Martin Altwegg

Whipple's Disease and “Tropheryma whippelii”

SUMMARY Whipples disease is a rare bacterial infection that may involve any organ system in the body. It occurs primarily in Caucasian males older than 40 years. The gastrointestinal tract is the most frequently involved organ, with manifestations such as abdominal pain, malabsorption syndrome with diarrhea, and weight loss. Other signs include low-grade fever, lymphadenopathy, skin hyperpigmentation, endocarditis, pleuritis, seronegative arthritis, uveitis, spondylodiscitis, and neurological manifestations, and these signs may occur in the absence of gastrointestinal manifestations. Due to the wide variability of manifestations, clinical diagnosis is very difficult and is often made only years or even decades after the initial symptoms have appeared. Trimethoprim-sulfamethoxazole for at least 1 year is usually considered adequate to eradicate the infection. The microbiological diagnosis of this insidious disease is rendered difficult by the virtual lack of culture and serodiagnostic methods. It is usually based on the demonstration of periodic acid-Schiff-positive particles in infected tissues and/or the presence of bacteria with an unusual trilaminar cell wall ultrastructure by electron microscopy. Recently, the Whipple bacteria have been characterized at the molecular level by amplification of their 16S rRNA gene(s). Phylogenetic analysis of these sequences revealed a new bacterial species related to the actinomycete branch which was named “Tropheryma whippelli.” Based on its unique 16S ribosomal DNA (rDNA) sequence, species-specific primers were selected for the detection of the organism in clinical specimens by PCR. This technique is currently used as one of the standard methods for establishing the diagnosis of Whipples disease. Specific and broad-spectrum PCR amplifications mainly but not exclusively from extraintestinal specimens have significantly improved diagnosis, being more sensitive than histopathologic analysis. However, “T. whippelii” DNA has also been found in persons without clinical and histological evidence of Whipples disease. It is unclear whether these patients are true asymptomatic carriers or whether differences in virulence exist among strains of “T. whippelii” that might account for the variable clinical manifestations. So far, six different “T. whippelii” subtypes have been found by analysis of their 16S-23S rDNA spacer region. Further studies of the pathogen “T. whippelii” as well as the host immune response are needed to fully understand this fascinating disease. The recent cultivation of the organisms is a promising major step in this direction.

Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.

Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.

Etiologic Diagnosis of Infective Endocarditis by Broad-Range Polymerase Chain Reaction: A 3-Year Experience

We analyzed surgically resected endocardial specimens from 49 patients by broad-range PCR. PCR results were compared with (1) results of previous blood cultures, (2) results of culture and Gram staining of resected specimens, and (3) clinical data (Duke criteria). Molecular analyses of resected specimens and previous blood cultures showed good overall agreement. However, in 18% of patients with sterile blood cultures, bacterial DNA was found in the resected materials. When data from patients with definite or rejected cases of infective endocarditis (IE) were included, the sensitivity, specificity, and positive and negative predictive values of broad-range PCR were 82.6%, 100%, 100%, and 76.5%, respectively, overall, and 94.1%, 100%, 100%, and 90%, for cases of native valve endocarditis. The sensitivity, specificity, and positive and negative predictive values of culture of resected specimens from patients with native valve endocarditis were 17.6%, 88.9%, 75%, and 36.4%. We recommend broad-range PCR of surgically resected endocardial material in cases of possible IE, in cases of suspected IE in which blood cultures are sterile, and in cases in which organisms grow in blood cultures but only Duke minor criteria are met. We propose to add molecular techniques to the pathologic criteria of the Duke classification scheme.

Deactivation of Macrophages with Interleukin-4 Is the Key to the Isolation of Tropheryma whippelii

Whipples disease is a systemic illness caused by a specific agent. Despite recognition of bacteria in lesions, efforts to isolate the causative agent remained futile. A novel strategy was devised to culture Whipple bacilli in deactivated mononuclear phagocytes. Infected tissue was inoculated into human phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone. Within 8-10 days, diastase-resistant periodic acid-Schiff-positive inclusions appeared, corresponding to intact and degenerating bacteria shown to be Tropheryma whippelii by electron microscopy and molecular analyses. T. whippelii was passaged several times in deactivated monocytes and a monoblastic cell line. Time-kinetics growth studies and comparative polymerase chain reaction analysis documented multiplication of T. whippelii in deactivated macrophages. Complementary studies showed that IL-4 rendered phagocytes permissive for T. whippelii, a strong indication that host factors contribute to the pathogenesis of Whipples disease. The propagation of T. whippelii will permit microbiologic, immunologic, seroepidemiologic, and therapeutic studies of this pathogen.

Ribosomal DNA Sequencing for Identification of Aerobic Gram-Positive Rods in the Clinical Laboratory (an 18-Month Evaluation)

ABSTRACT We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.

Quantitative Detection of Streptococcus pneumoniae in Nasopharyngeal Secretions by Real-Time PCR

ABSTRACT Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 106 cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient ofS. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae,Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were ≥106 cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.

Internal Transcribed Spacer Sequencing versus Biochemical Profiling for Identification of Medically Important Yeasts

ABSTRACT In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.

Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR.

M. pneumoniae is a common causative agent of community-acquired pneumonia in children. The diagnosis of such infections is usually based on serology using complement fixation or, more recently, enzyme-immuno assays. PCR has been shown to be a promising alternative. We have evaluated a real-time PCR assay targeting the P1 adhesion protein gene and compared it to a conventional semi-nested PCR assay with the 16S rDNA as target. Comparison of 147 specimens from 48 patients showed an overall agreement of 97.4%. Real-time PCR proved to be of equal value on clinical specimens as conventional PCR regarding sensitivity and specificity, but is clearly advantageous regarding speed, handling and number of samples that can be analyzed per run.

Whipple Endocarditis without Overt Gastrointestinal Disease: Report of Four Cases

Whipple disease is a systemic illness caused by the bacterium Tropheryma whippelii. The disorder mainly affects middle-aged white men and is characterized by diarrhea, weight loss, and migratory polyarthritis (1). Clinical diagnosis is confirmed by histologic detection in the intestinal mucosa of foamy macrophages loaded with inclusions that test positive on periodic acid-Schiff (PAS) staining (2). Cardiac manifestations with pericarditis, endocarditis, and myocarditis have been well described, mainly in postmortem studies (3). Endocarditis diagnosed before death has rarely been reported and has always been associated with classic gastrointestinal Whipple disease (4-7). We observed four patients with endocarditis caused by T. whippelii who needed valve replacement and had no histologic evidence of gastrointestinal involvement. Methods Data were collected on patients undergoing valve replacement who were examined for T. whippelii by polymerase chain reaction (PCR) because of clinical or histologic suspicion of Whipple disease (patients 1, 3, and 4) or as part of an ongoing study of broad-range PCR in culture-negative endocarditis (patient 2) (8). Four of the 21 valves examined by broad-range specific PCR tested positive for T. whippelii. Medical records were reviewed for the four identified patients by contacting their primary physicians when necessary. Heart valves were examined histologically, cultured in brain-heart infusion broth for 28 days, and processed for PCR. All patients had gastroduodenal endoscopy; deep biopsy of mucosa was done on the distal part of the duodenum. Biopsy specimens were examined by light microscopy, including special stains for PAS-positive inclusions. Broad-range and T. whippelii-specific PCR on material from the excised heart valves and specific PCR on material from the gastrointestinal biopsies were done as previously described (8-10). Results Case Reports Patient 1 A 64-year-old sales representative was hospitalized because of subacute severe aortic valve insufficiency. He had a 20-year history of severe refractory seronegative polyarthritis that involved the ankles, knees, hips, and wrists. He reported no weight loss, fever, or night sweats. His bowel movements included up to six loose stools per day. The patient had a prosthetic aortic valve replacement, and recovery was uneventful. Histologic examination revealed granulocytic endocarditis with few PAS-positive foamy cells and abundant bacteria seen in silver stains (Figure). Polymerase chain reaction on the valve was positive for T. whippelii. The patient was treated with penicillin and gentamicin for 6 weeks; cotrimoxazole was added and was given for 2 years. Bowel movements returned to normal, and arthritis symptoms disappeared within 2 weeks. The previously disabled patient was now able to go hiking. No signs of disease were observed after 36 months of follow-up. Figure. Microscopy of aortic heart valve with Tropheryma whippelii -endocarditis. A. B. C. D. Patient 2 A 53-year-old male postal worker was hospitalized for evaluation of progressive heart failure. For years, he had had transient arthralgia of the wrists and hips that responded promptly to nonsteroidal anti-inflammatory drugs. He reported no further symptoms other than intermittent night sweats. Transesophageal echocardiography showed a destroyed tricuspid aortic valve with characteristic vegetations and an annular abscess. Histologic examination of the excised valve revealed characteristics compatible with bacterial endocarditis. Broad-range PCR showed T. whippelii; this result was confirmed by specific PCR. Duodenal biopsy findings were microscopically normal, but PCR revealed T. whippelii DNA. The patient received a 1-year course of cotrimoxazole, which resulted in disappearance of his joint symptoms. No disease recurrence was noted during 32 months of follow-up. Patient 3 A 55-year-old politician was admitted to the hospital for recent onset of dyspnea and a new diastolic heart murmur. Eight months earlier, a work-up for chronic fatigue and knee, shoulder, and hand arthralgia led to the diagnosis of a myelodysplastic syndrome with normocytic anemia. The patient had lost 3 kg within the previous 2 months but reported no other symptoms. Transesophageal echocardiography revealed gross vegetations on the aortic valve and severe aortic insufficiency. The excised valve showed ulcerofibrinous endocarditis and a few gram-positive coccoid bacteria; abundant rods were seen on silver stain. Polymerase chain reaction of the valve tissue was positive for T. whippelii. The patients postoperative course was uneventful. He is currently being treated with oral cotrimoxazole. Patient 4 A 55-year-old female housekeeper had been followed for 3 years for recurrent embolic disease of the brain and an elevated erythrocyte sedimentation rate. Transthoracic and transesophageal echocardiography had repeatedly shown vegetations, but extensive studies showed no proof of infective endocarditis. She did not receive antibiotic treatment and showed no signs of progressive valvular disease. She had reported arthralgia for 2 years but had no gastrointestinal symptoms. Despite oral anticoagulation, another embolic event led to valve replacement. Microscopy showed granulocytic endocarditis with some gram-variable rods, few PAS-positive inclusions, and abundant rod-shaped bacteria in silver stains. Polymerase chain reaction identified T. whippelii in the valve. The patient received a 6-week course of intravenous ceftriaxone and is currently receiving cotrimoxazole. The arthralgia disappeared within days after antibiotic therapy was instituted. Clinical and Laboratory Findings Clinical and laboratory findings of all patients are summarized in the Table. Three patients presented with symptoms of progressive heart failure, and all had prolonged arthralgia. None reported gastrointestinal symptoms, but one patient reported up to six loose stools per day. Extensive work-up for infective endocarditis, which included many blood cultures and serologic studies, was negative in all patients, as was conventional culture of the excised valves. Histologic examination of duodenal mucosa was always normal, and PCR showed evidence of DNA for T. whippelii only in the intestinal tissue of patient 2. Laboratory values were notable only for mild anemia in all patients and elevated erythrocyte sedimentation rate in three patients. The valves of patients 1 and 2 provided material for the first successful propagation of T. whippelii in culture (10). Table. Clinical and Laboratory Characteristics in Four Patients with Tropheryma whippelii Endocarditis Discussion We describe four patients with T. whippelii endocarditis observed during a 20-month period. All patients had arthritis or arthralgia as the predominant extracardiac symptoms, but overt gastrointestinal disease was absent (with the exception of loose stools in one patient). Diagnosis was established by typical histologic examination of the excised heart valves and demonstration of genomic DNA of T. whippelii by broad-range or specific PCR. Histologic examination of gastrointestinal mucosa showed no changes typical of Whipple disease in any patient. In addition, PCR on intestinal biopsy, which is known to be more sensitive than histologic examination (9, 11), was positive for T. whippelii in only one of the four patients. Whipple disease is a rare, predominantly gastrointestinal illness characterized by severe diarrhea, malabsorption with consecutive weight loss, and polyarthralgia (1). In 1992, the suspected bacterial origin of the disease was confirmed when Relman and coworkers (12) found DNA corresponding to a bacterium later named Tropheryma whippelii. The bacterium could not be cultured until the organism was propagated in macrophages inactivated by cytokines (10). Cardiac involvement in Whipple disease is well established (3, 7, 13, 14). Postmortem studies have shown cardiac findings in more than half of the patients who die of the disease (3). We were able to find only a few case reports in which T. whippelii endocarditis was diagnosed before death (4-7), and all of these reports involved men with classic intestinal manifestation of Whipple disease. Whipple disease without overt involvement of the gastrointestinal tract has been described earlier (15), notably in cases affecting the central nervous system (16), eyes (17), or bone (9). The observation of four cases of endocarditis without overt gastrointestinal disease, however, is unique, as is the occurrence of this rare disease during a short period in a limited geographic area of Switzerland. Increased interest in and search for the organism, as well as frequent application of broad-range PCR in patients with endocarditis at our institutions, could be one explanation. However, because histologic findings on the valves are suggestive and the applied molecular methods are not limited to our institutions, the possibility of a greater incidence of this disease in eastern Switzerland cannot be dismissed. In a prospective blinded study by Ehrbar and colleagues (18) of PCR for T. whippelii in gastrointestinal biopsy samples of 105 patients without any sign of Whipple disease, positive results were found in 11.4% of gastric juice aspirates and 4.8% of duodenal biopsy specimens. Tropheryma whippelii-specific DNA was also detected in 25 of 38 samples of waste water in southern Germany (19). These findings suggest that T. whippelii may be a common environmental agent in certain geographic areas. Our observations add T. whippelii to the list of agents that cause culture-negative endocarditis (20). Before prospectively collected data are available, the relative frequency of this disorder cannot be estimated. Clinicians must be aware that Whipple endocarditis can occur even without clinical, histologic, or microbiological evidence of gastrointestinal disease. Because specific diagnostic tools (for example, serologic

Cellular Fatty Acid Composition as a Chemotaxonomic Marker for the Differentiation of Phenospecies and Hybridization Groups in the Genus Aeromonas

Ninety genotypically characterized Aeromonas strains, including members of all 14 currently established genospecies, were studied by performing gas-liquid chromatographic analysis of their cellular fatty acid methyl esters (FAMEs). A total of 44 fatty acids and two alcohols were found in members of the genus Aeromonas. All 90 strains contained 12:0, 13:0 iso, 14:0, 15:0 iso 3OH, 16:0, 16:1 ω7c, 17:0 iso, iso 17:1 ω9c, summed feature 3 (16:1 iso I and/or 14:0 3OH), and summed feature 7 (18:1 ω7c, 18:1 ω9t, and/or 18:1 ω12t), whereas all but one strain (99%) also contained 15:0 iso. Although the FAME profiles were very similar, minor quantitative variations could be used to differentiate phenospecies and/or hybridization groups. A cluster analysis of the mean data revealed five FAME clusters, which were compared with phenotypic and genotypic groups identified in the genus Aeromonas. Hybridization groups that constituted the Aeromonas hydrophila complex, the Aeromonas caviae complex, and the Aeromonas sobria complex were basically grouped into distinct FAME clusters. The taxonomic positions of hybridization groups 7 and 11 in these clusters, however, remained unclear. All of our results were highly reproducible. A new database of Aeromonas FAME fingerprints was generated, and this database can be used for rapid identification of unknown aeromonads. Using a large set of well-characterized aeromonads, we demonstrated for the first time that gas-liquid chromatographic FAME analysis can be used to differentiate the majority of the phenospecies and/or hybridization groups in the genus Aeromonas.