Pulmonary co-infections by Pneumocystis jirovecii and Herpesviridae: a seven-year retrospective study.

BACKGROUND: Pneumocystis jirovecii (P. jirovecii) is an opportunistic fungus responsible for Pneumocystis pneumonia (PCP) in deeply immunocompromised patients and for pulmonary colonization in individuals with mild immunosuppression or impaired respiratory function. PCP and Cytomegalovirus (CMV) co-infections have been widely described whereas those involving other Herpesviruses (HVs) such as Epstein-Barr virus (EBV), Herpes simplex virus type 1 and type 2 (HSV-1 and -2), and Varicella zoster virus (VZV) remain scarce. To date, no data are available concerning HVs co-infections in P. jirovecii colonization. METHODS: Our main objective was to evaluate the frequency of HVs in bronchoalveolar lavage fluid (BALF) samples from patients with PCP or with pulmonary colonization. The secondary objective was to assess the relationship between HVs and the mortality rate in PCP patients. A retrospective single-center study over a seven-year period was conducted. All patients with P. jirovecii detected using PCR in a BALF sample and for whom a PCR assay for HVs detection was performed were included in the study. RESULTS: One hundred and twenty-five patients were included, corresponding to 77 patients with PCP and 48 colonized patients. At least one HV was detected in 54/77 (70.1%) PCP patients and in 28/48 (58.3%) colonized patients. EBV was the most frequent in both groups. Furthermore, the 30-day survival rate in PCP patients was significantly lower with [EBV + CMV] co-infection than that with EBV co-infection, [EBV + HSV-1] co-infection and without HV co-infection. CONCLUSION: Our results show that the frequency of HV, alone or in combination is similar in PCP and colonization. They also suggest that [EBV + CMV] detection in BALF samples from PCP patients is associated with an increased mortality rate, underlying the significance to detect HVs in the course of PCP.

Design, synthesis, and characterization of novel aminoalcohol quinolines with strong in vitro antimalarial activity.

Malaria is the fifth most lethal parasitic infections in the world. Herein, five new series of aminoalcohol quinolines including fifty-two compounds were designed, synthesized and evaluated in vitro against Pf3D7 and PfW2 strains. Among them, fourteen displayed IC50 values below or near of 50.0 nM whatever the strain with selectivity index often superior to 100.17b was found as a promising antimalarial candidate with IC50 values of 14.9 nM and 11.0 nM against respectively Pf3D7 and PfW2 and a selectivity index higher than 770 whatever the cell line is. Further experiments were achieved to confirm the safety and to establish the preliminary ADMET profile of compound 17b before the in vivo study performed on a mouse model of P. berghei ANKA infection. The overall data of this study allowed to establish new structure-activity relationships and the development of novel agents with improved pharmacokinetic properties.

A Negative (1,3)-ß-D-Glucan Result Alone Is Not Sufficient to Rule Out a Diagnosis of Pneumocystis Pneumonia in Patients With Hematological Malignancies.

Background: Serum (1,3)-ß-D-glucan (BG) testing is increasingly being used in the diagnostic armamentarium for invasive fungal diseases. Given its high sensitivity, some studies suggest that a negative BG result contributes to rule out a diagnosis of Pneumocystis pneumonia (PCP). However, recent reports described a suboptimal sensitivity in HIV-negative immunocompromised patients. In this study, we evaluated the performance of BG assay for PCP diagnosis in HIV-negative patients with diverse PCP risk factors. We also assessed the correlation between Pneumocystis jirovecii load in pulmonary samples and serum BG levels. Methods: We retrospectively included HIV-negative patients with microscopically proven PCP and for whom a BG result was available. We also enrolled patients colonized by Pneumocystis as control group. Colonized patients were matched with PCP patients based on their underlying condition that exposed to PCP. Pulmonary fungal loads were determined by an in-house real-time PCR, and BG levels were measured by using the Fungitell® kit (Associates of Cape Cod, Inc.). Results: Thirty-nine patients were included in each of the two groups. Thirty-four of 39 PCP patients and one of 39 colonized patient had a positive BG test, resulting in a sensitivity of 0.87 (95% CI: 0.73-0.94), a specificity of 0.97 (95% CI: 0.87-0.99), a positive predictive value of 0.97 (95% CI: 0.85-0.99), and a negative predictive value of 0.88 (95% CI: 0.75-0.95) for BG assay. Nonetheless, median BG level differed according to the underlying condition. Among the PCP group, the lowest median level of 211 pg/ml was observed in patients with hematological malignancy (HM) and differed significantly from that observed either in solid organ transplants (3,473 pg/ml) or in patients with autoimmune or inflammatory disorder (3,480 pg/ml). Indeed, the sensitivity of BG assay was estimated at 0.64 (95% CI: 0.35-0.85) in HM patients and was lower than the one observed in the whole PCP group. Furthermore, BG level and fungal burden correlated poorly among all PCP patients. Conclusion: BG is not a reliable biomarker for ruling out PCP in HIV-negative patients with HM. Interpretation of a negative BG result should take into account, but not be limited to, the underlying condition predisposing to PCP.

It is still PCP that can stand for Pneumocystis pneumonia: Appeal for generalized use of only one acronym.

Twenty-years ago, considering the host specificity of Pneumocystis species, the human-derived Pneumocystis, Pneumocystis carinii formae specialis hominis, was renamed Pneumocystis jirovecii. Pneumocystis carinii formae specialis carinii was finally renamed Pneumocystis carinii and kept for the species derived from Rattus norvegicus. P. jirovecii is now widely used by most authors. The PCP acronym that initially referred to "Pneumocystis cariniipneumonia" was contemporaneously redefined to stand for Pneumocystispneumonia in order to avoid changing the acronym of the name of the disease that clinicians have used for several decades. Using analysis of multidata bases on PubMed, we have noted a recent acceleration in the use of PJP for Pneumocystis jiroveciipneumonia, which may be grammatically correct but not in accordance with retaining PCP, which was proposed in the early 2000s. Through this reminder, in order to standardize the literature on P. jirovecii, we plead for the use of only one acronym, PCP. LAY SUMMARY: Through this reminder on Pneumocystis nomenclature, we plead for the use of only one acronym, PCP, the retention of which was proposed in the early 2000s, and which currently stands for Pneumocystispneumonia.

Invasive Tracheobronchial Aspergillosis in Critically Ill Patients with Severe Influenza. A Clinical Trial.

Rationale: Invasive tracheobronchial aspergillosis (ITBA) is an uncommon but severe clinical form of invasive pulmonary aspergillosis in which the fungal infection is entirely or predominantly confined to the tracheobronchial tree.Objectives: To analyze the diagnostic and prognostic differences between tracheobronchial aspergillosis and pulmonary aspergillosis without tracheobronchial lesions among patients admitted to the ICU with severe influenza.Methods: This retrospective, observational study included critically ill patients with influenza associated with pulmonary aspergillosis from three hospital ICUs between 2010 and 2019. Patient characteristics and clinical and mycologic data at admission and during ICU stay were collected in a database to evaluate variables in the two groups.Measurements and Main Results: Thirty-five patients admitted to the ICU with severe influenza and pulmonary aspergillosis were included. Ten patients were included in the group with ITBA (n = 10 of 35; 28.6%), and 25 patients were included in the group without ITBA. The group with ITBA comprised more patients with active smoking, diabetes mellitus, and higher severity scores (Simplified Acute Physiology Score II). Ninety-day mortality rates in the groups with and without ITBA were 90% and 44%, respectively (P = 0.02). Moreover, significantly higher serum 1,3-ß-d-glucan and galactomannan and BAL fluid galactomannan concentrations were observed in the group with ITBA compared with the group without ITBA (P < 0.0001, P = 0.003, and P = 0.008, respectively).Conclusions: ITBA was associated with higher severity scores, mortality, and serum and BAL fluid galactomannan and 1,3-ß-d-glucan concentrations than invasive pulmonary aspergillosis without tracheobronchial lesions. ITBA should be systematically researched by bronchoscopic examination in ICU patients with concomitant pulmonary aspergillosis and influenza.Clinical trial registered with www.clinicaltrials.gov (NCT04077697).

Pneumocystis Infection Outbreaks in Organ Transplantation Units in France: A Nation-Wide Survey.

The burden of nosocomial Pneumocystis infections in transplantation units in France was evaluated through a retrospective survey. Over 12 years, 16 outbreaks occurred, including 13 among renal transplant recipients (RTRs). We performed Pneumocystis jirovecii genotyping in 5 outbreaks, which suggested that specific strains may have been selected by RTRs.

Diversity of Pneumocystis jirovecii Across Europe: A Multicentre Observational Study.

Pneumocystis jirovecii is an airborne human-specific ascomycetous fungus responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, affecting >500,000 patients per year (www.gaffi.org). The understanding of its epidemiology is limited by the lack of standardised culture. Recent genotyping data suggests a limited genetic diversity of P. jirovecii. The objective of the study was to assess the diversity of P. jirovecii across European hospitals and analyse P. jirovecii diversity in respect to clinical data obtained from the patients. Genotyping was performed using six already validated short tandem repeat (STR) markers on 249 samples (median: 17 per centre interquartile range [11-20]) from PCP patients of 16 European centres. Mixtures of STR markers (i.e., ≥2 alleles for ≥1 locus) were detected in 67.6% (interquartile range [61.4; 76.5]) of the samples. Mixture was significantly associated with the underlying disease of the patient, with an increased proportion in HIV patients (78.3%) and a decreased proportion in renal transplant recipients (33.3%) (p<0.001). The distribution of the alleles was significantly different (p<0.001) according to the centres in three out of six markers. In analysable samples, 201 combinations were observed corresponding to 137 genotypes: 116 genotypes were country-specific; 12 in two; six in three; and two in four and one in five countries. Nine genotypes were recorded more than once in a given country. Genotype 123 (Gt123) was significantly associated with France (14/15, p<0.001) and Gt16 with Belgium (5/5, p<0.001). More specifically, Gt123 was observed mainly in France (14/15/16 patients) and in renal transplant patient (13/15). Our study showed the wide population diversity across Europe, with evidence of local clusters of patients harbouring a given genotype. These data suggest a specific association between genotype and underlying disease, with evidence of a different natural history of PCP in HIV patients and renal transplant recipients.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study.

This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.

Diffusion of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis colonization: frequency and putative risk factors.

In a prospective bicentric study, Pneumocystis jirovecii excretion and diffusion was explored in air samples collected in the rooms occupied by 17 Pneumocystis-colonized patients. P. jirovecii DNA was detected by real-time PCR in the air collected from 3 patients' rooms (17.6%), with identical genotypes in corresponding clinical and air samples. Pneumocystis DNA was detected for 2/3 patients with autoimmune disease treated with corticosteroids versus 1/6 patients with hematologic disease and 0/5 kidney transplant recipients. These data confirm the possible excretion of the fungus by Pneumocystis-colonized patients and thus bring additional arguments for the prevention of airborne transmission in hospital wards.

Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization.

In this study, Pneumocystis jirovecii was detected and characterized in the air surrounding patients with Pneumocystis pulmonary colonization. Air samples were collected in the rooms of 10 colonized patients using Coriolis® µ air sampler at 1m and 5m from the patient's head. P. jirovecii DNA was amplified and genotyped in pulmonary and air samples at the mitochondrial large subunit ribosomal RNA gene. P. jirovecii DNA was detected in 5 of the 10 air samples collected at 1m and in 5 of the 10 other air samples collected at 5m. P. jirovecii genotyping was successful in 4 pairs or triplets of air and pulmonary samples. Full genotype matches were observed in 3 of the 4 pairs or triplets of air and pulmonary samples. These results provide original data supporting P. jirovecii exhalation from colonized patients and emphasize the risk of P. jirovecii nosocomial transmission from this patient population.

AIDS-related Pneumocystis jirovecii genotypes in French Guiana.

The study described Pneumocystis jirovecii (P. jirovecii) multilocus typing in seven AIDS patients living in French Guiana (Cayenne Hospital) and seven immunosuppressed patients living in Brest, metropolitan France (Brest Hospital). Archival P. jirovecii specimens were examined at the dihydropteroate synthase (DHPS) locus using a PCR-RFLP technique, the internal transcribed spacer (ITS) 1 and ITS 2 and the mitochondrial large subunit rRNA (mtLSUrRNA) gene using PCR and sequencing. Analysis of typing results were combined with an analysis of the literature on P. jirovecii mtLSUrRNA types and ITS haplotypes. A wild DHPS type was identified in six Guianese patients and in seven patients from metropolitan France whereas a DHPS mutant was infected in the remaining Guianese patient. Typing of the two other loci pointed out a high diversity of ITS haplotypes and an average diversity of mtLSUrRNA types in French Guiana with a partial commonality of these haplotypes and types described in metropolitan France and around the world. Combining DHPS, ITS and mtLSU types, 12 different multilocus genotypes (MLGs) were identified, 4 MLGs in Guianese patients and 8 MLGs in Brest patients. MLG analysis allows to discriminate patients in 2 groups according to their geographical origin. Indeed, none of the MLGs identified in the Guianese patients were found in the Brest patients and none of the MLGs identified in the Brest patients were found in the Guianese patients. These results show that in French Guiana (i) PCP involving DHPS mutants occur, (ii) there is a diversity of ITS and mtLSUrRNA types and (iii) although partial type commonality in this territory and metropolitan France can be observed, MLG analysis suggests that P. jirovecii organisms from French Guiana may present specific characteristics.

Cryptosporidiosis in Haiti: surprisingly low level of species diversity revealed by molecular characterization of Cryptosporidium oocysts from surface water and groundwater.

The protozoan parasite Cryptosporidium sp. has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrhoeal diseases worldwide. In Haiti, cryptosporidiosis is a frequent cause of diarrhoea in children under the age of five years, HIV-infected individuals, and people living in low socioeconomic conditions, mainly due to the consumption of water or food polluted by Cryptosporidium oocysts. The aim of this study was to detect and identify Cryptosporidium oocysts present in 12 water samples collected in Port-au-Prince and 4 water samples collected in Cap Haïtien. Initial detection consisted of immunomagnetic separation - immunofluorescence assay (IMS-IFA), which was confirmed by nested PCR, targeting the most polymorphic region of the 18S rRNA gene in 15/16 samples. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Under our working conditions, neither nested PCR-RFLP nor direct DNA sequencing revealed the expected species diversity, as only Cryptosporidium parvum was identified in the water samples studied. This study highlights the difficulty of detecting mixed populations of Cryptosporidium species in environmental samples.

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

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-ß-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 × 10(7) versus 3.4 × 10(3) copies/µl, P < 0.05). A lower cutoff value (1.6 × 10(3) copies/µl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 10(4) 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 × 10(3) and 2 × 10(4) copies/µl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Pneumocystis jirovecii haplotypes at the internal transcribed spacers of the rRNA operon in French HIV-negative patients with diverse clinical presentations of Pneumocystis infections.

Pneumocystis jirovecii, a transmissible fungus, is the causative agent of pulmonary infections. Its genomic diversity has appeared in reports from around the world but data on P. jirovecii genotypes in France are still limited. This study describes the typing of P. jirovecii isolates from 81 HIV-negative patients monitored at Brest University Hospital, Brittany, France, 40 of whom developed Pneumocystis pneumonia (PcP), and remaining 41 patients were colonized by the fungus. The isolates were assayed at the internal transcribed spacer (ITS)1 and ITS2 under improved amplification conditions to avoid in vitro ITS recombination. P. jirovecii ITS haplotypes were identified in 56/81 patients (31 PcP patients and 25 patients who were colonized) which revealed a high diversity in that 27 different haplotypes were identified. Eg was the most frequent haplotype (31/56, 55.3%), followed by Ec and Ai (5/56, 8.9% each). In contrast, Ne, usually the second most frequent haplotype in Europe and the USA, was observed in only 2/56 patients (3.6%). Mixed infections were detected in 18/56 patients (32.1%; 12 PcP patients and six who were colonized). No significant differences were observed in haplotype diversity, frequency of peculiar haplotypes, and mixed infection occurrence, between the two patient populations. The study, conducted with the largest HIV-negative patient population investigated so far, shows that ITS typing remains an efficient method for characterizing P. jirovecii among human populations, whatever their clinical presentation of Pneumocystis infections.

Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

Circulation of Pneumocystis dihydropteroate synthase mutants in France.

Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation <30 days, iii) infection incubation is assumed to last about 2 months, the results provide evidence of mutant circulation from Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France.