Epidemiology of Toxoplasma gondii in the pig industry of Yucatan, Mexico

Toxoplasmosis is a worldwide distributed parasitic disease caused by the zoonotic pathogen Toxoplasma gondii. Serological studies have estimated that more than 30% of the human population has been exposed to this protozoan. T. gondii is considered a leading cause of death attributed to foodborne illness. The consumption of infected pork meat is suggested to be an important source for human infection. However, the prevalence of T. gondii in pigs vary greatly between countries, the reasons for this heterogeneity has been addressed to the differences in climate distribution, environments, husbandry systems and fam management. The geographical location of this study is Yucatan, a state located in the south-east of Mexico. Yucatan is considered an endemic area of toxoplasmosis; the last National Seroepidemiological Health Survey (NSHS) revealed more than 70% of prevalence among the human population. Numerous studies suggested that the consumption of pork in this geographical area may be a major source of T. gondii. The aims of this study were to investigate the disease levels in the pig industry of Yucatan, assess an in-home ELISA widely used in this area (ELISA kit Human- GmbH, WB), study the risk factors associated with the disease in theses pig farms and evaluate the contamination with T. gondii in pork meat intended for human consumption. To do that, swine blood samples were collected through a cross-sectional age stratified opportunistic sampling of 12 farms across the state during the year 2014. Farm management and characteristics were obtained by interviewing farmers. In addition, meat and blood samples were collected from a local abattoir from 2013 to 2015. Anti - T. gondii IgG antibody levels were investigated with the well validated MAT (Modified Agglutination Test), with an ELISA (Enzyme-Linked Immunosorbent Assay) for use on pig serum (ID Screen ®, IDVet) and with the gold standard Dye test. The overall seroprevalence was 1.4% (95%CI: 0.6%-2.7%) among 632 pigs sampled. This seroprevalence increased with age (p < 0.05), reaching the 11.5% (95% CI: 2.5%-30%) in pigs older than 20 weeks. The seroprevalence was even higher, 17.8% (95% CI: 6%-37%), in slaughtered animals (n=34). In addition, T. gondii prevalence was investigated using a highly sensitive nested PCR protocol targeting the SAG1 gene. PCR diagnosis revealed the high frequency of 21.2% (CI: 18%-24.6%) of T. gondii DNA circulating in the blood of these pigs (n=632). Furthermore, MLST (Multi-Locus Sequence Typing) of four alleles (SAG1, SAG2, SAG3 and GRA6) allowed, for first time in Yucatan, genetic diversity to be assessed.Data revealed the presence of high genetic diversity among T. gondii strains of this geographical area with shared alleles to strains from both North and South/Central America origin. Moreover, a relatively high number of pigs presented multiple infections with different T. gondii strains suggesting high levels of T. gondii transmission on the intensive pig farms sampled. The frequency of T. gondii DNA was also investigated in pig tongues sampled at the abattoir of which 38.2% (95% CI: 22%-56.4%) were shown to harbour T. gondii DNA in their tissue. The viability of the parasite was also investigated in the tongues intended for human consumption and a total of 8.8% (95% CI: 1.8%-23%) were shown to have viable T. gondii using a mouse bioassay. However, the agreement between serology, PCR and mouse bioassay was low (k=0.12-0.29). Due to the risk of underestimating T. gondii infection by using solely one diagnostic method, a combination of indirect (serology) and direct methods (PCR or/and bioassay) is preferred for a robust diagnosis. This study was pioneering in using a serological test validated in pigs in the state of Yucatan and the data revealed a much lower prevalence than previously reported (95.8%-100%) in market age pigs (Ortega-Pacheco et al., 2013, Hernández- Cortazar et al., 2016a). Although a more optimistic view is obtained; due to the potential of T. gondii to lead to severe illness, measures to control the disease should still be taken. Furthermore, questionnaire results suggested the need for an improvement to the sanitary management of the pig farms of this geographical area not only to prevent T. gondii transmission but also that of other pathogens of zoonotic significance

Prevalence of neutralising antibodies against SARS-CoV-2 in acute infection and convalescence: A systematic review and meta-analysis.

BACKGROUND: Individuals infected with SARS-CoV-2 develop neutralising antibodies. We investigated the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how this proportion varies with selected covariates. METHODOLOGY/PRINCIPAL FINDINGS: This systematic review and meta-analysis examined the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how these proportions vary with selected covariates. Three models using the maximum likelihood method assessed these proportions by study group, covariates and individually extracted data (protocol CRD42020208913). A total of 983 reports were identified and 27 were included. The pooled (95%CI) proportion of individuals with neutralising antibodies was 85.3% (83.5-86.9) using the titre cut off >1:20 and 83.9% (82.2-85.6), 70.2% (68.1-72.5) and 54.2% (52.0-56.5) with titres >1:40, >1:80 and >1:160, respectively. These proportions were higher among patients with severe COVID-19 (e.g., titres >1:80, 84.8% [80.0-89.2], >1:160, 74.4% [67.5-79.7]) than those with mild presentation (56.7% [49.9-62.9] and 44.1% [37.3-50.6], respectively) and lowest among asymptomatic infections (28.6% [17.9-39.2] and 10.0% [3.7-20.1], respectively). IgG and neutralising antibody levels correlated poorly. CONCLUSIONS/SIGNIFICANCE: 85% of individuals with proven SARS-CoV-2 infection had detectable neutralising antibodies. This proportion varied with disease severity, study setting, time since infection and the method used to measure antibodies

A Novel Air-Dried Multiplex High Resolution Melt Assay for the Detection of Extended Spectrum Beta-Lactamase and Carbapenemase Genes.

OBJECTIVES: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended spectrum beta-Lactamase (ESBL) (blaSHV and blaCTXM groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that cause antimicrobial resistance to cephalosporins and carbapenems. METHODS: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility results. Assay reproducibility was evaluated across five different q-PCR instruments (Rotor-Gene Q, QuantStudioTM 5, CFX96, LightCycler® 480 and MIC). Assay stability was also assessed upon the assay storage in the refrigerator (6.2°C±0.9), room temperature (20.4°C±0.7) and oven (29.7°C±1.4) at six time points for eight months. RESULTS: The sensitivity and specificity for detecting the ESBL and carbapenemase genes in comparison to the reference gel-base PCR and sequencing was 94.7% (95%CI: 92.5%-96.5%) and 99.2% (95%CI: 98.8%-99.5%), and 98.5% (95%CI: 97.0%-99.4%) and 98.5% (95%CI: 98.0%-98.9%) when compared to the original HRM wet PCR mix format. The overall agreement was 91.1% (95%CI: 90.0%-92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay and no loss of sensitivity occurred under all storage conditions and time points. CONCLUSIONS: We present here a ready-to-use air-dried HRM-PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve AMR

Self-sampling of capillary blood for SARS-CoV-2 serology

Serological testing is emerging as a powerful tool to progress our understanding of COVID-19 exposure, transmission and immune response. Large-scale testing is limited by the need for in-person blood collection by staff trained in venepuncture, and the limited sensitivity of lateral flow tests. Capillary blood self-sampling and postage to laboratories for analysis could provide a reliable alternative. Two-hundred and nine matched venous and capillary blood samples were obtained from thirty nine participants and analysed using a COVID-19 IgG ELISA to detect antibodies against SARS-CoV-2. Thirty eight out of thirty nine participants were able to self-collect an adequate sample of capillary blood (≥ 50 µl). Using plasma from venous blood collected in lithium heparin as the reference standard, matched capillary blood samples, collected in lithium heparin-treated tubes and on filter paper as dried blood spots, achieved a Cohen’s kappa coefficient of > 0.88 (near-perfect agreement, 95% CI 0.738–1.000). Storage of capillary blood at room temperature for up to 7 days post sampling did not affect concordance. Our results indicate that capillary blood self-sampling is a reliable and feasible alternative to venepuncture for serological assessment in COVID-19

Detection of SARS-CoV-2 infection by saliva and nasopharyngeal sampling in frontline healthcare workers: An observational cohort study

Background The SARS-CoV-2 pandemic has caused an unprecedented strain on healthcare systems worldwide, including the United Kingdom National Health Service (NHS). We conducted an observational cohort study of SARS-CoV-2 infection in frontline healthcare workers (HCW) working in an acute NHS Trust during the first wave of the pandemic, to answer emerging questions surrounding SARS-CoV-2 infection, diagnosis, transmission and control. Methods Using self-collected weekly saliva and twice weekly combined oropharyngeal/nasopharyngeal (OP/NP) samples, in addition to self-assessed symptom profiles and isolation behaviours, we retrospectively compared SARS-CoV-2 detection by RT-qPCR of saliva and OP/NP samples. We report the association with contemporaneous symptoms and isolation behaviour. Results Over a 12-week period from 30th March 2020, 40∙0% (n = 34/85, 95% confidence interval 31∙3-51∙8%) HCW had evidence of SARS-CoV-2 infection by surveillance OP/NP swab and/or saliva sample. Symptoms were reported by 47∙1% (n = 40) and self-isolation by 25∙9% (n = 22) participants. Only 44.1% (n = 15/34) participants with SARS-CoV-2 infection reported any symptoms within 14 days of a positive result and only 29∙4% (n = 10/34) reported self-isolation periods. Overall agreement between paired saliva and OP/NP swabs was 93∙4% (n = 211/226 pairs) but rates of positive concordance were low. In paired samples with at least one positive result, 35∙0% (n = 7/20) were positive exclusively by OP/NP swab, 40∙0% (n = 8/20) exclusively by saliva and in only 25∙0% (n = 5/20) were the OP/NP and saliva result both positive. Conclusions HCW are a potential source of SARS-CoV-2 transmission in hospitals and symptom screening will identify the minority of infections. Without routine asymptomatic SARS-CoV-2 screening, it is likely that HCW with SARS-CoV-2 infection would continue to attend work. Saliva, in addition to OP/NP swab testing, facilitated ascertainment of symptomatic and asymptomatic SARS-CoV-2 infections. Combined saliva and OP/NP swab sampling would improve detection of SARS-CoV-2 for surveillance and is recommended for a high sensitivity strategy

Twelve lateral flow immunoassays (LFAs) to detect SARS-CoV-2 antibodies.

BACKGROUND: There are an abundance of commercially available lateral flow assays (LFAs) that detect antibodies to SARS-CoV-2. Whilst these are usually evaluated by the manufacturer, externally performed diagnostic accuracy studies to assess performance are essential. Herein we present an evaluation of 12 LFAs. METHODS: Sera from 100 SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR) positive participants were recruited through the FASTER study. A total of 105 pre-pandemic sera from participants with other infections were included as negative samples. RESULTS: At presentation sensitivity against RT-PCR ranged from 37.4 to 79% for IgM/IgG, 30.3-74% for IgG, and 21.2-67% for IgM. Sensitivity for IgM/IgG improved ≥ 21 days post symptom onset for 10/12 tests. Specificity ranged from 74.3 to 99.1% for IgM/IgG, 82.9-100% for IgG, and 75.2-98% for IgM. Compared to the EuroImmun IgG enzyme-linked immunosorbent assay (ELISA), sensitivity and specificity ranged from 44.6 to 95.4% and 85.4-100%, respectively. CONCLUSION: There are many LFAs available with varied sensitivity and specificity. Understanding the diagnostic accuracy of these tests will be vital as we come to rely more on the antibody status of a person moving forward, and as such manufacturer-independent evaluations are crucial

IgG Seroconversion and Pathophysiology in Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

We investigated the dynamics of seroconversion in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During March 29–May 22, 2020, we collected serum samples and associated clinical data from 177 persons in London, UK, who had SARS-CoV-2 infection. We measured IgG against SARS-CoV-2 and compared antibody levels with patient outcomes, demographic information, and laboratory characteristics. We found that 2.0%–8.5% of persons did not seroconvert 3–6 weeks after infection. Persons who seroconverted were older, were more likely to have concurrent conditions, and had higher levels of inflammatory markers. Non-White persons had higher antibody concentrations than those who identified as White; these concentrations did not decline during follow-up. Serologic assay results correlated with disease outcome, race, and other risk factors for severe SARS-CoV-2 infection. Serologic assays can be used in surveillance to clarify the duration and protective nature of humoral responses to SARS-CoV-2 infection

Genotyping of Toxoplasma gondii from pigs in Yucatan, Mexico

Toxoplasmosis is a zoonotic disease of worldwide distribution. The parasite exhibits strong geographical patterns of strain variation with contrasting high levels of diversity across South America and restricted variation across North America. Little is known about the diversity of strains in the transitional area between the two continents. Here we present data on the prevalance and diversity of Toxoplasma gondii in the Yucatan peninsula of Mexico, through a study in commercially reared pigs. A survey of 12 farms found evidence of circulating T. gondii DNA in 125 of 632 blood samples (19.8%, CI: 16.7%–23%). In addition, 46 tongue samples were collected from culled animals and 16 of these were positive for T. gondii DNA and 3 were positive in mouse bioassay. PCR-sequencing was used to generate genotyping data from blood and tissue samples. Four loci (SAG1, 2, 3 and GRA6) were reliably amplified and revealed a high diversity among Yucatan strains with evidence of recombination and novel alleles. Sequencing data from the four loci was achieved in eight samples each of which had a different genotype. The predominant allelic type was atypical, in relation to the dominant strain types (I, II, III), the number of allelic variants being 27 (I, II-III, u-1-25), 20 (I, III, u1-18), 6 (I, III, u1-4) and 11 (I, II, u1-9) for the SAG1, SAG2, SAG3 and GRA6 loci respectively. Phylogenetic analysis showed that T. gondii strains from Yucatan shared alleles with strains originating from both North and South America. Our findings are consistent with data from other regions of Central America and suggest the genetic population structure of the parasite, with significant levels of allelic variation and recombination, constitutes a reservoir from which new strains may emerge. Positive bioassay results (7.5%) indicate that consumption of undercooked pork could be a potential T. gondii infection risk to humans

A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum β-lactamase and carbapenemase genes

Objectives: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum β-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems. Methods: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months. Results: The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5–96.5%) and 99.2% (98.8–99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0–99.3%) and 98.5% (98.0–98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0–92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points. Conclusion: We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.</br