Evaluating the Efficacies of Carbapenem/β-Lactamase Inhibitors Against Carbapenem-Resistant Gram-Negative Bacteria in vitro and in vivo

BackgroundCarbapenem-resistant Gram-negative bacteria are a major clinical concern as they cause virtually untreatable infections since carbapenems are among the last-resort antimicrobial agents. β-Lactamases implicated in carbapenem resistance include KPC, NDM, and OXA-type carbapenemases. Antimicrobial combination therapy is the current treatment approach against carbapenem resistance in order to limit the excessive use of colistin; however, its advantages over monotherapy remain debatable. An alternative treatment strategy would be the use of carbapenem/β-lactamase inhibitor (βLI) combinations. In this study, we assessed the in vitro and in vivo phenotypic and molecular efficacies of three βLIs when combined with different carbapenems against carbapenem-resistant Gram-negative clinical isolates. The chosen βLIs were (1) Avibactam, against OXA-type carbapenemases, (2) calcium-EDTA, against NDM-1, and (3) Relebactam, against KPC-2.MethodsSix Acinetobacter baumannii clinical isolates were screened for blaOXA-23-like, blaOXA-24/40, blaOXA-51-like, blaOXA-58, and blaOXA-143-like, and eight Enterobacteriaceae clinical isolates were screened for blaOXA-48, blaNDM-1, and blaKPC-2. The minimal inhibitory concentrations of Imipenem (IPM), Ertapenem (ETP), and Meropenem (MEM) with corresponding βLIs for each isolate were determined. The efficacy of the most suitable in vitro treatment option against each of blaOXA-48, blaNDM-1, and blaKPC-2 was assessed via survival studies in a BALB/c murine infection model. Finally, RT-qPCR was performed to assess the molecular response of the genes of resistance to the carbapenem/βLI combinations used under both in vitro and in vivo settings.ResultsCombining MEM, IPM, and ETP with the corresponding βLIs restored the isolates’ susceptibilities to those antimicrobial agents in 66.7%, 57.1%, and 30.8% of the samples, respectively. Survival studies in mice revealed 100% survival rates when MEM was combined with either Avibactam or Relebactam against blaOXA-48 and blaKPC-2, respectively. RT-qPCR demonstrated the consistent overexpression of blaOXA-48 upon treatment, without hindering Avibactam’s activity, while blaNDM-1 and blaKPC-2 experienced variable expression levels upon treatment under in vitro and in vivo settings despite their effective phenotypic results.ConclusionNew carbapenem/βLI combinations may be viable alternatives to antimicrobial combination therapy as they displayed high efficacy in vitro and in vivo. Meropenem/Avibactam and Meropenem/Relebactam should be tested on larger sample sizes with different carbapenemases before progressing further in its preclinical development

Diagnosis of recent and relapsed cases of human brucellosis by PCR assay

BACKGROUND: Brucellosis affects human populations in many developing countries including the Middle East, and Latin America where it is still endemic. It has been prevalent in Jordan for years, where 7842 cases of human brucellosis were registered at the Ministry of Health during 10 year-period. This study was initiated by the recent increase in the number of human cases diagnosed in a rural area in the Northern Jordan to help assess the status of the disease in that area. For this purpose blood specimens from brucellosis suspected cases were tested by serology, culture and PCR. METHODS: Peripheral blood specimens from 50 healthy control subjects and 165 seropositive patients having compatible signs and symptoms that were clinically diagnosed to have brucellosis were tested by blood culture, and by PCR. The PCR assay used genus-specific primers from the conserved region of the 16S rRNA sequence, which showed high specificity for the Brucella spp. RESULTS: Diagnosis of Brucella was established by PCR in 120 cases (72.7%). All of them were seropositive and 20 were positive by culture. Forty-eight of 58 (82.8%) of the relapsed cases two months after completing the treatment with an increase in the previous serological titers were positive by PCR. The assay has 85.7% positive predicative value, 100% sensitivity and specificity since it correctly identified all cases that were positive by blood cultures, 95.8% by serology and none of the control group was positive. CONCLUSIONS: Results showed that PCR assay can be applied with serology for the diagnosis of brucellosis suspected cases and relapses regardless of the duration or type of the disease without relying on the blood cultures, especially in chronic cases

Poor performance of the rapid test for human brucellosis in health facilities in Kenya

Human brucellosis is considered to be an important but typically under-diagnosed cause of febrile illness in many low and middle-income countries. In Kenya, and throughout East Africa, laboratory diagnosis for the disease is based primarily on the febrile antigen Brucella agglutination test (FBAT), yet few studies of the diagnostic accuracy of this test exist. Assessment of the performance of the FBAT is essential for its appropriate clinical use, as well as for evaluating surveillance data reported by public health systems. To assess FBAT performance, we collected sera from people with symptoms compatible with brucellosis attending two health facilities in Busia County, Kenya. Sera were tested using the FBAT and results compared with those from the Rose Bengal Test (RBT), an assay with well-known performance characteristics. Positives on either test were confirmed using the classical serum agglutination test (SAT)-Coombs test combination and a rapid IgM/IgG lateral flow immunochromatography assay (LFA). A questionnaire focussing on known risk factors for exposure to Brucella spp. was also conducted, and relationships with FBAT positivity examined using logistic regression. Out of 825 recruited individuals, 162 (19.6%) were classified as positive using the FBAT. In contrast, only eight (1.0%) were positive using the RBT. Of the 162 FBAT positives, one (0.62%) had an atypical agglutination in SAT and three (1.9%) showed low Coombs titres. Out of 148 FBAT positive individuals tested using the LFA, five (3.4%) were IgM positive and none were IgG positive. Poor or no correlation was observed between FBAT results and most established risk factors for Brucella infection. We observed substantial disagreement between the FBAT and a number of well-known serological tests, with the majority of reactive FBAT results appearing to be false positives. Poor FBAT specificity, combined with a lack of confirmatory testing, strongly suggests overdiagnosis of brucellosis is common in this low prevalence setting. This is expected to have important economic impacts on affected patients subjected to the long and likely unnecessary courses of multiple antibiotics required for treatment of the disease