains had an amphotericin B (AMB)-MIC of 1, 2, and 4 µg/mL, respectively. The high percentage (66.9%, 79/118 strains) of non-wild-type clinical C. neoformans VNI strains, using an AMB-ECV of 0.5 µg/mL, was found. Moreover, 25 of 28 (89.3%) C. neoformans VNI strains from environmental and veterinary sources also had AMB-MICs above 0.5 µg/mL. In general, there was no significant difference in GM AMB-MIC of the clinical strains isolated from patients with (35 patients) and without (78 patients) prior AMB treatment (0.85 vs 0.76; p = 0.624). GM MIC of the environmental strains was not significantly different from that of the prior AMB-treatment strains (0.98 vs 0.76, p = 0.159) and the post-AMB-treatment strains (0.98 vs 0.85, p = 0.488). CONCLUSION: The high rate of non-wild-type among these otherwise naive isolates to amphotericin B is unexpected. Confirmation with more strains from a later era is needed.

Contribution of Laccase Expression to Immune Response against Cryptococcus gattii Infection.

Cryptococcosis is an infectious disease caused by two fungal species, Cryptococcus neoformans and Cryptococcus gattii While C. neoformans affects mainly immunocompromised patients, C. gattii infects both immunocompetent and immunocompromised individuals. Laccase is an important virulence factor that contributes to the virulence of C. neoformans by promoting pulmonary growth and dissemination to the brain. The presence of laccase in C. neoformans can shift the host immune response toward a nonprotective Th2-type response. However, the role of laccase in the immune response against C. gattii remains unclear. In this study, we characterized laccase activity in C. neoformans and C. gattii isolates from Thailand and investigated whether C. gattii that is deficient in laccase might modulate immune responses during infection. C. gattii was found to have higher laccase activity than C. neoformans, indicating the importance of laccase in the pathogenesis of C. gattii infection. The expression of laccase promoted intracellular proliferation in macrophages and inhibited in vitro fungal clearance. Mice infected with a lac1Δ mutant strain of C. gattii had reduced lung burdens at the early but not the late stage of infection. Without affecting type-1 and type-2 responses, the deficiency of laccase in C. gattii induced cryptococcus-specific interleukin-17 (IL-17) cytokine, neutrophil accumulation, and expression of the neutrophil-associated cytokine gene Csf3 and chemokine genes Cxcl1, Cxcl2, and Cxcl5 in vivo, as well as enhanced neutrophil-mediated phagocytosis and killing in vitro Thus, our data suggest that laccase constitutes an important virulence factor of C. gattii that plays roles in attenuating Th17-type immunity, neutrophil recruitment, and function during the early stage of infection.

A Predominance of Clade 17 Candida albicans Isolated From Hemocultures in a Tertiary Care Hospital in Thailand.

Candida albicans is one of the most common human fungal pathogens. Candidemia has significant mortality globally. No epidemiological study of C. albicans based on multilocus sequence typing (MLST) has been conducted in Thailand. Therefore, MLST was used to study the molecular epidemiology of C. albicans blood strains in a large Thai teaching hospital. In vitro virulence phenotypes and antifungal susceptibility testing by broth microdilution were also conducted. Forty-six C. albicans blood strains from 37 patients were collected from the Department of Microbiology, Siriraj Hospital, in 2016 and 2017. Most patients (71.8%) were more than 60 years old, and the case fatality rate was 54.8%. The male-to-female ratio was 5:3. Thirty-four diploid sequence types (DSTs), including six new DSTs, were identified, with DST2514 (8.7%) and DST2876 (8.7%) as the most common DSTs. Strains were clustered into nine clades. Unlike other studies of C. albicans blood strains in Asia, clade 17 was the most common (13 strains, 28.3%). Sequential allelic changes were evident in sequential strains from one patient. All strains produced phospholipase and hemolysin, while none produced proteinase. The ability to form biofilm was found in 82.6% of the strains. Clade 17 strains showed significantly stronger hemolytic activity than non-clade 17 strains (69.2% versus 27.3%; p = 0.022). However, no significant association existed between clades and patient mortalities. All were susceptible or wild type to anidulafungin (MIC range = 0.015-0.12 and GM = 0.030), micafungin (MIC range = ≤ 0.008-0.015 and GM = 0.008), caspofungin (MIC range = 0.008-0.12 and GM = 0.036), and amphotericin B (MIC range = 0.25-0.5 and GM = 0.381). Only one strain was resistant to voriconazole (MIC range = ≤ 0.008 to ≥ 8 and GM = 0.010) and fluconazole (MIC range = 0.12-16 and GM = 0.398). In conclusion, a high prevalence of clade 17 C. albicans blood strains was found in Thailand, in contrast to other Asian countries. This unique finding might be explained by the strong hemolytic activity that is required for bloodstream infection of C. albicans.

Association of ubiA mutations and high-level of ethambutol resistance among Mycobacterium tuberculosis Thai clinical isolates.

Ethambutol (EMB) is the first-line antituberculosis drug and a potential supplementary agent for a treatment regimen of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). It has long been known that mutations in embCAB operon, encoding EMB target, arabinosyltransferase, confer resistance to EMB. Recently, ubiA was additionally reported to be specifically associated with high-level EMB resistance in Mycobacterium tuberculosis. However, such information on ubiA is very limited. This study aimed to investigate correlations between mutations in ubiA and phenotypic EMB resistance among EMB-resistant (EMBR) M. tuberculosis Thai clinical isolates. Minimum inhibitory concentration (MIC) level of EMB and ubiA sequences were determined and analyzed. Of 68 EMBR-MDR isolates, 8.9% harbored mutations in ubiA. However, 10.0% and 46.6% of EMB-sensitive (EMBS)-MDR and pan-susceptible isolates also had ubiA mutations detected, respectively. Most nonsynonymous mutations, L31P, A35S, and V55M were only found in the EMBR-MDR isolates except E149D which was also found in EMBS-MDR and pan-susceptible isolates. A further phylogenetic analysis based on spoligotyping and IS6110-RFLP illustrated that E149D was in fact associated to EAI-families rather than EMB resistance. By excluding synonymous mutations and the E149D, we found a high correlation between ubiA mutations and high-level of EMB resistance with 100.0% specificity. In conclusion, despite its rare occurrence, mutations in ubiA can potentially be a marker for a detection of high level of EMB resistance at least in the MDR M. tuberculosis background.