A comparative analysis exposes an amplification delay distinctive to SARS-CoV-2 Omicron variants of clinical and public health relevance.

ABSTRACTMutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N, E, and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay's primers caused a > 4 cycle delay during amplification without impacting overall assay performance.Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.

Evaluating wildlife as a potential source of Salmonella serotype Newport (JJPX01.0061) contamination for tomatoes on the eastern shore of Virginia.

Eastern Shore of Virginia red, round tomatoes contaminated with Salmonella serotype Newport pattern JJPX01.0061 have been a source of several multistate outbreaks within the last 10 years. No source of the contamination has yet been identified. The goal of this study was to evaluate wildlife as a potential source of contamination. Faecal samples from deer, turtles and birds were collected between November 2010 and July 2011 from seventeen locations on the Eastern Shore of Virginia. A total of 262 samples were tested for the presence of Salmonella using an enzyme-linked immunosorbent assay (ELISA). A total of 23 (8.8%) samples tested positive for Salmonella spp. and were further characterized by serotyping and pulsed-field gel electrophoresis (PFGE) subtyping. Overall, twelve serotypes were identified, including Salmonella serotype Javiana, another common serotype associated with tomato-related outbreaks. Only one avian sample collected in July 2011 was determined to be positive for S. Newport pattern 61. This sample was collected from the ground at a site where birds, mostly gulls, were congregating. Although many of the avian samples from this site were dry, the site yielded eleven positive Salmonella samples. This suggests that certain Salmonella serotypes may persist in the environment despite extreme conditions. The recovery of one Newport pattern 61 isolate alone does not yield much information regarding the environmental reservoirs of this pathogen, but when combined with other data including the recovery of several isolates of Javiana from birds, it suggests that birds might be a potential source of Salmonella contamination for tomatoes on the Eastern Shore.

Investigation of a mumps outbreak among university students with two measles-mumps-rubella (MMR) vaccinations, Virginia, September-December 2006.

Following the clinical diagnosis of the first case of mumps on September 22, 2006 at the University of Virginia (UVA), 52 suspected cases were identified through active surveillance for mumps by the end of December 2006. Samples were collected from 47 students who presented with parotitis despite a documented history of two doses of measles, mumps, and rubella (MMR) vaccine. Six of 47 serum samples (13%) were positive for mumps IgM, and 46/47 specimens were positive for mumps IgG. Endpoint titration of acute phase serum samples from laboratory-confirmed cases did not provide evidence that elevated serum IgG is a consistent marker for infection among cases due to secondary vaccine failure. Buccal swab samples from 39 of the 47 students were tested by real-time reverse transcription-polymerase chain reaction (RT-PCR) and/or viral culture. Mumps virus or mumps RNA was detected in 12 of 39 buccal samples (31%). Genetic analysis of the virus from the outbreak at UVA indicated that the outbreak was not linked to the large mumps outbreak in the Midwestern US that occurred earlier in 2006. Our findings support the use of viral detection to improve laboratory diagnosis of mumps among persons who have received two doses of MMR.

A multistate outbreak of Escherichia coli O157:H7 infections linked to alfalfa sprouts grown from contaminated seeds.

A multistate outbreak of Escherichia coli O157:H7 infections occurred in the United States in June and July 1997. Two concurrent outbreaks were investigated through independent case-control studies in Michigan and Virginia and by subtyping isolates with pulsed-field gel electrophoresis (PFGE). Isolates from 85 persons were indistinguishable by PFGE. Alfalfa sprouts were the only exposure associated with E. coli O157:H7 infection in both Michigan and Virginia. Seeds used for sprouting were traced back to one common lot harvested in Idaho. New subtyping tools such as PFGE used in this investigation are essential to link isolated infections to a single outbreak.

Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp.

The effect of 100 polar and 100 nonpolar plant extract materials obtained from Southeast Asia were evaluated for amebicidal activity in vitro against three species of Acanthamoeba. A. culbertsoni, A. castellanii, and A. polyphaga, the causative agents of granulomatous amebic encephalitis and amebic keratitis, were studied in vitro to determine whether the plant extracts exhibited amebicidal activity or induced encystment of the amebae. Of the 200 plant extracts tested, extracts obtained from three plants (Ipomoea sp., Kaempferia galanga, and Cananga odorata) were amebicidal for all three species of Acanthamoeba and a fourth extract prepared from Gastrochilus panduratum was lytic for A. polyphaga and growth-inhibitory for A. castellanii and A. culbertsoni. Three plant extracts induced encystment of all three species of Acanthamoeba. Select plant extracts were tested as well for tumoricidal activity against B103 neuroblastoma cells. Some plant extracts that exhibited tumoricidal activity for B103 cells were not amebicidal for Acanthamoeba spp. Additionally, the polar and nonpolar extracts that exhibited amebicidal activity were also tested for activity against primary murine peritoneal macrophage cultures. Plant extracts that demonstrated tumoricidal or amebicidal activity were not lytic for normal macrophage cultures.

The interaction of Acanthamoeba spp. with activated macrophages and with macrophage cell lines.

Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B6C3F1 mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with 3H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chromium-51 from radiolabeled macrophages. Acanthamoeba culbertsoni, which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba-mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba-specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor alpha, interleukin 1 alpha, and interleukin 1 beta, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.

Resistance of Acanthamoeba species to complement lysis.

Acanthamoeba species were evaluated for susceptibility to complement lysis as determined by release of radiolabeled uridine. The 3 Acanthamoeba species tested, A. culbertsoni (ATCC 30171), A. castellanii (ATCC 30010), and A. polyphaga (ATCC 30461), depleted hemolytic complement activity from normal human serum (NHS), yet were resistant to its lytic effects. Examination of microtiter plates containing amoebae incubated in NHS demonstrated formation of a pellet in the wells. Pellet formation was not observed when amoebae were incubated in human cord serum, heat-inactivated serum, or C1q-deficient serum. Ultrastructural examination of serum-treated amoebae revealed the presence of a finely granular substance that surrounded the amoebae. Treatment of amoebae with enzymes or metabolic inhibitors prior to incubation in NHS was performed to investigate the mechanism of complement resistance. Cycloheximide or cytochalasin D pretreatment increased the susceptibility of A. culbertsoni and A. castellanii to complement lysis. Cytochalasin D treatment also increased the susceptibility of A. polyphaga to complement lysis. Inhibition of serine protease activity by phenylmethylsulfonylfluoride increased complement susceptibility of all 3 species of Acanthamoeba. Enzymatic removal of surface components from A. polyphaga or A. castellanii, with trypsin, neuraminidase, or phosphatidylinositol-specific phospholipase C (PIPLC), did not affect serum resistance. In contrast, PIPLC treatment of A. culbertsoni significantly increased lysis by complement. The ability of Acanthamoeba species to activate the alternative complement pathway yet resist complement-mediated cellular lysis can be attributed to both the release of a transport-dependent extracellular matrix as well as the presence of complement inhibitory surface proteins.

Dependence of growth, metabolic expression, and pathogenicity of Naegleria fowleri on exogenous porphyrins.

The pathogenicity of the free-living amoeba Naegleria fowleri is modulated by the composition of the medium used for cultivation. The constituents that determine the level of pathogenicity of N. fowleri, however, have not been definitively established. The present study examined the effects of selected porphyrins on N. fowleri amoebae. The iron-containing porphyrins, hemin or hematin, or the iron-free porphyrin, protoporphyrin IX, were effective in supporting growth of N. fowleri in Cline medium lacking serum. Iron-binding proteins, including hemoglobin, could not satisfy the growth requirement of the amoebae for exogenous porphyrin. Expression of biological functions including azocaseinase activity, agglutination, mobility, complement susceptibility, and virulence were altered by the composition of the growth medium. Amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX displayed greater mobility and were more resistant to lysis by complement than those grown in Nelson medium. Similarly, amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX were more pathogenic for B6C3F1 mice than those grown in Nelson medium. The addition of protoporphyrin IX to Nelson medium resulted in a modest increase in mobility, resistance to complement lysis and virulence when compared to N. fowleri amoebae grown in Nelson medium without added porphyrin.

Anandamide inhibits macrophage-mediated killing of tumor necrosis factor-sensitive cells.

Anandamide (arachidonoylethanolamide) was shown to inhibit macrophage-mediated killing of tumor necrosis factor-sensitive murine L929 fibroblasts. Scanning electron microscopy (SEM) demonstrated that L929 cells, co-cultured with Propionibacterium acnes (P. acnes)-activated peritoneal macrophages from mice treated with vehicle, were either disrupted or had surface abnormalities and numerous punctate lesions. In contrast, L929 cells co-cultured with macrophages from mice receiving P. acnes in concert with Anandamide (20 mg/kg-80 mg/kg) or the exogenous cannabinoid delta-9-tetrahydrocannabinol (THC; 80 mg/kg) did not exhibit ultrastructural abnormalities. Cytotoxicity assays were performed in parallel with SEM in order to determine whether ultrastructural observations correlated with target cell killing as measured by release of radiolabel from L929 target cells. P. acnes-activated macrophages from vehicle-treated mice elicited 41% specific release of radiolabel from [51Cr]-labeled L929 cells. In contrast, macrophages from animals treated with P. acnes and with 20, 40, or 80 mg/kg Anandamide exhibited 38%, 25%, or 28% specific release of radiolabel, respectively. Similarly, macrophages from animals treated with P. acnes and with 80 mg/kg THC exhibited 21% specific release of radiolabel. In vitro cytotoxicity studies using radiolabeled L929 target cells and conditioned medium from RAW264.7 murine macrophage-like cells allowed for determination of the time interval over which Anandamide exerted its inhibitory effect. Maximal inhibition of target cell killing occurred when conditioned medium was obtained from macrophages exposed to Anandamide for 1 hr prior to activation. In contrast, conditioned medium from THC-treated macrophages exerted its maximal inhibition of target cell killing when obtained from RAW264.7 cells pretreated for 24hr-48hr prior to activation. These results indicate that Anandamide and THC exert a similar inhibition of killing of TNF-sensitive target cells. However, the time interval over which these two substances elicit their suppressive effect differs.

Modulation of complement resistance and virulence of Naegleria fowleri amoebae by alterations in growth media.

Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant. The present study evaluates the effect of complement on N. fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media. Pathogenic N. fowleri maintained in "enriched" Cline medium are virulent for mice and resistant to complement lysis. A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N. fowleri are grown in Nelson medium lacking hemin. N. fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement. Cycloheximide treatment of N. fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium. Proteins were radiolabeled with [35S] during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement.

Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage.

Pathogenic and nonpathogenic Naegleria amoebae activate the alternative C pathway; however, only pathogenic amoebae are resistant to C-mediated damage. The present study was undertaken to determine the mechanism by which highly pathogenic N. fowleri amoebae resist C-mediated damage. Nomarski optics microscopy and electron microscopy of Naegleria amoebae revealed membrane blebbing on the surface of C-resistant N. fowleri, but not on C-sensitive N. gruberi, in response to incubation in normal human serum diluted 1:4 to 1:16. Immunofluorescent staining of pathogenic amoebae, by using antiserum to human C proteins comprising the membrane attack complex, C5b through C9, and FITC-labeled goat anti-rabbit IgG, confirmed that the membrane attack complex was concentrated on the membrane blebs. Binding studies with the use of radioiodinated C9 demonstrated a decrease in the 125I-labeled C9 cpm associated with N. fowleri amoebae and an increase in the 125I-labeled C9 cpm associated with the released membrane vesicles after increasing incubation periods in normal human serum. Treatment of pathogenic, C-resistant N. fowleri with cytochalasin D or cytochalasin B to inhibit actin-dependent exocytic processes increased the susceptibility of the amoebae to C damage. In contrast, incubation of nonpathogenic, C-sensitive amoebae with cytochalasins did not alter their susceptibility to C lysis. These data indicate that pathogenic N. fowleri use membrane vesiculation to remove membrane-deposited C proteins, specifically the membrane attack complex (C5b-C9). The ability to remove surface-associated membrane attack complexes serves as one mechanism by which pathogenic N. fowleri resist C lysis.

Modulation of biological functions of Naegleria fowleri amoebae by growth medium.

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri, LEE, and a highly pathogenic strain, LEEmpC1, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri, LEE and LEEmpC1, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.

Alterations in protein expression and complement resistance of pathogenic Naegleria amoebae.

Highly pathogenic strains of Naegleria fowleri activate the alternative complement pathway but are resistant to lysis. In contrast, weakly pathogenic and nonpathogenic Naegleria spp. activate the complement pathway and are readily lysed. The present study was undertaken to determine whether surface components on amoebae accounted for resistance to complement lysis. Enzymatic removal of surface components from highly pathogenic N. fowleri with phosphatidylinositol-specific phospholipase C or with endoglycosidase H increased the susceptibility of these amoebae to complement-mediated lysis. Similar treatment of nonpathogenic amoebae had no effect on susceptibility to complement. Tunicamycin treatment of highly and weakly pathogenic N. fowleri increased susceptibility to lysis by complement in a dose-related manner. Tunicamycin treatment did not alter the susceptibility of nonpathogenic amoebae to complement. Proteins of 234 and 47 kDa were detected in supernatant fluid from phosphatidylinositol-specific phospholipase C-treated highly pathogenic amoebae but not in supernatant fluid from phosphatidylinositol-specific phospholipase C-treated weakly pathogenic amoebae. Electrophoretic analysis of iodinated surface proteins of highly pathogenic N. fowleri revealed species of 89, 60, 44, and 28 kDa. Western immunoblots of lysates from surface-iodinated amoebae were stained with biotinylated concanavalin A or biotinylated Ulex europaeus agglutinin I. Surface proteins, identified in highly pathogenic amoebae by iodination, were shown to be glycoproteins by lectin analysis specific for the detection of mannose and fucose residues.

Immunomodulation by various nitrosoureas and its effect on the survival of the murine host bearing a syngeneic tumor.

Chemotherapeutic efficacies of the nitrosoureas 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), chlorozotocin (CLZ), and streptozotocin (STZ) were investigated against the LSA tumor which is syngeneic to C57BL/6 mice. It was observed that a single injection of 20 mg/kg body weight of BCNU or CLZ, even at an advanced stage of tumor growth, completely cured greater than 90% of the tumor-bearing mice. Furthermore, BCNU-cured or CLZ-cured mice could specifically reject secondary rechallenge with the LSA tumor. In contrast, a single dose treatment with STZ at 20-200 mg/kg body weight failed to cure the tumor-bearing mice (0% survival). The failure of STZ to cure tumor-bearing mice was next addressed considering three possible mechanisms: (a) STZ was less tumoricidal; (b) STZ suppressed the immunity of the host; and (c) STZ failed to eliminate tumor-specific suppressor T-cells. The failure of STZ to cure tumor-bearing mice was not totally related to its tumoricidal properties since STZ at higher doses did possess significant tumoricidal activity in vitro and in vivo, comparable to that of BCNU or CLZ. When spleen cells from normal mice treated with BCNU, CLZ, or STZ were assayed for their responsiveness to the T-cell mitogens concanavalin A or phytohemagglutinin, it was observed that STZ was in fact less immunosuppressive than BCNU or CLZ. The fact that STZ did not suppress the immunity of the host was also suggested by the findings that BCNU-cured mice treated with STZ or CLZ could still reject secondary rechallenge with the specific tumor LSA. Following treatment of tumor-bearing mice with BCNU or CLZ, tumor-specific delayed type hypersensitivity responses were demonstrable in these mice but not in STZ-treated mice. The inability of STZ-treated tumor-bearing mice to elicit a delayed type hypersensitivity response was not due to selective depletion of delayed type hypersensitivity-inducing CD4+ T-cells but was probably due to failure of STZ to eliminate tumor-specific suppressor cells. Together these findings suggested that the failure of STZ to cure LSA tumor-bearing mice was not due to lack of tumoricidal activity or related to suppression of tumor-specific effector T-cell function but may be due to the failure of STZ to eliminate tumor-specific T suppressor cells. The present study suggests that the outcome of chemotherapy with nitrosoureas depends, in addition to the tumoricidal activity of the drug, on the immunomodulating action on the immune mechanisms of the host.