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Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle., Pathak et al. identify LDB3, a striated muscle PDZ-LIM protein, as a signaling adaptor in a major mechanosensor assembly through interactions with filamin C, HSPA8, and PKCα. When LDB3 is mutated, PKCα and TSC2-mTOR mediated homeostasis is impaired, leading to protein aggregation myopathy.

Background: In countries with a high HIV/TB burden like Nigeria, HIV/TB and emerging infectious diseases like coronavirus (COVID-19) epidemics are anticipated to overlap. The objective of this study is to determine the public health impact of the COVID-19 pandemic on HIV/TB diagnosis in Abuja, Nigeria. Methodology: This study was carried out in HIV/TB testing laboratories within the Federal Capital Territory. The study population comprised all the full-time laboratory professionals working in the selected laboratories. A structured questionnaire was used to collect data via Google form from September to December, 2020. Data collected were analyzed using IBM-SPSS version 25.0. Results: The study comprised 164 laboratory staff from seven TB and HIV diagnostic laboratories in Abuja. COVID-19 crisis has significantly contributed to job layoffs (68.2%) and financial challenges (69.8%) within the laboratories. The negative impact of COVID-19 on staff availability at work was rated 81/100. Availability of appropriate and timely provision of PPE was poorly rated (50.2%). Also, respondents rated regular diagnosis of HIV/TB during COVID-19 very poor (37.0%). The negative effects of the pandemic on the supply of consumables and commodities (78.4%) and the purchase of consumables and reagents (76.0%) were rated high (p>0.05). Other negative diagnostic impacts of COVID-19 include lack of proper collection of diagnostic materials (61.4%), delay in the receipt of specimens (64.0%), and long turnaround time for HIV/TB test results (63.2%). Conclusion: Measures should be taken to ensure proper and regular sample collections during pandemic rather than focusing on COVID-19 and neglecting other diseases.

Objective There is a need for reliable serological assays to determine accurate estimates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence. Most single target antigen assays have shown some limitations in Africa. To assess the performance of a multi-antigen assay, we evaluated a commercially available SARS-CoV-2 Multi-Antigen IgG assay for human coronavirus disease 2019 (COVID-19) in Nigeria. Methods Validation of the xMAP SARS-CoV-2 Multi-Antigen IgG assay was carried out using well-characterized SARS-CoV-2 reverse transcription polymerase chain reactive positive (97) and pre-COVID-19 pandemic (86) plasma panels. Cross-reactivity was assessed using pre-COVID-19 pandemic plasma specimens (213) from the 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS). Results The overall sensitivity of the xMAP SARS-CoV-2 Multi-Antigen IgG assay was 75.3% [95% CI: 65.8%– 82.8%] and specificity was 99.0% [95% CI: 96.8%– 99.7%]. The sensitivity estimate increased to 83.3% [95% CI: 70.4%– 91.3%] for specimens >14 days post-confirmation of diagnosis. However, using the NAIIS pre-pandemic specimens, the false positivity rate was 1.4% (3/213). Conclusions Our results showed overall lower sensitivity and a comparable specificity with the manufacturer’s validation. There appears to be less cross-reactivity with NAIIS pre-pandemic COVID-19 specimens using the xMAP SARS-CoV-2 Multi-Antigen IgG assay. In-country SARS-CoV-2 serology assay validation can help guide the best choice of assays in Africa.

Background: Accurate SARS-CoV-2 serological assays are critical for COVID-19 serosurveillance. However, previous studies have indicated possible cross-reactivity of these assays, including in malaria-endemic areas.Methods: We tested 213 well-characterized pre-pandemic samples from Nigeria using two SARS-CoV-2 serological assays: Abbott Architect IgG and Euroimmun NCP IgG assay, both targeting SARS-CoV-2 nucleocapsid protein. To assess antibody binding strength, an avidity assay was performed on these samples and on plasma from SARS-CoV-2 PCR-positive persons.Results: Thirteen (6.1%) of 212 samples run on the Abbott assay and 38 (17.8%) of 213 run on the Euroimmun assay were positive. Anti-Plasmodium IgG levels were significantly higher among false-positives for both Abbott and Euroimmun; no association was found with active P. falciparum infection. An avidity assay using various concentratIons of urea wash in the Euroimmun assay reduced loosely-bound IgG: of 37 positive/borderline pre-pandemic samples, 46%, 86%, 89%, and 97% became negative using 2M, 4M, 5M, and 8M urea washes, respectively. The wash slightly reduced avidity of antibodies from SARS-CoV-2 patients within 28 days of PCR confirmation; thereafter avidity increased for all urea concentrations except 8M.Conclusions: This validation found moderate to substantial cross-reactivity on two SARS-CoV-2 serological assays using samples from a malaria-endemic setting. A simple urea wash appeared to alleviate issues of cross-reactivity.

Wound infections due to Clostridium botulinum were not recognised in the UK and Republic of Ireland before 2000. C. botulinum produces a potent neurotoxin which can cause paralysis and death. In 2000 and 2001, ten cases were clinically recognised, with a further 23 in 2002, 15 in 2003 and 40 cases in 2004. All cases occurred in heroin injectors. Seventy cases occurred in England; the remainder occurred in Scotland (12 cases), Wales (2 cases) and the Republic of Ireland (4 cases). Overall, 40 (45%) of the 88 cases were laboratory confirmed by the detection of botulinum neurotoxin in serum, or by the isolation of C. botulinum from wounds. Of the 40 cases in 2004, 36 occurred in England, and of the 12 that were laboratory confirmed, 10 were due to type A. There was some geographical clustering of the cases during 2004, with most cases occurring in London and in the Yorkshire and Humberside region of northeast England.

UNLABELLED The Listeria genus comprises 10 recognized species. Listeria monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. Listeria ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of nonpathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of Listeria seeligeri, Listeria welshimeri, L. monocytogenes, L. ivanovii, Listeria grayi and Listeria innocua. The assay consists of two triplexes that were evaluated using 53 cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food-processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY The identification of species of Listeria from foods is important to monitor pathogenic strains and facilitates the implementation of control measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes and L. ivanovii, L. grayi, L. innocua. The developed assay proved to be specific, rapid and reproducible and therefore could be implemented in busy specialist reference laboratories.

Traumatic childhood experiences are a major risk factor for anxiety disorders and depression. Brain-derived neurotrophic factor (BDNF) is critical to the developing brain; reduced BDNF levels have been observed in these disorders. Studies show that early life stress programs the development of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in sustained activation of the HPA axis and behavioral changes. A link between elevated levels of corticosteroids (CORT), and decreased BDNF protein also has been observed. Thus, early life trauma and stress may contribute to functional changes in brain structure and neuroplasticity via alteration in BDNF protein levels. Our laboratory developed a model for short duration, early life, maternal separation (MS) stress using Balb/CByJ mice, employing a split-litter design, where half the pups were removed from their dam for 1 hr./day from PND 2 to PND 7 and exposed for 30 minutes to cold (4oC) or hot (37o C) stress on alternating days. Control group included littermates (LMC) who stayed with the dam and age-matched (AMC) litters raised without any intervention. This MS paradigm resulted in altered CORT release, anxiety and social behavior, as well as changes in brain structure and plasticity. Altered levels of mature BDNF (mBDNF) in the cortex and hippocampus of stressed (STR) mice were seen as well. This thesis, examines if BDNF level changes can be seen in adolescence, a typical age of onset for depression; altered social behavior in adolescence is seen in our model. We hypothesized that mBDNF levels would be lower and precursor BDNF (pro-BDNF) would be higher in cortex and hippocampus of stressed adolescent mice, at postnatal day 30, compared to the control group. I have examined the cortex and hippocampus of STR (N=20), LMC (N=10), and AMC (N=18) male and female mice who were sacrificed at PND 30. Brains were extracted and dissected on ice and stored at -70o C until they were processed for Western blot analysis of mBDNF and pro-BDNF. Multiple technical difficulties were encountered throughout this study hampering data generation and results returned inconclusive. I will discuss this process of trouble shooting my experiments and lessons learned from this study.

There are three main, naturally occurring, epidemiological types of botulism: food-borne, intestinal colonization (infant botulism) and wound botulism. The neurological signs and symptoms are the same for all three epidemiological types and may include respiratory paralysis. Wound botulism is caused by growth of cells and release of toxin in vivo, is associated with traumatic wounds and abscesses and has been reported in drug users, such as those injecting heroin or sniffing cocaine. Up to the end of 1999 there were no confirmed cases of wound botulism in the UK. Between the beginning of 2000 and the end of December 2002, there were 33 clinically diagnosed cases of wound botulism in the UK and Ireland. All cases had injected heroin into muscle or by 'skin popping'. The clinical diagnosis was confirmed by laboratory tests in 20 of these cases. Eighteen cases were caused by type A toxin and two by type B toxin.

Infant botulism is a rare disease in the UK, with the first case being recognized in 1978 and only five subsequent cases being reported before 2007. This study reports two unlinked cases of infant botulism, caused by two distinct strains of Clostridium botulinum (toxin types A and B, respectively), that occurred within a single month in the south-east of England in October 2007. The use of real-time PCR to detect C. botulinum neurotoxin genes in clinical specimens to improve the diagnostic procedure and to follow carriage of the causative organism in the infant gut is described. The laboratory investigation of these two cases demonstrated that a combination of the mouse bioassay, real-time PCR assays and conventional microbiological culture can provide rapid confirmation of a clinical diagnosis and affect patient management. Both infants (aged 4 and 8 months) were previously healthy prior to the onset of symptoms, and in both cases, a diagnosis of infant botulism was delayed for at least 10 days after initial admission to hospital. Once diagnosed, one of the infants was the first in the UK to be treated with human-derived botulism immunoglobulin. Real-time PCR was used to demonstrate that C. botulinum was excreted in the infants' faeces for up to 68 and 81 days, respectively. Despite the infrequency of infant botulism in the UK, clinicians should be aware of this rare but serious condition and should seek microbiological advice when presented with young infants with compatible symptomologies.

Surmmary The purpose of this study was to investigate the incidence of cases of sporadic diarrhoea associated with enterotoxigenic Clostridium perfringens. Cases were identified by detection of C. perfringens enterotoxin with the Oxoid RPLA kit, with confirmation by ELISA, in faecal specimens from isolated incidents of diarrhoea and from which no other enteropathogen had been isolated. In a 2-month study, 65 (18%) of 370 specimens were enterotoxin positive. There was no predominant age group or sex in the enterotoxin-positive group, but a higher proportion (79 %) was resident in the community than were enterotoxin-negative cases (34%). Only four of the 65 enterotoxin-positive patients had received antibiotic therapy. Spore counts in most enterotoxin-positive patients were > 105 /g, indicating that detection of high numbers of C. perfringens is not useful in determining the aetiology of sporadic diarrhoea. Diagnosis should be confirmed by the detection of enterotoxin, but further work is required to assess whether an acceptable accuracy is obtained with the RPLA kit or whether ELISA is needed in all cases.

Wound infections due to Clostridium botulinum were not recognised in the UK and Republic of Ireland before 2000. C. botulinum produces a potent neurotoxin which can cause paralysis and death. In 2000 and 2001, ten cases were clinically recognised, with a further 23 in 2002, 15 in 2003 and 40 cases in 2004. All cases occurred in heroin injectors. Seventy cases occurred in England; the remainder occurred in Scotland (12 cases), Wales (2 cases) and the Republic of Ireland (4 cases). Overall, 40 (45%) of the 88 cases were laboratory confirmed by the detection of botulinum neurotoxin in serum, or by the isolation of C. botulinum from wounds. Of the 40 cases in 2004, 36 occurred in England, and of the 12 that were laboratory confirmed, 10 were due to type A. There was some geographical clustering of the cases during 2004, with most cases occurring in London and in the Yorkshire and Humberside region of northeast England.

The aims of the study were to determine the prevalence of Listeria monocytogenes in the feces of healthy Austrians and to characterize the isolates by various typing methods. Stool specimens from 505 healthy volunteers from the Tyrol were tested for the presence of L. monocytogenes using cold enrichment for 6 months and five different detection methods: conventional plating onto Palcam and Rapid'L.MONO agar, immunomagnetic separation (IMS) followed by conventional plating, enzyme-linked fluorescent immunoassay (ELFA), ELISA, and PCR. L. monocytogenes was isolated by conventional plating from one specimen (0.2%), and a further three were positive on immunomagnetic separation (0.8%). Only one specimen tested positive with ELFA and EIA, although it tested negative by conventional culture, IMS, and PCR. Eighteen of 505 samples were positive by PCR (3.6%), and this included three of the four culture-confirmed specimens. Serotyping, phage-typing, arsenic cadmium, antimicrobial-resistance typing and pulsed-field gel electrophoresis showed that multiple L. monocytogenes isolates from three of the four carriers were indistinguishable. Our data indicate that the Austrian fecal carriage rate is at least 0.8%. In view of a listeriosis incidence of 0.16/100,000 per year, the chances of fecal carriage developing into listeriosis appear to be very low.