Christelle Pomares

Importance of worldwide asymptomatic carriers of Leishmania infantum (L. chagasi) in human

Leishmaniasis due to Leishmania infantum (syn. L. chagasi) infection is a zoonotic disease present mainly in Mediterranean basin, central Asia and Brazil. Besides a limited number of human cases of clinical visceral leishmaniasis, a great number of infections remains asymptomatic. In this review, the prevalence of asymptomatic carriers of L. infantum was evaluated worldwide using parasitological methods or indirect testing such as a skin test or serology. The consequences of the presence of asymptomatic carriers on parasite transmission by blood donation or the development of clinical visceral leishmaniasis in immunocompromised individuals and its possible role as reservoir are discussed.

Toxoplasmosis and Horse Meat, France

To the Editor: Toxoplasma gondii parasites are obligate intracellular apicomplexans that can infect virtually all warm-blooded animals; felids are definitive hosts. The most common sources of human infection are ingestion of tissue cysts in undercooked meat or of food or water contaminated with oocysts shed by felids and transplacental transmission. Acquired toxoplasmosis in immunocompetent humans is frequently asymptomatic but is associated with cervical or occipital lymphadenopathy in ≈10% of patients. Severe or fatal outcomes for immunocompetent patients have been attributed to the virulence of specific T. gondii genotypes (1). We describe 3 cases of toxoplasmosis caused by atypical strains probably acquired by from ingestion of raw horse meat imported from Canada and Brazil. Patient 1, a 74-year-old man, was hospitalized locally (Antibes-Juan Les Pins, southern France) in March 2009 for asthenia and persistent febrile bronchitis. His medical history included severe smoking-related chronic obstructive pulmonary disease and coronary artery disease. He received broad-spectrum antimicrobial drugs and methylprednisolone. On day 23 after admission, he was transferred to our teaching hospital in Nice because of clinical deterioration and persistent fever. Disseminated toxoplasmosis was diagnosed on the basis of serologic evidence of recent primary T. gondii parasite infection and quantitative PCR detection of high Toxoplasma DNA levels in peripheral blood. Despite specific antitoxoplasma therapy with sulfadiazine and pyrimethamine, he remained febrile, his respiratory function worsened, and he died on day 27. Patient 2, a 24-year-old pregnant woman, was hospitalized in Draguignan, France, in December 2009 for full-term delivery. Three weeks earlier, routine serologic testing showed T. gondii parasite infection seroconversion. The newborn’s and mother’s ophthalmologic examinations were unremarkable. Congenital toxoplasmosis was diagnosed on the basis immunoglobulin M in the infant’s serum, positive quantitative PCR of samples from the placenta, and strain isolation after inoculation of mice with a placental preparation. Sulfadiazine and pyrimethamine were started. We performed a retrospective epidemiologic investigation of an unusual case of toxoplasmosis that occurred in March 1991. Patient 3, a 21-year-old pregnant woman living in the Nice area, was treated with spiramycine because routine serologic testing had shown T. gondii parasite infection seroconversion at 22 weeks’ gestation. Amniocentesis showed T. gondii tachyzoites in amniotic fluid by microscopic examination. At 26 weeks’ gestation, the woman underwent termination of pregnancy for ultrasonography-detected fetal severe abnormalities. Fetal necropsy showed numerous cerebral, cardiac, and hepatic abscesses with T. gondii tachyzoites. A few days after pregnancy termination, the woman experienced cervical lymphadenopathy, which lasted 3 years. She reported having eaten raw horse meat regularly during her pregnancy. Genetic analyses with microsatellite markers of the Toxoplasma spp. strains isolated from the 3 patients found 3 different atypical genotypes. Atypical strains are common in South America but unusual in France, where >95% of reported strains collected from human and animal toxoplasmosis cases belonged to the type II clonal lineage (2,3). Hence, isolation of an atypical Toxoplasma genotype from a patient in France strongly suggests contamination by a non-European strain, either during residence abroad or after ingestion of imported meat. Epidemiologic investigation of our case-patients that included questioning relatives, patients, and butchers found that eating raw horse meat imported from Canada (patient 1) or Brazil (patient 2) was the most likely source of the parasites. The geographic origin of the horse meat eaten by patient 3 is unknown. Moreover, an atypical T. gondii strain was isolated after mouse inoculation with horse meat from the first patient’s butcher. In all 3 cases, close relatives encouraged the patients to eat raw horse meat regularly because the practice is traditionally thought to reinforce health. Human toxoplasmosis cases associated with horse meat consumption are rarely reported but are probably underestimated (2). In the European Union, France and Italy account for more than two thirds of all horse meat eaten, predominantly raw, thereby increasing the likelihood of infection by various parasites, including Trichinella spp. and Toxoplasma spp (4). Under natural conditions, serologic prevalence of T. gondii parasites in horses worldwide may range from 0% to 80% (5). Many factors could account for this variation, including the sensitivity and specificity of the serologic test, ages of animals, geographic area and hygienic condition of farm management (5). The only prevalence survey of horses slaughtered for food that we are aware of was conducted in Canada and the United States and found antibodies to T. gondii parasites in 124 (6.9%) of 1,788 serum samples (6). T. gondii tissue cysts in meat are immediately killed by reaching an internal temperature of 67°C in all parts of meat during cooking (7). Deep freezing (<–12°C for at least 3 days) of meat before cooking is recommended because it reduces the risk for infection by inactivating most tissue cysts (7). These precautions are often not applied to horse meat because these imported carcasses are usually shipped as “fresh meat” and frequently eaten raw. Eating raw horse meat imported from non-European countries may expose consumers to high inocula of highly virulent atypical Toxoplasma spp. strains, which may cause life-threatening primary infection (case-patient 1) or severe congenital toxoplasmosis with atypical outcome of acquired toxoplasmosis in the mother (case-patient 3). Risk assessment for toxoplasmosis from horses slaughtered for food and imported into the European Union, as was recently done in France for ovine meat, is urgently needed (3).

Pneumocystis jirovecii Pneumonia in Patients with or without AIDS, France

Immunosuppressed patients without AIDS had longer time to treatment and a higher rate of death than did patients with AIDS.

Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota

Alterations in the composition of commensal bacterial populations, a phenomenon known as dysbiosis, are linked to multiple gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome, or to infections by diverse enteric pathogens. Blastocystis is one of the most common single-celled eukaryotes detected in human faecal samples. However, the clinical significance of this widespread colonization remains unclear, and its pathogenic potential is controversial. To address the issue of Blastocystis pathogenicity, we investigated the impact of colonization by this protist on the composition of the human gut microbiota. For that purpose, we conducted a cross-sectional study including 48 Blastocystis-colonized patients and 48 Blastocystis-free subjects and performed an Ion Torrent 16S rDNA gene sequencing to decipher the Blastocystis-associated gut microbiota. Here, we report a higher bacterial diversity in faecal microbiota of Blastocystis colonized patients, a higher abundance of Clostridia as well as a lower abundance of Enterobacteriaceae. Our results contribute to suggesting that Blastocystis colonization is usually associated with a healthy gut microbiota, rather than with gut dysbiosis generally observed in metabolic or infectious inflammatory diseases of the lower gastrointestinal tract.

Mucosal Leishmania infantum leishmaniasis: Specific pattern in a multicentre survey and historical cases

OBJECTIVE Leishmania infantum mucosally restricted leishmaniasis was rarely reported, so that diagnostic and treatment strategies remain debated. A long-term multicentric survey appeared thereby necessary. METHODS Cases were prospectively collected over 12 years in 3 academic hospitals of Southern France. Predisposing factors, clinical findings, diagnostic procedures, treatment and outcome were compared to medical literature. RESULTS Ten new cases and 40 historical reports were collected. Respectively 10/10 and 35/40 patients were adult males. Immunodeficiency was frequent (5/10 and 18/40). No previous cutaneous lesion was reported. Leishmaniasis affected mostly larynx (5/10 and 19/40), but also mouth (2/10 and 19/40) and nose (3/10 and 5/40). Lesions were highly polymorph. Mucosa histological examination provided respectively 1/10 and 2/40 false negative results, contrary to serum immunoblotting and PCR on mucosal biopsy. Although local response was always satisfactory even using topical treatment, subsequent visceral spreading was observed in 2/10 and 1/40 cases. CONCLUSION L. infantum mucosally restricted leishmaniasis exhibits a specific pattern, marked by tropism for adult males, high clinical and histological polymorphism. Immunoblot screening and PCR confirmation of suspected lesions are necessary because of direct examination occasional false negative results. The risk of visceral spreading sustains systemic therapy. SUMMARY Leishmania infantum mucosal leishmaniasis mostly affects adult males, half of them immunodeficient. Clinical and histological polymorphism makes the diagnosis difficult, stressing the need for immunoblot screening and mucosa PCR analysis of suspected cases. Possible visceralization sustains systemic therapy.

Reduced in vitro susceptibility to artemisinin derivatives associated with multi-resistance in a traveller returning from South-East Asia.

Decreased in vitro susceptibility to dihydroartemisinin (21.2 nM) and artesunate (16.3 nM) associated with decreased susceptibility or resistance to quinine (1131 nM), mefloquine (166 nM), lumefantrine (114 nM), pyronaridine (70.5 nM) and piperaquine (91.1 nM) is reported in a patient returning from South-East Asia after trekking along the Mekong from the south of Laos to the north of Thailand. Decreased in vitro susceptibility to artemisinin derivatives did not appear to be mediated by the number of copies of pfmdr1 or pfATPase6, pfcrt, pfmdr1 or pfmrp polymorphism. The high IC50 to mefloquine of this Asian isolate was not associated with pfmdr1 copy number. Pfnhe-1 microsatellite ms4760 showed a profile 7 (ms4760-7) with three repeats of DNNND and one repeat of DDDNHNDNHNN, which is associated with high quinine reduced susceptibility. The patient recovered in three days without relapse after treatment with the association of quinine and doxycycline. Decreased in vitro susceptibility to quinine and the delayed effect of doxycycline may both have contributed to the delayed parasite clearance time, D4 (0.5%) and D7 (0.004%). The in vitro data, with IC50 for dihydroartemisinin and artesunate were up to ten times those of the reference clone W2, which suggests that this isolate may be resistant to artemisinin derivatives, associated with a decreased susceptibility to quinine.

Significance of a positive Toxoplasma Immunoglobulin M test result in the United States

ABSTRACT A positive Toxoplasma immunoglobulin M (IgM) result is often interpreted as a marker of an acute infection. However, IgM can persist for several years, and Toxoplasma commercial IgM diagnostic test kits can yield a number of false-positive results. For these reasons, a chronic Toxoplasma infection can be erroneously classified as an acute infection, resulting in serious adverse consequences, especially in pregnant women, leading to emotional distress and unnecessary interventions, including termination of pregnancy. Interpretation of Toxoplasma serology at a reference laboratory can help differentiate a recently acquired infection from a chronic infection. Serological test results for 451 patients with positive Toxoplasma IgM and IgG test results obtained at nonreference laboratories (NRLs) that were referred to Palo Alto Medical Foundation Toxoplasma Serology Laboratory (PAMF-TSL) to determine whether the patient was acutely or chronically infected were retrospectively reviewed. PAMF-TSL results established that of the 451 patients, 335 (74%) had a chronic infection, 100 (22%) had an acute infection, and 7 (2%) were not infected, and for 9 (2%), results were indeterminate. Positive Toxoplasma IgM and IgG test results obtained at NRLs cannot accurately distinguish between acute and chronic infections. To do so, testing at reference laboratories is required, as mandated in 1997 in a letter from the Food and Drug Administration (FDA) to clinicians and laboratories in the United States.

Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro.

Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice—the liver and spleen being mainly studied—or in vitro. Whatever stationary phase L. infantum promastigotes population—wild type or engineered to express luciferase—the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues—liver and spleen—being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection.

Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients

ABSTRACT Pneumocystis jirovecii pneumonia (PCP) is an acute and life-threatening lung disease caused by the fungus Pneumocystis jirovecii. The presentation of PCP in HIV-positive patients is well-known and consists of a triad of dyspnea, fever, and cough, whereas the presentation of PCP in HIV-negative patients is atypical and consists of a sudden outbreak, O2 desaturation, and a rapid lethal outcome without therapy. Despite the availability of direct and indirect identification methods, the diagnosis of PCP remains difficult. The cycle threshold (CT ) values obtained by quantitative PCR (qPCR) allow estimation of the fungal burden. The more elevated that the fungal burden is, the higher the probability that the diagnosis is pneumonia. The purposes of the present study were to evaluate the CT values to differentiate colonization and pneumonia in a population of immunocompromised patients overall and patients stratified on the basis of their HIV infection status. Testing of bronchoalveolar lavage (BAL) fluid samples from the whole population of qPCR-positive patients showed a mean CT value for patients with PCP of 28 (95% confidence interval [CI], 26 to 30) and a mean CT value for colonized patients of 35 (95% CI, 34 to 36) (P < 10−3). For the subgroup of HIV-positive patients, we demonstrated that a CT value below 27 excluded colonization and a CT value above 30 excluded PCP with a specificity of 100% and a sensitivity of 80%, respectively. In the subgroup of HIV-negative patients, we demonstrated that a CT value below 31 excluded colonization and a CT value above 35 excluded PCP with a specificity of 80% and a sensitivity of 80%, respectively. Thus, qPCR of BAL fluid samples is an important tool for the differentiation of colonization and pneumonia in P. jirovecii-infected immunocompromised patients and patients stratified on the basis of HIV infection status with different CT values.

Laboratory diagnosis of congenital toxoplasmosis

ABSTRACT Recent studies have demonstrated that screening and treatment for toxoplasmosis during gestation result in a decrease of vertical transmission and clinical sequelae. Early treatment was associated with improved outcomes. Thus, laboratory methods should aim for early identification of infants with congenital toxoplasmosis (CT). Diagnostic approaches should include, at least, detection of Toxoplasma IgG, IgM, and IgA and a comprehensive review of maternal history, including the gestational age at which the mother was infected and treatment. Here, we review laboratory methods for the diagnosis of CT, with emphasis on serological tools. A diagnostic algorithm that takes into account maternal history is presented.