We evaluated the clinical performance of Check-Direct CPE for carbapenemase detection directly from 301 perirectal swabs (258 patients) in a nonoutbreak setting. Culture of a PCR-confirmed, ...carbapenemase-containing organism, or history of colonization with such organism within the previous 2 weeks, was used as the reference standard. Check-Direct CPE demonstrated a sensitivity value, specificity value, positive predictive value (PPV), and negative predictive value (NPV) of 100% (all
bla
KPC
), 88%, 21%, and 100%, respectively. False positives accounted for 79% (
n
= 34) of samples for which a cycle threshold (
C
T
) value was reached. Simulated studies to evaluate specimen pooling as an approach to minimize costs showed no difference in
C
T
values for pooled groups of three or five that each contained a single specimen spiked with ∼1,500 CFU
bla
KPC
Klebsiella pneumoniae
; however, the detection rate dropped to 60% at a seeded concentration of ∼150 CFU. When data were pooled,
C
T
values for
bla
KPC
were higher for heavy-feces-containing than for light-feces-containing liquid-suspended specimens. Furthermore,
C
T
values for liquid-suspended specimens were 4 to 5
C
T
values lower (i.e., represented greater sensitivity) than those seen in direct swab analysis. Culture was equivalent to or better than Check-Direct CPE for 13/15 (87%) isolates tested in a limit-of-detection analysis. Detection of a carbapenemase gene at a
C
T
cutoff value of ≤35 was culture confirmed in 23/24 (96%) of cases; however,
C
T
values of >35 overlapped broadly between culture-positive (
n
= 21) and culture-negative (
n
= 36) specimens. Check-Direct CPE will likely prove most useful in high-prevalence areas or in outbreak settings where rapid carbapenemase detection is critical for infection control management.
OXA-181 is the second most common global OXA-48-like carbapenemase and is endemic in the Indian subcontinent. Molecular studies have shown that
Enterobacterales
with OXA-181 are often introduced into ...regions of nonendemicity. Distinguishing OXA-181 from other OXA-48-like enzymes often requires sequencing, which is rather expensive and time-consuming.
OXA-181 is the second most common global OXA-48-like carbapenemase and is endemic in the Indian subcontinent. Molecular studies have shown that
Enterobacterales
with OXA-181 are often introduced into regions of nonendemicity. Distinguishing OXA-181 from other OXA-48-like enzymes often requires sequencing, which is rather expensive and time-consuming. A specific PCR (i.e., OXA181PCR) for the detection of
bla
OXA-181
was validated using a global collection (
n
= 315) of bacteria with well-characterized carbapenemases and showed 100% sensitivity and specificity (95% confidence interval CI, 94.1 to 100 and 98.6 to 100, respectively) for detecting bacteria with OXA-181. The OXA181PCR subsequently gave positive results on 58/160 (36%)
Enterobacterales
with OXA-48-like carbapenemases from the 2015 INFORM surveillance program. The
bla
OXA-181
-positive
Enterobacterales
were present in 9 countries spanning 5 continents, illustrating the global distribution of OXA-181. This methodology can easily be incorporated into molecular surveillance programs to provide accurate information about the prevalence of OXA-181. A loop-mediated isothermal amplification (LAMP)-OXA48 assay overall performed well for detecting OXA-48-like enzymes but showed poor specificity due to false-positive results with non-OXA carbapenemases.
A prospective multicenter clinical study involving subjects from 21 sites across the United States was conducted to validate the performance of a new
in vitro
diagnostic nucleic acid amplification ...test (NAAT) for the detection of
Mycoplasma genitalium
.
A prospective multicenter clinical study involving subjects from 21 sites across the United States was conducted to validate the performance of a new
in vitro
diagnostic nucleic acid amplification test (NAAT) for the detection of
Mycoplasma genitalium
. Seven urogenital specimen types (
n
= 11,556) obtained from 1,778 females, aged 15 to 74 years, and 1,583 males, aged 16 to 82 years, were tested with the Aptima
Mycoplasma genitalium
assay, an investigational transcription-mediated amplification (TMA) NAAT for the detection of
M. genitalium
16S rRNA. Infected status for enrolled subjects was established using results obtained from testing either self-collected vaginal swab or clinician-collected male urethral swab specimens with a composite reference method consisting of three transcription-mediated amplification NAATs targeting unique regions of
M. genitalium
16S or 23S rRNA.
M. genitalium
prevalence was 10.2% in females and 10.6% in males; prevalence was high in both symptomatic and asymptomatic subjects for both sexes. Compared to the subject infected status standard, the investigational test had sensitivity and specificity estimates, respectively, of 98.9% and 98.5% for subject-collected vaginal swabs, 92.0% and 98.0% for clinician-collected vaginal swabs, 81.5% and 98.3% for endocervical swabs, 77.8% and 99.0% for female urine, and 98.2% and 99.6% for male urethral swabs, 88.4% and 97.8% for self-collected penile meatal swabs, and 90.9% and 99.4% for male urine specimens. For all seven specimen types, within-specimen positive and negative agreements between the investigational test and the composite reference standard ranged from 94.2% to 98.3% and from 98.5 to 99.9%, respectively. These results provide clinical efficacy evidence for the first FDA-cleared NAAT for
M. genitalium
detection in the United States.
In this issue of the
, Hustmyer and colleagues describe a new method for rapidly generating reporter libraries (Hustmyer citation). This RAIL technique (
apid
rbitrary PCR
nsertion
ibraries) uses ...arbitrary PCR and isothermal DNA assembly to insert random fragments of promoter regions into reporter plasmids, resulting in libraries that can be screened to identify regions required for gene expression. This technique will likely be useful for a number of different genetic applications.
As carbapenemase-producing Gram-negative bacilli (CP-GNB) (
,
, and
spp.) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The Amplidiag ...CarbaR+VRE assay is a multiplex nucleic acid-based
diagnostic test intended for the detection of CP-GNB and vancomycin-resistant enterococci (VRE) from cultured colonies. We have evaluated its ability to detect carbapenemase genes in 100 well-characterized GNB and in 200 consecutive enterobacterial isolates with reduced susceptibility to carbapenems that were referred to the French National Reference Center for carbapenem resistance. The assay has been validated on purified DNA but also directly on colonies. The Amplidiag CarbaR+VRE assay could detect all KPC, NDM, VIM, IMP, and OXA-48-like variants tested and all acquired carbapenem-hydrolyzing oxacillinases from
(OXA-23, OXA-24/-40, and OXA-58) as well as the overexpressed chromosomally encoded OXA-51-like β-lactamase associated with an upstream inserted IS
However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES-5, and GES-14), GIM-1, AIM-1, SPM-1, DIM-1, OXA-198 in
, or OXA-143-like in
were not detected. Amplidiag CarbaR+VRE's performance values were high (100% sensitivity and 99% specificity) as it could detect the five major carbapenemases-NDM, VIM, IMP, KPC, and OXA-48-as well as OXA-type carbapenemases from
spp. that are currently emerging also among
and other enterobacterial isolates. It can provide a result directly from colonies growing on Mueller-Hinton (MH) agar or on selective screening medium in less than 2 h. Further evaluations are now necessary to determine the performance values directly on rectal swabs.
In alpha-complementation, inactive N-terminal (alpha-domain) and C-terminal (omega-domain) fragments of beta-galactosidase associate to reconstitute the active protein. To date, the effect of ...alpha-domain size on alpha-complementation activity has not been systematically investigated. In this study, we compared the complementation activities of alpha-domains of various sizes using an in vitro system. We found that the complementation activities are similar for alpha-domains comprising between 45 and 229 N-terminal residues but are significantly decreased for those containing less than 37 residues. However, these smaller alpha-domains (15 and 25 residues) exhibited sufficient alpha-complementation activity for application as reporters.
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