Development of novel broad-range pan-genus PCR assays for the detection of Tropheryma species.

Introduction. Tropheryma whipplei is responsible for the classical Whipple's disease. Recently, a new Tropheryma species was described in a Belgian immunocompromised patient with pleuritis.Gap Statement. There is currently no specific molecular diagnostic test detecting other Tropheryma species than Tropheryma whipplei.Aim. To develop and validate two broad-range pan-Tropheryma genus PCRs detecting both T. whipplei and new Tropheryma species.Methodology. From shotgun sequencing data of the lung tissue biopsy of the Belgian subject, we designed two PCRs targeting the 23S rRNA and rnpB genes. Prospectively, requests for T. whipplei PCR were tested with T. whipplei-specific PCRs and the two Tropheryma broad-range PCRs from January 2019 to November 2022.Results. In total, 2605 samples were tested using both the pan-Tropheryma 23S rRNA PCR and the T. whipplei-specific PCR. In addition, 833 of the 2605 samples were also tested using the pan-Tropheryma rnpB PCRs. Sensitivity was 78.8% and 79.7% for 23S rRNA and rnpB PCRs, as compared with the species-specific T. whipplei PCR. Specificity was 99.9% and 99.7% for the 23S rRNA and the rnpB PCRs, respectively. We identified a patient whose bronchoalveolar lavage tested positive with the two broad-range PCRs with >105 copies ml-1. Specific T. whipplei PCRs were negative. Known for panuveitis, this 49-year-old male presented with an eye inflammation recurrence, and a CT scan showed multiple mediastino-hilar necrotic adenopathies. Doxycyclin (1 year), hydroxychloroquin (1 year) and co-trimoxazol (1 month) treatments led to a favourable outcome.Conclusion. Specific T. whipplei PCR exhibited better sensitivity than the pan-Tropheryma PCRs. However, both broad-range pan-Tropheryma PCRs demonstrated excellent specificity and were pivotal to identifying a new probable case of Tropheryma infection due to another species-level lineage.

Tropheryma whipplei pneumonia revealed by Metagenomic next-generation sequencing: Report of two cases.

Tropheryma whipplei is the causative agent of Whipple's disease, which is a rare multiorgan systemic disease. We report two cases of Tropheryma whipplei infection, all routine tests were negative and it was finally detected by mNGS. This may help clinicians increase awareness of the diagnosis and treatment of acute severe pneumonia and interstitial pneumonia caused by Tropheryma whipplei.

Tropheryma whipplei detected by metagenomic next-generation sequencing in bronchoalveolar lavage fluid.

Whipple's disease is a chronic systemic infectious disease that mainly affects the gastrointestinal tract. In some cases, Tropheryma whipplei can cause infection at the implant site or even throughout the body. In this study, we collected alveolar lavage fluid samples from patients with Tropheryma whipplei from 2020 to 2022, and retrospectively analyzed the clinical data of Tropheryma whipplei positive patients. Patient's past history, clinical manifestations, laboratory examinations, chest CT findings, treatment, and prognosis were recorded. 16 BALFs (70/1725, 4.0 %) from 16 patients were positive for Tropheryma whipplei. 8 patients were male with an average age of 50 years. The main clinical symptoms of patients included fever (9/16), cough (7/16), dyspnea (7/16), and expectoration (5/16), but neurological symptoms and arthralgia were rare. Cardiovascular and cerebrovascular diseases were the most common comorbidity (n=8). The main laboratory characteristics of the patient are red blood cell count, hemoglobin, total protein and albumin below normal levels (11/16), and/or creatinine above normal levels(14/16). Most chest computed tomography mainly show focal or patchy heterogeneous infection (n=5) and pleural effusion (n=8). Among the 6 samples, Tropheryma whipplei was the sole agent, and Klebsiella pneumoniae was the most common detected other pathogens. Metagenomic next-generation sequencing technology has improved the detection rate and attention of Tropheryma whipplei. Further research is needed to distinguish whether Tropheryma whipplei present in respiratory samples is a pathogen or an innocent bystander.

Novel Patterns in High-Resolution Computed Tomography in Whipple Pneumonia.

With the use of metagenomic next-generation sequencing, patients diagnosed with Whipple pneumonia are being increasingly correctly diagnosed. We report a series of 3 cases in China that showed a novel pattern of movable infiltrates and upper lung micronodules. After treatment, the 3 patients recovered, and lung infiltrates resolved.

Prevalence estimation of Tropheryma whipplei in duodenal biopsy tissues of Koreans.

Whipple's disease caused by Tropheryma whipplei is difficult to diagnose because of a broad spectrum of manifestations and non-specific clinical signs. In the current global era, the incidence of duodenal infection/inflammation caused by T. whipplei in Korea may has been underestimated. Here we estimated the prevalence of T. whipplei in duodenal biopsy tissues of Koreans using real-time PCRs (RT-PCRs). A total of 252 duodenal biopsy tissues were collected from Korean patients who underwent esophagogastroduodenoscopy and duodenal biopsy. DNA extracted from the duodenal biopsy tissues was analyzed using three RT-PCRs targeting T. whipplei-specific regions of the 16S-23S rRNA intergenic spacer, hsp65, and Dig15 in parallel. In the samples positive in RT-PCRs, direct sequencing was performed for each RT-PCR target. The prevalence of T. whipplei was estimated based on the RT-PCR and sequencing results. Among the analyzed samples, T. whipplei was not detected. The prevalence of T. whipplei in duodenal biopsy tissues of Koreans was estimated to be less than 0.4%. This is the first study to attempt to detect T. whipplei in duodenal biopsy tissues of Koreans and estimate its prevalence. Our findings infer that while T. whipplei carriers exist in Korea, the incidence of duodenal infection/inflammation caused by T. whipplei is extremely rare.

Ophthalmic manifestations of Whipple's disease.

PURPOSE OF REVIEW: Whipple's disease is an infectious cause of uveitis that may present with nonspecific findings of intraocular inflammation, which can precede the development of neurologic symptoms and signs. Whipple's disease, then, may evade consideration in the differential diagnosis for uveitis. RECENT FINDINGS: Molecular tests can be helpful in identifying the presence of Tropheryma whipplei from ocular specimens. The application of metagenomic sequencing for ocular specimens is promising, as it offers the opportunity to identify the pathogen when suspicion for an intraocular infection is high. Whipple's disease demonstrates the ability to abrogate the host immune response, which gives some insight into its pathogenesis. SUMMARY: Whipple's disease should be suspected in patients who have uveitis refractory to anti-inflammatory therapy. Knowledge of this important pathogen can help direct the timely implementation of diagnostic testing.

[Chronic Tropheryma whipplei infection: an important differential diagnosis of refractory polyarthritis]. / Chronische Tropheryma-whipplei-Infektion: Eine wichtige Differentialdiagnose der therapierefraktären Polyarthritis.

BACKGROUND: Refractory arthritis is a common problem in routine rheumatology practice, and can be a diagnostic challenge. In these cases, chronic Tropheryma whipplei (T. whipplei) infection is an important differential diagnosis that should be considered. OBJECTIVE: Based on five clinical cases, this case-based review describes the diagnostic and therapeutic principles in the management of chronic T. whipplei infection. RESULTS: Whipple's disease is a multisystemic infectious disease caused by the bacterium T. whipplei. The disease typically manifests with arthralgia, weight loss and diarrhoea. Joint involvement often develops years before gastrointestinal symptoms occur. In addition to systemic manifestations ("classic Whipple's disease"), T. whipplei can also lead to localized joint infections without gastrointestinal involvement. Articular manifestations of systemic and localized T. whipplei infections are commonly misdiagnosed as a sign of various forms of autoimmmune arthritis. DISCUSSION: Whipple's disease and localized T. whipplei joint infection should be considered in the diagnostic work-up of refractory arthritis. Synovial fluid analysis by means of specific polymerase chain reaction-based testing for T. whipplei is diagnostically ground-breaking.

Genomic characterization of two metagenome-assembled genomes of Tropheryma whipplei from China.

Whipple's disease is a rare chronic systemic disease that affects almost any organ system of the body caused by the intracellular bacterium Tropheryma whipplei, which is found ubiquitously in the environment. Sequencing of the T. whipplei genome has revealed that it has a reduced genome (0.93 Mbp), a characteristic shared with other intracellular bacteria. Until our research started, 19 T. whipplei strains had been sequenced from cultures originated in France, Canada, and Germany. The genome of T. whipplei bacterium has not been studied in Asia yet. Here, two metagenome-assembled genomes (MAGs) of T. whipplei from China were reconstructed through metagenomic next-generation sequencing (mNGS) and genome binning. We also provided genomic insights into the geographical role and genomic features by analyzing the whole genome. The whole-genome phylogenetic tree was constructed based on single-nucleotide polymorphism (SNP) distance calculations and then grouped by distance similarity. The phylogenetic tree shows inconsistencies with geographic origins, thus suggesting that the variations in geographical origins cannot explain the phylogenetic relationships among the 21 T. whipplei strains. The two Chinese strains were closely related to each other, and also found to be related to strains from Germany (T. whipplei TW08/27) and France (T. whipplei Bcu26 and T. whipplei Neuro1). Furthermore, the Average Nucleotide Identity (ANI) matrix also showed no association between geographic origins and genomic similarities. The pan-genome analysis revealed that T. whipplei has a closed pan-genome composed of big core-genomes and small accessory genomes, like other intracellular bacteria. By examining the genotypes of the sequenced strains, all 21 T. whipplei strains were found to be resistant to fluoroquinolones, due to the genetic mutations in genes gyrA, gyrB, parC, and parE. The 21 T. Whipplei strains shared the same virulence factors, except for the alpC gene, which existed in 7 out of the 21 T. whipplei strains. When comparing 21 entire T. whipplei pan-genomes from various nations, it was discovered that the bacterium also possessed a closed genome, which was a trait shared by intracellular pathogens.

Tropheryma whipplei detection by metagenomic next-generation sequencing in bronchoalveolar lavage fluid: A cross-sectional study.

Tropheryma whipplei is the bacterium associated with Whipple's disease (WD), a chronic systemic infectious disease primarily involving the gastrointestinal tract. T. whipplei can also be detected in different body site of healthy individuals, including saliva and feces. Traditionally, Tropheryma whipplei has a higher prevalence in bronchoalveolar lavage fluid (BALF) of immunocompromised individuals. Few studies have explored the significance of the detection of T. whipplei in BALF. Herein, we retrospectively reviewed 1725 BALF samples which detected for metagenomic next-generation sequencing (mNGS) from March 2019 to April 2022 in Zhuhai, China. Seventy BALs (70/1725, 4.0%) from 70 patients were positive for T. whipplei. Forty-four patients were male with an average age of 50 years. The main symptoms included cough (23/70), expectoration (13/70), weight loss (9/70), and/or dyspnea (8/70), but gastrointestinal symptoms were rare. Chronic liver diseases were the most common comorbidity (n=15, 21.4%), followed by diabetes mellitus (n=13, 18.6%). Only nine patients (12.9%) were immunocompromised. Twenty-four patients (34.3%) were finally diagnosed with reactivation tuberculosis and 15 patients (21.4%) were diagnosed with lung tumors, including 13 primary lung adenocarcinoma and two lung metastases. Fifteen patients (21.4%) had pneumonia. Among the 20 samples, T. whipplei was the sole agent, and Mycobacterium tuberculosis complex was the most common detected other pathogens. Among the non-tuberculosis patients, 31 (31/46, 67.4%) had ground glass nodules or solid nodules on chest CT. Our study indicates that T. whipplei should be considered as a potential contributing factor in some lung diseases. For non-immunocompromised patients, the detection of T. whipplei also needs attention. The mNGS technology improves the detection and attention of rare pathogens. In the future, the infection, colonization, and prognosis of T. whipplei in lung still need to be studied.

Tropheryma whipplei Intestinal Colonization in Migrant Children, Greece.

We obtained fecal samples from migrant children <12 years of age throughout hotspots in Greece and tested them for Tropheryma whipplei by using a quantitative PCR assay. We identified 6 genotypes of T. whipplei, 4 of which are newly described. Our findings suggest a high prevalence of T. whipplei in these regions.

Tropheryma whipplei in the stool samples of children with acute diarrhea: a study from Tehran, Iran.

BACKGROUND: Recently, Tropheryma whipplei has been suggested as one of the causative agents of diarrhea among children worldwide. Limited data is available on the prevalence of T. whipplei among children with diarrhea in most countries such as Iran. This study was conducted to evaluate the prevalence of T. whipplei in children with acute diarrhea in Iran. METHODS: In this study, the stool samples were collected from 130 children under 10 years old with acute diarrhea from children's hospitals in Tehran city. Genomic DNA was extracted from stool samples and was tested for the presence of DNA of T. whipplei using the SYBR Green Real-time PCR method. Positive T. whipplei samples were finally confirmed by PCR Product sequencing. RESULTS: The mean age of participants was 32.5 months, and 54.6% of children were female. Using the SYBR Green Real-time PCR, 9.23% (12/130) of samples were positive for T. whipplei, which were confirmed by sequencing. 66.67% of positive cases were males. The duration of diarrhea in infected children with T. whipplei (83.3%) was significantly longer (OR: 5.93, 95% CI 1.24-28.22) compared to children with negative results (45.8%). Other demographic factors and clinical signs had not a statistically significant relationship with T. whipplei infection. CONCLUSIONS: In this study, T. whipplei was detected in stool samples of children with acute diarrhea. The results indicated that T. whipplei could be associated with childhood diarrhea in Iran. The health care system and physicians should be aware of the presence of T. whipplei infection in Iran, especially in childhood diarrhea.

Severe Pneumonia Caused by Infection With Tropheryma whipplei Complicated With Acinetobacter baumannii Infection: A Case Report Involving a Young Woman.

Whipple's disease is a very rare systemic infectious disease, and very few cases have been reported. However, it can be fatal if not diagnosed and treated appropriately. The major clinical manifestations of this disease are usually digestive and nervous system symptoms. The majority of patients are male and between 40 and 50 years old. Although respiratory symptoms of this disease have rarely been reported, they pose a serious threat to the lives of the patients, especially when they progress to severe pneumonia. During admission to the hospital, Acinetobacter baumannii infection makes treatment more difficult. While most patients are middle-aged men, more attention should be given to the diagnosis and treatment of affected young women. To our knowledge, the case presented in the study is the first case of Tropheryma whipplei infection that resulted in severe pneumonia and was complicated by A. baumannii infection during treatment. We hope that our study can serve as a reference for the diagnosis and treatment of related cases in the future.

Tropheryma whipplei intestinal colonization in immunocompromised children in Iran: a preliminary study.

Aim & method:Tropheryma whipplei causes Whipple's disease. Children are reservoirs of this bacterium. The aim of this study was to investigate the presence of T. whipplei in children with immunodeficiency in central Iran from July 2018 to February 2019. Stool samples were tested by SYBR Green and Taq-Man real-time PCR assays. For confirmation, the isolated DNA was sequenced. Results: One hundred and thirty children were enrolled. Acute lymphocytic leukemia was the most reported immunodeficient disease (77%), followed by non-Hodgkin lymphoma and retinoblastoma. Thirteen (10%) children had T. whipplei DNA in the stool; 11.4% of the children under 5 years old were positive. Conclusion: This is the first study showing the circulation of T. whipplei in Iran.

Case Report: Tropheryma whipplei Hide in an AIDS Patient With Pneumocystis Pneumonia.

Introduction:Pneumocystis pneumonia (PCP) is one of the most common opportunistic infections in HIV-infected patients. However, coinfection with Tropheryma whipplei is infrequent in AIDS patients with PCP. Case Presentation: We report a 28-year-old male AIDS patient coinfected with T. whipplei and Pneumocystis jirovecii diagnosed in the bronchoalveolar lavage. After sulfamethoxazole-trimethoprim and meropenem treatment, the patient showed clinical improvement in 2 weeks. Conclusion: Clinicians need to be alert to the occurrence of T. whipplei infection in AIDS patients with PCP and timely diagnosis and antibacterial treatments are essential. This case may help clinicians for timely diagnosis of the coinfection of T. whipplei and P. jirovecii in AIDS patients.

Phenotypic diversity of Tropheryma whipplei clinical isolates.

Tropheryma whipplei is a bacterial pathogen responsible for a wide range of infections in humans, covering asymptomatic carriage, acute infections, chronic isolated infections and classic Whipple's disease. Although the bacterium is commonly found in the environment, it very rarely causes disease. Genetic comparison of clinical isolates has revealed that main variations were found in region encoding T. whipplei surface glycoproteins called WiSP. However, no association has been made between the genetic diversity and the clinical manifestations of the infection. In this study we evaluated the phenotypic diversity of 26 clinical isolates from different origins and taken from patient with different infection outcomes. MRC5 and macrophages cells were infected, and bacterial uptake, survival and the pro-and anti-inflammatory potential of the different clinical isolates was assessed. No significant difference of phagocytosis was found between the different isolates; however, we found that bacterial replication was increased for bacteria expressing high molecular weight WiSP. In addition, we found that the expression of the genes coding for IL-1ß and TGF-ß was significantly higher when MRC5 cells were stimulated with isolates from chronic infections compared to isolates from localized infections while no significant differences were observed in macrophages. Overall, our study revealed that, as previously observed at the genetic level, phenotypic diversity of T. whipplei isolates is associated with the expression of different WiSP, which may result in subtle differences in host responses. Other host factors or genetic predisposition may explain the range of clinical manifestations of T. whipplei infections.

Whipple's disease: the great masquerader-a high level of suspicion is the key to diagnosis.

BACKGROUND: Whipple's disease is a chronic infectious disease that primarily affects the small intestine, but several organs can simultaneously be involved. The disease is caused by a gram-positive bacterium called Tropheryma whipplei. The disease is difficult to suspect because it is rare with unspecific and long-term symptoms; it can be lethal if not properly treated. CASE PRESENTATION: We here present three patients who presented with a plethora of symptoms, mainly long-standing seronegative arthritis and gastrointestinal symptoms in the form of diarrhea with blood, weight loss, fever, and lymphadenopathy. They were after extensive investigations diagnosed with Whipple's disease, in two of them as long as 8 years after the first occurrence of joint manifestations. The diagnosis was made by PCR targeting the T. whipplei 16S rRNA gene from small bowel specimen in all three patients, and, besides from histopathologic findings from the duodenum and distal ileum in one and mesenteric lymph nodes in another patient. CONCLUSIONS: This report aims to raise awareness of a very rare disease that presents with a combination of symptoms mimicking other and significantly more common diseases.