Assessing students with dyslexia and other reading difficulties through multiple diagnostic assessments.

This article aims to assist practitioners in understanding dyslexia and other reading difficulties and assessing students' learning needs. We describe the essential components of language and literacy, universal screening, diagnostic assessments, curriculum-based measurement and eligibility determination. We then introduce four diagnostic assessments as examples, including norm-referenced assessments (i.e. the Comprehensive Test of Phonological Processing second edition and the Woodcock-Johnson IV Tests of Achievement) and criterion-referenced assessments (i.e. the Gallistel-Ellis Test of Coding Skills and the Dynamic Indicators of Basic Early Literacy Skills). Finally, We use a makeup case as a concrete example to illustrate how multiple diagnostic assessments are recorded and how the results can be used to inform intervention and eligibility for special education services.

Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics.

The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

Update on methods used for mycological testing: wide diversity and opportunities for improvement persist.

Past analysis of laboratory methods used for mycology specimens revealed significant variation in practices, many of which fell short of recommended procedures. In 2016 these findings led to a set of recommendations for laboratories to consider modification of their methods where appropriate, to analyse current laboratory methods used by participants in the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) Mycology module, and to compare these to the 2016 recommendations. Seven test items, with 105-107 participants each, were analysed. Several laboratories (7-12%) did not handle specimens as recommended in an appropriate biological safety cabinet. Direct microscopy was not performed on tissue specimens 23-25% of the time. The most used staining method was potassium hydroxide with an optical brightener for fluorescent microscopy (49%) followed by Gram stain (33%). While 17-25% of laboratories used three or more media, use of four or more was uncommon (<3%). Between 9-13% of participants used only a single non-inhibitory medium for cultures. Urine specimens were incubated longer than recommended with 57% of laboratories incubating for >7days and 24% >21 days. Duration of incubation was shorter than recommended for several specimen types with 36% of skin specimens and 37-48% of tissue specimens being kept ≤21 days. For cultures kept >7 days, 13% were inspected daily but for those incubating >14 days only 3%. The methods of several laboratories remain outside recommended practice. An updated set of recommendations are made.

Species distribution and susceptibility of Nocardia isolates in New Zealand 2002-2021.

The aim was to record the distribution and susceptibility of Nocardia species in New Zealand. Local and referred isolates were identified by an evolving approach over the study period including conventional phenotypic methods, susceptibility profiles, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF) and molecular sequencing. Isolates previously identified as a Nocardia sp. or part of the N. asteroides complex were reidentified by MALDI-TOF and/or molecular methods. Antimicrobial susceptibility to eight antibiotics was performed by standard microbroth dilution. The site of isolation, susceptibility profiles and species distribution were analysed. A total of 383 isolates were tested: N. brasiliensis 23 (6%), N. cyriacigeorgica 42 (11%), N. farcinica 41 (11%), N. nova complex 226 (59%), and 51 (13%) other species/complexes. The respiratory tract was the most common site of infection (244, 64%), with skin and soft tissue the second most common site (104, 27%). All 23 N. brasiliensis isolates were from skin and soft tissue specimens. Almost all isolates (≥98%) were susceptible to amikacin, linezolid and trimethoprim-sulfamethoxazole; 35% and 77% were resistant to clarithromycin and quinolones, respectively. The expected susceptibility profiles of the four common species and complex were observed for most agent-organism parings. Multi-drug resistance was uncommon (3.4%). The spectrum of Nocardia species in New Zealand is similar to overseas reports and our most common group is the N. nova complex. While amikacin, linezolid and trimethoprim-sulfamethoxazole remain good empiric treatment choices, other agents should have their activity confirmed before use.

The emergence of azole resistance in Aspergillus fumigatus complex in New Zealand.

BACKGROUND: Azole resistance in Aspergillus fumigatus (A. fumigatus) is increasing globally. A pan-azole-resistant isolate prompted genetic analysis of local azole-resistant isolates to determine resistance genotypes. METHODS: All A. fumigatus complex isolates were tested by the broth colorimetric micro-dilution method, Sensititre® YeastOne® (SYO) (TREK Diagnostic Systems, West Sussex, England). Epidemiological cutoff values derived from the Clinical & Laboratory Standards Institute method were used to determine the proportion of non-wild-type (non-WT) isolates (ie, those with an increased likelihood to harbour acquired mechanisms of resistance). Non-WT isolates were identified by ß-tubulin gene sequencing and the genotype for azole resistance was determined. The history of the patient with the first pan-resistant isolate was reviewed along with the treatment history of patients with azole-resistant strains. RESULTS: From January 2001 to August 2020, antifungal susceptibility testing was performed on 260 A. fumigatus complex isolates: six isolates were non-WT for one or more azole agent, two A. fumigatus sensu stricto and four other members within the species complex: two A. fischeri and two A. lentulus. There were three non-WT isolates for amphotericin B, three for itraconazole, five for posaconazole and five for voriconazole. All six non-WT strains were isolated in the past nine years (P<0.01), and four in the past three years. Azole-resistance genotyping for the A. fumigatus sensu stricto isolates detected amino acid changes at hot spots in the cyp51A gene: one at G54E and one at G138C. All six isolates were WT for caspofungin. Five of the six patients with azole-resistant strains had previous azole treatment, and the patient with the pan-azole-resistant strain had been on continuous azole treatment for 42 months preceding strain isolation. CONCLUSIONS: New Zealand can be added to the growing list of countries with azole-resistant A. fumigatus complex isolates, including pan-azole resistance in A. fumigatus sensu stricto. While uncommon and mostly found in cryptic species within the complex, azole resistance is increasing. The results provide a baseline for monitoring this emerging antifungal resistance trend in A. fumigatus in New Zealand.

Antifungal susceptibility of clinical mould isolates in New Zealand, 2001-2019.

The objective of this study was to review the antifungal susceptibility of clinical mould isolates performed by the New Zealand Mycology Reference Laboratory. Isolates were either local or referred for testing from other New Zealand laboratories. All isolates were tested by the broth colorimetric microdilution method, Sensititre YeastOne (SYO). Epidemiological cut-off values (ECVs) derived from either the Clinical and Laboratory Standards Institute (CLSI) method or SYO were used to determine the proportion of non-wild type (non-WT) isolates, i.e., those with an increased likelihood to harbour acquired mechanisms of resistance. A total of 614 isolates were tested. Most isolates (55%) were from the respiratory tract followed by musculoskeletal tissue (17%), eye (10%) and abdomen (5%). The azoles had similar activity except for voriconazole which was less active against the Mucorales. The echinocandins had good activity against Aspergillus spp., other hyaline moulds and dematiaceous isolates but were inactive against Fusarium spp., Lomentospora prolificans and the Mucorales. Amphotericin B had best activity against the Mucorales. The two least susceptible groups were Fusarium spp. and L. prolificans isolates. Three Aspergillus isolates were non-WT for amphotericin B, and four non-WT for azoles. Non-WT were not encountered for caspofungin. Non-Aspergillus isolates in New Zealand have susceptibility patterns similar to those reported elsewhere. In contrast to a growing number of other countries, azole resistance was rare in A. fumigatus sensu stricto. Non-WT isolates were uncommon. The results provide a baseline for monitoring emerging antifungal resistance in New Zealand and support current Australasian treatment guidelines for invasive fungal infections.

Antifungal susceptibility testing results of New Zealand yeast isolates, 2001-2015: Impact of recent CLSI breakpoints and epidemiological cut-off values for Candida and other yeast species.

OBJECTIVES: We reviewed the antifungal susceptibility testing results of local yeast isolates (2001-2015) to record the impact of recently updated interpretive criteria and epidemiological cut-off values (ECVs) for yeast species. METHODS: Susceptibility testing was performed using Sensititre® YeastOne®. The results were interpreted following CLSI criteria or YeastOne-derived ECVs. RESULTS: A total of 2345 isolates were tested; 62.0% were from sterile body sites or tissue. Application of new CLSI interpretative criteria for fluconazole increased the proportion of non-susceptible isolates of Candida parapsilosis, Candida tropicalis and Candida glabrata (P≤0.03 for all species). For voriconazole, the greatest increase was for C. tropicalis (P<0.0001). Application of new CLSI interpretive criteria for caspofungin increased the proportion of non-susceptible isolates for C. glabrata and Pichia kudriavzevii (P<0.0001 for both). The new amphotericin ECV (≤2mg/L) did not reveal any non-wild-type (non-WT) isolates in the five species covered. YeastOne itraconazole ECVs detected 2%, 5% and 6% non-WT isolates for P. kudriavzevii, C. tropicalis and C. glabrata, respectively. No itraconazole non-WT isolates of Clavispora lusitaniae were detected. CONCLUSIONS: Whilst most results are similar to other large surveys of fungal susceptibility, the new CLSI interpretive criteria significantly altered the proportion of non-susceptible isolates to fluconazole, voriconazole and caspofungin for several Candida spp. Application of CLSI and YeastOne-derived ECVs revealed the presence of a low proportion of non-WT isolates for many species. The results serve as a baseline to monitor the susceptibility of Candida and other yeast species in New Zealand over time.

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples.

The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose.

Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection.

Epidemiological studies, using the probe Ca3, have shown that in a given patient population a single cluster of genetically related Candida albicans isolates usually predominates. The authors have investigated whether these local clusters are part of a single group, geographically widespread and highly prevalent as an aetiological agent of various types of candidiasis. An unrooted neighbour-joining tree of 266 infection-causing C. albicans isolates (each from a different individual) from 12 geographical regions in 6 countries was created, based on genetic distances generated by Ca3 fingerprinting. Thirty-seven per cent of all isolates formed a single genetically homogeneous cluster (cluster A). The remainder of isolates were genetically diverse. Using the maximum branch length within cluster A as a cut-off, they could be divided into 37 groups, whose prevalence ranged between 0.3% and 9%. Strains from cluster A were highly prevalent in all but one geographical region, with a mean prevalence across all regions of 41%. When isolates were separated into groups based on patient characteristics or type of infection, strains from cluster A had a prevalence exceeding 27% in each group, and their mean prevalence was 43% across all patient characteristics. These data provide evidence that cluster A constitutes a general-purpose genotype, which is geographically widespread and acts as a predominant aetiological agent of all forms of candidiasis in all categories of patients surveyed.