Molecular Tests That Target the RTX Locus Do Not Distinguish between Kingella kingae and the Recently Described Kingella negevensis Species
Authors: Elsa Prudent
Pierre-Edouard Fournier
Stéphane Bonacorsi
Pablo Yagupsky
Nawal El Houmami
Janek Bzdrenga
Didier Raoult
Philippe Minodier
Hervé Seligmann
Sofiane Bakour
Guillaume André Durand
Journal: Journal of Clinical Microbiology (2017)
Discipline: MICROBIOLOGY
Keywords: IS1 family; Kingella kingae; Kingella negevensis; RTX toxins; osteoarticular infections
Affiliations: Aix-Marseille Université, Research Unit on Infectious and Emerging Tropical Diseases (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée Infection, Assistance Publique-Hôpitaux de Marseille, Marseille, France ., Aix-Marseille Université, Research Unit on Infectious and Emerging Tropical Diseases (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée Infection, Assistance Publique-Hôpitaux de Marseille, Marseille, France., Université Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France., Department of Pediatric Emergency, North Hospital, Marseille, France., Inserm, IAME, UMR 1137, Université Paris-Diderot, Sorbonne Paris Cité, Laboratoire de Microbiologie, Hôpital Robert-Debré, Assistance Publique-Hôpitaux de Paris, Paris, France., Clinical Microbiology Laboratory, Soroka University Medical Center, Beer-Sheva, Israel.

Abstract:  Kingella kingae is an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis of K. kingae infections. However, the present study shows that Kingella negevensis, a Kingella species newly identified in young children, harbors an identical Kingella RTX locus, raising the question of whether K. negevensis can be misidentified as K. kingae by clinical microbiology laboratories. In silico comparison of Kingella sp. RTX and groEL genes and in vitro studies provided evidence that targeting the rtxA and rtxB genes could not differentiate between strains of K. kingae and K. negevensis, whereas targeting the groEL gene could. This prompted the design of a highly specific and sensitive qPCR assay targeting K. negevensis groEL (kngroEL). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay and Kingella-specific rtxB, K. kingae-specific groEL (kkgroEL), and kngroEL qPCR assays. Forty-two specimens were rtxB positive, including 41 that were also kkgroEL positive and 1 (the remaining one) that was kngroEL positive. Thus, this study discloses an invasive infection caused by K. negevensis in humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis of K. kingae infections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused by K. negevensis in humans. 

Retracted: No

PubPeer comments:
 This paper is part of a set of 14 papers relating to the protocol approved by the Ethics Committee of Institut Hospitalier Universitaire Mu00e9diterranu00e9e Infection under the number 2016-024. Readers will find some of the same authors or authors from the same institution in the 14 papers. **One protocol approved by the ethic committee under the number no. 2016-024** Authors mention in each paper that the study has been approved by the ethics committee of of Institut Hospitalier Universitaire Mu00e9diterranu00e9e Infection under the number 2016-024. According to other papers published by the Institut Hospitalier Universitaire Mu00e9diterranu00e9e Infection, it appears that the number 2016-024 is related to one protocol only. The 14 papers should be considered as part of a unique protocol nu00b02016-024. **Ethics approval obtained on October 19th, 2016.** 2 papers (articles #3 and #14) mention that the ethics approval of the protocol 2016-024 has been obtain on October 19th, 2016 **When the recruitment has been done?** Depending on the paper, the recruitment started before or after the ethics approval -t**Before the ethics approval:** In 3 articles, the recruitment starts June 2016 and ends for the later one in September 2016 (articles #1, #6, #7) -t**After the ethics approval:** in 5 articles, the recruitment starts after the ethics approval (articles #2, #4, #10, #12, #13). Depending on the paper, the recruitment is done in 2017 and or 2018. The others papers donu2019t mention the recruitment dates. **Retrospective ethical approval?** Among the 3 articles describing a recruitment before the ethics approval (June-September 2016), 2 are case reports and one is a study on 11 children. This study in children collected oropharyngeal swabs in order to develop a modified multilocus sequence typing (MLST) method to genotype K. kingae strains from oropharyngeal samples without prior culture (article #1). Retrospective ethics committee approval for a prospective interventional protocol is a serious breach of the Helsinki Declaration. In accordance with Editors guidances and Helsinki Declaration, if a prospective study has not been granted ethics committee approval prior to commencing, retrospective ethics approval cannot be obtained and it may not be possible to consider the manuscript for peer review. Accepting retrospective ethics approval for interventional prospective study would promote these practices and this risk should be considered very seriously by Editors. **Prospective study mislabeled?** In the article #10, the authors mention the study on 327 participants is retrospective. *u201cWe retrospectively included in this study all the CSF specimens submitted to one of the two point-of-care (POC) laboratories (referred to as POC-A and POC-B) of the IHU Mu00e9diterrannu00e9e Infection, Marseille, France for the diagnosis of infectious meningitis over two 8-week periods (from November 1, 2017 to December 31, 2017 (ufb01rst8-week period, period 1) and from November 1, 2018 to December 31, 2018 (second 8-week period, period 2))*. The recruitment period is after the ethics approval. The data have not been collected yet when the protocol was approved by the IHU ethics committee. As a consequence, in the article #10, the recruitment is not retrospective. It is a prospective recruitment. Furthermore, an another paper (article #4) from the same authors describe this time a prospective recruitment with the same type of sample (CSF) and on a similar recruitment period (November 2017 to September 2018) *u201cAll the CSF submitted to the point-of-care laboratory of the IHU Mu00e9diterrannu00e9e Infection laboratory, Marseille, France, for the diagnosis of infectious meningitis over a 43-week period (from 24 November 2017 to 16 September 2018) were **prospectively** included in the studyu201d*. **In France, only the Comitu00e9 de Protection des Personnes (IRB) are qualified to approve prospective study in humans.** The IHU ethics committee is not on the list of the certified IRB in France. It is not qualified to validate a prospective study in humans. None of the 14 papers mention any approval from a Comitu00e9 de Protection des Personnes. Could the authors clarify if the protocol 2016-024 has obtained an ethics approval from a Comitu00e9 de Protection des Personnes before to start to recruit the first participant as it is mandatory in France? **Different type of objectives:** Depending on the paper, the objective of the study is : -ta case report on 1 single patient only and on different diseases or medical targets. -tdifferent studies on hundreds or a thousand of participants to develop different new diagnostic techniques for different diseases -tstudies on diseases in dozens of participants. How a unique protocol can cover so many different situations? **Different types of participants and different type of samples** Depending on the papers: -tParticipants are different. They can be children only, or adults and children. S -tType of samples are different. **How the same protocol can cover so many different interventions, different study designs and different types of participants?** Could the authors disclose the protocol of the study 2016-024 ? Or, at least, explain how a unique protocol can cover the diversity of situations described by the 14 papers ? The Editors should also clarify how the same protocol can cover so many different type of study design, inclusion criteria and exclusion criteria. Editors should also note that using one protocol number and one Ethics Committee approval number 2016-0XX for different types of design, interventions and participants are something already noted concerning some articles of the authors. - [Protocol 2016-010 - 34 papers] - [Protocol 2016-011 - 38 papers] **Salami publication** According to the article  Salami publication: definitions and examples : *u201cSalami publication can be roughly defined as a publication of two or more articles derived from a single study. Articles of such type report on data collected from a single study split into several segments just large enough to gain reasonable results and conclusions, also known as u201cminimal publishable unitu201d.* (...) * From the publication ethics point of view, it gives undeserved credit to authors, misleads the scientific community and directly influences clinical practice by distorting medical evidence. * **LIST OF THE 14 PAPERS WITH THE SAME PROTOCOL NUMBER 2016-024** 1.t[BMC microbiology - A modified multilocus sequence typing protocol to genotype Kingella kingae from oropharyngeal swabs without bacterial isolation] 2.t[Emerging infectious diseases - Routine Cultureu2013Resistant Mycobacterium tuberculosis Rescue and Shell-Vial Assay, France] 3.t[European Journal of Clinical Microbiology Infectious Diseases - Bead-captured Mycobacterium tuberculosis for next-generation sequencing diagnosis of uncultured tuberculosis] 4.t[European Journal of Clinical Microbiology Infectious Diseases - Point-of-care multiplexed diagnosis of meningitis using the FilmArrayu00ae ME panel technology] 5.t[Frontiers in cellular and infection microbiology - High concentrations of serum soluble E-cadherin in patients with Q fever] 6.t[Frontiers in pediatrics - Acute septic arthritis of the knee caused by Kingella kingae in a 5-year-old Cameroonian boy] 7.t[Infection - Low antibodies titer and serological cross-reaction between Coxiella burnetii and Legionella pneumophila challenge the diagnosis of mediastinitis, an emerging Q fever clinical entity] 8.t[Journal of clinical microbiology - Molecular tests that target the RTX locus do not distinguish between Kingella kingae and the recently described Kingella negevensis species] 9.t[Journal of Hand Surgery (European Volume) - Kingella kingae hand and wrist tenosynovitis in young children] 10.t[Letters in Applied Microbiology - Preventing contamination of PCR-based multiplex assays including the use of a dedicated biosafety cabinet] 11.t[Medicine - Transplanted lungs and the u201cwhite plagueu201d: a case-report and review of the literature] 12.t[Medicine - Fluorescence in situ hybridization microscopic detection of Bacilli Calmette Guu00e9rin mycobacteria in aortic lesions: a case report] 13.t[Open forum infectious diseases - Granulocyte macrophage colony-stimulating factor-specific autoantibodies and cerebral nocardia with pulmonary alveolar proteinosis] 14.t[The Pediatric Infectious Disease Journal - SHELL-VIAL ASSAY IN DIAGNOSIS OF DISSEMINATED BCG INFECTION IN AN IMMUNODEFICIENT CHILD] ,