Clinical performance of Roche cobas 6800, Luminex ARIES, MiRXES Fortitude Kit 2.1, Altona RealStar, and Applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.

We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on nasopharyngeal swab samples: Roche "cobas," Luminex "ARIES," MiRXES "Fortitude," Altona "RealStar," and Thermo Fisher Scientific "TaqPath." A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2-14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS-CoV-2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re-optimization before go-live at the testing laboratory.

Determination of hand grip strength and its correlates during pregnancy: a cross-sectional study.

BACKGROUND: Pregnancy results in many changes, including reduced hand grip strength (HGS). However, good HGS is required for physical functions such as carrying and breastfeeding the baby after birth. The aim of this study was to determine the factors that may predict HGS during pregnancy. METHODS: The study was a cross-sectional study approved by the Research Ethics Committees of Kano State Ministry of Health and Aminu Kano Teaching Hospital in Kano, north-west, Nigeria. Pregnant women at the designated hospitals were included in the study if they had no serious comorbidities or any known neurological condition that affects the hands and the neck. Demographic characteristics and independent (predictor) variables (age, weight, height, BMI, maternity leave status, number of full-term deliveries, number of preterm deliveries, number of live births, number of abortuses, gravidity, trimester, systolic blood pressure, diastolic blood pressure, inter arm systolic BP difference [IASBP], inter arm diastolic BP difference [IADBP], and heart rate) of each of the participants were recorded by experienced therapists. The data were analysed using descriptive statistics, t-test, Pearson correlation coefficient and standard multiple regression. RESULT: One hundred and sixty-one pregnant women with mean age, 25.04 ± 4.83 years participated in the study. In the dominant hand, 120 participants (74.5%) had weak grip strength. In the non-dominant hand, 135 participants (83.9%) had weak grip strength. For the dominant hand, the total variance explained by the whole model was significant, 28.5%, F(11, 161) = 1.187, R2 = 0.081, p = 0.300 . In the final model, none of the variables significantly predicted HGS. However, systolic blood pressure contributed to the model more than any other variable (Beta = -0.155). For the non-dominant hand, the total variance explained by the whole model was not significant, 33.1%, F(11, 161) = 1.675, R2 = 0.111, p = 0.089 . In the final model, only systolic blood pressure (Beta = -0.254, p = 0.023) significantly predicted hand grip strength. CONCLUSION: Cardiovascular events or changes during pregnancy (such as change in systolic blood pressure) may be related to HGS in pregnant women. It is therefore, important for clinicians to pay attention to this, in planning rehabilitation strategies for pregnant women.

Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected.

Vitreous cryo-section-induced compression influences the interpretation and the reliability of electron microscopy images and tomographic reconstructions. Previous studies of this deformation have been focused at the cellular level where considerable compression occurs, yet the degree of possible intracellular macromolecular deformation has remained unclear. Here, electron cryo-tomographic reconstructions of vitreous cryo-sections show that 80S ribosomes, both intracellular and in an isolated state, appear able to resist section-induced compression. Our observations indicate that vitreous section-induced compression is non-uniform between whole cells that have been sectioned and their intracellular macromolecular complexes. We conclude that electron cryo-tomography of vitreous cryo-sections, in spite of section-induced compression, is a suitable technique for charting the structural organization of cellular nanomachines, such as ribosomes, in a cellular environment.

The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri.

The type III secretion apparatus (T3SA) is a central virulence factor of many Gram-negative bacteria. Its overall morphology consists of a cytoplasmic region, inner- and outer-membrane sections and an extracellular needle. In Shigella, the length of the needle is regulated by Spa32. To understand better the role of Spa32 we searched for its interacting partners using a two-hybrid screen in yeast. We found that Spa32 interacts with the 33 C-terminal residues (CC*) of Spa40, a member of the conserved FlhB/YscU family. Using a GST pull-down assay we confirmed this interaction and identified additional interactions between Spa40 and the type III secretion components Spa33, Spa47, MxiK, MxiN and MxiA. Inactivation of spa40 abolished protein secretion and led to needleless structures. Genetic and functional analyses were used to investigate the roles of residues L310 and V320, located within the CC* domain of Spa40, in the assembly of the T3SA. Spa40 cleavage, at the conserved NPTH motif, is required for assembly of the T3SA and for its interaction with Spa32, Spa33 and Spa47. In contrast, unprocessed forms of Spa40 interacted only with MxiA, MxiK and MxiN. Our data suggest that the conformation of the cytoplasmic domain of Spa40 defines the multi-step assembly process of the T3SA.

Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism.

The effectors of enterocyte invasion by Shigella are dependent on a type III secretion system that contains a needle whose length average does not exceed 50 nm. Previously, we reported that Spa32 is required for needle length control as well as to switch substrate specificity from MxiH to Ipa proteins secretion. To identify functional domains of Spa32, 11 truncated variants were constructed and analysed for their capacity (i) to control the needle's length; (ii) to secrete the Ipa proteins; and (iii) to invade HeLa cells. Deletion at either the N-terminus or C-terminus affect Spa32 function in all cases, but Spa32 variants lacking internal residues 37-94 or 130-159 retained full Spa32 function. Similarly, a Spa32 variant obtained by inserting of the YscP's ruler domain retained Spa32 function although it programmed slightly elongated needles. Using the GST pull-down assay, we show that residues 206-246 are required for Spa32 binding to the C-terminus of Spa40, an inner membrane protein required for Ipa proteins secretion. Our data clearly demonstrate that shortening Spa32 affects the length of the needle in a comparable manner to the spa32 mutant, indicating that the control of needle length does not require a molecular ruler mechanism.

Toward visualization of nanomachines in their native cellular environment.

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be 'docked' or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as 'high pressure freezing' with 'freeze-substitution followed by room temperature plastic sectioning' or 'cryo-sectioning of unperturbed vitreous fully hydrated samples' have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported.

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus.

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.

Structural organization of the needle complex of the type III secretion apparatus of Shigella flexneri.

The secretion apparatus known as the needle complex (NC) from the bacterium Shigella flexneri was studied by single particle electron microscopy. The isolated intact NC appears in projection to be composed of a basal body consisting of seven rings and a protruding needle appendage. A comparison of averaged projections of the intact NC and its fragments revealed the organization of the NC into several major subcomplexes. One of these lacks an inner membrane ring of the basal body but still presents the needle appendage attached to four upper rings. The position of the needle appendage within these rings is variable, suggesting that the dissociated component is necessary for stabilizing the needle appendage. Averaged images of the subcomplex lacking the inner membrane basal rings show a thicker extension at the base of the needle appendage, called the socket. This socket was also found to be present in images of the basal body fragment isolated from mutants lacking the mxiH and mxiI genes. This suggests that the socket is not composed of MxiH and MxiI subunits, which form the needle appendage. A symmetry analysis of the basal body top view projections indicated that a peripheral protein component of the inner membrane ring is present in a ring with 24 copies, in contrast to the Salmonella typhimurium NC. A model is presented in which the NC is only associated to the outer- and inner-membranes with its first and seventh ring, respectively.

The Iron Status of Sickle Cell Anaemia Patients in Ilorin, North Central Nigeria.

Objectives. Sickle cell anaemia (SCA) is one of the commonest genetic disorders in the world. It is characterized by anaemia, periodic attacks of thrombotic pain, and chronic systemic organ damage. Recent studies have suggested that individuals with SCA especially from developing countries are more likely to be iron deficient rather than have iron overload. The study aims to determine the iron status of SCA patients in Ilorin, Nigeria. Methods. A cross-sectional study of 45 SCA patients in steady state and 45 non-SCA controls was undertaken. FBC, blood film, sFC, sTfR, and sTfR/log sFC index were done on all subjects. Results. The mean patients' serum ferritin (589.33 ± 427.61 ng/mL) was significantly higher than the mean serum ferritin of the controls (184.53 ± 119.74 ng/mL). The mean serum transferrin receptor of the patients (4.24 ± 0.17 µg/mL) was higher than that of the controls (3.96 ± 0.17 µg/mL) (p = 0.290). The mean serum transferrin receptor (sTfR)/log serum ferritin index of the patients (1.65 ± 0.27 µg/mL) was significantly lower than that of the control (1.82 ± 0.18 µg/mL) (p = 0.031). Conclusion. Iron deficiency is uncommon in SCA patients and periodic monitoring of the haematological, biochemical, and clinical features for iron status in SCA patients is advised.

Malaria Parasitaemia among Blood Donors in Ilorin, Nigeria.

BACKGROUND: The prevalence of malaria parasitaemia among blood donors in Ilorin has not been documented. In this study, we determined the prevalence of malaria parasitaemia among blood donors in Ilorin, as well as, the sociodemographic and other factors associated with it. METHOD: This was a hospital-based cross sectional study involving 308 consenting blood donors. The sociodemographic characteristics of participants as well as blood donation history were obtained using structured questionnaires specifically designed for this purpose. Giemsastained thick and thin blood films to identify malaria parasites were performed using standard method. ABO blood grouping and haemoglobin electrophoresis tests were also done using standard methods. RESULTS: The prevalence of malaria parasitaemia among blood donors in Ilorin was 27.3%. The parasite species found were more of Plasmodium falciparum(85.7%) than Plasmodium malariae(14.3%) . There was no age or sex difference in malaria parasitaemia. (p-value of 0.8 and 0.32 respectively). A greater proportion of blood group O individuals had malaria parasitaemia than groups A and B but this difference was not significant (p-value = 0.13). There was also no significant difference among haemoglobin genotypes. CONCLUSION: The prevalence of malaria parasites among blood donors in Ilorin is considerably high and lack of routine screening of blood puts recipients at risk. We recommend that routine screening for malaria parasites be commenced in our blood banks. Treatment of donor blood with riboflavin and UV light to inactivate malaria parasites and other infectious pathogens before they are transfused to patients may also be considered in our blood banks.

Autotransporter ß-domains have a specific function in protein secretion beyond outer-membrane targeting.

Autotransporters (ATs) of Gram-negative bacteria contain an N-proximal passenger domain that is transported to the extracellular milieu and a C-terminal ß-domain that inserts into the outer membrane (OM) in a ß-barrel conformation. This ß-domain facilitates translocation of the passenger domain across the OM and has long been considered to be the translocation pore. However, available crystal structures of ß-domains show that the ß-barrel pore is too narrow for the observed transport of folded elements within the passenger domains. ATs have recently been shown to interact with the ß-barrel assembly machinery. These findings questioned a direct involvement of the ß-domain in passenger translocation and suggested that it may only target the passenger to the ß-barrel assembly machinery pore. To address the function of the ß-domain in more detail, we have replaced the ß-domain of the Escherichia coli AT hemoglobin protease by ß-domains originating from other OM proteins. Furthermore, we have modified the diameter of the ß-domain pore. The mutant proteins were analyzed for their capacity to insert into the OM and for surface display of the passenger. Our results show that efficient passenger secretion requires a specific ß-domain that not only functions as a targeting device but also is directly involved in the translocation of the passenger to the cell surface.

Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus.

Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices.