Female RNA concussion (FeRNAC) study: assessing hormone profiles and salivary RNA in females with concussion by emergency departments in New Zealand: a study protocol.

BACKGROUND: Females of reproductive age with concussion report a greater number of symptoms that can be more severe and continue for longer than age matched males. Underlying mechanisms for sex differences are not well understood. Short non-coding Ribonucleic Acids (sncRNAs) are candidate salivary biomarkers for concussion and have been studied primarily in male athletes. Female sex hormones influence expression of these biomarkers, and it remains unclear whether a similar pattern of sncRNA expression would be observed in females following concussion. This study aims to evaluate recovery time, the ratio of salivary sncRNAs and symptom severity across different hormone profiles in females presenting to emergency departments (ED) with concussion and, to investigate the presence of low energy availability (LEA) as a potential modifier of concussion symptoms. METHODS: This prospective cohort study recruits participants from New Zealand EDs who are biologically female, of reproductive age (16-50 years) and with a confirmed diagnosis of concussion from an ED healthcare professional. Participants are excluded by ED healthcare professionals from study recruitment as part of initial routine assessment if they have a pre-diagnosed psychiatric condition, neurological condition (i.e., epilepsy, cerebral palsy) or more than three previously diagnosed concussions. Participants provide a saliva sample for measurement of sncRNA's, and online survey responses relating to hormone profile and symptom recovery at 7-day intervals after injury until they report a full return to work/study. The study is being performed in accordance with ethical standards of the Declaration of Helsinki with ethics approval obtained from the Health and Disability Ethics Committee (HDEC #2021 EXP 11655), Auckland University of Technology Ethics Committee (AUTEC #22/110) and locality consent through Wellington hospital research office. DISCUSSION: If saliva samples confirm presence of sncRNAs in females with concussion, it will provide evidence of the potential of saliva sampling as an objective tool to aid in diagnosis of, and confirmation of recovery from, concussion. Findings will determine whether expression of sncRNAs is influenced by steroid hormones in females and may outline the need for sex specific application and interpretation of sncRNAs as a clinical and/or research tool. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) registration number ACTRN12623001129673.

Mycoplasma genitalium Antimicrobial Resistance in Community and Sexual Health Clinic Patients, Auckland, New Zealand.

Our retrospective study compared genotypic antimicrobial resistance in Mycoplasma genitalium-positive specimens collected from 48 community and 33 sexual health clinic (SHC) patients. Macrolide resistance was similar in community (75%) and SHC (76%) patients. We observed no significant difference in fluoroquinolone resistance between community (19%) and SHC (27%) patients (p = 0.66).

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.

Evaluation of ssrA-targeted real time PCR for the detection of Bartonella species in human clinical samples and reflex sequencing for species-level identification.

The genus Bartonella includes species capable of causing disease in animals and humans. Due to its fastidious nature, direct detection of Bartonella causing human infection relies largely on molecular microbiological methods. Thus, it is imperative that diagnostic assays in use have the ability to detect a range of Bartonella species associated with human disease. In this study, we compared the performance of a real time polymerase chain reaction (PCR) assay targeting the ssrA gene to conventional rpoB-targeted PCR and sequencing for detection and differentiation of Bartonella species in human clinical samples. The real time ssrA PCR performed better for non-Bartonella henselae species, detecting B. clarridgeiae and B. quintana DNA in heart valve specimens that were not detected by rpoB PCR, and improved the sensitivity of B. henselae detection in blood specimens. Our findings suggest the real time ssrA PCR assay is suitable for detection and identification of Bartonella species in human clinical specimens.

Epidemiology of carbapenem resistant Acinetobacter baumannii in New Zealand.

AIM: Carbapenem resistant Acinetobacter baumannii have limited treatment options and a propensity to cause hospital outbreaks. In recent years an increase in their detection has been observed in New Zealand. This study aimed to describe the molecular epidemiology of these isolates. METHOD: This study utilised carbapenem resistant A. baumannii complex isolates identified across New Zealand between January 2010 to April 2018. Whole genome sequence analysis and associated demographic information was used to contextualise local isolates within the global epidemiology and establish the relationship between isolates. RESULTS: Thirty-three carbapenem resistant A. baumannii complex isolates (31 A. baumannii sensu stricto) were identified. Twenty-four (73%) were from January 2015 onwards. Twenty-four (73%) had an identifiable epidemiological link to overseas hospitalisation. Twenty-three (74%) of 31 A. baumannii sensu stricto were sequence type (ST) 2 (Pasteur scheme). Phylogenetic analysis identified three ST2 clusters. The largest cluster, of 12 isolates, was from 2015 onwards; with nine (75%) associated with recent hospitalisation in Fiji or Samoa. CONCLUSION: Increasing numbers of carbapenem resistant A. baumannii are being identified in New Zealand. Our data show that this is in large part associated with transnational spread of a single A. baumannii sensu stricto ST 2 strain between Fiji, Samoa and New Zealand.

Evaluation of extraction and amplification assays for the detection of SARS-CoV-2 at Auckland Hospital laboratory during the COVID-19 outbreak in New Zealand.

Utilising diverse molecular platforms has formed a solid foundation in New Zealand's COVID-19 response. We evaluated multiple extraction and PCR assays for the detection of SARS-CoV-2. We included 65 positive samples which were run on the Panther Fusion using a laboratory developed test (LDT, E gene target). Where viral RNA was extracted by MagNA Pure (MP) 96 extraction platform or EpMotion 5075/Geneaid extraction kit, SARS-CoV-2 detection was performed on Light Cycler (LC) 480 using a LDT (E gene) or 3 commercial assays; Certest Viasure (Orf1ab, N genes) GenePro (E, RdRp genes) and A* Star Fortitude (proprietary target). Median Cts on LC 480 LDT for specimens (n = 9) extracted on MP 96 (26.6) were lower than on EpMotion (31.6) whereas median Cts for specimens (n = 10) extracted on the Panther Fusion LDT (23.1) were comparable with MP 96 /LC480 LDT (23.6). Specimens tested on Panther Fusion LDT (n = 28), extracted by MP 96, and amplified using commercial assays showed good concordance with a few exceptions; lower median Ct values were seen for 2 targets on GenePro (16.9, 21.5) and Viasure (19.5, 21.1) than for the Panther Fusion LDT (24.2) and A* Star Fortitude (25.6). Specimens tested on MP 96 (n = 18) had comparable results using commercial assays, with lower median Cts for Viasure (22.2, 23.7) compared with the LC 480 LDT (24.7), GenePro (24.7,25.7) and A*Fortitude (25.1) assays. The study provides an early assessment of the performance characteristics of 3 extraction methods for viral RNA and 5 PCR assays for the detection of SARS-CoV-2.

Helicobacter cinaedi bacteremia in a returning traveler.

Of the non-Helicobacter pylori Helicobacter (NHPH) species, Helicobacter cinaedi is an emerging cause of infection in humans. Here we report a novel clinical presentation of H. cinaedi infection: a case of fever in a returning traveler. A 31 year old previously fit and well male presented with onset of fever 24 h after returning from travel in Singapore and Indonesia. Associated symptoms consisted of sore throat, mild shortness of breath, generalized myalgia and arthralgia, headache, and four episodes of loose stools. The patient recovered spontaneously without treatment and was discharged. After 4 days of incubation, blood cultures grew H. cinaedi. H. cinaedi is a slow-growing fastidious organism poorly detected by some commonly used automated blood culture systems, and difficult to identify using commercial or traditional biochemical identification systems. This case illustrates the importance of H. cinaedi as an emerging pathogen in immunocompetent patients, with a wide variety of possible clinical presentations. The challenges in the microbiological diagnosis of H. cinaedi infections lead us to speculate that H. cinaedi is an underdiagnosed cause of febrile illness, both in returning travelers and in other clinical settings.

Utility of whole genome sequencing for multidrug resistant Mycobacterium tuberculosis isolates in a reference TB laboratory in New Zealand.

New Zealand has a low burden of multi-drug resistant TB (MDR-TB), but with increased mobility within the population, rapid detection and treatment of MDR-TB is a priority from the public health point of view. Mycobacterium Reference Laboratory in LabPLUS, Auckland City Hospital receives referred Mycobacterium tuberculosis complex (MTBC) isolates from all over New Zealand for second-line drug susceptibility testing (DST) and 24-loci MIRU VNTR genotyping. Between 2002 and 2013, 38 multidrug resistant Mycobacterium tuberculosis (MDR-TB) isolates were recorded by culture-based DST. A retrospective study revealed that in 12 of these 38 MDR-TB isolates (28%) there was a discrepancy between the genotypic and the phenotypic results. In order to address this, whole genome sequencing (WGS) was performed on the discrepant MDR-TB isolates. Reported here are the additional information on the drug resistant markers from WGS, which shed light on the discordance between results from the culture-based DST and the molecular diagnostic tests. These results underscore the utility of WGS in a reference mycobacterium laboratory in New Zealand to supplement other molecular tests and to assist in a rapid but accurate diagnosis and appropriate management of MDR-TB.

Differential carriage of virulence-associated loci in the New Zealand Rangipo outbreak strain of Mycobacterium tuberculosis.

BACKGROUND: The Rangipo strain of Mycobacterium tuberculosis achieved notoriety in New Zealand due to its role in several tuberculosis (TB) outbreaks. Why this strain should be the source of relatively large clusters of the disease is unknown. In this work, we performed an in-depth analysis of the genome of the Rangipo strain to determine whether it offers clues to understanding its prevalence. METHODS: Next-generation sequencing was performed on nine isolates which matched the Rangipo genotypic profile. Sequence reads were assembled against the H37Rv reference genome and single-locus variants identified. Unmapped reads were compared against the genome sequences of other M. tuberculosis strains, in particular CDC1551, Haarlem and Erdman. RESULTS: Across the nine Rangipo strains, a total of 727 single-locus variants were identified with respect to H37Rv, of which 700 were common to all Rangipo strains sequenced. Within the common variants, 386 were non-synonymous, with 12 occurring in genes associated with M. tuberculosis virulence. Next-generation and Sanger sequencing determined the presence of three genes in the Rangipo isolates, which are absent in H37Rv, but which have been reported to be important for the pathogenicity of M. tuberculosis. The differentially encoded Rangipo genes consisted of transcriptional regulator EmbR2, and molybdopterin cofactor biosynthesis proteins A and B. The Rangipo strain also harbours an extended DNA helicase and an additional adenylate cyclase. CONCLUSIONS: Our study provides new insights into the genomic content of the New Zealand Rangipo strain of M. tuberculosis and highlights the presence of additional virulence-related loci not found in H37Rv.

Draft Genome Sequence of a New Zealand Rangipo Strain of Mycobacterium tuberculosis.

The Rangipo genotype of the Mycobacterium tuberculosis complex has been associated with a number of tuberculosis (TB) outbreaks in New Zealand. We report here the draft whole-genome sequence of a representative isolate of this strain.

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Draft Genome Sequence of a Drug-Susceptible New Zealand Isolate of Mycobacterium tuberculosis Lineage 3.

We report here the draft whole-genome sequence of a drug-susceptible lineage 3 (East-African Indian) isolate of Mycobacterium tuberculosis from New Zealand (NZ3DS1) and compare it to a multidrug-resistant lineage 3 isolate (NZ3MDR1) with an identical 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat profile.

Lymphogranuloma venereum in men who have sex with men: evidence of local transmission in New Zealand.

Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by Chlamydia trachomatis. Five laboratory confirmed cases of LGV were detected in MSM (men who have sex with men) in the upper North Island; four in Auckland between September and December 2013 and a fifth case was detected in Waikato in June 2014. The absence of a recent travel history for four cases supports the likelihood of local transmission of this uncommon infection.

Draft Genome Sequence of the First Isolate of Extensively Drug-Resistant Mycobacterium tuberculosis in New Zealand.

Extensively drug-resistant (XDR) tuberculosis has now been described in >90 countries worldwide. The first case of XDR tuberculosis (XDR-TB) in New Zealand was recorded in 2010. We report the draft whole-genome sequence of the New Zealand isolate, NZXDR1, and describe a number of single-nucleotide polymorphisms that relate to drug resistance.

Improved detection of toxigenic Clostridium difficile using the Cepheid Xpert C difficile assay and impact on C difficile infection rates in a tertiary hospital: a double-edged sword.

Recent recommendations suggest implementation of a 2-step diagnostic algorithm for the laboratory detection of toxigenic Clostridium difficile. We found that the rate of toxigenic C difficile detection in our laboratory doubled after the introduction of an algorithm that incorporated polymerase chain reaction testing. This led to an abrupt 70% increase in the overall rate of C difficile infection in our institution.

An evaluation of the Xpert MTB/RIF assay and detection of false-positive rifampicin resistance in Mycobacterium tuberculosis.

Recent reports suggest that false-positive rifampicin resistance may be assigned by the Xpert MTB/RIF assay. We analysed 169 specimens using the MTB/RIF assay. Using culture as the gold standard, we found that the assay had 100% sensitivity and specificity for detecting M. tuberculosis. However, we found that the assay incorrectly assigned rifampicin resistance in 4/13 (31%) of cases.

Crystal structures of the staphylococcal toxin SSL5 in complex with sialyl Lewis X reveal a conserved binding site that shares common features with viral and bacterial sialic acid binding proteins.

Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (Fc alphaRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe(X)), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5-sLe(X) complex at resolutions of 1.65 and 2.75 A for crystals at two pH values. In both structures, sLe(X) bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.