A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp

BACKGROUND: Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. METHODS: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. RESULTS AND CONCLUSIONS: The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Genetic heterogeneity within the genospecies Leptospira weilii

Barnett, LJ ORCiD: 0000-0003-4345-3766Leptospirosis is a significant but lesser known disease in Queensland. Management of the disease is by vaccination of domestic host animals. Current vaccines for cattle incorporate the serovars pomona and hardjo genotype bovis. For successful management of leptospirosis, vaccines must be targeted for serovars enzootic in local regions. Identification of serovars implicated in human and animal disease is generally done serologically using the microscopic agglutination test (MAT) with a panel of antigens for serovars known to be present in Australia. Isolation of leptospires is difficult and can take long periods of time. Traditional serotyping of strains may not identify infecting leptospires adequately for the development of suitably targeted vaccines

Cercariae of intertidal nassariid gastropods: Taxonomy, specificity and ecology

Barnett, LJ ORCiD: 0000-0003-4345-3766"The biodiversity and ecology of marine cercariae in Australia is poorly known. Although nassariid gastropods are widely distributed in Australia and are expected to have a diverse parasite fauna similar to that of other nassariids worldwide, their cercariae have not been characterised. Nassarius dorsatus, N. olivaceus and N. pullus were collected at three separate estuarine localities in central Queensland between August 2004 and August 2006 and their emergent cercariae characterised using morphological and molecular methods. Fifteen morphotypes of cercariae from six families that infect fishes and birds as definitive hosts were characterised. The highest richness was within the Acanthocolpidae (nine morphotypes) which comprised three cercariae interpreted as species of Stephanostomum and a complex of six unusual cercariae with a ventral keel. Molecular characterisation of these six cercariae supported their placement in the Acanthocolpidae and indicated that they comprised only three species, two of which displayed marked phenotypic plasticity. Molecular analysis supported the characterisation of the three species of Stephanostomum and suggested that one of the unusual cercariae was a species of Stephanostomoides. Dissection of fish from Sandy Point provided a single match; the sequence for a metacercaria in Valamugil buchanani, the blue-tail mullet, was identical to that of one of the species of Stephanostomum. There were two cercariae from the Echinostomatidae and one from each of the Lepocreadiidae, Microphallidae, Opecoelidae and Zoogonidae. Molecular analyses supported the placement of the cercariae from the Echinostomatidae, Lepocreadiidae, Opecoelidae and Zoogonidae within their respective families. The cercaria from the Lepocreadiidae was identified by comparison to known adult data and putatively corresponds to Prodistomum keyam, a parasite of Monodactylus argenteus, the silver moony or butter bream, common in the study region. Morphological analysis supported the characterisation of both of the Echinostomatidae as species of Himasthla. Although no other cercariae could be characterised to genus level, the opecoelid cercaria was determined to be in the subfamily Opecoelinae. Nassarius olivaceus was infected by three acanthocolpid cercariae and one cercaria from each of the other families. Nassarius dorsatus was infected by five acanthocolpid cercariae and one each from the Echinostomatidae and Lepocreadiidae. Nassarius pullus was infected by a single cercaria from the Echinostomatidae. Most cercariae were specific to a single species of gastropod; only four infected two species and none infected all three. Cercariae that infect fish as a definitive host were found at all three localities; but cercariae that infect birds were restricted to a single locality, Sandy Point at Corio Bay, important for domestic and migratory bird species. These results emphasise the relative significance of Sandy Point as an important bird habitat. In total, 126 infections were diagnosed (115 by cercarial emergence), with both the highest frequency of infection and number of infections in N. olivaceus (frequency 5.3%; n=102; 81% of infections), followed by N. dorsatus (1.1%; 20; 16%) and N. pullus (0.2%; 4; 3%). The highest number of infections occurred at Sandy Point (66; 52% of infections), followed by Cawarral Creek (38; 30%) and then Ross Creek (22; 17%). The highest number of infections was by the Acanthocolpidae (54; 43%). Diagnosis of infection by emergence was suitable for all cercariae except the opecoelid, which was underdiagnosed. Nassarius olivaceus had the longest survival in captivity (~4.5 y), followed by N. dorsatus (~0.8 y) and N. pullus (~0.3 y). Almost all infections remained patent for the life of the gastropod. Families commonly found in nassariids worldwide include Acanthocolpidae, Echinostomatidae, Lepocreadiidae and Zoogonidae. Nassariid gastropods are also the dominant marine first intermediate hosts for the Acanthocolpidae, Lepocreadiidae and Zoogonidae and one of the three most common first intermediate host families for species of marine Himasthla. Molecular relationships also indicate that the Acanthocolpidae and Brachycladiidae may actually be comprised of three discrete groups, the Stephanostomum + Monostephanostomum group, the Brachycladiidae, and a group of non-Stephanostomum species comprising Pleorchis, Tormopsolus and Stephanostomoides. This research adds information about the parasite assemblages of nassariid gastropods in Australia, adding 12 new cercarial species and 16 new host-parasite records. Cercarial reports for the Acanthocolpidae, Echinostomatidae, Lepocreadiidae, Opecoelidae and Zoogonidae have been comprehensively reviewed and patterns of association between nassariids and their common parasite families presented. As well, this research has provided a potential avenue for a review of the Acanthocolpidae, a family which is currently considered a ‘catch-all’ group.

A review of the currently recognised opecoelid cercariae, including the identification and emergence ecology of Cercaria capricornia XII (Digenea: Opecoelidae) from Nassarius olivaceus (Gastropoda: Nassariidae) in Central Queensland, Australia

A new opecoelid cercaria, Cercaria capricornia XII, is reported from Nassarius olivaceus in Capricornia, Central Queensland, Australia. Combined molecular and morphological data indicate that this cercaria is a member of the subfamily Opecoelinae. Cercaria capricornia XII is the first known opecoeline cercaria reported from a nassariid gastropod. Cercaria capricornia XII can be distinguished from other opecoelid cercariae by the combination of the presence of a 2-pointed stylet, body length and width, and the size of the tail. The emergence pattern for C. capricornia XII in captivity was erratic; rapid emergences of thousands of cercariae were interspersed by periods that sometimes exceeded a month in which no emergence occurred. There was no detectable pattern to or stimulus of the emergence. The molluscan host range of opecoelids is analysed in detail. Gastropods from the Buccinoidea, Cerithioidea and Rissooidea are hosts to both opecoeline and plagioporine cercariae, but the dominant subfamily infecting the Cerithioidea and Rissooidea is the Plagioporinae. The dominant marine host gastropod superfamily for opecoeline cercariae is the Buccinoidea; the family Nassariidae is contained in the Buccinoidea. The range of gastropod superfamilies known as hosts of plagioporines is much broader than that for opecoelines, which may be explained by the relative size of the two opecoelid families and perhaps by indications that the Plagioporinae is polyphyletic

Gestational diabetes mellitus screening and diagnosis criteria before and during the COVID-19 pandemic: a retrospective pre–post study

Objective: To determine whether perinatal outcomes after excluding gestational diabetes mellitus (GDM) on the basis of fasting venous plasma glucose (FVPG) assessment during the coronavirus disease 2019 (COVID-19) pandemic in 2020 were similar to those during the preceding year after excluding GDM using the standard oral glucose tolerance test (OGTT) procedure. Design: Retrospective pre–post study. Setting, participants: All women who gave birth in Queensland during 1 July – 31 December 2019 and 1 July – 31 December 2020. Main outcome measures: Perinatal (maternal and neonatal) outcomes for pregnant women assessed for GDM, by assessment method (2019: OGTT/glycated haemoglobin [HbA1c] assessment; 2020: GDM could be excluded by an FVPG value below 4.7 mmol/L). Results: 3968 of 29 113 pregnant women in Queensland during 1 July – 31 December 2019 (13.6%) were diagnosed with GDM, and 4029 of 28 778 during 1 July – 31 December 2020 (14.0%). In 2020, FVPG assessments established GDM in 216 women (1.1%) and excluded it in 1660 (5.8%). The frequencies of most perinatal outcomes were similar for women without GDM in 2019 and those for whom it was excluded in 2020 on the basis of FVPG values; the exception was caesarean delivery, for which the estimated probability increase in 2020 was 3.9 percentage points (95% credibility interval, 2.2–5.6 percentage points), corresponding to an extra 6.5 caesarean deliveries per 1000 births. The probabilities of several outcomes — respiratory distress, neonatal intensive care or special nursery admission, large for gestational age babies — were about one percentage point higher for women without GDM in 2020 (excluding those diagnosed on the basis of FVPG assessment alone) than for women without GDM in 2019. Conclusions: Identifying women at low absolute risk of gestational diabetes-related pregnancy complications on the basis of FVPG assessment as an initial step in GDM screening could reduce the burden for pregnant women and save the health system substantial costs.</p

Leptospirosis serology in the Common Brushtail Possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic serogroup Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea

Additional file 1 of Process evaluation of a tailored nudge intervention to promote appropriate care and treatment of older patients at the end-of-life

Supplementary Material 1