Chembio DPP COVID-19 IgM/IgG Serological Testing: Laboratory Validation, Guidelines, and Recommendations.

BACKGROUND: On January 30, 2020, WHO declared COVID-19 a pandemic. In this article we describe our experience at Richmond University Medical Center with Chembio serological IgM, IgG testing. METHODS: In this prospective cohort study of patients and hospital employees, we utilized Chembio COVID-19 IgM/IgG serological testing in addition to Cepheid RT-PCR analysis. RESULTS: We evaluated the performance of Chembio serological test for IgM and IgG as an employee screening tool in a community hospital setting. The total number of currently asymptomatic employees screened was 1,866 from the Richmond University Medical Center. The non-exposed group included 1,253 (67.1%) employees with no significant clinical history and non-reactive IgM and IgG antibodies. The convalescent group included 255 (13.7%) of the employees with elevation of IgG only, 18 (1%) employees with past history of positive PCR and COVID-19 who currently have non-reactive IgM and IgG antibodies or demonstrate elevated IgG only, followed by 3 employees (< 1%) with no past clinical history who demonstrated reactive IgM and IgG antibodies and negative follow up by PCR. The reported 14.9% exposure/convalescent rate is lower than the reported 20% by the Department of Health and Governor Andrew Cuomo and may represent a better utilization of personal protective equipment, better hand washing techniques, and better disinfection procedures combined with strict social distancing. CONCLUSIONS: Chembio's performance is satisfactory; however, hospitals must design their own policies addressing: who needs to be screened and who will interpret the results as well as constructing management algorithms for employees with no previous history and current double positive antibodies.

Evidence that mycobacterial PE_PGRS proteins are cell surface constituents that influence interactions with other cells.

The elucidation of the genomic sequence of Mycobacterium tuberculosis revealed the presence of a novel multigene family designated PE/PE_PGRS that encodes numerous, highly related proteins of unknown function. In this study, we demonstrate that a transposon insertion in a PE_PGRS gene (1818(PE_PGRS)) found in Mycobacterium bovis BCG Pasteur, which is the BCG homologue of the M. tuberculosis H37Rv gene Rv1818c, introduces new phenotypic properties to this BCG strain. These properties include dispersed growth in liquid medium and reduced infection of macrophages. Complementation of the 1818(PE_PGRS)::Tn5367 mutant with the wild-type gene restores both aggregative growth (clumping) in liquid medium and reestablishes infectivity of macrophages to levels equivalent to those for the parent BCG strain. Western blot analysis using antisera raised against the 1818(PE_PGRS) protein shows that PE_PGRS proteins are found in cell lysates of BCG and M. tuberculosis H37Ra and in the cell wall fraction of M. tuberculosis H37Rv. Moreover, immunofluorescent labeling of mycobacteria indicates that certain PE_PGRS proteins are localized at the cell surface of BCG and M. tuberculosis. Together these results suggest that certain PE_PGRS proteins may be found at the surface of mycobacteria and influence both cell surface interactions among mycobacteria as well as the interactions of mycobacteria with macrophages.

Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico.

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.

Transposon mutagenesis in mycobacteria using conditionally replicating mycobacteriophages.

Genetic analyses of pathogenic mycobacteria such as Mycobacterium tubeculosis and Mycobacterium bovis required improvement of existing methodologies for the generation of large representative libraries of mutants. Two basic methodologies have been used to generate mutant libraries in both fast- and slow-growing mycobacteria: chemical mutagenesis and transposon mutagenesis. Chemical mutagenesis has successfully been used to produce different auxotrophic mutants in the fast growing mycobacteria Mycobacterium phlei (1,2) and Mycobacterium smegmatis (3,4). A detailed chemical mutagenesis protocol for the generation of mutant libraries in the fast-growing mycobacteria can be found in the previous volume of this manual (5). Chemical mutagenesis is not the ideal method for producing large representative mutant libraries for the slow-growing mycobacteria because: (1) the mutation frequency is relatively low,(2) multiple mutations may occur in the same cells,(3) clumping of the mycobacteria makes the identification and purification of the mutant clones very difficult,and (4) no generalized transducing phage has been described for the slow-growing mycobacteria to allow transfer of the point mutations and construction of isogenic strains.

Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis.

Attenuated mutants of Mycobacterium tuberculosis represent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph of M. tuberculosis was created by allelic exchange, replacing wild-type leuD (Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was <10(-11). In contrast to wild-type M. tuberculosis, the DeltaleuD mutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-type leuD abolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the DeltaleuD mutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-type M. tuberculosis, were immunized with the DeltaleuD mutant and subsequently challenged with virulent M. tuberculosis by both the intravenous and aerosol routes. A comparison group of mice was immunized with conventional Mycobacterium bovis BCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the DeltaleuD mutant of M. tuberculosis exhibited enhanced and statistically equivalent survival curves. However, the leuD auxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection against M. tuberculosis challenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains of M. tuberculosis should persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.

Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box.

Detecting antibiotic resistance in Mycobacterium tuberculosis is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25 M. tuberculosis reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents.

Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.

TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.

Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis.

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30 degrees C but not at 37 degrees C (TM4) or 38.5 degrees C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette-Guérin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.

Characterization of PPD protein antigens in whole cell lysates of Mycobacterium bovis BCG.

The low molecular mass protein antigens in PPD from M. bovis BCG were chemically oligomerized using sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (S-SMPB) as a crosslinking agent. Protein oligomers with molecular mass over 90 kDa were obtained and used for the preparation of hyperimmune polyclonal rabbit antiserum. Using this antiserum four protein bands with molecular mass 120, 90, 75 and 65 kDA were detected in immunoblotting analysis of sonic extract from M. bovis BCG separated in SDS-polyacrylamide gel. We suggest that these immunoreactive proteins in the sonic extract represent the native forms of the heat stable low molecular mass protein antigens in PPD.

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica.

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.

Chemical synthesis and characteristics of a hybrid phage T5-lac promoter.

A new P1O1R9 inducible promoter (where P1 is a promoter sequence analogous to that of the phage T5 early promoter; O1 is lac-operator; and R9 is a ribosome binding site) was synthesized. We studied the efficiency of this promoter in controlling and inducible gene expression using two model genes: human calcitonin tetrameric (hCT[4]) and human interferon alpha 1 (hIFN alpha 1). The synthetic lac-operator O1 was found to repress P1 activity in media free of lac-operon inducers which was derepressed in the presence of IPTG. The levels of expression of both genes (evaluated by quantitating mRNA and protein) in the presence of lac-inducers were close to those obtained with the P1R9 constitutive promoter.

Expression of repetitive human calcitonin genes in Escherichia coli.

In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E. coli under the control of a constitutive synthetic phage promoter. The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc. The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene). This effect held true as well for the proteins coded by the corresponding repetitive hCT genes. The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats. The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E. coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics). One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene. The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E. coli strains in order to find the optimal host for production of rhCT. The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1.

Molecular cloning of mycobacterial promoters in Escherichia coli.

Mycobacterium bovis BCG chromosomal DNA, digested with EcoR1 and HindIII, was used to construct a promoter library in Escherichia coli using the promoter probe plasmid pKO-1. DNA inserts of various sizes showed promoter activity judged by the level of galactokinase (galK) whose synthesis they activate (between 50 and 850 galactokinase units). No correlation between the length of the DNA insert and the level of the galactokinase was found suggesting that the multicopy pool of the promoters does not influence the level of the transcription of the galK gene.

Is the nuclear matrix the site of DNA replication in eukaryotic cells?

Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.