[show abstract][hide abstract] ABSTRACT: Tropheryma whipplei, which causes Whipple disease, is found in human feces and may cause gastroenteritis. To show that T. whipplei causes gastroenteritis, PCRs for T. whipplei were conducted with feces from children 2-4 years of age. Western blotting was performed for samples from children with diarrhea who had positive or negative results for T. whipplei. T. whipplei was found in samples from 36 (15%) of 241 children with gastroenteritis and associated with other diarrheal pathogens in 13 (33%) of 36. No positive specimen was detected for controls of the same age (0/47; p = 0.008). Bacterial loads in case-patients were as high as those in patients with Whipple disease and significantly higher than those in adult asymptomatic carriers (p = 0.002). High incidence in patients and evidence of clonal circulation suggests that some cases of gastroenteritis are caused or exacerbated by T. whipplei, which may be co-transmitted with other intestinal pathogens.
[show abstract][hide abstract] ABSTRACT: Tropheryma whipplei is the etiologic pathogenic agent of Whipple disease (WD), characterized by various clinical signs, such as diarrhea, weight loss, lymphadenopathy, and polyarthritis. PCR-based methods for diagnosis of WD have been developed. T. whipplei has been identified in saliva and stool samples from patients with WD and from healthy persons. T. whipplei DNA has also been found in bronchoalveolar lavage (BAL) samples of a child with pneumonia. We detected DNA of T. whipplei in 6 (3%) of 210 BAL samples collected in intensive care units by using 16S rDNA and specific quantitative PCR. We identified 4 novel genotypes of T. whipplei. In 1 case, T. whipplei was the only bacterium; in 4 others, it was associated with buccal flora. We suggest that T. whipplei should be investigated as an etiologic agent of pneumonia.
[show abstract][hide abstract] ABSTRACT: Q fever is a worldwide zoonotic disease caused by Coxiella burnetii. Epidemiologically, animals are considered reservoirs and humans incidental hosts.
We investigated Q fever in rural Senegal. Human samples (e.g., sera, saliva, breast milk, feces) were screened in the generally healthy population of two villages of the Sine-Saloum region. Ticks were collected in four regions. Seroprevalence was studied by immunofluorescence, and all other samples were tested by two qPCR systems for detection of C. burnetii. Positive samples were genotyped (multispacer typing) by amplification and sequencing of three spacers. Strains were isolated by cell culture. We found that the seroprevalence may be as high as 24.5% (59 of 238 studied) in Dielmo village. We identified spontaneous excretion of C. burnetii by humans through faeces and milk. Hard and soft ticks (8 species) were infected in 0-37.6%. We identified three genotypes of C. burnetii. The previously identified genotype 6 was the most common in ticks in all studied regions and the only one found in human samples. Three strains of genotype 6 of C. burnetii were also recovered from soft tick Ornithodoros sonrai. Two other genotypes found in ticks, 35 and 36, were identified for the first time.
Q fever should be considered a significant public health threat in Senegal. Humans, similar to other mammals, may continuously excrete C. burnetii.
[show abstract][hide abstract] ABSTRACT: To estimate the relationship between vaginal quantification of the main microorganisms related with bacterial vaginosis and the risk of preterm delivery among women with preterm labor.
Molecular methods were used to prospectively quantify Lactobacillus species, Gardnerella vaginalis, Atopobium vaginae, and Mycoplasma hominis in vaginal fluid samples from women admitted for spontaneous preterm labor with intact membranes from July 2007 through July 2008. The primary outcome measure was the relationship between bacterial concentration at admission and preterm delivery, before 37 weeks of gestation. Sensitivity and specificity of molecular cutoff values and 95% confidence intervals (CIs) were calculated using the University of British Columbia Bayesian Calculator type 2.
Of the 90 women included, 36 delivered before 37 weeks of gestation (40%). Preterm delivery was not associated with the presence of Lactobacillus species, G vaginalis, A vaginae, or M hominis. In contrast, molecular quantification detected high concentrations of A vaginae (10(6)/mL or more: 25.0% in the preterm group and 9.3% in the term group, P=.04) and G vaginalis (10(7)/mL or more: 16.7% and 3.7%, P=.03) more often in women with preterm deliveries compared with term deliveries. Moreover, high vaginal concentrations of these two microorganisms together were associated with a significantly (P=.03) shorter interval between preterm labor and delivery (46 days, 95% CI 30-61) than were lower concentrations (85 days, 95% CI 75-95). The hazard ratio for a short preterm labor-to-delivery interval was three times higher for high vaginal fluid concentrations of A vaginae and G vaginalis than for lower concentrations (hazard ratio 3.3, 95% CI 1.1-9.5, P=.03).
The risk of preterm delivery is significantly associated with high vaginal concentrations of A vaginae and G vaginalis in women with preterm labor.
ClinicalTrials.gov, www.clinicaltrials.gov, NCT00484653.
Obstetrics and Gynecology 01/2010; 115(1):134-40. · 4.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rickettsioses are one of the most important causes of systemic febrile illness among travelers from developed countries, but little is known about their incidence in indigenous populations, especially in West Africa.
Overall seroprevalence evaluated by immunofluorescence using six rickettsial antigens (spotted fever and typhus group) in rural populations of two villages of the Sine-Saloum region of Senegal was found to be 21.4% and 51% for spotted fever group rickettsiae for Dielmo and Ndiop villages, respectively. We investigated the role of tick-borne rickettsiae as the cause of acute non-malarial febrile diseases in the same villages. The incidence of rickettsial DNA in 204 blood samples from 134 (62M and 72F) febrile patients negative for malaria was studied. DNA extracted from whole blood was tested by two qPCR systems. Rickettsial DNA was found in nine patients, eight with Rickettsia felis (separately reported). For the first time in West Africa, Rickettsia conorii was diagnosed in one patient. We also tested 2,767 Ixodid ticks collected in two regions of Senegal (Niakhar and Sine-Saloum) from domestic animals (cows, sheep, goats, donkeys and horses) by qPCR and identified five different pathogenic rickettsiae. We found the following: Rickettsia aeschlimannii in Hyalomma marginatum rufipes (51.3% and 44.8% in Niakhar and Sine-Saloum region, respectively), in Hyalomma truncatum (6% and 6.8%) and in Rhipicephalus evertsi evertsi (0.5%, only in Niakhar); R. c. conorii in Rh. e. evertsi (0.4%, only in Sine-Saloum); Rickettsia massiliae in Rhipicephalus guilhoni (22.4%, only in Niakhar); Rickettsia sibirica mongolitimonae in Hyalomma truncatum (13.5%, only in Sine-Saloum); and Rickettsia africae in Rhipicephalus evertsi evertsi (0.7% and 0.4% in Niakhar and Sine-Saloum region, respectively) as well as in Rhipicephalus annulatus (20%, only in Sine-Saloum). We isolated two rickettsial strains from H. truncatum: R. s. mongolitimonae and R. aeschlimannii.
We believe that together with our previous data on the high prevalence of R. africae in Amblyomma ticks and R. felis infection in patients, the presented results on the distribution of pathogenic rickettsiae in ticks and the first R. conorii case in West Africa show that the rural population of Senegal is at risk for other tick-borne rickettsioses, which are significant causes of febrile disease in this area.
[show abstract][hide abstract] ABSTRACT: In this study, we describe 13 patients with prosthetic infections due to Finegoldia magna (2% of our tested series). Patients presented with either polymicrobial infection after an open fracture or nosocomial infection after recent prosthesis implantation. Molecular techniques are critical for diagnosis, and recommended antibiotic prophylaxis has poor activity against F. magna.
[show abstract][hide abstract] ABSTRACT: A patient with classic Whipple's disease developed erythema nodosum leprosum-like lesions and fever one month after the beginning of an accurate therapy with trimethoprim-sulfamethoxazole. Immune reconstitution inflammatory syndrome was suspected, but corticosteroid therapy failed to improve the patient. Finally, thalidomide was used and successfully induced rapid improvement.
The Journal of infection 10/2009; 60(1):79-82. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tropheryma whipplei is a bacterium that causes Whipple disease. However, T. whipplei can be carried in the gut of asymptomatic people, which may lead to difficulty in the interpretation of positive stool sample test results.
This study included 60 patients with classic Whipple disease at the time of diagnosis and 26 T. whipplei carriers. Western blots testing for total immunoglobulin (Ig), IgG, IgM, and IgA were performed using glycosylated and deglycosylated T. whipplei. A blind test involving 10 patients and 10 carriers was performed. Sera samples from 32 treated patients were tested for total immunoglobulin.
Total immunoglobulin from patients with classic Whipple disease exhibited either a lack of reaction (23 [38%] of 60 patients) or a decrease in reaction (33 [55%] of 60 patients) with a T. whipplei glycoprotein of 110 kDa after deglycosylation. Only 4 patients exhibited a stronger immune response than that which was observed for carriers (21 [81%] of 26 carriers). Five carriers presented a response profile similar to that for the patients. IgM (4 [7%] of 60 patients) or IgA (1 [2%] of 60 patients) responses were rarely observed but were exclusive to patients. Overall, results were consistent and reproducible. Antibiotic therapy had no effect on the serological profiles of the patients.
Western blot serology is useful to distinguish between carriers and patients; paradoxical responses of the antibodies were investigated.
[show abstract][hide abstract] ABSTRACT: Whipple's disease is a chronic multisystemic infection caused by Tropheryma whipplei. Host factors likely predispose for the establishment of an infection, and macrophages seem to be involved in the pathogenesis of Whipple's disease. However, macrophage activation in Whipple's disease has not been studied systematically so far.
Samples from 145 Whipple's disease patients and 166 control subjects were investigated. We characterized duodenal macrophages and lymphocytes immunohistochemically and peripheral monocytes by flow cytometry and quantified mucosal and systemic cytokines and chemokines indicative for macrophage activation. In addition, we determined duodenal nitrite production and oxidative burst induced by T whipplei and by other bacteria.
Reduced numbers of duodenal lymphocytes, increased numbers of CD163(+) and stabilin-1(+), reduced numbers of inducible nitric synthase+ duodenal macrophages, and increased percentages of CD163(+) peripheral monocytes indicated a lack of inflammation and a M2/alternatively activated macrophage phenotype in Whipple's disease. Incubation with T whipplei in vitro enhanced the expression of CD163 on monocytes from Whipple's disease patients but not from control subjects. Chemokines and cytokines associated with M2/alternative macrophage activation were elevated in the duodenum and the peripheral blood from Whipple's disease patients. Functionally, Whipple's disease patients showed a reduced duodenal nitrite production and reduced oxidative burst upon incubation with T whipplei compared with healthy subjects.
The lack of excessive local inflammation and alternative activation of macrophages, triggered in part by the agent T whipplei itself, may explain the hallmark of Whipple's disease: invasion of the intestinal mucosa with macrophages incompetent to degrade T whipplei.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Whipple's disease (WD) is a chronic infection caused by Tropheryma whipplei. A 1-year treatment of oral trimethoprim/sulfamethoxazole (SXT) is commonly used. Advances in the culture of T. whipplei have allowed for full genome sequencing and antibiotic susceptibility testing, which has demonstrated resistance of T. whipplei to trimethoprim. Several mutations in the folP gene that encodes dihydropteroate synthase, the target of sulphonamides, has been reported for one patient with clinically acquired resistance to SXT. Here we report three new patients who experienced clinically acquired resistance to SXT during treatment and one patient with biological failure. Sixty-two folP sequences from DNA samples of 59 WD patients were also obtained. Among the detected amino acid changes, two positions (N4S and S234F) significantly predicted secondary sulfamethoxazole failure (four of five). We suggest that these mutations should be detected at the time of WD diagnosis by sequencing folP in order to avoid sulfamethoxazole monotherapy.
International journal of antimicrobial agents 05/2009; 34(3):255-9. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The proteome of Tropheryma whipplei, the intracellular bacterium responsible for Whipple's disease (WD), was analyzed using two complementary approaches: 2-DE coupled with MALDI-TOF and SDS-PAGE with nanoLC-MS/MS. This strategy led to the identification of 206 proteins of 808 predicted ORFs, resolving some questions raised by the genomic sequence of this bacterium. We successfully identified antibiotic targets and proteins with predicted N-terminal signal sequences. Additionally, we identified a family of surface proteins (known as T. whipplei surface proteins (WiSPs)), which are encoded by a unique group of species-specific genes and serve as both coding regions and DNA repeats that promote genomic recombination. Comparison of the protein expression profiles of the intracellular facultative host-associated WD bacterium with other host-associated, intracellular obligate, and environmental bacteria revealed that T. whipplei shares a proteomic expression profile with other host-associated facultative intracellular bacteria. In summary, this study describes the global protein expression pattern of T. whipplei and reveals some specific features of the T. whipplei proteome.
[show abstract][hide abstract] ABSTRACT: Tropheryma whipplei is a bacterium commonly found in people with Whipple's disease, a rare systemic chronic infection. In the present study, we hypothesized that bacterium glycosylation may impair the immune response.
Bacterial extracts were analyzed by glycostaining, and reactive proteins, identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectometry, were purified to generate antibodies that could be used in immunofluorescence studies. The reactivity of serum samples obtained from patients and asymptomatic carriers was tested against native or deglycosylated bacteria, for which the fate in macrophages was also investigated.
To our knowledge, we evidenced, for the first time in T. whipplei, a 110-kDa glycoprotein containing sialic acid. This protein, identified as an Wnt1-inducible signaling pathway (WiSP) protein, is associated with periodic acid-Schiff (PAS) staining in infected intramacrophage biofilm. Consistent with the lack of enzymes required for the glycosylation pathway in this bacterium, the glycoproteins disappear during in vitro axenic subcultures, whereas human transcriptome analysis reveals the up-regulation of corresponding genes within infected macrophages. Proteic antigens are not recognized by the serum samples obtained from patients compared with those obtained from nonsick carriers, and T. whipplei that exhibits a low glycosylation profile does not efficiently multiply in macrophages in vitro.
T. whipplei glycosylation is likely to impair antibody-mediated immune recognition in patients. Such an intracellular antigen masking system in bacteria has not previously been described.
The Journal of Infectious Diseases 03/2009; 199(7):1043-52. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Whipple disease (WD) is a chronic infectious disease caused by Tropheryma whipplei. WD DNA has been found in stool and saliva specimens from patients and asymptomatic carriers.
A total of 4418 samples that were sent to our center for determination of WD were tested by a T. whipplei-specific quantitative polymerase chain reaction (PCR) based on repetitive sequences. Definite WD was diagnosed in 71 patients, including 55 patients with classic WD (defined by positive results of periodic acid-Schiff staining and/or specific immunohistochemistry of small-bowel biopsy specimens) and 16 patients with localized WD (including patients with endocarditis, neurologic infection, and uveitis).
Of the persons without WD, 2.3% had stool specimens positive for T. whipplei by PCR and 0.2% had saliva specimens positive for T. whipplei by PCR. Diagnosis of WD was likely in patients with positive results of both PCR of saliva specimens and PCR of stool specimens (positive predictive value, 95.2%). When the bacterial load was >10(4) colony-forming units per g of stool, the positive predictive value was 100%. A negative result of PCR of a saliva or stool specimen had a negative predictive value of 99.2% for classic WD. For localized WD, positive results of both PCR of saliva specimens and PCR of stool specimens had a sensitivity of 58% (compared with 94% for classic WD). The positive predictive value of testing of blood, cerebrospinal fluid, and urine specimens was 100% for each, and the positive predictive value for testing of duodenal biopsy specimens was 97.5%.
T. whipplei-specific quantitative PCR of saliva and stool specimens should be performed as first-line noninvasive screening for WD. When the results for both types of specimens are positive, diagnosis of classic WD should be highly suspected, especially if a high bacterial load is detected. Because PCR of saliva and stool specimens lacks sensitivity for determination of localized WD, invasive samples should be tested on the basis of clinical manifestations.
[show abstract][hide abstract] ABSTRACT: Bacterial vaginosis (BV) is a poorly detected public health problem that is associated with preterm delivery and for which no reliable diagnostic tool exists.
Molecular analysis of 231 vaginal samples, classified by Gram stain-based Nugent score, was used to propose molecular criteria for BV; these criteria were prospectively applied to 56 new samples. A quantitative molecular tool targeting 8 BV-related microorganisms and a human gene was developed using a specific real-time polymerase chain reaction assay and serial dilutions of a plasmid suspension. The targeted microorganisms were Gardnerella vaginalis, Lactobacillus species, Mobiluncus curtisii, Mobiluncus mulieris, and Candida albicans (which can be identified by Gram staining), as well as Atopobium vaginae, Mycoplasma hominis, and Ureaplasma urealyticum (which cannot be detected by Gram staining).
With use of the Nugent score, 167 samples were classified as normal, 20 were classified as BV, and 44 were classified as intermediate. Except for U. urealyticum, M. mulieris, and Lactobacillus species, DNA of the tested bacteria was detected more frequently in samples demonstrating BV, but the predictive value of such detection was low. The molecular quantification of A. vaginae (DNA level, > or = 10(8) copies/mL) and G. vaginalis (DNA level, > or = 10(9) copies/mL) had the highest predictive value for the diagnosis of BV, with excellent sensitivity (95%), specificity (99%), and positive (95%) and negative (99%) predictive values; 25 (57%) of the samples demonstrating intermediate flora had a BV profile. When applied prospectively, our molecular criteria had total positive and negative predictive values of 96% and 99%, respectively.
We report a highly reproducible, quantitative tool to objectively analyze vaginal flora that uses cutoff values for the concentrations of A. vaginae and G. vaginalis to establish the molecular diagnosis of BV.
[show abstract][hide abstract] ABSTRACT: Whipple disease is a chronic infection caused by Tropheryma whipplei. Trimethoprim-sulfamethoxazole is recommended for treatment of Whipple disease but is associated with treatment failure. T. whipplei is resistant in vitro to trimethoprim, because the gene targeted by this agent is missing.
A patient experienced clinical failure during treatment with trimethoprim-sulfamethoxazole. The gene encoding the enzyme putatively believed to be dihydropteroate synthase (DHPS), the target of sulfamethoxazole, was amplified and sequenced for 20 T. whipplei strains from our laboratory and for isolates recovered from a case patient at the time of diagnosis and the time of treatment failure. An Escherichia coli knockout strain for this gene was complemented with the sequences from a susceptible strain and from isolates recovered from the case patient. Susceptibilities of complemented E. coli to sulfamethoxazole were tested.
The target gene was identified among genes encoding a unique trifunctional enzyme in which DHPS is combined with the 2 preceding enzymes of the folate biosynthesis pathway. Changes in the amino acid sequence of putative DHPS were detected in the case patient. Gene complementation showed that the gene encoding putative DHPS restored the folate biosynthesis pathway and susceptibility to sulfamethoxazole, whereas the mutated sequence was associated with sulfamethoxazole resistance.
Antibiotic susceptibility of fastidious bacteria such as T. whipplei can be evaluated by means of gene complementation techniques. Mutations in the target gene of sulfamethoxazole appear during treatment.
The Journal of Infectious Diseases 08/2008; 198(1):101-8. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conventional methods such as microbiological cultures may lack the sensitivity and specificity to establish definitive diagnosis of osteoarticular infections. Herein, we review the general principles and the usefulness of broad-range PCR to improve the etiological diagnosis of osteoarticular infections.
Broad-range PCR followed by sequencing has been successfully developed to identify microorganisms involved in infections when patients have previously received antibiotics or in the presence of slow-growing or intracellular microorganisms. For osteoarticular infections, the studies have shown that the use of this molecular tool increased mainly the identification of Kingella kingae, anaerobic bacteria, and Streptococcus spp. However, it is very important to underline that the interpretation of this molecular tool is critical because of several pitfalls, including contamination causing false-positive results.
Broad-range PCR followed by sequencing offers several advantages when used to complement culture results for the diagnosis of fastidious bacteria and for patients taking antibiotics. However, its use should be restricted mainly for culture-negative cases when infection is suspected on the basis of clinical signs and symptoms or inflammatory syndrome. Future developments will include the use of real-time PCR in a closed system and pathogen-specific PCR for the molecular diagnosis of osteoarticular infections.
Current opinion in rheumatology 07/2008; 20(4):463-70. · 4.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: The purpose of this study, which involved 276 patients, was to report the importance of Propionibacterium acnes in shoulder infections. The proportion of patients with shoulder infection who had infection due to P. acnes was significantly greater than the proportion of patients with lower limb infection who had infection due to P. acnes (9 of 16 patients vs. 1 of 233 patients; P < .001). This bacterium requires a prolonged incubation period and should not be considered to be a contaminant.