Short Signature rpoB Gene Sequence to Differentiate Species in Mycobacterium abscessus Group.

Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that consist of three closely related species: M. abscessus (Ma), M. bolletii (Mb), and M. massiliense (Mm). Differentiation of these species can be difficult but is increasingly requested owing to recent infectious outbreaks and their differential drug resistance. We developed a novel and rapid pyrosequencing method using short signature sequences (35 to 45 bp) at a hypervariable site in the rpoB gene to differentiate the three MAG species, along with M. chelonae (Mc), and M. immunogenum (Mi). This method was evaluated using 111 M. chelonae-abscessus complex (MCAC) isolates, including six reference strains. All isolates were successfully differentiated to the species level (69 Ma, four Mb, six Mm, 23 Mc, and nine Mi). The species identifications by this method had 100% agreement with Sanger sequencing as well as an in-silico rpoB typing method. This short signature sequencing (SSS) method is rapid (6 to 7 h), accurately differentiates MAG species, and is useful for informing antimicrobial therapy decision. IMPORTANCE Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that include three species: M. abscessus, M. massiliense, and M. bolletii. These species are among the leading causes of nontuberculosis mycobacteria infections in humans but difficult to differentiate using commonly used methods. The differences of drug resistance among the species shape the treatment regimens and make it significant for them to be differentiated accurately and quickly. We developed and evaluated a novel short signature sequencing (SSS) method utilizing a gene called rpoB to differentiate the three MAG species, as well as other two species (M. chelonae and M. immunogenum). The identification results had 100% agreement with both the reference method of Sanger sequencing and rpoB typing method via a computer-simulated analysis. This SSS method was accurate and quick (6 to 7 h) for species differentiation, which will benefit patient care. The technology used for this method is affordable and easy to operate.

Recovery and Its Dynamics of Filamentous Fungi from Clinical Specimen Cultures: An Extensive Study.

The culture method remains vital in diagnosing fungal infections, but extensive data-based evaluation of the method, especially for filamentous fungi (molds), is minimal. The purpose of this study was to characterize mold recoveries from fungal cultures and the impact of media and incubation duration. Clinical specimens for fungal cultures were submitted primarily from the eastern and central United States, and mold isolation data were prospectively collected and analyzed. A total of 1,821 molds in 59 genera were isolated from 1,687 positive specimens, accounting for approximately 5.6% of our cohort of 30,000 fungal cultures. Within 2 weeks, nearly 90% of molds and 97.3% of Aspergillus fumigatus complex were recovered (>95% confidence interval [CI]). All Mucorales fungi were recovered within 11 days of incubation. The recovery peak time was day 3 for Mucorales fungi, day 4 for hyaline molds, day 5 for dematiaceous molds, and day 7 for Onygenales fungi. The recovery of Histoplasma capsulatum and Trichophyton species in the fourth week of incubation reveals that a 3-week incubation time is insufficient. Inhibitory mold agar was the best medium for recovering all mold types among all tested specimen types, yielding nearly 78% of mold growth overall, indicating the necessity of selective medium for fungal cultures. IMPORTANCE Fungal culture is the gold standard method of diagnosing fungal infections, but important information, such as the impact of media and incubation times on fungal recovery, is not well documented. This study addressed these gaps using extensive data-based evaluation focused on molds. We identified the best medium types and incubation times for better fungal culture practice. We analyzed 1,821 molds from 1,687 positive specimens in our cohort of approximately 30,000 fungal cultures. Mold recovery peaked between 3 and 7 days of incubation, dependent upon the type of mold. Some well-defined fungal pathogens, such as Histoplasma capsulatum and Trichophyton species, were isolated in the fourth week of incubation. Inhibitory mold agar was identified as the best medium for recovering all mold types among all tested specimen sources. As we are aware, this is the largest study of fungal culture methods and supports 4 weeks of incubation for optimal mold recovery.

Retrospective Performance Analyses of over Two Million U.S. QuantiFERON Blood Sample Results.

Both the QuantiFERON-TB Gold Plus (QFT-Plus) and the QuantiFERON-TB Gold In-Tube (QFT-GIT) tests are interferon gamma (IFN-γ) release assays (IGRAs) intended to detect in vitro cell-mediated immune responses to Mycobacterium tuberculosis antigens. In this study, we retrospectively analyzed performance data for both the QFT-GIT and QFT-Plus test systems from over 2 million samples. QFT-Plus and QFT-GIT testing was performed as specified in the respective package inserts at 23 Quest Diagnostics sites. Blood specimens were collected from individuals in all 50 states from November 2018 through December 2019. Retrospective analyses compared the proportion of positive, indeterminate, and conversion/reversion results. The overall proportion of QFT-positive results was 7% for both the QFT-Plus and QFT-GIT. The proportion of positive results was highest for QFT-GIT (7.5%) followed by the heparin 1-tube QFT-Plus (7.2%); a lower proportion of positives was observed with the 4-tube (all four QFT tubes were used in blood collection) QFT-Plus (6.0%). The proportions of indeterminate results for the 1-tube (heparin-only tube collection) and 4-tube QFT-Plus methods were less than 1% and 4%, respectively. This study indicates a higher proportion of positive results for M. tuberculosis than data from other studies. Additionally, the proportion of indeterminate QFT results were markedly lower when the sample was transported in one lithium-heparin tube instead of direct inoculation into 4 QFT-Plus tubes at the site of blood collection. IMPORTANCE In this study, we retrospectively analyzed results from both the QFT-GIT and QFT-Plus test systems from over 2 million blood specimens. The variables analyzed were (i) QFT positivity rates among various U.S. populations, (ii) indeterminate rates among various types of blood draws and how often an indeterminate result was resolved within 30 days after the initial draw, and (iii) the association of TB1 and TB2 antigen tubes with IGRA reversion and conversion events from serial QFT testing. This is, to our knowledge, the largest QFT study representing patients from an extensive geographic coverage across the United States and U.S. territories.

Current significance of the Mycobacterium chelonae-abscessus group.

Organisms of the Mycobacterium chelonae-abscessus group can be significant pathogens in humans. They produce a number of diseases including acute, invasive and chronic infections, which may be difficult to diagnose correctly. Identification among members of this group is complicated by differentiating at least eleven (11) known species and subspecies and complexity of identification methodologies. Treatment of their infections may be problematic due to their correct species identification, antibiotic resistance, their differential susceptibility to the limited number of drugs available, and scarcity of susceptibility testing.

Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method.

AIMS: Acid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated. METHODS: The method was validated using spiked cell-clotted paraffin blocks before use with patients' specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB. RESULTS: Both sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq. CONCLUSIONS: The PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.

Antibiotic Resistance among Urinary Isolates from Female Outpatients in the United States in 2003 and 2012.

A retrospective analysis was performed using The Surveillance Network, USA, to examine the prevalence of antibiotic resistance among urine isolates from U.S. female outpatients in 2012 and assessed trends in antibiotic resistance comparing data from 2003 and 2012. The most common pathogen identified in 2012 (n = 285,325) was Escherichia coli (64.9% of isolates). In 2012, E. coli resistance to nitrofurantoin was low (<3%) across all age groups. E. coli resistance to ciprofloxacin was high among adults (11.8%) and elderly outpatients (29.1%). When comparing the 2003 and 2012 data from isolates from adults, E. coli resistance to nitrofurantoin changed only slightly (from 0.7% to 0.9%), whereas increases in resistance to ciprofloxacin (3.6% to 11.8%) and trimethoprim-sulfamethoxazole (17.2% to 22.2%) changed substantially. In the United States, E. coli has become increasingly resistant to ciprofloxacin and trimethoprim-sulfamethoxazole (TMP-SMX) in adult female outpatients. Nitrofurantoin retains high levels of antibiotic activity against urinary E. coli.

Direct detection and identification of acid-fast bacteria from smear-positive broth cultures using a pyrosequencing method.

Broth culture is a standard method for detection of acid-fast bacteria (AFB) (e.g., Mycobacterium and Nocardia) from patient specimens. Direct nucleic acid-based identification from smear-positive broths expedites the infectious disease diagnosis. We developed and evaluated the performance of a pyrogram-based technique (direct-broth-pyrosequencing [DBP]) to identify AFB directly from smear-positive broths. One hundred thirteen AFB-positive broths from patient specimens were tested. Bacterial DNA was amplified by polymerase chain reaction and sequenced using the PyroMark ID system. The DBP method correctly identified the AFB species/group in 109 (97%) of the 113 broths, including 15 Mycobacterium species and 4 Nocardia species. Three broths that yielded indeterminate results were found to be AFB-AFB mixed broths and required purified colonies on solid media for definite identification. The 4th broth was repeatedly identified by sequencing to be Mycobacterium intracellulare, even though the organism was not isolated and the AccuProbe was negative. This method did not identify the AFB organisms from broths containing 2 AFB organisms, but did not produce false identification. No cross-reaction was observed when AFB-positive broths were spiked with non-AFB microorganisms, indicating that the DBP method was specific to AFB. The DBP method gives rapid (within 8 h), accurate AFB identification directly from broth cultures and provides another useful AFB identification tool in a clinical laboratory.

Escherichia coli antimicrobial resistance increased faster among geriatric outpatients compared with adult outpatients in the USA, 2000-10.

OBJECTIVES: Few studies have examined Escherichia coli antimicrobial resistance across age groups over time. The objective of this study was to compare urinary E. coli antimicrobial resistance trends among adult and geriatric outpatients from 2000 to 2010. METHODS: Antimicrobial susceptibility results for E. coli urine isolates from adult (aged 16-64 years) and geriatric (aged ≥65 years) outpatients were analysed using data from The Surveillance Network Database-USA. RESULTS: Susceptibility test results from adult (n = 6 412 025) and geriatric (n = 3 395 297) outpatients showed that E. coli antimicrobial resistance increased faster among geriatric outpatients for all agents studied. The greatest increases in resistance over the study time period were for ciprofloxacin (9.4% and 23.5% increases among adult and geriatric individuals, respectively), trimethoprim/sulfamethoxazole (4.3% and 10.5%) and ampicillin (2.0% and 13.6%). CONCLUSIONS: Urinary E. coli antimicrobial resistance increased faster among geriatric outpatients than adult outpatients in the USA. Rising antimicrobial resistance disproportionately affects geriatric populations and presents a threat to public health.

Recent trends in resistance to cell envelope-active antibacterial agents among key bacterial pathogens.

Cell envelope-active agents, particularly ß-lactams, play a pivotal role in the treatment of bacterial infections and the extent to which their activity is affected by the emergence of multidrug-resistant organisms is of concern. We analyzed the Surveillance Network (TSN) database to evaluate resistant trends for key cell envelope-active drugs among ESKAPE pathogens. Analysis demonstrated that the activity of these drugs has been notably influenced by the emergence of multidrug resistance; this was especially evident for the ß-lactam drugs. For example, Acinetobacter baumannii resistance to imipenem increased from 23.9% to 34.3%, and resistance to piperacillin-tazobactam increased from 37.0% to 49.7% between 2007 and 2011. During the same time period Klebsiella pneumoniae resistance to imipenem increased from 0.8% to 3.8%. As ß-lactams are a cornerstone of anti-infective therapy, it is important to closely monitor the activity of the agents being used today and to aggressively pursue new strategies that can augment current drugs and thwart ever-emerging ß-lactam resistance mechanisms that are continuously encountered.

Klebsiella pneumoniae antimicrobial drug resistance, United States, 1998-2010.

We studied antimicrobial-resistant Klebsiella pneumoniae for 1998-2010 by using data from The Surveillance Network. Susceptibility results (n = 3,132,354) demonstrated significant increases in resistance to all antimicrobial drugs studied, except tetracycline. Cross-resistance among carbapenem-resistant K. pneumoniae was lower for tetracycline and amikacin.

In vitro antimicrobial resistance of urinary Escherichia coli isolates among U.S. outpatients from 2000 to 2010.

This study examines in vitro antimicrobial resistance data from Escherichia coli isolates obtained from urine samples of U.S. outpatients between 2000 and 2010 using The Surveillance Network (TSN). Antimicrobial susceptibility results (n = 12,253,679) showed the greatest increases in E. coli resistance from 2000 to 2010 for ciprofloxacin (3% to 17.1%) and trimethoprim-sulfamethoxazole (TMP-SMX) (17.9% to 24.2%), whereas nitrofurantoin (0.8% to 1.6%) and ceftriaxone (0.2% to 2.3%) showed minimal change. From 2000 to 2010, the antimicrobial resistance of urinary E. coli isolates to ciprofloxacin and TMP-SMX among outpatients increased substantially.

Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory.

Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.

Analysis of resistance, cross-resistance and antimicrobial combinations for Pseudomonas aeruginosa isolates from 1997 to 2009.

Pseudomonas aeruginosa is a nosocomial and community-acquired pathogen associated with considerable patient morbidity and mortality. Multidrug resistance in P. aeruginosa is a concern owing to the limited therapeutic options available to treat infections due to this organism. In this study, rates of antimicrobial resistance of P. aeruginosa isolates collected by The Surveillance Network Database-USA (Eurofins Medinet, Chantilly, VA) from 1997 to 2009 were examined. The patient population and specimens were stratified according to patient setting and age as well as specimen source. Multidrug resistance was defined as resistance to three or more of the following antimicrobial agents: aztreonam; cefepime; ciprofloxacin; imipenem; gentamicin; and piperacillin/tazobactam (TZP). A total of 924740 P. aeruginosa isolates were examined in this study. Changes in resistance rates to individual antimicrobial agents were <5% for all agents except ciprofloxacin. There was a statistically significant decreasing rate of multidrug-resistant P. aeruginosa to four, five and six antimicrobial agents. For isolates resistant to imipenem, aztreonam and gentamicin, ciprofloxacin had the highest cross-resistance rates. The greatest coverage against P. aeruginosa was by the combination of TZP plus amikacin (94%) followed by aztreonam plus amikacin (90%). Pseudomonas aeruginosa resistance rates remained steady or minimally declined to all antimicrobials from 1997 to 2009. Amongst the ß-lactams, TZP has the greatest activity against P. aeruginosa.

Methicillin-resistant Staphylococcus aureus resistance to non-beta-lactam antimicrobials in the United States from 1996 to 2008.

We report the resistance rates of Staphylococcus aureus to non-beta-lactam antimicrobials from The Surveillance Network Database-USA (Eurofins-Medinet, Chantilly, VA). Specimens studied were from lower respiratory tract, wounds, and blood. Patients were stratified by age group and patient setting. There were 2,053,219 isolates of S. aureus and 973,116 of methicillin-resistant S. aureus (MRSA). The MRSA rate increased until 2004 and then leveled off. MRSA showed decreasing resistance to tetracycline and trimethoprim-sulfamethoxazole (TMP-SMX). By age group, the greatest MRSA rate increase was for individuals 17 years and younger. Non-beta-lactam antimicrobials and particularly TMP-SMX should be considered therapeutic options for staphylococcal infections.

Identification of acid-fast bacilli using pyrosequencing analysis.

Pyrosequence identification of 117 isolates of acid-fast bacilli (AFB) was compared to both routine phenotypic methods and Sanger sequencing. Two (2) vendor-provided pyrosequencing primers specific for AFB were used for the study. Pyrosequence analysis correctly identified 114 (98%) of the tested 117 AFB isolates. Among the test Mycobacterium spp., 18 of 20 Mycobacterium spp. were identified correctly to the species level. All rapidly growing mycobacteria were correctly identified to species by pyrosequencing. Other slowly growing mycobacteria such as Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium-intracellulare, and others were easily identified by pyrosequencing. Only Mycobacterium simiae and Mycobacterium scrofulaceum were not identifiable by the pyrosequence method. Among the 25 Nocardia isolates, all were correctly identified to the genus level. Identification of AFB by pyrosequence analysis provides both a rapid and accurate method for this group of organisms.

Tracking the implementation of NCCLS M100-S12 expanded-spectrum cephalosporin MIC breakpoints for nonmeningeal isolates of Streptococcus pneumoniae by clinical laboratories in the United States during 2002 and 2003.

BACKGROUND: The Performance Standards for Antimicrobial Susceptibility Testing, Twelfth Informational Supplement, M100-S12, published by the National Committee for Clinical Laboratory Standards (NCCLS) in January 2002 introduced distinct minimum inhibitory concentration (MIC) interpretative breakpoints for ceftriaxone, cefotaxime, and cefepime for nonmeningeal isolates of Streptococcus pneumoniae. Previously, a single set of interpretative breakpoints was used for both meningeal and nonmeningeal isolates. METHODS: To estimate the rate of adoption of the M100-S12 interpretive breakpoints by clinical laboratories, antimicrobial susceptibility test results for ceftriaxone and cefotaxime from nonmeningeal S. pneumoniae isolates were studied using data collected from January 2002 to June 2003 by The Surveillance Network Database--USA (TSN, an electronic surveillance database. RESULTS: Of the 262 laboratories that provided data that could be evaluated, 67.6% had adopted the M100-S12 breakpoints one and one-half years after they were published. CONCLUSIONS: The NCCLS M100-S12 recommendation to interpret MICs to expanded-spectrum cephalosporins using two distinct sets of breakpoints for meningeal and nonmeningeal isolates of S. pneumoniae was steadily implemented by clinical microbiology laboratories in the United States following their initial publication in January 2002. The use of these new breakpoints more accurately reflects the clinical activities of expanded-spectrum cephalosporins than did the single set of interpretative breakpoints previously used for both meningeal and nonmeningeal isolates.

TSN Database Australia, a new tool to monitor antimicrobial resistance in Australia.

An electronic network of Australian microbiology laboratories was established to monitor the emergence and occurrence of antimicrobial resistance among clinically relevant bacteria. It is believed that the data network collected approximately 42 per cent of all antibacterial susceptibility test results generated by Australian laboratories. The network comprised 94 hospitals and 9 private commercial laboratories. Selected data elements were extracted and electronically transmitted to a central location. Upon receipt, all data were first normalised and thereafter examined for errors. Duplicate results for the same patient were identified to prevent skewing of the data toward resistance. All data passing quality assessment was staged for release of a new database release that occurred monthly. Unusual test results were first validated prior to their inclusion into the database. Using an Internet-based query tool, individual institutions could query their own data, but could only query aggregated data for other regional or national analyses. Individual patient results could be examined nor could the results of any individual institution other than their own. As of March 2002, TSN Database Australia contained 14,648,752 test results, from 2,000,394 strains (453 different taxa) and 1,213,605 patients. Since the same database concept has been established in 10 other countries (United States of America, Europe, and Canada), observations made in Australia may be compared to those observed elsewhere in the world. This article will describe TSN in greater detail, describe the query tool and some of the analyses that are possible.

Low levels of fluoroquinolone resistance in Escherichia coli. A five-year trend in Australia measured through the use of TSN Database Australia.

In many countries, fluoroquinolones are among the most commonly used antibacterial drugs. Concerns about bacterial resistance to these and other frequently used drugs have been raised by the medical and scientific communities. While fluoroquinolone resistance has not yet developed among many bacteria, emergence of resistance in Escherichia coli would be a problem as multiple resistances to other antibiotics is now a common problem. This paper examines trends in resistance to fluoroquinolones in Escherichia coli through analysis of data collected from Australian institutions between 1997 and 2001. During the study period, norfloxacin and ciprofloxacin were the most frequently tested fluoroquinolones in Australian laboratories. An examination of results for strains tested simultaneously against both drugs indicated that testing against either drug accurately predicted resistance or susceptibility for the other (99.7% agreement). Over 400,000 tests were performed to determine the fluoroquinolone susceptibility of E. coil. Data were analysed by the test method used (Calibrated Dichotomous Sensitivity (CDS) or National Committee for Clinical Laboratory Standards (NCCLS)). The data indicate that fluoroquinolone resistance in E. coli has not yet emerged as a significant problem in Australia, but there are some indications of low level increases in resistance rates. Norfloxacin results are likely to be a better guide to fluoroquinolone resistance in this species using this method of surveillance.