First evidence for the existence of Simkania negevensis in the genitalia of human females.

INTRODUCTION: Simkania negevensis has been linked to some respiratory and non-respiratory diseases. However, there is still a serious lack of clinical investigations that attempt to determine possible body sites that could be inhabited by this microorganism and evaluate its true pathogenic capacity. The goal of this study was to examine the potential presence of Simkania and its prevalence in the genital tract of human adult females. METHODS: Lower vaginal swabs from 169 Jordanian adult females who attended Obstetrics and Gynecology clinic were collected and tested for Simkania DNA by PCR method. RESULTS: The presence of bacterial nucleic acids was confirmed in the genital system of adult females with an overall prevalence of 24.26% (41/169). Interestingly, the positivity of Simkania DNA was significantly higher in women of reproductive age than females of non-reproductive age (28.03% versus 10.81%; p≤0.05). Moreover, the presence of S. negevensis was evident in approximately 43% of females suffering from vaginal itching and/or abnormal discharge, exhibiting about two-fold increase in the positivity rate compared to detection rates assessed for women who attended the clinic for routine checkup or menstruation problems. However, the current work failed to find any link between the bacterial agent and spontaneous abortion (miscarriage). CONCLUSIONS: This study showed for the first time the presence of S. negevensis in the genitalia of human females. These novel data could provide a basis to clarify the exact role of S. negevensis in the female genitalia and its potential involvement in genital system disorders.

Development of a duplex q-PCR for the simultaneous detection of Parachlamydia acanthamoebae and Simkania negevensis in environmental and clinical samples.

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

First evidence for the existence of Simkania negevensis in the genitalia of human females / Primera evidencia de la existencia de Simkania negevensis en los genitales femeninos humanos

Introduction: Simkania negevensis has been linked to some respiratory and non-respiratory diseases. However, there is still a serious lack of clinical investigations that attempt to determine possible body sites that could be inhabited by this microorganism and evaluate its true pathogenic capacity. The goal of this study was to examine the potential presence of Simkania and its prevalence in the genital tract of human adult females. Methods: Lower vaginal swabs from 169 Jordanian adult females who attended Obstetrics and Gynecology clinic were collected and tested for Simkania DNA by PCR method. Results: The presence of bacterial nucleic acids was confirmed in the genital system of adult females with an overall prevalence of 24.26% (41/169). Interestingly, the positivity of Simkania DNA was significantly higher in women of reproductive age than females of non-reproductive age (28.03% versus 10.81%; p≤0.05). Moreover, the presence of S. negevensis was evident in approximately 43% of females suffering from vaginal itching and/or abnormal discharge, exhibiting about two-fold increase in the positivity rate compared to detection rates assessed for women who attended the clinic for routine checkup or menstruation problems. However, the current work failed to find any link between the bacterial agent and spontaneous abortion (miscarriage). Conclusions: This study showed for the first time the presence of S. negevensis in the genitalia of human females. These novel data could provide a basis to clarify the exact role of S. negevensis in the female genitalia and its potential involvement in genital system disorders.(AU)

Introducción: Simkania negevensis se ha asociado a algunas enfermedades respiratorias y no respiratorias. Sin embargo, todavía existe una importante carencia de investigaciones clínicas para intentar determinar posibles lugares del cuerpo que puedan ser colonizados por este microorganismo, y evaluar su auténtica capacidad patogénica. El objetivo de este estudio era examinar la potencial presencia de Simkania y su prevalencia en el tracto genital de mujeres adultas. Métodos: Se recogieron hisopos vaginales de 169 mujeres adultas de Jordania que acudieron a una clínica de obstetricia y ginecología y se analizaron para detectar ADN de Simkania mediante PCR. Resultados: Se confirmó la presencia de ácidos nucleicos bacterianos en el aparato genital femenino adulto, con una prevalencia global del 24,26% (41/169). Una observación interesante fue que la positividad a ADN de Simkania fue significativamente superior en mujeres en edad reproductiva en comparación con las mujeres en edad no reproductiva (28,03 frente al 10,81%; p≤0,05). Además, la presencia de S. negevensis era evidente en aproximadamente el 43% de las mujeres que sufrían picor vaginal y/o flujo vaginal anormal, mostrando un incremento cercano al doble en la tasa de positividad en comparación con las tasas de detección evaluadas para mujeres que acudían a la clínica para revisiones de rutina o por problemas menstruales. Sin embargo, la investigación no logró encontrar una asociación entre el agente bacteriano y el aborto espontáneo (aborto natural). Conclusiones: Este estudio mostró por primera vez la presencia de S. negevensis en los genitales femeninos humanos. Estos nuevos datos pueden sentar una base para clarificar con precisión el papel de S. negevensis en los genitales femeninos humanos y su potencial implicación en trastornos del aparato genital.(AU)

First evidence for the existence of Simkania negevensis in the genitalia of human females.

INTRODUCTION: Simkania negevensis has been linked to some respiratory and non-respiratory diseases. However, there is still a serious lack of clinical investigations that attempt to determine possible body sites that could be inhabited by this microorganism and evaluate its true pathogenic capacity. The goal of this study was to examine the potential presence of Simkania and its prevalence in the genital tract of human adult females. METHODS: Lower vaginal swabs from 169 Jordanian adult females who attended Obstetrics and Gynecology clinic were collected and tested for Simkania DNA by PCR method. RESULTS: The presence of bacterial nucleic acids was confirmed in the genital system of adult females with an overall prevalence of 24.26% (41/169). Interestingly, the positivity of Simkania DNA was significantly higher in women of reproductive age than females of non-reproductive age (28.03% versus 10.81%; p≤0.05). Moreover, the presence of S. negevensis was evident in approximately 43% of females suffering from vaginal itching and/or abnormal discharge, exhibiting about two-fold increase in the positivity rate compared to detection rates assessed for women who attended the clinic for routine checkup or menstruation problems. However, the current work failed to find any link between the bacterial agent and spontaneous abortion (miscarriage). CONCLUSIONS: This study showed for the first time the presence of S. negevensis in the genitalia of human females. These novel data could provide a basis to clarify the exact role of S. negevensis in the female genitalia and its potential involvement in genital system disorders.

Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis.

Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion.

Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Simkania negevensis in Crohn's Disease.

BACKGROUND: Simkania negevensis is an obligate intracellular Gram-negative bacterium (family Simkaniaceae, order Chlamydiales) that has been isolated from domestic and mains water supplies, is able to infect human macrophages, and can induce an inflammatory response in the host. METHODS: From June to December 2016, in a single-center observational study, colonic Crohn's disease patients and controls (subjects undergoing screening for colorectal cancer) underwent blood tests to identify serum-specific immunoglobulin G (IgG) and immunoglobulin A (IgA) to S. negevensis and a colonoscopy with biopsies for detection of S. negevensis DNA by polymerase chain reaction (PCR). RESULTS: Forty-three Crohn's disease patients and 18 controls were enrolled. Crohn's disease patients had higher prevalence of IgA antibodies to S. negevensis compared with controls (20.9% versus 0%, p = 0.04). Simkaniaceae negevensis DNA was detected in 34.9% and 5.6% of intestinal biopsies in Crohn's disease patients and controls, respectively (p = 0.02). All Crohn's disease patients with PCR-positive biopsies for S. negevensis were IgG seropositive, with specific IgA in 60% of them (p < 0.001). Immunosuppressive therapies, extraintestinal manifestations, or disease activity did not influence the presence of S. negevensis in the Crohn's disease population. CONCLUSIONS: We identified S. negevensis in Crohn's disease patients by demonstrating the presence of S. negevensis mucosal DNA and seropositivity to the bacterium. These results could support the presence of an acute or persistent S. negevensis infection and suggest a possible role in the pathogenesis of Crohn's disease.

Seroprevalence of a "new" bacterium, Simkania negevensis, in renal transplant recipients and in hemodialysis patients.

BACKGROUND: Simkania negevensis is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydiales order. It is considered an ubiquitous microorganism and aquatic environments may be involved as a source of infection for humans. It was just isolated in samples from domestic water supplies and from mains water supplies, like spa water or swimming pool water, confirming its ability to resist to the common chlorination treatments. Evidence indicates a possible role of the microorganism in respiratory tract infections, in gastroenteric disorders and in the pathogenesis of cardiovascular disease, furthermore it has hypothesized that it could play a role in lung transplant rejection. Prevalence and possible effects in nephrology are unknown. METHODS: We examined the occurrence of Simkania negevensis in two differents populations, both characterized by a high susceptibility to infectious complications: 105 hemodialysis patients, 105 renal transplant recipients and 105 healthy subjects through the IgG and IgA response to Simkania negevensis in their sera. Serum antibodies to Simkania negevensis were detected by a homemade ELISA performed according to the Kahane's protocol. Furthermore water samples from hemodialytic circuit were collected, to evaluate Simkania negevensis resistance to usual treatment of disinfection. RESULTS: Our results were unexpected, showing a higher seroprevalence of antibodies against Simkania negevensis in the hemodialysis patients, compared to renal transplant patients (IgG 22% vs 9% - IgA 9% vs 3%). S. negevensis was isolated in all water samples analyzed. CONCLUSIONS: Our study detected for the first time the occurrence of S. negevensis in hemodialysis and in renal transplant patients. Our findings suggest that water used in hemodialysis could be one of the possible sources of S. negevensis infection, without clinical involvement risk for patients.

Interaction of Chlamydiae with human macrophages.

The phylum Chlamydiae contains several members that are well-known human pathogens, like Chlamydia trachomatis and C. pneumoniae. Establishing a chronic bacterial infection requires the active evasion of the host immune response. A major arm of the innate immune defence is constituted by macrophages, which fight infections by removing bacteria and triggering an adaptive immune response. However, some pathogenic Chlamydia infect and survive in macrophages at least for a certain period of time. Therefore, macrophages can serve as vehicles for the dissemination of bacterial infections from the primary infection site via the urogenital or respiratory tract to distant sites in the body. The capacity to infect macrophages seems to depend on the chlamydial strain and the source of macrophages. In vitro infections of macrophages with C. trachomatis, C. psittaci and C. pneumoniae reveal low efficiency of infection and progeny formation, as well as failure to develop mature inclusions. In contrast, the emerging pathogen, Simkania negevensis, actively replicates in macrophages. Here we summarize the current knowledge of the intracellular and molecular key mechanisms of C. trachomatis, C. pneumoniae and S. negevensis infections in human macrophages.

Simkania negevensis: Is it a real respiratory pathogen?

Simkania negevensis, a recently discovered Chlamydia-like organism, has been associated with respiratory infections such as pneumonia, bronchiolitis and chronic obstructive pulmonary disease in children and adults. The aim of the present study was to evaluate S. negevensis in the etiology of pediatric community-acquired pneumonia, bronchiolitis and asthma exacerbation in our region. Overall, 102 patients and 46 healthy controls were included in the study. S. negevensis was investigated by real time PCR (Primer Design, UK) in nasopharyngeal swab samples. It failed to be detected in either the study or control group. In conclusion, our results suggest that S. negevensis is not an important respiratory pathogen in our region.

Purification and proteomics of pathogen-modified vacuoles and membranes.

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

Simkania negevensis and acute cellular rejection in lung transplant recipients.

Simkania negevensis infection has been hypothesized to play a role in lung transplant rejection. The incidence of S. negevensis infection and its association with acute cellular rejection (ACR) were determined in a prospective cohort study of 78 lung transplant recipients (LTRs) in Toronto, Canada, and Pittsburgh, USA, from July 2007 to January 2010. Simkania negevensis testing was detected by quantitative polymerase chain reaction (PCR) on bronchoalveolar lavage fluid. The relationship between S. negevensis and ACR was examined using Cox proportional hazards models and generalized linear and latent mixed models. Cumulative incidence estimates for time-to-ACR in S. negevensis PCR-positive vs. PCR-negative LTRs were 52.7% vs. 31.1% at six months and 68.9% vs. 44.6% at one yr, respectively. Although not statistically significant, there was a trend toward a higher risk of ACR among S. negevensis PCR-positive vs. PCR-negative LTRs in all statistical models.

Prevalence of Simkania negevensis in chlorinated water from spa swimming pools and domestic supplies.

AIMS: This study aimed to investigate the prevalence of Simkania negevensis in the chlorinated water of spa swimming pools and domestic network systems. METHODS AND RESULTS: A total of 10 and 36 samples were collected from two domestic water supplies and four spa facilities, respectively. Simkania negevensis was isolated in Acanthamoeba castellanii monolayers and detected by Gimenez staining and immunofluorescence test. Simkania negevensis DNA, extracted from the positive samples, was confirmed by a nested PCR assay followed by sequencing. Simkania negevensis was detected in 41·3% of samples (domestic water: 50%; untreated spa supply water: 25%; chlorinated spa pool water: 42·9%) from all the examined water systems in successive samplings performed in 1 year. The presence of S. negevensis was not correlated with the counts of Heterotrophic Bacteria and Pseudomonas aeruginosa. Unlike Legionella spp., which were never isolated from the swimming-pool water samples, S. negevensis was also detected in chlorinated spa water. CONCLUSIONS: This investigation shows that Simkania is widespread in natural and man-made aquatic environments, which may represent possible sources of infection: in the swimming pools, in particular, the aerosol generated by the water movement could increase the risk of inhalation of infected particles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first evidence of the presence of S. negevensis in spa pool water. Sero-epidemiological surveys on spa users could help to clarify its transmission in this environment.

High seroprevalence of Simkania negevensis in Jordan

The bacterium Simkania negevensis is a germ associated with respiratory diseases. This study aims at estimating the prevalence of Simkania in the Jordanian population. Serum samples from 664 Jordanian males and females, aged 2 to 86 years were collected. IgG and IgM Simkania-specific antibodies were detected using an indirect immunofluorescence test. Seropositivity titers for IgG and IgM were defined as 1:8 and 1:10, respectively. The overall prevalence of IgG antibody in all examined Jordanian nationals was 58.4%. IgG seropositivity was low in children under the age of 10 years (34.2%), and increased rapidly with age and ranged between 49.4% and 72%. Simkania-specific IgM was detected in 24.8% of subjects. IgM prevalence in children under 10 years was lowest (10.5%) and increased in older ages and remained above 20%. Overall detection rates of both IgG and IgM were significantly higher in females than males (60.7% vs. 54.5% for IgG and 26.7% vs. 21.7% for IgM). These data indicate that Simkania infection is highly prevalent in Jordan. The high level of seropositivity is most likely maintained by re-infections or chronic infections. Our data may serve as a basis to elucidate the pathogenesis of Simkania in Jordan.

High seroprevalence of Simkania negevensis in Jordan.

The bacterium Simkania negevensis is a germ associated with respiratory diseases. This study aims at estimating the prevalence of Simkania in the Jordanian population. Serum samples from 664 Jordanian males and females, aged 2 to 86 years were collected. IgG and IgM Simkania-specific antibodies were detected using an indirect immunofluorescence test. Seropositivity titers for IgG and IgM were defined as 1:8 and 1:10, respectively. The overall prevalence of IgG antibody in all examined Jordanian nationals was 58.4%. IgG seropositivity was low in children under the age of 10 years (34.2%), and increased rapidly with age and ranged between 49.4% and 72%. Simkania-specific IgM was detected in 24.8% of subjects. IgM prevalence in children under 10 years was lowest (10.5%) and increased in older ages and remained above 20%. Overall detection rates of both IgG and IgM were significantly higher in females than males (60.7% vs. 54.5% for IgG and 26.7% vs. 21.7% for IgM). These data indicate that Simkania infection is highly prevalent in Jordan. The high level of seropositivity is most likely maintained by re-infections or chronic infections. Our data may serve as a basis to elucidate the pathogenesis of Simkania in Jordan.