Fluorescent in situ hybridization can be used as a complementary assay for the diagnosis of Tropheryma whipplei infection.

BACKGROUND: Immunohistochemistry and Periodic acid-Schiff (PAS) staining have been routinely used for the diagnosis of Whipple's disease (WD). However, these methods present limitations. As a result, the last years, Fluorescence in situ hybridization (FISH) has been increasingly used as a complementary tool for the diagnosis of WD from various tissue samples. CASE REPORT: In this study, we visualized, by FISH, Tropheryma whipplei within macrophages of a lymph node from a patient with WD. Moreover, we report in this study a patient with a pulmonary biopsy compatible with WD by PAS, immunostaining and FISH, although the specific molecular assays for T. whipplei were negative. Sequencing analysis of the 16S rDNA revealed a T. whipplei-related species with unknown classification. CONCLUSION: FISH can be a valuable method for the detection of Tropheryma species in formalin-fixed paraffin-embedded tissues. FISH cannot replace the other already approved diagnostic techniques for WD, it can be used as a complementary tool and can provide supplementary information in a relatively short time.

Novel Tropheryma species in a lung biopsy sample from a kidney transplant recipient.

OBJECTIVES: A kidney transplant recipient with recurrent pleuritis underwent an open lung biopsy, the results of which revealed multiple nodular infiltrates. Grocott and periodic acid-Schiff staining were positive. Fungal and Tropheryma whipplei PCR were, however, negative. Further identification was needed. METHODS: Formalin-fixed, paraffin-embedded (FFPE) extraction was performed using an FFPE extraction kit. T. whipplei was searched for using a real-time PCR targeting the noncoding repeat specific for T. whipplei. Identification of the bacteria in the extract was done using 16S rDNA and 23S rDNA sequencing and BLAST analysis. Internal transcribed spacer PCR was used for fungal DNA identification. RESULTS: The FFPE extract was negative for fungi and T. whipplei. 16S rDNA sequence analysis of a 1375 bp fragment gave T. whipplei as the best match with 26 mismatches, resulting in only 98% agreement. Sequence analysis of the 23S rDNA gene again gave T. whipplei as the best match, but with only 91% agreement. A pan-Tropheryma 16S rDNA real-time PCR was developed, and both the biopsy sample and a respiratory sample of the patient were strongly positive. The patient received antimicrobial treatment targeting T. whipplei with good clinical outcome. CONCLUSIONS: 16S and 23S rDNA sequencing gave T. whipplei as the best hit, although with limited agreement. These findings suggest that a novel Tropheryma species that lacks the noncoding repeat, most frequently used for molecular detection of Whipple disease, might be the cause of the pulmonary disease. Adaptation of current PCR protocols is warranted in order to detect all Tropheryma species.

Longitudinal analysis of the lung microbiota of cynomolgous macaques during long-term SHIV infection.

BACKGROUND: Longitudinal studies of the lung microbiome are challenging due to the invasive nature of sample collection. In addition, studies of the lung microbiome in human disease are usually performed after disease onset, limiting the ability to determine early events in the lung. We used a non-human primate model to assess lung microbiome alterations over time in response to an HIV-like immunosuppression and determined impact of the lung microbiome on development of obstructive lung disease. Cynomolgous macaques were infected with the SIV-HIV chimeric virus SHIV89.6P. Bronchoalveolar lavage fluid samples were collected pre-infection and every 4 weeks for 53 weeks post-infection. The microbiota was characterized at each time point by 16S ribosomal RNA (rRNA) sequencing. RESULTS: We observed individual variation in the composition of the lung microbiota with a proportion of the macaques having Tropheryma whipplei as the dominant organism in their lungs. Bacterial communities varied over time both within and between animals, but there did not appear to be a systematic alteration due to SHIV infection. Development of obstructive lung disease in the SHIV-infected animals was characterized by a relative increase in abundance of oral anaerobes. Network analysis further identified a difference in community composition that accompanied the development of obstructive disease with negative correlations between members of the obstructed and non-obstructed groups. This emphasizes how species shifts can impact multiple other species, potentially resulting in disease. CONCLUSIONS: This study is the first to investigate the dynamics of the lung microbiota over time and in response to immunosuppression in a non-human primate model. The persistence of oral bacteria in the lung and their association with obstruction suggest a potential role in pathogenesis. The lung microbiome in the non-human primate is a valuable tool for examining the impact of the lung microbiome in human health and disease.

Sewage workers with low antibody responses may be colonized successively by several Tropheryma whipplei strains.

OBJECTIVES: Asymptomatic faecal carriage of Tropheryma whipplei, the agent of Whipple's disease, is reported among sewage workers. However, the potential development of such carriage is unknown. A 7-year follow-up of T. whipplei-carrying sewage workers is reported. METHODS: Nineteen sewage workers previously detected as faecal carriers of T. whipplei were followed to ascertain the chronicity of their carriage. Faeces were tested by molecular assays using quantitative real-time PCR specifically targeting T. whipplei. Serological anti-T. whipplei Western blotting was also performed. RESULTS: Seventy-nine percent (15/19) of workers exhibited a strong immune response against T. whipplei. Among these, five were followed for more than 1 year. Four maintained a strong response, with three carrying the same strain and one becoming negative. The fifth exhibited a decreased immune response, a negative faeces result, and subsequent carriage of another strain. Three individuals with low immune responses were also followed. Two never developed a response, with one carrying the same strain and one becoming negative and then positive with another strain; the third developed a strong response and became negative. CONCLUSIONS: Chronic T. whipplei carriers appear to be protected against reinfection, but those with low or decreasing antibody levels may be re-colonized by another strain.

High prevalence of Tropheryma whipplei in Lao kindergarten children.

BACKGROUND: Tropheryma whipplei is a bacterium commonly found in feces of young children in Africa, but with no data from Asia. We estimated the prevalence of T. whipplei carriage in feces of children in Lao PDR (Laos). METHODS/PRINCIPAL FINDINGS: Using specific quantitative real-time PCR, followed by genotyping for each positive specimen, we estimated the prevalence of T. whipplei in 113 feces from 106 children in Vientiane, the Lao PDR (Laos). T. whipplei was detected in 48% (51/106) of children. Those aged ≤ 4 years were significantly less frequently positive (17/52, 33%) than older children (34/54, 63%; p< 0.001). Positive samples were genotyped. Eight genotypes were detected including 7 specific to Laos. Genotype 2, previously detected in Europe, was circulating (21% of positive children) in 2 kindergartens (Chompet and Akad). Genotypes 136 and 138 were specific to Chompet (21% and 15.8%, respectively) whereas genotype 139 was specific to Akad (10.55%). CONCLUSIONS/SIGNIFICANCE: T. whipplei is a widely distributed bacterium, highly prevalent in feces of healthy children in Laos. Further research is needed to identify the public health significance of this finding.

Tropheryma whipplei as a commensal bacterium.

Tropheryma whipplei is the bacterial agent of the well-known and rare Whipple's disease, mainly observed among Caucasians. This bacterium has recently been involved in other chronic and acute infections. For a long time, the only known source of the bacterium was patients with Whipple's disease; however, thanks to the advent of molecular biology, T. whipplei has now been detected in specimens from healthy individuals, mainly in stool and saliva samples. The prevalence of carriage depends on several factors, such as age, exposure and geographical area, reaching 75% in stool specimens from children less than 4 years old in rural Africa. T. whipplei is a commensal bacterium that only causes Whipple's disease in a subset of individuals, probably those with a still-uncharacterized specific immunological defect.

Endocarditis due to Tropheryma whipplei: rapid detection, limited genetic diversity, and long-term clinical outcome in a local experience.

The characteristic features of Whipple's disease include abdominal pain, diarrhoea, wasting, and arthralgias, with the causative agent, Tropheryma whipplei, being detected mainly in intestinal biopsies. PCR technology has led to the identification of T. whipplei in specimens from various other locations, including the central nervous system and the heart. T. whipplei is now recognized as one of the causes of culture-negative endocarditis, and endocarditis can be the only manifestation of the infection with T. whipplei. Although it is considered a rare disease, the true incidence of endocarditis due to T. whipplei is not clearly established. With the increasing use of molecular methods, it is likely that T. whipplei will be more frequently identified. Questions also remain about the genetic variability of T. whipplei strains, optimal diagnostic procedures and therapeutic options. In the present study, we provide clinical data on four new patients with documented endocarditis due to T. whipplei in the context of the available published literature. There was no clinical involvement of the gastrointestinal tract. Genetic analysis of the T. whipplei strains with DNA isolated from the excised heart valves revealed little to no genetic variability. In a selected case, we describe acridine orange staining for early detection of the disease, prompting early adaptation of the antibiotic therapy. We provide long-term follow-up data on the patients. In our hands, an initial 2-week course of intravenous antibiotics followed by cotrimoxazole for at least 1 year was a suitable treatment option for T. whipplei endocarditis.

Multisegmental spondylitis due to Tropheryma whipplei: case report.

We report a patient who presented with inflammatory back pain due to multisegmental spondylitis. Following a vertebral biopsy which failed to detect an infectious organism, the patient was treated with etanercept, a tumor necrosis factor (TNF)-alpha inhibitor, for suspected undifferentiated spondyloarthritis. The back pain worsened and the spondylitic lesions increased. Only in a vertebral rebiopsy with polymerase chain reaction (PCR) amplification of Tropheryma whipplei, the causative agent of Whipple's disease was identified. Tropheryma whipplei should be considered as a cause of spondylitis even with multisegmental involvement and in the absence of gastrointestinal symptoms. In this clinical setting, routine PCR for Tropheryma whipplei from vertebral biopsies is recommended.

Tropheryma whipplei in fecal samples from children, Senegal.

We tested fecal samples from 150 healthy children 2-10 years of age who lived in rural Senegal and found the prevalence of Tropheryma whipplei was 44%. Unique genotypes were associated with this bacterium. Our findings suggest that T. whipplei is emerging as a highly prevalent pathogen in sub-Saharan Africa.

Prosthetic hip infection caused by Tropheryma whipplei.

We report a case of prosthetic hip infection due to Tropheryma whipplei in a 74-year-old man not previously known to have Whipple's disease. Diagnosis was based on systematic 16S rRNA gene amplification and sequencing of samples obtained during revision hip arthroplasty.

Genotyping reveals a wide heterogeneity of Tropheryma whipplei.

Tropheryma whipplei, the causative agent of Whipple's disease, is associated with various clinical manifestations as well as an asymptomatic carrier status, and it exhibits genetic heterogeneity. However, relationships that may exist between environmental and clinical strains are unknown. Herein, we developed an efficient genotyping system based on four highly variable genomic sequences (HVGSs) selected on the basis of genome comparison. We analysed 39 samples from 39 patients with Whipple's disease and 10 samples from 10 asymptomatic carriers. Twenty-six classic gastrointestinal Whipple's disease associated with additional manifestations, six relapses of classic Whipple's disease (three gastrointestinal and three neurological relapses), and seven isolated infections due to T. whipplei without digestive involvement (five endocarditis, one spondylodiscitis and one neurological infection) were included in the study. We identified 24 HVGS genotypes among 39 T. whipplei DNA samples from the patients and 10 T. whipplei DNA samples from the asymptomatic carriers. No significant correlation between HVGS genotypes and clinical manifestations of Whipple's disease, or asymptomatic carriers, was found for the 49 samples tested. Our observations revealed a high genetic diversity of T. whipplei strains that is apparently independent of geographical distribution and unrelated to bacterial pathogenicity. Genotyping in Whipple's disease may, however, be useful in epidemiological studies.

Comparative genomic analysis of Tropheryma whipplei strains reveals that diversity among clinical isolates is mainly related to the WiSP proteins.

BACKGROUND: The aim of this study was to analyze the genomic diversity of several Tropheryma whipplei strains by microarray-based comparative genomic hybridization. Fifteen clinical isolates originating from biopsy samples recovered from different countries were compared with the T. whipplei Twist strain. For each isolate, the genes were defined as either present or absent/divergent using the GACK analysis software. Genomic changes were then further characterized by PCR and sequencing. RESULTS: The results revealed a limited genetic variation among the T. whipplei isolates, with at most 2.24% of the probes exhibiting differential hybridization against the Twist strain. The main variation was found in genes encoding the WiSP membrane protein family. This work also demonstrated a 19.2 kb-pair deletion within the T. whipplei DIG15 strain. This deletion occurs in the same region as the previously described large genomic rearrangement between Twist and TW08/27. Thus, this can be considered as a major hot-spot for intra-specific T. whipplei differentiation. Analysis of this deleted region confirmed the role of WND domains in generating T. whipplei diversity. CONCLUSION: This work provides the first comprehensive genomic comparison of several T. whipplei isolates. It reveals that clinical isolates originating from various geographic and biological sources exhibit a high conservation rate, indicating that T. whipplei rarely interacts with exogenous DNA. Remarkably, frequent inter-strain variations were dicovered that affected members of the WiSP family.