Gilles Nevez

MOLECULAR CHARACTERIZATION OF CRYPTOSPORIDIUM ISOLATES OBTAINED FROM HUMANS IN FRANCE

ABSTRACT Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification ofCryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whomCryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes ofCryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range ofCryptosporidium species and genotypes.

A Cluster of Pneumocystis Infections Among Renal Transplant Recipients: Molecular Evidence of Colonized Patients as Potential Infectious Sources of Pneumocystis jirovecii

BACKGROUND Eighteen renal transplant recipients (RTRs) developed Pneumocystis jirovecii infections at the renal transplantation unit of Brest University Hospital (Brest, Brittany, France) from May 2008 through April 2010, whereas no cases of P. jirovecii infection had been diagnosed in this unit since 2002. This outbreak was investigated by identifying P. jirovecii types and analyzing patient encounters. METHODS The identification of P. jirovecii internal transcribed spacer (ITS) types was performed on P. jirovecii isolates from the 18 RTRs (12 patients with Pneumocystis pneumonia [PCP], 6 colonized patients), 22 unlinked control patients (18 patients with PCP, 4 colonized patients), and 69 patients (34 patients with PCP, 35 colonized patients) with contemporaneously diagnosed P. jirovecii infections in the Brest geographic area. A transmission map was drawn up. Its analysis was combined with the results of P. jirovecii typing. RESULTS P. jirovecii ITS type identification was successful in 14 of 18 RTRs, 15 of 22 control patients, and 48 of the 69 patients. Type Eg was the most frequent type in the 3 patient groups. However, its frequency was significantly higher in the first patient group than in the 2 other groups (P < .05 and P < .01, respectively). Fourteen encounters between RTRs who harbored an identical type were observed. Ten patients were considered as possible index patients, of whom 3 were colonized by the fungus, and 7 presented PCP. CONCLUSIONS The results provide to our knowledge the first data on the role of colonized patients as potential sources of P. jirovecii in a context of nosocomial acquisition of the fungus.

Pulmonary Colonization with Pneumocystis carinii in Human Immunodeficiency Virus-Negative Patients: Assessing Risk with Blood CD4 + T Cell Counts

Use of PCR analysis has led to detection of low numbers of Pneumocystis carinii organisms, which were undetectable by microscopy, in bronchoalveolar lavage (BAL) fluid specimens from immunosuppressed patients who showed no evidence of acute P. carinii pneumonia (PCP) (1). These low levels of par- asites were usually considered to reflect pulmonary coloniza- tion, but their significance was a subject of controversy. Cases of colonization were described mainly in patients positive for HIV who had blood CD4 1 T cell counts of /L (2). 6 ! 400 3 10 There were little data concerning HIV-negative patients (3). This study investigates the presence of P. carinii DNA in BAL fluid specimens from HIV-negative patients with no evidence of PCP and examines the link between colonization and decreases in blood CD4 1 T cell counts in this patient population.

Strain Typing Methods and Molecular Epidemiology of Pneumocystis Pneumonia

Several typing methods, with different strengths and weaknesses, are available for studies of Pneumocystis pneumonia.

Genotypes at the Internal Transcribed Spacers of the Nuclear rRNA Operon of Pneumocystis jiroveci in Nonimmunosuppressed Infants without Severe Pneumonia

ABSTRACT The frequency of Pneumocystis jiroveci (human-derived Pneumocystis) in immunocompetent infants developing acute respiratory syndromes has recently been evaluated and has been shown to be close to 25%. Until now, there have been no data on the genomic characteristics of the fungus in these patients, while molecular typing of P. jiroveci organisms was mostly performed with samples from immunosuppressed patients with pneumocystosis (Pneumocystiscarinii pneumonia [PCP]). The present report describes the genotypes of P. jiroveci organisms in 26 nonimmunosuppressed infants developing a mild Pneumocystis infection contemporaneously with an episode of bronchioloalveolitis. The typing was based on sequence analysis of internal transcribed spacers (ITSs) 1 and 2 of the rRNA operon, followed by the use of two typing scores. By use of the first score, 11 P. jiroveci ITS types were identified: 10 were previously reported in immunosuppressed patients with PCP, while 1 was newly described. By use of the second score, 13 types were identified, of which 2 were newly described. The most frequent type was identified as type B1a3 (first score), which corresponds to type Eg (second score). Mixed infections were diagnosed in three infants. The occurrence of such diversity of P. jiroveci ITS types, an identical main type, and mixed infections has previously been reported in immunosuppressed patients with PCP. Thus, the P. jiroveci ITS genotypes detected in immunocompetent infants and immunosuppressed patients developing different forms of Pneumocystis infection share characteristics, suggesting that both groups of individuals make up a common human reservoir for the fungus. Finally, the frequency of P. jiroveci in nonimmunosuppressed infants with acute respiratory syndromes and the genotyping results provide evidence that this infant population is an important reservoir for the fungus.

Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1→3) β-D-glucan for differential diagnosis of Pneumocystis pneumonia and Pneumocystis colonization.

ABSTRACT This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 107 versus 3.4 × 103 copies/μl, P < 0.05). A lower cutoff value (1.6 × 103 copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 104 copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 103 and 2 × 104 copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Pneumocystis carinii detection using nested-PCR in nasopharyngeal aspirates of immunocompetent infants with bronchiolitis.

Sero-epidemiological data indicate that first contacts with Pnewnocysris carinii (P. carinii) occur in more than 90% of immunocompetent infants before 2 years of age [l]. Until recently. primary infection by P. carinii was thought to be essentially asymptomatic [6 ] . In fact, there is little data concerning clinical profiles related to primary infection occurring in inununocompctcnt infant populations [3]. Indeed, for ethical reasons it is difficult to carry out prospective studies among healthy infants. Several infants and preschool children presenting acute respiratory illness related to bronchiolitis are hospitalized each year in our hospital (University Hospital, Amiens, France). Nasopharyngcal aspirates (NPAs) are routinely performed in this patient population in order to diagnose viral and bacterial infections. However, the specimens are not usually examined for P. carinii. We hypothesized that some of these pulmonary syndromes could be due to P. carinii infection. To test this hypothesis, all NPAs obtained from immunocompctent infants and preschool children with acute pulmonary syndrome who were admitted to our hospital over a 2-year period, were examined for P. carinii. The aim of this study was to desaibc the dctcction of P . carinii in NPAs from immunocompetent infants diagnosed with bronchiolitis.

Pneumocystis jiroveci Dihydropteroate Synthase Genotypes in Immunocompetent Infants and Immunosuppressed Adults, Amiens, France

To date, investigations of Pneumocystis jiroveci circulation in the human reservoir through the dihydropteroate synthase (DHPS) locus analysis have only been conducted by examining P. jirovecii isolates from immunosuppressed patients with Pneumocystis pneumonia (PCP). Our study identifies P. jirovecii genotypes at this locus in 33 immunocompetent infants colonized with P. jirovecii contemporaneously with a bronchiolitis episode and in 13 adults with PCP; both groups of patients were monitored in Amiens, France. The results have pointed out identical features of P. jirovecii DHPS genotypes in the two groups, suggesting that in these two groups, transmission cycles of P. jirovecii infections are linked. If these two groups represent sentinel populations for P. jirovecii infections, our results suggest that all persons parasitized by P. jirovecii, whatever their risk factor for infection and the form of parasitism they have, act as interwoven circulation networks of P. jirovecii.

Pneumocystis jiroveci Internal Transcribed Spacer Types in Patients Colonized by the Fungus and in Patients with Pneumocystosis from the Same French Geographic Region

ABSTRACT Pneumocystis jiroveci (human-derived Pneumocystis) infections can display a broad spectrum of clinical presentations, of which pulmonary colonization with the fungus may represent an important part, occurring frequently in patients with various underlying diseases and presenting alternative diagnoses of acute pneumocystosis (Pneumocystis carinii pneumonia [PCP]). There are few data concerning the P. jiroveci genotypes involved in pulmonary colonization, whereas several genotypes responsible for PCP in immunocompromised patients have been described. In this study, P. jiroveci genotypes have retrospectively been investigated and compared in 6 colonized patients and in 11 patients with PCP who were in the same hospital. Seventeen archival bronchoalveolar lavage samples were genotyped at internal-transcribed spacer 1 (ITS1) and ITS2 of the nuclear rRNA operon. Fourteen different genotypes were identified, of which 1 was found only in colonized patients, 10 were found only in patients with PCP, and 3 were found in both patient populations. Mixed infections were diagnosed in 2 of the 6 colonized patients and in 6 of the 11 patients with PCP. The results show that similar genotypes can be responsible for PCP as well as pulmonary colonization. There is a high diversity of genotypes in colonized patients and in patients with PCP. Mixed infections may occur in these two patient populations. These shared features of P. jiroveci ITS genotypes in colonized patients and patients with PCP suggest that human populations infected by P. jiroveci, whatever the clinical manifestation, may play a role as a common reservoir for the fungus.

Apparent Absence of Pneumocystis jirovecii in Healthy Subjects

We prospectively investigated 30 healthy subjects with normal CD4+ T cell counts in blood and normal findings of spirometry and chest radiography for the presence of Pneumocystis jirovecii, by performing polymerase chain reaction on sputum specimens. Fifty patients with chronic obstructive pulmonary disease were investigated at the same time in the same manner; this group was used as controls for the diagnosis of pulmonary colonization with P. jirovecii. None of the healthy subjects had positive test results, whereas the fungus was detected in 8 patients with chronic obstructive pulmonary disease. The results suggest that in our region (Amiens, France), P. jirovecii is apparently uncommon in healthy subjects and that this population, therefore, plays a minor role in circulation of the fungus within human communities.