Demonstrating a Statistically Significant Association Between Anal High-Grade Squamous Intraepithelial Lesion and Positive OncoE6 Anal Test in Men Who Have Sex With Men and Are Living With HIV.

OBJECTIVES: The aim of the study is to determine whether a positive OncoE6 Anal Test result has statistically significant higher odds of being associated with high-grade squamous intraepithelial lesion (HSIL) and to calculate sensitivity and specificity of this test for predicting HSIL in adult men who have sex with men and are living with HIV (MSMLWH). MATERIALS AND METHODS: Men living with HIV 18 years or older having ≥atypical squamous cells of undetermined significance-grade anal cytology results were eligible to enroll in this cross-sectional study. Anal samples were collected just before the high-resolution anoscopy procedure. OncoE6 Anal Test results were compared with histology, the reference standard. Sensitivity, specificity, and odds ratio were calculated using HSIL as the threshold. RESULTS: Two hundred seventy-seven consented MSMLWH were enrolled between June 2017 and January 2022. Of these, 219 (79.1%) had biopsies obtained and histology performed; 81 of 219 participants (37%) had 1 or more biopsies with HSIL results while the remaining 138 of 219 (63%) had only low-grade squamous intraepithelial lesion or were negative for dysplasia. Anal samples from 7 participants (8.6%, 7/81) with HSIL and 3 (2.2%, 3/138) with low-grade squamous intraepithelial lesion had positive OncoE6 Anal Test results. Odds of having HSIL were 4.26 times higher among participants testing positive for HPV16/HPV18 E6 oncoprotein(s) (OR = 4.26, 95% CI = 1.07-16.95, p = .04). The OncoE6 Anal Test demonstrated excellent specificity, 97.83% (93.78-99.55), but poor sensitivity, 8.64% (3.55-17.0). CONCLUSIONS: In this highest-risk population for anal cancer, one could combine the OncoE6 Anal Test, having excellent specificity, with the anal Pap test, having higher sensitivity. Patients found having both an abnormal anal Pap and positive OncoE6 Anal Test result could be triaged for rapid scheduling of their high-resolution anoscopy.

Cervical Cancer Prevention and High-Risk HPV Self-Sampling Awareness and Acceptability among Women Living with HIV: A Qualitative Investigation from the Patients' and Providers' Perspectives.

Routine cervical cancer screening is important for women living with HIV (WLH) due to the greater incidence and persistence of high-risk HPV (HR-HPV) infection. HR-HPV self-sampling has been proposed to overcome barriers to in-office cervical cancer screening in underserved populations. However, little is known about baseline knowledge of HR-HPV and the acceptability of HR-HPV self-sampling among WLH. This paper describes WLH's experiences and needs regarding cervical cancer screening, specifically HR-HPV self-sampling, and seeks to reconcile their experiences with the views of their providers. In total, 10 providers and 39 WLH participated in semi-structured interviews and group discussions, respectively. Knowledge of cervical cancer and HR-HPV was generally limited among WLH; when present, it was often due to personal experience of or proximity to someone affected by cervical cancer. Most WLH were not familiar with HR-HPV self-sampling but, despite some of the providers' skepticism, expressed their willingness to participate in a mail-based HR-HPV self-sampling intervention and highlighted convenience, ease of use, and affordability as facilitators to the uptake of HR-HPV self-sampling. The experiences identified can be used to guide patient-centered communication aimed at improving cervical cancer knowledge and to inform interventions, such as HR-HPV self-sampling, designed to increase cervical cancer screening among under-screened WLH.

Characterization of HIV Risk Behaviors and Clusters Using HIV-Transmission Cluster Engine Among a Cohort of Persons Living with HIV in Washington, DC.

Molecular epidemiology (ME) is one tool used to end the HIV epidemic in the United States. We combined clinical and behavioral data with HIV sequence data to identify any overlap in clusters generated from different sequence datasets; to characterize HIV transmission clusters; and to identify correlates of clustering among people living with HIV (PLWH) in Washington, District of Columbia (DC). First, Sanger sequences from DC Cohort participants, a longitudinal HIV study, were combined with next-generation sequences (NGS) from participants in a ME substudy to identify clusters. Next, demographic and self-reported behavioral data from ME substudy participants were used to identify risks of secondary transmission. Finally, we combined NGS from ME substudy participants with Sanger sequences in the DC Molecular HIV Surveillance database to identify clusters. Cluster analyses used HIV-Transmission Cluster Engine to identify linked pairs of sequences (defined as distance ≤1.5%). Twenty-eight clusters of ≥3 sequences (size range: 3-12) representing 108 (3%) participants were identified. None of the five largest clusters (size range: 5-12) included newly diagnosed PLWH. Thirty-four percent of ME substudy participants (n = 213) reported condomless sex during their last sexual encounter and 14% reported a Syphilis diagnosis in the past year. Seven transmission clusters (size range: 2-19) were identified in the final analysis, each containing at least one ME substudy participant. Substudy participants in clusters from the third analysis were present in clusters from the first analysis. Combining HIV sequence, clinical and behavioral data provided insights into HIV transmission that may not be identified using traditional epidemiological methods alone. Specifically, the sexual risk behaviors and STI diagnoses reported in the substudy survey may not have been disclosed during Partner Services activities and the survey data complemented clinical data to fully characterize transmission clusters. These findings can be used to enhance local efforts to interrupt transmission and avert new infections.

Cross comparison of AmpFire HPV genotyping assay and Roche human papillomavirus (HPV) linear array for HPV genotyping of anal swab samples.

Although anal cancers represent just 0.5 % of all new cancer cases in U.S., rates have increased markedly, with highest rates in HIV-infected MSM. American Cancer Society estimates there will be ∼9090 new cases and ∼1420 deaths in 2021. We compared Roche Linear Array HPV Genotyping (Roche) and AmpFire HPV Genotyping (AmpFire) assays for concordance and agreement to detect 15 hr-HPV types from 151 anal specimens. Within run precision of AmpFire was assessed on 50 anal specimens. Specimens with Roche Combo-positive and HPV33, HPV35 and/or HPV58-positive results were further tested using HPV52-specific TaqMan assay. AmpFire generated valid results on 149/151 (98.7 %) specimens; 135/149 (90.6 %) and 134/149 (89.9 %) had detectable HR-HPV DNA by AmpFire or Roche, respectively. Overall concordance was 89.8 % (2007/2235, κ = 0.65). HPV16 showed highest overall concordance at 93.3 % (139/149, κ = 0.84). HPV68 had lowest overall concordance at 77.2 % (115/149, κ = 0.28). Kappa values were interpreted as being moderate or good for all other HR-HPV types. Within run precision generated 744/750 concordant results; R2 value = 0.97 (p < 0.0001) (Mantel Test). In conclusion, AmpFire and Roche demonstrated good inter-assay agreement for detecting most HR-HPV types from anal samples, with AmpFire detecting a broader range of HPV68 subtypes and detecting HPV52 without the need for confirmatory testing.

Author Correction: A cross-sectional study to characterize local HIV-1 dynamics in Washington, DC using next-generation sequencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A cross-sectional study to characterize local HIV-1 dynamics in Washington, DC using next-generation sequencing.

Washington, DC continues to experience a generalized HIV-1 epidemic. We characterized the local phylodynamics of HIV-1 in DC using next-generation sequencing (NGS) data. Viral samples from 68 participants from 2016 through 2017 were sequenced and paired with epidemiological data. Phylogenetic and network inferences, drug resistant mutations (DRMs), subtypes and HIV-1 diversity estimations were completed. Haplotypes were reconstructed to infer transmission clusters. Phylodynamic inferences based on the HIV-1 polymerase (pol) and envelope genes (env) were compared. Higher HIV-1 diversity (n.s.) was seen in men who have sex with men, heterosexual, and male participants in DC. 54.0% of the participants contained at least one DRM. The 40-49 year-olds showed the highest prevalence of DRMs (22.9%). Phylogenetic analysis of pol and env sequences grouped 31.9-33.8% of the participants into clusters. HIV-TRACE grouped 2.9-12.8% of participants when using consensus sequences and 9.0-64.2% when using haplotypes. NGS allowed us to characterize the local phylodynamics of HIV-1 in DC more broadly and accurately, given a better representation of its diversity and dynamics. Reconstructed haplotypes provided novel and deeper phylodynamic insights, which led to networks linking a higher number of participants. Our understanding of the HIV-1 epidemic was expanded with the powerful coupling of HIV-1 NGS data with epidemiological data.

Validation of publicly-available software used in analyzing NGS data for HIV-1 drug resistance mutations and transmission networks in a Washington, DC, Cohort.

The DC Cohort is an ongoing longitudinal observational study of persons living with HIV. To better understand HIV-1 drug resistance and potential transmission clusters among these participants, we performed targeted, paired-end next-generation sequencing (NGS) of protease, reverse transcriptase and integrase amplicons. We elected to use free, publicly-available software (HyDRA Web, Stanford HIVdb and HIV-TRACE) for data analyses so that laboratory personnel without extensive bioinformatics expertise could use it; making the approach accessible and affordable for labs worldwide. With more laboratories transitioning away from Sanger-based chemistries to NGS platforms, lower frequency drug resistance mutations (DRMs) can be detected, yet their clinical relevance is uncertain. We looked at the impact choice in cutoff percentage had on number of DRMs detected and found an inverse correlation between the two. Longitudinal studies will be needed to determine whether low frequency DRMs are an early indicator of emerging resistance. We successfully validated this pipeline against a commercial pipeline, and another free, publicly-available pipeline. RT DRM results from HyDRA Web were compared to both SmartGene and PASeq Web; using the Mantel test, R2 values were 0.9332 (p<0.0001) and 0.9097 (p<0.0001), respectively. PR and IN DRM results from HyDRA Web were then compared with PASeq Web only; using the Mantel test, R2 values were 0.9993 (p<0.0001) and 0.9765 (p<0.0001), respectively. Drug resistance was highest for the NRTI drug class and lowest for the PI drug class in this cohort. RT DRM interpretation reports from this pipeline were also highly correlative compared to SmartGene pipeline; using the Spearman's Correlation, rs value was 0.97757 (p<0.0001). HIV-TRACE was used to identify potential transmission clusters to better understand potential linkages among an urban cohort of persons living with HIV; more individuals were male, of black race, with an HIV risk factor of either MSM or High-risk Heterosexual. Common DRMs existed among individuals within a cluster. In summary, we validated a comprehensive, easy-to-use and affordable NGS approach for tracking HIV-1 drug resistance and identifying potential transmission clusters within the community.

A Randomized Controlled Trial Comparing the Treatment of Patients Tested for Chlamydia and Gonorrhea After a Rapid Polymerase Chain Reaction Test Versus Standard of Care Testing.

BACKGROUND: We tested the effect of a rapid molecular test for Chlamydia trachomatis (CT)/Neisseria gonorrhoeae (NG) diagnosis on clinical emergency department decision making compared with standard care. The new test presents an opportunity to improve antibiotic management and patient outcomes. METHODS: We conducted a randomized controlled trial of 70 consenting patients 18 years or older presenting to an urban emergency department with sexually transmitted infections complaints (vaginal/penile discharge, dysuria, vaginal/penile itching/pain, dyspareunia). Participants were randomized to rapid testing or standard care if a sexually transmitted infection was suspected. Follow-up phone calls were performed 7 to 10 days postdischarge. The primary outcomes included: antibiotic overtreatment rates, partner notification, and health care utilization. RESULTS: A total of 12.9% tested positive for CT or NG and received antibiotics. Test patients with negative results were less likely to receive empirical antibiotic treatment than control patients, absolute risk difference [RD], 33.4 (95% confidence interval [CI], 7.9%-58.9%), risk ratio [RR], 0.39 (95% CI, 0.19-0.82). Thirty-seven participants (53%) were contacted for follow-up 7 to 10 days postdischarge. Test patients were less likely to report missed antibiotic doses (RD, -51.3%; 95% CI, -84.4% to -18.2%; RR, 0.23; 95% CI, 0.06-0.88). Test patients were more likely to be notified of their results (RD, 50.6%; 95% CI, 22.7%-78.5%; RR, 2.72; 95% CI, 1.26-5.86). There were no significant differences in charges or health care utilization measures. CONCLUSIONS: We found a significant reduction in unnecessary antibiotic treatment for CT/NG in subjects receiving the rapid molecular test compared with those receiving nucleic acid amplification test.

Staphylococcus aureus Nasal Carriage among Beefpacking Workers in a Midwestern United States Slaughterhouse.

Occupational contact with livestock is an established risk factor for exposure to livestock-associated methicillin-resistant Staphylococcus aureus (MRSA), particularly among industrial swine workers. While S. aureus is known to infect cattle, livestock-associated S. aureus carriage among workers in the beef production chain has received limited attention. Beefpacking workers, who slaughter, butcher and process cattle, have intensified exposure to potentially infectious animal materials and may be at risk of livestock-associated S. aureus exposure. We conducted a cross-sectional study of beefpacking workers (n = 137) at an industrial slaughterhouse in the Midwestern United States to evaluate prevalence and characteristics of S. aureus nasal colonization, specifically the absence of the scn gene to identify putative association with livestock, antibiotic susceptibility, presence of Panton-Valentin leukocidin (PVL) genes lukS-PV and lukF-PV, and spa type. Overall prevalence of S. aureus nasal carriage was 27.0%. No workers carried livestock-associated MRSA. Methicillin-sensitive S. aureus isolates (MSSA) recovered from five workers (3.6%) lacked the scn gene and were considered putative livestock-associated S. aureus (pLA-SA). Among pLA-SA isolates, spa types t338, t748, t1476 and t2379 were identified. To our knowledge, these spa types have not previously been identified as associated with livestock. Prevalence of human-adapted MRSA carriage in workers was 3.6%. MRSA isolates were identified as spa types t002, t008 and t024, and four of five MRSA isolates were PVL-positive. To date, this is the first study to indicate that industrial beefpacking workers in the United States may be exposed to livestock-associated S. aureus, notably MSSA, and to spa types not previously identified in livestock and livestock workers. Occupational exposure to livestock-associated S. aureus in the beef production chain requires further epidemiologic investigation.

Comparison of antibiotic susceptibility of Escherichia coli in urinary isolates from an emergency department with other institutional susceptibility data.

PURPOSE: The antibiotic susceptibility of Escherichia coli in isolates from patients with uncomplicated urinary tract infection (UTI) in an emergency department (ED) was compared with susceptibility data from the associated hospital. METHODS: Patients eligible for study participation included women age 18-65 years with one or more symptoms consistent with a UTI for whom a urine dipstick, urinalysis, or urine culture was ordered. Clinical decision-making, including the decision to order a urine culture, was at the discretion of the individual healthcare provider; however, a deidentified urine culture and antimicrobial susceptibility testing were performed for those study participants for whom a urine culture was not ordered. We compared the E. coli-specific antibiogram for uncomplicated UTI to the antibiogram based on all urine cultures in the ED regardless of patient disposition, non-intensive care unit (ICU) hospital inpatients, and the hospitalwide antibiogram. RESULTS: Of the 578 ED patients screened for study eligibility, 119 met the inclusion criteria. E. coli, detected in 53 (74%) of the 72 pathogen-positive cultures, was the most common pathogen isolated. For E. coli, ciprofloxacin nonsusceptibility was significantly less common in isolates from ED patients with uncomplicated cystitis and pyelonephritis than in isolates from non-ICU inpatients or from the hospitalwide population. E. coli nonsusceptibility to ciprofloxacin was significantly less common in ED isolates from patients with uncomplicated UTI than in isolates from all ED patients with clinician-ordered urine cultures. CONCLUSION: Antibiotic susceptibility of E. coli in an ED and its associated hospital depended on factors such as whether patients were hospitalized and whether ED isolates were from patients with uncomplicated UTI.

Treatment Failure Outcomes for Emergency Department Patients with Skin and Soft Tissue Infections.

INTRODUCTION: Skin and soft tissue infections (SSTIs) are commonly evaluated in the emergency department (ED). Our objectives were to identify predictors of SSTI treatment failure within one week post-discharge in patients with cutaneous abscesses, as well as to identify predictors of recurrence within three months in that proportion of participants. METHODS: This was a sub-analysis of a parent study, conducted at two EDs, evaluating a new, nucleic acid amplification test (NAAT) for Staphylococcus aureus in ED patients. Patients≥18 years receiving incision and drainage (I&D) were eligible. Patient-reported outcome data on improvement of fever, swelling, erythema, drainage, and pain were collected using a structured abstraction form at one week, one month, and three months post ED visit. RESULTS: We enrolled 272 participants (20 from a feasibility study and 252 in this trial), of which 198 (72.8%) completed one-week follow up. Twenty-seven additional one-week outcomes were obtained through medical record review rather than by the one-week follow-up phone call. One hundred ninety-three (73%) patients completed either the one- or three-month follow up. Most patients recovered from their initial infection within one week, with 10.2% of patients reporting one-week treatment failure. The odds of treatment failure were 66% lower for patients who received antibiotics following I&D at their initial visit. Overall SSTI recurrence rate was 28.0% (95% CI [21.6%-34.4%]) and associated with contact with someone infected with methicillin resistant S. aureus (MRSA), previous SSTI history, or clinician use of wound packing. CONCLUSION: Treatment failure was reduced by antibiotic use, whereas SSTI recurrence was associated with prior contact, SSTI, or use of packing.

A Randomized Clinical Trial Comparing Use of Rapid Molecular Testing for Staphylococcus aureus for Patients With Cutaneous Abscesses in the Emergency Department With Standard of Care.

OBJECTIVE: To determine whether real-time availability of rapid molecular results of Staphylococcus aureus would impact emergency department clinician antimicrobial selection for adults with cutaneous abscesses. DESIGN: We performed a prospective, randomized controlled trial comparing a rapid molecular test with standard of care culture-based testing. Follow-up telephone calls were made at between 2 and 7 days, 1 month, and 3 months after discharge. SETTING: Two urban, academic emergency departments. PATIENTS: Patients at least 18 years old presenting with a chief complaint of abscess, cellulitis, or insect bite and receiving incision and drainage were eligible. Seven hundred seventy-eight people were assessed for eligibility and 252 met eligibility criteria. METHODS: Clinician antibiotic selection and clinical outcomes were evaluated. An ad hoc outcome of test performance was performed. RESULTS: We enrolled 252 patients and 126 were randomized to receive the rapid test. Methicillin-susceptible S. aureus-positive patients receiving rapid test results were prescribed beta-lactams more often than controls (absolute difference, 14.5% [95% CI, 1.1%-30.1%]) whereas methicillin-resistant S. aureus-positive patients receiving rapid test results were more often prescribed anti-methicillin-resistant S. aureus antibiotics (absolute difference, 21.5% [95% CI, 10.1%-33.0%]). There were no significant differences between the 2 groups in 1-week or 3-month clinical outcomes. CONCLUSION: Availability of rapid molecular test results after incision and drainage was associated with more-targeted antibiotic selection. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01523899.

Evaluation of real-time PCR and pyrosequencing for screening incubating blood culture bottles from adults with suspected bloodstream infection.

Several molecular platforms can identify bacteria associated with bloodstream infections but require positive culture bottles as starting material. Here, we describe results of screening 1140 blood cultures at 8h postinoculation, from 918 eligible adults being evaluated for bloodstream infection. DNA was extracted and analyzed by 16S and/or 23S rRNA real-time PCR/pyrosequencing. Compared to culture, PCR/pyrosequencing displayed 90.9% sensitivity, 99.6% specificity, 95.7% positive predictive value, and 99.1% negative predictive value. Overall concordance rate was 98.9% (1127/1140). In 4 cases with molecular-positive/culture-negative results, medical chart reviews provided evidence of identical bacteria from subsequent blood or concomitant urine/sputum cultures. Nine culture-positive/molecular-negative cases were associated with either polymicrobial growth, grew only in the anaerobic bottle of the clinical pair, and/or were detected by PCR/pyrosequencing after 8h. In summary, this approach accurately detected and identified bacteria in ~91% of culture-confirmed cases significantly sooner than the phenotypic identification was available, having the potential to improve antibiotic stewardship.

Prevalence of bacterial vaginosis and Candida among postmenopausal women in the United States.

OBJECTIVES: To describe the prevalence of bacterial vaginosis (BV) and Candida among community-dwelling postmenopausal women in the United States and determine their change with age, using estimates based on Waves 1 and 2 of the National Social Life, Health and Aging Project (NSHAP). METHOD: Self-administered vaginal swabs were collected in-home from women aged 57-85 (n = 1,016) in Wave 1 and again 5 years later in Wave 2 (n = 883). Gram-stained specimens were evaluated for BV using the Nugent score as well as presence of Candida. RESULTS: BV was prevalent in 23% and 38% of postmenopausal women in Waves 1 and 2 and increased with age. Women initially categorized with BV in Wave 1 were more than 10 times as likely to be categorized with BV in Wave 2, relative risk ratio (RRR) = 10.5; 95% confidence interval (CI) (4.45-24.7); p < .001, whereas women initially categorized as intermediate in Wave 1 were five times more likely to have a BV categorization, RRR = 5.0; 95% CI (2.56-9.75); p < .001. Although the presence of Candida was similar in both waves (6% and 5%), its relationship with age only became evident in Wave 2, with odds of detecting Candida decreasing by 7% with each year of age, OR = 0.93, 95% CI (0.88, 0.98); p = .010. DISCUSSION: In Wave 2, the prevalence of BV was higher and increased with age while the prevalence of Candida was low and declined with age. A 5-year age increase contributed to the prevalence change across waves. Methods refinements in Wave 2 improved the detection of BV and Candida and clarified their relationship with age.

Evaluation of MolYsis™ Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing.

Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P<0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.

Molecular detection of culture-confirmed bacterial bloodstream infections with limited enrichment time.

Conventional blood culturing using automated instrumentation with phenotypic identification requires a significant amount of time to generate results. This study investigated the speed and accuracy of results generated using PCR and pyrosequencing compared to the time required to obtain Gram stain results and final culture identification for cases of culture-confirmed bloodstream infections. Research and physician-ordered blood cultures were drawn concurrently. Aliquots of the incubating research blood culture fluid were removed hourly between 5 and 8 h, at 24 h, and again at 5 days. DNA was extracted from these 6 time point aliquots and analyzed by PCR and pyrosequencing for bacterial rRNA gene targets. These results were then compared to those of the physician-ordered blood culture. PCR and pyrosequencing accurately identified 92% of all culture-confirmed cases after a mean enrichment time of 5.8 ± 2.9 h. When the time needed to complete sample processing was included for PCR and pyrosequencing protocols, the molecular approach yielded results in 11.8 ± 2.9 h compared to means of 27.9 ± 13.6 h to obtain the Gram stain results and 81.6 ± 24.0 h to generate the final culture-based identification. The molecular approach enabled accurate detection of most bacteria present in incubating blood culture bottles on average about 16 h sooner than Gram stain results became available and approximately 3 days sooner than the phenotypic identification was entered in the Laboratory Information System. If implemented, this more rapid molecular approach could minimize the number of doses of unnecessary or ineffective antibiotics administered to patients.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

BACKGROUND: In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. OBJECTIVES: Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. STUDY DESIGN: 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. RESULTS: The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. CONCLUSIONS: Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay.

A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture.

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples.

Multicenter study evaluating performance of the Smart Group B Streptococcus (GBS) assay using an enrichment protocol for detecting GBS colonization in patients in the antepartum period.

Group B streptococcus (GBS) remains the leading cause of infectious morbidity and mortality in infants born in the United States, especially among black infants. Because a newborn can acquire GBS during and after delivery, the Centers for Disease Control and Prevention (CDC) recommends that pregnant women be screened for rectovaginal GBS colonization during the antepartum period between weeks 35 and 37 of gestation and, if they are colonized, that intrapartum antibiotic prophylaxis be administered. A prospective investigational study was undertaken from 2 May 2006 to 14 August 2006 at three sites to establish the performance characteristics of the Smart GBS LB assay on the SmartCycler II system for detecting GBS colonization in subjects in the antepartum period from combined vaginal/rectal swab-based specimens after broth enrichment. Results were compared to broth enrichment culture and to the predicate device, the BD GeneOhm StrepB direct assay. The collected specimens were randomized for swab testing order. Each swab sample was processed simultaneously by culture, Smart GBS LB assay, and the BD GeneOhm StrepB assay. A total of 310 subjects were enrolled, with 306 subject results included in the study. Compared to enrichment culture, the Smart GBS LB assay demonstrated a sensitivity, specificity, positive predictive value, and negative predictive value of 98.6%, 90.4%, 77.1%, and 99.5%, respectively. The Smart GBS LB assay demonstrated substantially equivalent or better performance than culture or the predicate device. Screening of broth enrichment fluids by nucleic acid amplification testing requires careful handling during sample processing to avoid possible contamination.

Molecular diagnosis of neonatal sepsis.

Several molecular testing options are now or will soon be available for diagnosing bloodstream infections in the neonate. The advantages include the speed at which results would be available and the ability to use those results to tailor empirical therapy and reduce the amount of unnecessary or ineffective antibiotics an infant receives. However, there are still difficult challenges before this potential can be realized. A variety of technological advances are needed, including (1) improved recovery of microorganisms in whole blood extractions, (2) increased assay sensitivity, (3) simpler testing platforms that could be run 24/7, and (4) more assays to detect antibiotic resistance genes to reduce reliance on culture-based protocols for antimicrobial susceptibility testing. Although considerable hurdles remain, this challenge is now a priority for investigators in academia and industry.