Coinfection with "Rickettsia sibirica subsp. mongolotimonae" and Rickettsia conorii in a Human Patient: a Challenge for Molecular Diagnosis Tools.

Rickettsioses are zoonoses transmitted by vectors. More than one agent can coexist in vectors. Although vectors may transmit more than one microorganism to humans, information on dual infections is scarce. We present a case of a patient with an atypical rickettsiosis diagnosis in whom two species of Rickettsia were detected.

Shell-vial culture and real-time PCR applied to Rickettsia typhi and Rickettsia felis detection.

Murine typhus is a zoonosis transmitted by fleas, whose etiological agent is Rickettsia typhi. Rickettsia felis infection can produces similar symptoms. Both are intracellular microorganisms. Therefore, their diagnosis is difficult and their infections can be misdiagnosed. Early diagnosis prevents severity and inappropriate treatment regimens. Serology can't be applied during the early stages of infection because it requires seroconversion. Shell-vial (SV) culture assay is a powerful tool to detect Rickettsia. The aim of the study was to optimize SV using a real-time PCR as monitoring method. Moreover, the study analyzes which antibiotics are useful to isolate these microorganisms from fleas avoiding contamination by other bacteria. For the first purpose, SVs were inoculated with each microorganism. They were incubated at different temperatures and monitored by real-time PCR and classical methods (Gimenez staining and indirect immunofluorescence assay). R. typhi grew at all temperatures. R. felis grew at 28 and 32 °C. Real-time PCR was more sensitive than classical methods and it detected microorganisms much earlier. Besides, the assay sensitivity was improved by increasing the number of SV. For the second purpose, microorganisms and fleas were incubated and monitored in different concentrations of antibiotics. Gentamicin, sufamethoxazole, trimethoprim were useful for R. typhi isolation. Gentamicin, streptomycin, penicillin, and amphotericin B were useful for R. felis isolation. Finally, the optimized conditions were used to isolate R. felis from fleas collected at a veterinary clinic. R. felis was isolated at 28 and 32 °C. However, successful establishment of cultures were not possible probably due to sub-optimal conditions of samples.

Serosurvey of Rickettsia typhi and Rickettsia felis in HIV-infected patients.

Consistent with the effects of HIV on cell-mediated immunity, an increased susceptibility to intracellular microorganisms has been observed. Rickettsiae are obligate intracellular microorganisms. The aim of this study was to examine Rickettsia typhi and Rickettsia felis infections in HIV+ population. Sera of 341 HIV+ patients were evaluated by indirect immunofluorescent assay. Age, sex, residential locality, risk behavior, stage according to criteria of the Center for Disease Control and Prevention, CD4+/CD8+ T cells, Hepatitis B antigen, and Hepatitis C serology were surveyed. Seroprevalences of R. typhi and R. felis infection were 7.6% and 4.4%, respectively. No associations were found between seropositivities and the assessed variables. Findings were similar to those obtained in healthy subjects from the same region.

Seroprevalence of Bartonella spp. infection in HIV patients in Catalonia, Spain.

BACKGROUND: Although the first clinical descriptions of Bartonella infection were associated with immunocompromised patient with bacillary angiomatosis, we currently know that this organism is directly involved in diseases affecting a large number of patients, regardless of their immune status. Cat scratch disease, hepatic peliosis, and some cases of bacteraemia and endocarditis, are directly caused by some species of the genus Bartonella. The purpose of this study was to determinate the prevalence of IgG antibodies against Bartonella henselae and B. quintana in HIV patients and to identify the epidemiological factors involved. METHODS: Serum samples were collected from HIV patients treated at Hospital de Sabadell. Antibodies to B. henselae and B. quintana from 340 patients were examined by indirect immunofluorescence assay (IFA). Significance levels for univariate statistical test were determined by the Mann-Whitney U test and chi2 test. RESULTS: Of 340 patients, 82 were women and 258 men, with a median age of 42.21 +/- 10.35 years (range 16-86 years). Seventy-six (22.3%) patients reacted with one or more Bartonella antigens. Of all the factors concerning the seroprevalence rate being studied (age, sex, intravenous drugs use, alcohol consumption, CD4 levels, AIDS, HCV, HBV, residential area), only age was statistically significant. CONCLUSION: A high percentage of HIV patients presents antibodies to Bartonella and is increasing with age.

Shell-vial culture, coupled with real-time PCR, applied to Rickettsia conorii and Rickettsia massiliae-Bar29 detection, improving the diagnosis of the Mediterranean spotted fever.

Rickettsia conorii and Rickettsia massiliae-Bar29 are related to Mediterranean spotted fever (MSF). They are intracellular microorganisms. The Shell-vial culture assay (SV) improved Rickettsia culture but it still has some limitations: blood usually contains low amount of microorganisms and the samples that contain the highest amount of them are non-sterile. The objectives of this study were to optimize SV culture conditions and monitoring methods and to establish antibiotic concentrations useful for non-sterile samples. 12 SVs were inoculated with each microorganism, incubated at different temperatures and monitored by classical methods and real-time PCR. R. conorii was detected by all methods at all temperatures since 7th day of incubation. R. massiliae-Bar29 was firstly observed at 28°C. Real-time PCR allowed to detected it 2-7 days earlier (depend on temperature) than classical methods. Antibiotics concentration needed for the isolation of these Rickettsia species from non-sterile samples was determined inoculating SV with R. conorii, R. massiliae-Bar29, biopsy or tick, incubating them with different dilutions of antibiotics and monitoring them weekly. To sum up, if a MSF diagnosis is suspected, SV should be incubated at both 28°C and 32°C for 1-3 weeks and monitored by a sensitive real-time PCR. If the sample is non-sterile the panel of antibiotics tested can be added.

Prevalence of Bartonella henselae in cats in Catalonia, Spain.

Bartonella henselae, an emerging pathogen bacterium, is the main causative agent of the cat scratch disease. While the first clinical descriptions were associated with immunosupressed patients, it is now more frequently observed in patients with normal immune status (endocarditis and bacteremia). Cats were found to be the only known reservoir of B. henselae. In this paper, we report the results obtained in the first study made to investigate the prevalence of B. henselae bacteremia and antibodies in domestic cats in Catalonia, Spain. Serum samples from 115 cats were tested for antibodies to B. henselae by immunofluorescent antibody testing, and 29.6% had a titer >or= 1:64. Seven B. henselae strains were isolated using standard culture techniques and amplification by a polymerase chain reaction and subsequent sequencing was performed on the intergenic spacer region between the 16 and 23S ribosomal RNA genes. Of all factors concerning the studied bacteremia rate (age, sex, habitat, presence of antibodies, contact with animals, parasites), only the presence of antibodies to B. henselae was statistically significant.

The role of cats in the eco-epidemiology of spotted fever group diseases.

BACKGROUND: Mediterranean Spotted Fever (MSF), whose etiological agent is R. conorii, is one of the oldest described vector-borne infectious diseases. Although it is endemic in the Mediterranean area, clinical cases have also been reported in other regions. R. massiliae-Bar29 is related to MSF cases. This strain is distributed worldwide. R. conorii and R. massiliae-Bar29 are transmitted by ticks. Dogs are considered the sentinel of R. conorii infection. Cats could also be involved in their transmission. Rickettsia felis, etiological agent of Flea-borne spotted fever, is mainly transmitted by the cat flea, Ctenocephalides felis. Up to now, the role of cats in its transmission is not entirely elucidated. The aim of the study is to analyze the infection in cats by these microorganisms. METHODS: The study was undertaken in Northeastern Spain. Twenty municipalities of seven regions participated in the study. 212 cats (pets and stray cats) were analyzed. Variables surveyed were: date of collection, age, sex, municipality, source, living place, outdoor activities, health status, type of disease, contact with other animals, and ectoparasite infestation. Sera were evaluated by indirect immunofluorescence antibody assay (IFA). Molecular detection (real-time PCR and sequencing) and cultures were performed on blood samples. RESULTS: There were 59 (27.8%) cats seroreactive to one or more microorganisms. Considering cross-reactions, the seroprevalences were 15.6%-19.5% (R. massiliae-Bar29), 1.9%-6.2% (R. conorii), and 5.2%-7.5% (R. felis). A weak association was observed between SFG seropositivity and tick infestation. Ticks found on seropositive cats were Rhipicephalus pusillus, R. sanguineus and R. turanicus. DNA of Rickettsia was detected in 23 cats. 21 of them could be sequenced. Sequences obtained were identical to those sequences of SFG rickettsiae similar to R. conorii and R. massiliae. No amplification of R. felis was obtained. CONCLUSIONS: Cats can be infected by SFG rickettsiae and produce antibodies against them. Cats may play a role in the transmission cycle of R. conorii and R. massiliae-Bar29, although the role in the R. felis cycle needs further analysis.

The role of dogs in the eco-epidemiology of Rickettsia typhi, etiological agent of Murine typhus.

Rickettsia typhi, etiological agent of Murine typhus (MT), is transmitted to humans from an animal reservoir through two cycles: a classic rat-flea-rat cycle, and a peridomestic animal cycle. There are not many studies concerning which animals are involved in the peridomestic cycle, and most of them are focused on cats. The aim of this study was to determine the presence of R. typhi in dogs, not only by serological methods but also by direct methods such as culture and molecular detection. Two hundred and one dog blood samples were collected from Veterinary clinics, kennels, and shelters in Northeastern Spain (2006-2008). Age, sex, municipality, living place, healthy status, contact with animals, and ectoparasite infestations were surveyed. IgG was measured by IFA. Titers ≥ 1/64 were considered positive. Cultures were carried out using samples of dogs with titers ≥ 1/128. The molecular detection was performed by real-time PCR. Nine dogs (4.5%) were positive according to IFA (5: 1/64; 3: 1/128; 1: 1/512). There were no significant differences in the rates of antibodies related to any of the variables. Rickettsial DNA was detected in two cultures. Sequences obtained were identical to those of R. typhi. The results show direct and indirect evidences of the presence of R. typhi infection in dogs.

Molecular detection of Rickettsia typhi in cats and fleas.

BACKGROUND: Rickettsiatyphi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R. typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R. typhi in our location analyzing its presence in cats and fleas. METHODOLOGY/PRINCIPAL FINDINGS: 221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R. typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant. Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R. typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis. R. typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R. felis was observed. CONCLUSIONS: Although no clinical case has been described in this area, the presence of R. typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R. typhi is detected in naturally infected cats.

Rickettsia felis in fleas from Catalonia (Northeast Spain).

INTRODUCTION: Rickettsia felis produces a syndrome indistinguishable from murine typhus, which has been described in Spain. R. felis is transmitted to humans by fleas. Although no clinical case has been described so far, serologic evidence of infections in humans, cats, and dogs has been obtained in our area. However, no study has been conducted regarding its presence in vectors. Recognition of routes of transmission is of great importance to prevent infection in humans. Taking into account these results, R. felis seems to be present in animals that are in contact with humans. The aim of this study was to determine the presence of R. felis in the fleas of cats and dogs from Northeast Spain, to show the presence of peridomestic cycle in our area. MATERIALS AND METHODS: Between May 2006 and July 2008, 78 fleas were collected. Sixty-three fleas were recovered from kennels. Most of them were collected from cages and a few of them on dogs and cats living in kennels. Fifteen fleas were collected from dogs and cats attended at a veterinary clinic. Fleas were rinsed with ethanol, dried, identified, and stored at 4°C. DNA was extracted from each flea individually. Rickettsial DNA was determined by quantitative real-time polymerase chain reaction. OmpB-specific primers and molecular beacon probes targeting specifically R. felis were used. RESULTS: All 78 fleas were identified as Ctenocephalides felis. R. felis was detected in 34 (43.6%) fleas. No nucleic acids were amplified from negative controls and expected results were obtained from positive controls. Eight positive samples were also confirmed by sequencing. CONCLUSIONS: R. felis was found in a high percentage of Ct. felis from cats and dogs. It seems that there is a peridomestic cycle in Northeast Spain, which would allow contact of R. felis with humans.

[Microbiological diagnosis of emerging bacterial pathogens: Anaplasma, Bartonella, Rickettsia, and Tropheryma whipplei]. / Diagnóstico microbiológico de las infecciones por patógenos bacterianos emergentes: Anaplasma, Bartonella, Rickettsia, Tropheryma whipplei.

Ehrlichia/Anaplasma, Bartonella, Rickettsia and Tropheryma whipplei (formerly called whippelii) are fastidious bacterial organisms, considered the causative agents of potentially severe emerging and re-emerging diseases with repercussions on public health. The recent availability of advanced molecular biology and cell culture techniques has led to the implication of many of these species in human pathologies. These issues are extensively covered in number 27 of the SEIMC microbiological procedure: Diagnóstico microbiológico de las infecciones por patógenos bacterianos emergentes: Anaplasma, Bartonella, Rickettsia y Tropheryma whippelii (Microbiological diagnosis of Anaplasma, Bartonella, Rickettsia and Tropheryma whippelii infections) (2nd ed., 2007) (www.seimc.org/documentos/protocolos/microbiologia/).

Evaluation of the BacT/Alert and VITAL blood culture systems for the diagnosis of bacteremia.

OBJECTIVE: To evaluate the detection of bacterial growth in the BacT/Alert (Organon Teknika) and VITAL (bioMérieux) automated blood culture systems. METHODS: In accordance with the protocol of study, 1021 blood sample pairs for culture were obtained from adult patients admitted to the Emergency Room and Intensive Care Unit. RESULTS: In total, 139 (13.6%) clinically significant blood cultures were detected, of which 79 (56.8%) were detected by both systems, 48 (34.5%) only by BacT/Alert and 12 (8.6%) only by VITAL (P cent0.0001). The BacT/Alert system detected positive blood cultures more rapidly for all groups of microorganisms. The VITAL system showed six false-negative blood cultures, while the BacT/Alert system showed none (P50.03). There was no significant difference between the number of false-positive blood cultures detected by the two systems. CONCLUSIONS: In our study, overall the BacT/Alert system achieved a better recovery of microorganisms than the VITAL system.

Endemic meningococcal disease in Cerdanyola, Spain, 1987--93: molecular epidemiology of the isolates of Neisseria meningitidis.

OBJECTIVE: To establish the relationships between 30 Neisseria meningitidis strains isolated in Cerdanyola (Spain) from 30 out of 36 sporadic cases of meningococcal disease (MD) during 1987--93 and their spread in this population by multilocus enzyme electrophoresis (MEE) and by pulsed-field gel electrophoresis (PFGE), and to evaluate the usefulness of PFGE versus serologic typing methods and MEE as an alternative epidemiologic marker to study meningococcal infection. METHODS: Serotyping, electrophoretic mobility of seven isoenzymes determined by MEE and chromosomal DNA macrorestriction with NheI resolved by PFGE were analyzed. RESULTS: Of these 30 strains, 25 were serogroup B and the remaining five were serogroup C, with the 4:P1.15 and the 2b:NT as the most common antigenic phenotypes, respectively. There were 13 electrophoretic types (ETs) by MEE, with 14 isolates showing an identical ET, 8. Sixteen pulse types (PTs) were generated by PFGE. The 14 ET 8 isolates were clustered into six PTs, A1, A2, A4, A5, A6 and A8. However, by combining both methods, 19 genetically distinct groups were obtained. Eleven of these groups (20 serogroup B strains) and two of these (four serogroup C strains) were genetically related. CONCLUSIONS: We conclude that, according to the clonal population structure, these 30 N. meningitidis strains are heterogeneous although a great number are related. Moreover, PFGE is a useful method to establish clonal structure in N. meningitidis strains under endemic conditions. Finer discrimination of these strains was achieved by combining both MEE and PFGE methods.