Diagnostic accuracy of the alpha defensin lateral flow device (Synovasure) for periprosthetic infections in microbiologically complex situations: A study of 42 cases in a French referral centre.

BACKGROUND: Joint aspiration is currently the reference standard test for diagnosing periprosthetic joint infection (PJI) despite the high rate of false-negative results, of which a major cause is the fastidious nature of some microorganisms. A rapid diagnostic test that detects alpha defensin (Synovasure™, Zimmer, Warsaw, IN, USA) in joint fluid can provide the diagnosis of PJI within a few minutes across the full spectrum of causative organisms (including mycobacteria and yeasts). Its performance in detecting bacterial infections is unaltered by concomitant antibiotic therapy. Few studies of Synovasure™ have been conducted by groups that were involved in designing the test, which has not been validated in France. Assessments in referral centres where complex microbiological situations are common hold considerable interest. The objective of this prospective study was to determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and causes of error of Synovasure™ used to diagnose periprosthetic infection in complex microbiological situations. HYPOTHESIS: The rapid diagnostic test Synovasure™ has greater than 90% NPV for detecting periprosthetic infections in complex microbiological infections. MATERIAL AND METHODS: Synovasure™ was used 42 times in 39 patients between October 2015 and October 2017 in challenging microbiological situations [discordant joint aspiration results (n=20), negative cultures with clinical or laboratory evidence of infection, (n=21), and concomitant antibiotic therapy (n=1)]. Of the 39 patients, 23 had total knee prostheses, 13 total hip prostheses, and 3 total femoral prostheses. The reference standard to which the Synovasure™ results were compared was the PJI criteria set developed by the Musculoskeletal Infection Society (MSIS). RESULTS: Synovasure™ was negative in 30 cases with negative joint fluid cultures (30/42, 71.4%). Of the 12 (28.6%) cases with positive Synovasure™ results, only 7 (7/12, 58.3%) had positive joint fluid cultures. According to the MSIS criteria 9 cases were infected, including 8 with positive and 1 with negative Synovasure™ results. Of the 33 cases that were not infected according to MSIS criteria, 29 had negative and 3 positive Synovasure™ results; the remaining case had a positive Synovasure™ result but was excluded when metallosis was found intra-operatively. NPV was 96.7%, PPV 72.7%, sensitivity 88.9%, and specificity 90.6%. DISCUSSION: The high NPV of Synovasure™ suggests a role for this test in microbiologically complex situations as a new tool for ruling in and, most importantly, ruling out infection in doubtful cases. LEVEL OF EVIDENCE: III, prospective study of diagnostic accuracy.

Reversion in Chinese hamster lines amplified at the AMPD2 locus: spontaneous and benzamide-stimulated gradual loss of amplified alleles of marker genes.

The HC47 and HC474 cell lines of Chinese hamster fibroblasts resist coformycin through the intrachromosomal amplification of the AMP deaminase 2 (AMPD2) gene. Due to the coamplification of a mu glutathione S-transferase (GST) gene, these mutant lines are more sensitive than GMA32 wild-type parental cells to buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis. This property was exploited to select revertants of amplification from HC474 cells. Reversion in that line is frequently a gradual process that does not involve extrachromosomal intermediates. The terminal products of this process are commonly cells with a complete deletion of the amplified allele of marker genes and are therefore haploid for these loci on the homologous chromosome. Exposing HC474 cells to benzamide (BA), an inhibitor of polyADP-ribosylation, increased the recovery of revertants to an extent allowing the detection of reverting cells without BSO selection. This effect of BA was used to isolate revertant cells from the HC47 line that is extremely stable and to demonstrate that the mechanism of gradual reversion also occurs in this line. The gradual deletion of amplified copies within the chromosomes suggests that breakage-fusion-bridge (BFB) cycles drive this process.

A glutathione depletion selectively imposed on mu glutathione S-transferase overproducing cells increases nitrogen mustard toxicity.

Glutathione (GSH) contributes to the detoxification of anticancer drugs through the operation of specific glutathione S-transferases (GST) and innate, or acquired, overexpression of this enzyme family has been frequently observed in tumor cell lines. In the GMA32 line of Chinese hamster fibroblasts, we showed that GSH starvation produced by exposing cells to buthionine sulfoximine (BSO) increased the toxicity of chlorambucil and melphalan, but not that of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), cisplatine and doxorubicin. This indicates that efficient mechanisms of detoxification using GSH operate for chlorambucil and melphalan, but not for the other drugs in these cells. We then showed that GSH depletion could be selectively and transiently induced in the mu GST overexpressing cell line derived from GMA32, HC474, by exposing cells to substrates specific to the overexpressed isozyme. Exposing cells to such a substrate, trans-stilbene oxide, does not alter the sensibility of GMA32 cells to melphalan and chlorambucil, but increases that of HC474 cells to these drugs, to an extent comparable to that obtained with BSO. This observation highlights the possibility of exploiting GST overexpression, a frequent feature of tumor cells, to selectively sensitize these undesirable cells to anticancer drugs.

Coamplification of mu class glutathione S-transferase genes and an adenylate deaminase gene in coformycin-resistant Chinese hamster fibroblasts.

In Chinese hamster fibroblasts, we previously detected an expressed gene located near the AMP deaminase gene. This gene was named Y1. Upon selection for resistance to coformycin, an inhibitor of AMP deaminase activity, both genes were amplified in several mutants. We have determined the complete nucleotide sequence of Y1 cDNA and identified the Y1 gene as a mu class glutathione S-transferase gene by comparison with sequences present in a data bank. Accordingly, Y1-amplified mutants express an increased glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene; this activity, as well as the abundance of the corresponding RNA, appears, however, to reach a limit despite further increase in the Y1 gene copy number during successive amplification steps. Southern blot experiments showed that Y1 belongs to a multigene family, all or part of which has been amplified in mutant lines. These data provide a method to amplify and to overexpress the mu class of the glutathione S-transferase gene family on the basis of its linkage with the AMP deaminase gene.

Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (approximately 800 times) are due to the esterase B1 gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase B1 protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase B1 gene. It is suggested that these repetitive sequences may have a role in the amplification process.

The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

The amplified DNA of HC50474, a Chinese hamster fibroblast cell line selected in three steps for high resistance to coformycin, consists chiefly of 150 copies of a large inverted duplication including the adenylate deaminase gene. Most if not all of these units are more than 2 x 120 kb long. The inverted duplication was first detected in the cells recovered from the second selection step, at the same chromosomal location as the first step amplified units. Its formation and amplification appear to be coupled since the second step cell line already contained 40 copies of this novel structure. Reamplification of the inverted duplication occurred at the third step of selection concomitant with the loss of amplified DNA acquired during the first step. The head-to-head junction has been formed by recombination within a recombinational hotspot described previously [Hyrien, O., Debatisse, M., Buttin, G. and Robert de Saint Vincent, B. (1987) EMBO J., 6, 2401-2408]. Sequences at the joint and in the corresponding wild-type region reveal that the crossover sites, one of which occurs in the putative promoter region of B2 repeat, are located at the top of significant stem-loop structures and that patchy homologies between the parental molecules on one side of the breakpoints allow alignment of these crossover sites. We present a model which explains the formation and amplification of this and other large inverted duplications by errors in DNA replication.

A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA.

We have identified, in the amplified domain of adenylate deaminase (AMPD) overproducing Chinese hamster fibroblasts, a 2.6 kb recombinogenic DNA region which is frequently involved in amplification-associated rearrangements. The nucleotide sequence reveals a mosaic organization of four Alu-equivalent repeats of the B1 and B2 families and eight long A + T-rich DNA segments. Part of this region is enriched with long imperfect palindromes. The center of one palindrome contains a putative topoisomerase I cleavage site and this site defines the position of a novel junction which was formed by illegitimate recombination with anther A + T-rich DNA sequence located far apart on the amplified DNA. These findings and their significance are discussed in the context of related data from other systems and in the light of current models for eukaryotic DNA recombination, replication and organization.

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito.

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase.

Four genes encoding proteins designated as W, X, Y1, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Y1 gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes--in a particular order--in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.

Expression of several amplified genes in an adenylate-deaminase overproducing variant of Chinese hamster fibroblasts.

Unstable variants with increasing amounts of adenylate-deaminase (AMPD) have been stepwise recovered from Chinese hamster fibroblasts plated in selective medium containing increasing coformycin concentrations; several polypeptides accumulate in the variants in parallel to AMPD: they are no longer detectable in cells which reverted to the wild-type enzyme level. We report here the molecular cloning of cDNA sequences complementary to mRNAs coding for four such polypeptides. The plasmidic probes have been exploited to characterize their complementary mRNAs and to quantify the copies of these cognate genes in a variant and in two revertant clones. The results show that different mRNAs code for the four polypeptides; their accumulation is accounted for by amplification of their specific genes; these observations suggest that cells overproducing AMPD are characterized by the presence of amplification units comprising several expressed genes.

Effect of chromosomal position on amplification of transfected genes in animal cells.

A single protein (CAD) contains the first three enzymatic activities of de novo uridine biosynthesis. The chromosomal location of CAD genes introduced into Chinese hamster ovary cells significantly affects the frequency and cytogenetic result of their amplification. The amplification frequency in one transformant is 100-fold that of the others; in another, amplification of donated genes inserted near a centromere results in chromosome instability and rearrangements.

Analysis of CAD gene amplification using a combined approach of molecular genetics and cytogenetics.

CAD is a multifunctional protein which catalyzes the first three steps of de novo uridine biosynthesis. Rodent cells resistant to PALA, a specific inhibitor of the ATCase activity of CAD, overproduce the CAD protein and CAD mRNA as a direct result of the amplification of the CAD gene. In order to study the mechanism of CAD gene amplification, a functional Syrian hamster CAD gene was inserted into a cosmid vector using molecular cloning techniques. The cloned genes were assayed for biological function by fusing CAD-deficient Chinese hamster ovary (CHO) cell mutants with protoplasts of E. coli containing the CAD cosmids. Two clones with functional CAD genes were isolated and shown to contain inserts 40 and 45 kb long. The cloned genes could also be introduced into wild type CHO cells by selecting for cells which became resistant to high PALA concentrations in a single step. Transformations of mutant and wild type CHO cells contained multiple active copies of the donated Syrian hamster CAD genes in addition to their endogenous CHO CAD genes. The cloned genes in all transformants analyzed are integrated into host cell chromosomes at single locations defined by in situ hybridization. Independently isolated transformants contain the donated genes in different chromosomes. Co-transformation of CHO cells with two different genes by protoplast fusion is also shown to be possible.

Homologous recombination in mammalian cells mediates formation of a functional gene from two overlapping gene fragments.

Chinese hamster cells with a lesion in the CAD gene (cell line Urd-A) require exogenous uridine to survive. Uridine prototrophs could be isolated after introducing two recombinant plasmids containing overlapping fragments of a cloned Syrian hamster CAD gene. In contrast, no uridine prototrophs were obtained after introducing a plasmid containing only one of the two overlapping fragments. DNA restriction analysis showed that the prototrophic transformants contain a functional CAD gene which was formed by a recombination event in the overlapping region of the two clones. Most of the recombination events involved homologous exchanges, and some of them apparently were reciprocal. In situ hybridization analysis revealed that the donated sequences were integrated at a single chromosomal site which was different in each transformant. These results demonstrate the existence of a recombination system(s) in mammalian cells that can catalyze homologous exchanges. Recombination between donated sequences is a means by which this system can be characterized and also utilized for the production of novel gene fusions.

The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker.

Rodent cells resistant to PALA, a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional CAD protein, overproduce CAD as a result of amplification of the CAD gene. We cloned a functional CAD gene from Syrian hamster cells using a cosmid vector. Two independently isolated cosmids containing CAD genes have inserts 40 and 45 kb long. We introduced the cloned genes into CAD-deficient Chinese hamster ovary (CHO) cell mutants by fusing them with protoplasts of Escherichia coli containing the cosmids. We also introduced the cloned genes into wild-type CHO cells by selecting cells that became resistant to high concentrations of PALA following protoplast fusion. The transformants of the mutant and wild-type CHO cells contain multiple active copies of the donated Syrian hamster CAD genes. The cloned genes in three independent transformants are integrated into host-cell chromosomes at single locations identified by in situ hybridization. In two of these transformants, the genes are located in one X chromosome or in a chromosome resembling the X. In the third case, the genes are located in a small metacentric or rearranged chromosome.