Clinical Specimen-Direct LAMP: A Useful Tool for the Surveillance of <i>bla</i>_OXA-23-Positive Carbapenem-Resistant <i>Acinetobacter baumannii</i>

<div><p>Healthcare-associated infections are a leading cause of morbidity and mortality worldwide. Treatment is increasingly complicated by the escalating incidence of antimicrobial resistance. Among drug-resistant pathogens, carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAb) is of increasing concern because of the limited applicable therapies and its expanding global distribution in developed countries and newly industrialized countries. Therefore, a rapid detection method that can be used even in resource-poor countries is urgently required to control this global public health threat. Conventional techniques, such as bacterial culture and polymerase chain reaction (PCR), are insufficient to combat this threat because they are time-consuming and laborious. In this study, we developed a loop-mediated isothermal amplification (LAMP) method for detecting <i>bla</i>_OXA-23-positive CRAb, the most prevalent form of CRAb in Asia, especially in Thailand, and confirmed its efficacy as a surveillance tool in a clinical setting. Clinical samples of sputum and rectal swabs were collected from patients in a hospital in Bangkok and used for LAMP assays. After boiling and centrifugation, the supernatants were used directly in the assay. In parallel, a culture method was used for comparison purposes to evaluate the specificity and sensitivity of LAMP. As a first step, a total of 120 sputum samples were collected. The sensitivity of LAMP was 88.6% (39/44), and its specificity was 92.1% (70/76) using the culture method as the “gold standard”. When surveillance samples including sputum and rectal swabs were analyzed with the LAMP assay, its sensitivity was 100.0%. This method enables the direct analysis of clinical specimens and provides results within 40 minutes of sample collection, making it a useful tool for surveillance even in resource-poor countries.</p></div

Genotypes of carbapenem-resistant <i>A</i>. <i>baumannii</i> (CRAb) isolates from hospital patients in Bangkok.

<p>Total^a: Total number of isolates with each resistance gene</p><p>%^b: The proportion of isolates with each resistance gene</p><p>Genotypes of carbapenem-resistant <i>A</i>. <i>baumannii</i> (CRAb) isolates from hospital patients in Bangkok.</p

Real-time turbidity assays under various conditions using a turbidimeter.

<p>(A) To determine the optimal reaction conditions, a LAMP assay was performed on extracted bacterial DNA at temperatures ranging from 62°C to 67°C. At 65°C, the reaction finished within the shortest period of time, and the negative control remained transparent after 60 minutes of incubation. (B) To determine the detection limit, the extracted DNA templates were serially diluted 10 times (from 2 pg to 2×10⁻⁶ pg) and used in the LAMP assay. The turbidity was evaluated with a turbidimeter every 5 minutes.</p

LAMP primers used for <i>bla</i>_oxa-23 and the ITS sequence.

<p>F3: outer forward primer; B3: backward inner primer; LF/LB: loop primersouter backward primer; FIP: forward inner primer; BIP:</p><p>LAMP primers used for <i>bla</i>_oxa-23 and the ITS sequence.</p