Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5′ end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.     

Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans

A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and was able to detect all homozygous mutants included in the collection of strains, demonstrating that it is a robust, quick, and labor-saving method that is suitable for a routine clinical diagnostic laboratory.     

Development of a Multiplex Primer Extension Assay for Rapid Detection of Salmonella Isolates of Diverse Serotypes

Food-borne salmonellosis is a major manifestation of gastrointestinal disease in humans across the globe. Accurate and rapid identification methods could positively impact the identification of isolates, enhance outbreak investigation, and aid infection control. The SNaPshot multiplex system is a primer extension-based method that enables multiplexing of single nucleotide polymorphisms (SNPs). Here the method has been developed for the identification of five Salmonella serotypes, commonly detected in the United Kingdom, based on serotype-specific SNPs identified in the multilocus sequence typing (MLST) database of Salmonella enterica. The SNPs, in genes hemD, thrA, purE, and sucA, acted as surrogate markers for S. enterica serovars Typhimurium, Enteritidis, Virchow, Infantis, and Braenderup. The multiplex primer extension assay (MPEA) was conducted in two separate panels and evaluated using 152 Salmonella enterica isolates that were characterized by MLST. The MPEA was shown to be 100% specific and sensitive, within this collection of isolates. The MPEA is a sensitive and specific method for the identification and detection of Salmonella serotypes based upon SNPs seen in MLST data. The method can be applied in less than 6 h and has the potential to improve patient care and source tracing. The utility of the assay for identification of Salmonella serotypes directly from clinical specimens and food samples warrants further investigation.Food-borne salmonellosis is an important public health problem, causing substantial morbidity and mortality worldwide. Transmission of salmonella to humans has been linked to multiple sources, including contaminated or undercooked food. Therefore, development of rapid and sensitive methods for detection of Salmonella serovars directly from clinical specimens and environmental samples may have a significant impact on the disease burden caused by this pathogen. Tools developed for such purposes could help both in preventing the spread of outbreaks and in clinical diagnosis. Traditional methods for diagnosis of Salmonella strains, including culture on selective media and biochemical and serological identification methods, have been successful in clinical diagnostic laboratories and in epidemiological surveillance (1, 10, 13, 26). However, these methods may require up to 3 days to provide results and are not as amenable to high-throughput analyses as some more-recently proposed molecular typing methods. Many molecular methods have been described for detection of Salmonella serovars (4, 6, 24, 25, 31, 33); however, these methods are not always rapid assays and generally do not permit serovar identification. In addition, these approaches cannot be applied directly to clinical and environmental samples and require a bacterial culture, which increases analysis time and can lead to culture-based biases.Multilocus sequence typing (MLST) is a high-resolution genotyping technique that has emerged as a powerful tool for determining the global epidemiology and population structure of many bacterial pathogens, including Salmonella enterica (18, 32). MLST detects sequence variability within particular genes to determine the genetic relatedness of organisms (12). The method is reproducible, and the data generated are easy to interpret and directly comparable among laboratories (9). However, MLST remains expensive and impractical for routine examination of bacterial genotype. Therefore, the use of informative single nucleotide polymorphisms (SNPs) has been recently suggested as a cost-effective alternative to full MLST characterization (2, 3). SNP typing, using various genetic targets, has been reported to be a robust method for analysis of relationships of S. enterica serovar Typhi (20, 22, 23). An advantage of the SNPs observed across MLST loci is that they represent neutral genetic variation in the bacterial genome (12) and thus could be used for developing accurate, rapid, economical, and phylogenetically meaningful typing methods that may lead to significant improvements in diagnosis and surveillance for Salmonella serotypes.The SNaPshot multiplex system (Applied Biosystems) can be used to genotype multiple known SNPs simultaneously using a multiplex primer extension assay (MPEA) approach. These assays monitor the single-base extension of unlabeled oligonucleotide primers, which bind to a cDNA template adjacent to target SNPs. DNA polymerase extends the primer at the polymorphic site, incorporating a fluorescently labeled dideoxynucleoside triphosphate (ddNTP), and the fluorescently labeled extension products are separated and visualized using capillary electrophoresis. The SNaPshot approach has been demonstrated to be efficient in various fields of research, such as forensic investigation (5, 21) and clinical diagnosis (11). The approach has been applied in microbiological investigations, though until recently, this has been for analysis of mammalian systems when interacting with bacteria (8, 14-16, 19, 28).In the present study, we have developed an MPEA for rapid “molecular serotyping” of Salmonella isolates by targeting 15 SNPs identified by interrogation of the MLST database for salmonella.(Some of this work was presented as a poster at the Health Protection Agency Conference, Warwick, United Kingdom, September 2009.)     

Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing.     

Microelectronic DNA Assay for the Detection of BRCA1 Gene Mutations

Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).     

Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay

Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.     

Multiplex PCR-Ligation Detection Reaction Assay for Simultaneous Detection of Drug Resistance and Toxin Genes from Staphylococcus aureus,Enterococcus faecalis,and Enterococcus faecium

A multiplex PCR-ligation detection reaction (PCR-LDR) assay was developed for rapid detection of methicillin, tetracycline, and vancomycin resistance, as well as toxic shock toxin and Panton-Valentine leukocidin. The assay was tested on 470 positive blood culture bottles containing Staphylococcus aureus or enterococci. PCR-LDR exhibited a sensitivity and specificity of ≥98% for all components except tetracycline resistance, which had a sensitivity of 94.7%. Rapid and sensitive detection of antimicrobial resistance and virulence genes could help guide therapy and appropriate infection control measures.Previous studies have demonstrated the ability of multiplex PCR-ligation detection reaction (PCR-LDR) assays to detect and identify a variety of clinically significant bacteria and flaviviruses (4, 12, 14). We have now developed a multiplex PCR-LDR assay to directly determine antibiotic resistance profiles, as well as the toxic shock syndrome (Tsst-1) and Panton-Valentine leukocidin (PVL) toxins, of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium present in positive blood culture bottles. Rapid identification of antimicrobial resistance and virulence determinants could have a significant impact on reducing morbidity, mortality, and health care costs. Methicillin and vancomycin resistance genes were included in the assay because of the clinical importance of vancomycin-resistant enterococci (VRE), methicillin-resistant S. aureus (MRSA), and most recently vancomycin-resistant S. aureus (VRSA) infections (5, 15, 18). Tetracycline resistance genes were included because of renewed interest in this group of antibiotics due to their activity against several biothreat agents and the increase in community-acquired MRSA (7, 8, 21, 22).Positive blood cultures containing S. aureus, E. faecalis, and/or E. faecium were obtained from patients at Weill Cornell Medical Center, New York Presbyterian Hospital (WCMC-NYPH). Blood cultures were collected and incubated in a BacT/Alert system (bioMérieux, Durham, NC) in accordance with the manufacturer''s instructions. When a blood culture was read as positive by the BacT/Alert, 100-μl aliquots of the blood culture were transferred in quadruplicate to a 96-deep-well plate for subsequent extraction of DNA; negative controls containing only Tris-EDTA buffer were incorporated into each 96-well plate, and the plates were stored at −70°C. An additional 400 μl of the positive blood culture was stored for retesting of any discordant results. Bacteria were identified using standard methods. Additional clinical isolates of S. aureus, E. faecalis, and E. faecium were collected from wound, urine, respiratory, or autopsy samples. These isolates were spiked into negative clinical blood culture bottles as described previously (12). The use of human clinical samples in this research has complied with all relevant federal guidelines and WCMC-NYPH institutional policies.Bacterial isolates previously shown by sequencing to contain the mecA, vanA, vanB, tetK, tetL, tetM, or lukS-lukF (PVL) gene(s) were obtained from the WCMC-NYPH clinical microbiology collection. E. faecium N97-0330 (vanD3) and E. faecium N03-0072 (vanD5) (1) were provided by Michael Mulvey. E. faecium NEF1 (vanD1) (9) was provided by Albert Sotto and Jean Phillippe Lavigne.DNA from clinical specimens was extracted using an ABI 6100 nucleic acid prep system (Applied Biosystems, Foster City, CA) as described previously (12). The procedure used fine grade charcoal to remove sodium polyanetholesulfonate (SPS), an essential component of blood culture media and a potent inhibitor of PCR.PCR and LDR primers were designed based on sequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov) as previously described (12). In instances where sequences did not share 100% identity, multiple PCR/LDR primers were designed over the selected region. To prevent failure of PCR amplification, two PCR primer pairs were designed for each of the gene amplicons. The 44 PCR primers used in the multiplex PCR assay are presented in Table SA1 in the supplemental material. The 71 LDR primers used in the multiplex LDR assay are presented in Table SA2 in the supplemental material. Two to three LDR primer pairs were designed for each of the PCR amplicons. Identification of at least two LDR signals for any gene was required for a positive result. The assay also incorporated two previously defined positive amplification controls specific to the 16S rRNA genes, identifying the bacteria as Staphylococcus species, E. faecium, or E. faecalis (12). PCRs and LDR were performed as described in Table SA3 in the supplemental material. Capillary electrophoresis (CE) was performed and analyzed as previously described (4, 12, 14) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) Capillary electrophoresis (CE) trace for the antibiotic resistance, and toxin PCR-LDR assay of a vancomycin-resistant E. faecium isolate. The x axis shows the LDR product size, and the y axis shows fluorescence intensity of the bands. Red arrows indicate signals that are unique to the 16S rRNA genes of E. faecium and the vanA gene. (B) The CE data are displayed as a reconstructed gel image generated by a software program developed in our laboratory. The left panel represents the size and fluorescence of the 16S rRNA gene LDR products which act as a positive control and differentiate between the bacteria E. faecalis, E. faecium, and Staphylococcus species. The middle panel represents the LDR signals that identify the presence of methicillin resistance (mecA), vancomycin resistance (vanA, vanB, vanD), tetracycline resistance (tetL tetK, tetM), the toxic shock toxin gene (tst), and the PVL toxin genes (lukS-lukF). Data in the right panel represent two blood cultures. In one, the 16S rRNA gene control identified E. faecium, and the vanA gene was detected, indicating a VRE; in the other the 16S rRNA gene control identified S. aureus, and the mecA and lukS-lukF genes were detected, indicating an MRSA gene encoding the PVL toxin.The antibiotic susceptibility of each organism was determined by VITEK2 (bioMerieux, Durham, NC). If an intermediate or discrepant call was made by the VITEK2, secondary/confirmatory testing was performed by Etest (AB Biodisk, Solna, Sweden). MICs were interpreted based on CLSI guidelines (3). Production of Tsst-1 was confirmed by reverse passive latex agglutination, using the TST-RPLA kit (Oxoid, United Kingdom). S. aureus ATCC 51651 and ATCC 13566 were used as positive and negative controls, respectively. The presence of the PVL toxin genes was confirmed by amplification and sequencing by using the primers luk-PV-1 and luk-PV-2 (10).The resistance and toxin genotypes of 470 blood culture samples (193 positive blood cultures and 277 clinical isolates spiked into negative blood cultures) were examined using the multiplex PCR-LDR assay. Twenty of the positive blood cultures were polymicrobial; fourteen of these contained at least one S. aureus, E. faecium, or E. faecalis resistance gene.Correlation of the multiplex PCR-LDR assay with standard susceptibility testing for pure cultures is presented in Table ​Table1.1. The PCR-LDR assay detected methicillin resistance in S. aureus with 99.2% sensitivity and 99.1% specificity. One isolate was mecA positive and oxacillin susceptible, and one was mecA negative and oxacillin resistant. Vancomycin resistance was detected in E. faecium and E. faecalis with 98.6% sensitivity and specificity compared to susceptibility testing. Almost all (98.4%) vancomycin resistance in E. faecium was due to vanA, whereas vanB was responsible for an appreciable portion (44.4%) of resistance in E. faecalis (see Table SA4 in the supplemental material). Two isolates of E. faecalis were vancomycin susceptible but were positive for vanA or vanB. Low-level expression of the van genes may have failed to raise the MIC above the breakpoint for resistance. One isolate of E. faecium was vancomycin resistant but was negative for vanA, vanB, and vanD. Resistance may have been due to gene sequences not incorporated into this assay, such as vanE or vanG (5).

TABLE 1.

Antibiotic resistance phenotypes and PCR-LDR gene identification in blood cultures containing S. aureus or enterococci

Development of Whole-Virus Multiplex Luminex-Based Serological Assays for Diagnosis of Infections with Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Homologs in Macaques

Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a tumorigenic rhadinovirus that is associated with all forms of Kaposi''s sarcoma. Current serological detection of KSHV is based on enzyme-linked immunosorbent or immunofluorescence assays that suffer from a variety of problems, including the lack of defined standards for test comparison. While KSHV is the only known human rhadinovirus, two lineages of KSHV-like rhadinoviruses are found in Old World primates: the RV1 lineage includes KSHV and retroperitoneal fibromatosis herpesvirus (RFHV) in macaques, and the RV2 lineage includes RRV and MneRV2 from different macaque species. To develop animal models of KSHV-associated diseases, we developed quantitative multiplex bead-based serological assays to detect antibodies against rhadinovirus antigens. Proteins from KSHV (RV1) and MneRV2 (RV2) virions were coupled to spectrally distinct fluorescent beads and used in Luminex flow cytometry-based assays to detect immune responses in macaques. Both assays showed large dynamic ranges with high levels of seroreactivity to both KSHV and MneRV2 proteins. A large set of macaque serum samples from the Washington National Primate Research Center was screened, and most of the samples (82%) were positive in both assays, consistent with the high level of RV1-RV2 coinfection detected by PCR. The macaque sera showed broad, variable, and unique serological responses to the different viral antigens, allowing an initial seroprevalence to be determined for the macaque viruses. The Luminex assays offer a novel multiplexed approach to assess rhadinovirus infection patterns in both humans and nonhuman primates. This will help advance our understanding of rhadinovirus biology and associated host immunological responses.     

Ciprofloxacin Resistance in Campylobacter jejuni Isolates: Detection of gyrA Resistance Mutations by Mismatch Amplification Mutation Assay PCR and DNA Sequence Analysis

The gyrA quinolone resistance determining region was sequenced from 13 ciprofloxacin-resistant and 20 ciprofloxacin-susceptible Campylobacter jejuni isolates. All isolates resistant to ciprofloxacin had Thr-86-to-Ile mutations, a mutation frequently associated with the acquisition of resistance to fluoroquinolones. A mismatch amplification mutation assay (MAMA) PCR protocol was developed that detects this gyrA mutation in quinolone-resistant isolates. The MAMA PCR provides a means for routine detection of the gyrA mutation without the need for sequencing the gyrA gene.     

Development of a PCR Assay for Rapid Detection of Enterococci

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.     

4株蚊媒病毒多重RT-PCR快速检测方法的建立

目的建立登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒的多重RT-PCR快速检测方法。方法参照病毒核酸序列设计多重RT-PCR引物,并检索GenBank国际基因序列数据库初步验证其特异性,随后对Mg2＋、dNTP及引物浓度,RT-PCR反应条件进行优化,建立稳定、特异的多重RT-PCR快速检测4株病毒方法 ,并以同属于黄病毒科的登革3型病毒、登革4型病毒及甲病毒属基孔肯亚病毒、辛德毕斯病毒为对照,验证其特异性。结果应用多重RT-PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;采用多重RT-PCR引物对登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒进行扩增,分别获得574、251、879、422 bp片段,与设计相符,而对照病毒组均无非特异性扩增条带。结论实验证明,所建立的多重RT-PCR方法能够快速地检测登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒,为其检测提供了一种方便易行的方法 。     

Microsatellite marker analysis as a typing system for Candida glabrata

Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.