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Background Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. Objectives To develop a rapid method for identifying patients infected with MCV via swab sampling. Study Design Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. Results Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. Conclusions These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.

A multiplex PCR assay was used to detect the erythromycin (EM) and clindamycin (CM) antibiotic resistance genes, ermB, ermTR, and mefA/E, in Group B Streptococcal (GBS) clinical isolates and in DNA extracted from the corresponding cervicovaginal-rectal (CVR) swabs. We compared these results to the standard EM/CM double disk diffusion assay of 46 isolates. Given that these genes are present in other CVR flora and are found on mobile genetic elements, the PCR assay was unable to predict GBS resistance directly from the swabs. Therefore, PCR can only accurately detect resistance genes and predict the resistance phenotype from purified GBS isolates.

Background: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. Objective: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. Study design: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5′ biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. Results: The quantitative range of the assay extended from 10 8 through 10 0 copies of each virus ( r 2 > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. Conclusions: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.

A real-time PCR and pyrosequencing method was developed to detect Aspergillus fumigatus in whole blood by amplifying the cyp51A gene and sequencing the codon for glycine 54. Mutations in this codon can result in amino acid substitutions that confer reduced susceptibility to itraconazole and posaconazole.

A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non- albicans species with increases in age.

Objective.To present the detection rates ofCandidaspecies in vaginal samples from patients visiting physicians.Methods.The presence ofC. albicans, C. glabrata, C. parapsilosisandC. tropicalisin 3978 vaginal swabs from patients in six US states was detected by PCR amplification.Results.Candida DNA was detected in 33.1% of the population studied. Of the 1316 positive samples, 80.2% containedC. albicans, 14.3% containedC. glabrata, 5.9% containedC. parapsilosisand 8.0% containedC. tropicalis. Comparing samples by patients' state of residence revealed an association with the detection ofC. glabrata(p=0.029). Comparing samples by patients' age revealed a decrease in the overall detection ofCandida(p< 0.001) andC. albicans(p< 0.001), concomitant with an increase in the detection ofC. glabrata(p< 0.001) andC. parapsilosis(p= 0.025).Conclusions.These results provide geographic- and age-specific data on fourCandidaspecies associated with vaginitis.

Real-time polymerase chain reaction (PCR) is currently considered the most sensitive method to detect low abundance DNA of pathogens in clinical samples. Furthermore, obtaining DNA sequence is the 'gold standard' of precise molecular detection. Here we combine species-specific real-time PCR and pyrosequencing to rapidly amplify and sequence ribosomal DNA from Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, which are commonly associated with candida vaginitis (CV). A standard curve was developed from plasmids containing the target DNA for each of the Candida species. A minimum real-time PCR and pyrosequencing detection limit of 100 copies per reaction was achieved. The combined technique was applied to the identification of the four Candida species in DNA extracts from vaginal samples. The results from 231 samples were compared with conventional PCR methods of identification. The results of both methods agreed on all but two samples, which were determined by both methods to contain C. albicans, but real-time PCR and pyrosequencing identified a second species that went undetected by conventional PCR. This is the first application of real-time PCR and pyrosequencing to DNA from vaginal samples for identification of four Candida species associated with CV, without the need for time-consuming culture methods.

The results from the pathogen stability experiments from Day 0 through Day 5 postpathogen inoculation are shown; conventional PCR and real-time PCR experiments are presented as gel photographs and bar graphs, respectively. The HPV conventional PCR reaction contained two negative (‘-’ ; water substituted for DNA) controls. Each realtime PCR reaction contained three concentrations of a positive vector control and one negative template control (NTC) which consisted of the substitution of water for DNA. The vector controls were designed to contain the amplicon of each real-time PCR reaction and were used to generate a standard curve based upon CT value (cycle in which the amplification curve passes a threshold level) and input concentration. The Rotor-Gene software (version 5.0.60) was used to generate an automated threshold and to calculate a concentration for each of the unknowns at daily time points. Means and standard deviations for each pathogen are presented in the bar charts. The copies per reaction were calculated by applying the following formula: (copies/mL) = (calculated copies / 2.5 μl input material) x (20 μl elution volume / 0.470 mL sample volume). This analysis demonstrated that for all pathogens tested under the described conditions, it was possible to detect the corresponding DNA using real-time PCR or conventional PCR for up to five days postinoculation. AMENDED ABSTRACT