Axenic Long-Term Cultivation of Pneumocystis jirovecii.

Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.

First molecular detection of Pneumocystis spp. in red foxes (Vulpes vulpeslinnaeus, 1758) and raccoon dogs (Nyctereutes procyonoidesgray, 1834).

Fungal organisms of the genus Pneumocystis may cause Pneumocystis pneumonia (PCP) in humans, but also domestic and wild mammals. Almost every animal species hosts its own genetically distinct Pneumocystis species, however information is sparse. In this study, 62 red foxes (Vulpes vulpes) and 37 raccoon dogs (Nyctereutes procyonoides) were collected in North-East Germany. The lung tissues of the animals were analysed by a new designed specific pan-Pneumocystis mtLSU rRNA gene PCR and sequencing. With this PCR, detection and discrimination of all known Pneumocystis spp. in a single step should be possible. This first detection of Pneumocystis spp. in 29/62 (46.8%) red foxes and 29/37 (78.4%) raccoon dogs indicated, that they harbour two dissimilar strains, as seen by specific single nucleotide position changes (SNPs). Nevertheless, five samples with contrary SNPs showed a probable inter-species transmission.

Occurrence of Gastrointestinal Parasites in Small Mammals from Germany.

An increase in zoonotic infections in humans in recent years has led to a high level of public interest. However, the extent of infestation of free-living small mammals with pathogens and especially parasites is not well understood. This pilot study was carried out within the framework of the "Rodent-borne pathogens" network to identify zoonotic parasites in small mammals in Germany. From 2008 to 2009, 111 small mammals of 8 rodent and 5 insectivore species were collected. Feces and intestine samples from every mammal were examined microscopically for the presence of intestinal parasites by using Telemann concentration for worm eggs, Kinyoun staining for coccidia, and Heidenhain staining for other protozoa. Adult helminths were additionally stained with carmine acid for species determination. Eleven different helminth species, five coccidians, and three other protozoa species were detected. Simultaneous infection of one host by different helminths was common. Hymenolepis spp. (20.7%) were the most common zoonotic helminths in the investigated hosts. Coccidia, including Eimeria spp. (30.6%), Cryptosporidium spp. (17.1%), and Sarcocystis spp. (17.1%), were present in 40.5% of the feces samples of small mammals. Protozoa, such as Giardia spp. and amoebae, were rarely detected, most likely because of the repeated freeze-thawing of the samples during preparation. The zoonotic pathogens detected in this pilot study may be potentially transmitted to humans by drinking water, smear infection, and airborne transmission.

IL-37 Causes Excessive Inflammation and Tissue Damage in Murine Pneumococcal Pneumonia.

Streptococcus pneumoniae infections can lead to severe complications with excessive immune activation and tissue damage. Interleukin-37 (IL-37) has gained importance as a suppressor of innate and acquired immunity, and its effects have been therapeutic as they prevent tissue damage in autoimmune and inflammatory diseases. By using RAW macrophages, stably transfected with human IL-37, we showed a 70% decrease in the cytokine levels of IL-6, TNF-α, and IL-1ß, and a 2.2-fold reduction of the intracellular killing capacity of internalized pneumococci in response to pneumococcal infection. In a murine model of infection with S. pneumoniae, using mice transgenic for human IL-37b (IL-37tg), we observed an initial decrease in cytokine expression of IL-6, TNF-α, and IL-1ß in the lungs, followed by a late-phase enhancement of pneumococcal burden and subsequent increase of proinflammatory cytokine levels. Additionally, a marked increase in recruitment of alveolar macrophages and neutrophils was noted, while TRAIL mRNA was reduced 3-fold in lungs of IL-37tg mice, resulting in necrotizing pneumonia with augmented death of infiltrating neutrophils, enhanced bacteremic spread, and increased mortality. In conclusion, we have identified that IL-37 modulates several core components of a successful inflammatory response to pneumococcal pneumonia, which lead to increased inflammation, tissue damage, and mortality.

Pneumocystis pneumonia (PCP) and Pneumocystis jirovecii carriage in renal transplantation patients: a single-centre experience.

BACKGROUND: The Pneumocystis pneumonia is an increasing problem in transplanted patients: up to 25% suffer from Pneumocystis pneumonia, occurring during the first 6 months after transplantation. METHODS: From 2001 to 2009, we investigated 21 patients with pneumonia after renal transplantation for the presence of Pneumocystis jirovecii. The laboratory diagnosis was established by Grocott and Giemsa staining methods and Pneumocystis-specific mitochondrial transcribed large subunit nested polymerase chain reaction (PCR). The PCR was also used for the differentiation of Pneumocystis pneumonia from Pneumocystis carriage. RESULTS: Of 21 patients, 7 had a Pneumocystis pneumonia, 6 were Pneumocystis carriers and 8 patients were negative. Four out of seven Pneumocystis pneumonia patients and two out of six patients with Pneumocystis carriage had a delayed graft function. An acute cytomegalovirus infection after transplantation was not detectable in the patients with Pneumocystis pneumonia, but in three patients with Pneumocystis carriage. CONCLUSIONS: Pneumocystis pneumonia was present in 33.3% of transplanted patients with suspected pneumonia. An association between acute rejection or co-infections and Pneumocystis pneumonia or carriage in patients after renal transplantation cannot be excluded. In three out of seven Pneumocystis pneumonia patients, an overlapping of hospitalisation times and an onset of Pneumocystis pneumonia 6 months after transplantation was found. Thus, person-to-person transmission seems probable in these cases.

Tick-borne encephalitis virus habitats in North East Germany: reemergence of TBEV in ticks after 15 years of inactivity.

The incidence of tick-borne encephalitis has risen in Europe since 1990 and the tick-borne encephalitis virus (TBEV) has been documented to be spreading into regions where it was not previously endemic. In Mecklenburg-West Pomerania, a federal state in Northern Germany, TBEV was not detectable in over 16,000 collected ticks between 1992 and 2004. Until 2004, the last human case of TBE in the region was reported in 1985. Following the occurrence of three autochthonous human cases of TBE after 2004, however, we collected ticks from the areas in which the infections were contracted. To increase the chance of detecting TBEV-RNA, some of the ticks were fed on mice. Using nested RT-PCR, we were able to confirm the presence of TBEV in ticks for the first time after 15 years. A phylogenetic analysis revealed a close relationship between the sequences we obtained and a TBEV sequence from Mecklenburg-East Pomerania published in 1992 and pointed to the reemergence of a natural focus of TBEV after years of low activity. Our results imply that natural foci of TBEV may either persist at low levels of activity for years or reemerge through the agency of migrating birds.

Clinical trial to evaluate the safety and immunogenicity of a trivalent surface antigen seasonal influenza vaccine produced in mammalian cell culture and administered to young and elderly adults with and without A(H1N1) pre-vaccination.

UNLABELLED: Vaccination against influenza is an important means of reducing morbidity and mortality in subjects at risk. The prevalent viral strains responsible for seasonal epidemics usually change annually, but the WHO recommendations for the 2011/2012-season in the Northern hemisphere included the same antigens as for the previous season. We conducted a single-center, single-arm study involving 62 younger (18-60 years) and 64 older (>60 years) adults to test the immunogenicity, safety and tolerability of a trivalent surface antigen, inactivated influenza vaccine produced in mammalian cell-culture. The vaccine contained 15 µg hemagglutinin of each of the virus strains recommended for the 2011-2012 Northern hemisphere winter season (A/California/7/09 (H1N1)-; A/Perth/16/09 (H3N2)-; B/Brisbane/60/08-like strain) in a non-adjuvanted preservative-free formulation. Antibody response was measured by hemagglutination inhibition 21 days after immunization. Adverse events and safety were assessed using subject diary cards and telephone interviews. Seroconversion or a 4-fold antibody increase in antibody titers was detectable against A(H1N1) in 68% of both younger and older adults, against A(H3N2) in 53% and 27%, and against the B influenza strain in 35% and 17%. Antibody titers of 40 or more were observed against A(H1N1) in 87% and 90% of younger and older adults, against A(H3N2) in 98% and 98%, and against the B influenza strain in 93% and 90%. Pre-vaccination antibody titers were protective against A(H1N1), A(H3N2) and B in 38%, 58% and 58%, respectively, of younger and in 43%, 88% and 70% of older adults. Among subjects with previous A(H1N1) vaccination only 48% of younger and 47% of older adults had protective A(H1N1) antibodies at inclusion. Adverse reactions were generally mild. The most frequently reported reactions were pain at the injection site, myalgia and fatigue. The vaccine generated protective antibodies against all three viral strains and had an acceptable safety profile in both younger and older adults. TRIAL REGISTRATION: ClinicalTrials.govNCT01422512.

Pneumocystis jirovecii colonization among renal transplant recipients.

AIM: Renal transplant recipients are at risk of developing Pneumocystis pneumonia (PcP), especially in the first 2 years after transplantation, with a mortality rate of up to 50%. No data are available on pulmonary colonization with Pneumocystis jirovecii in renal transplant recipients. The aim of this study was to determine the prevalence of pulmonary colonization with Pneumocystis jirovecii in renal transplant recipients and to find related risk factors. METHODS: We investigated the induced sputa of 70 renal transplant recipients for the presence of Pneumocystis jirovecii using nested polymerase chain reaction. RESULTS: Thirteen of 70 patients (18.6%) were colonized with Pneumocystis jirovecii. There was no significant correlation between colonization and immunosuppressive medication or regimens. However, colonized subjects had undergone transplantation longer ago than non-colonized subjects. 30.8% of those whose transplantation had taken place more than 8 years previously were colonized, in contrast to 11.4% of those whose transplantation had taken place less than 8 years ago (P = 0.059; odds ratio = 3.467, 95% confidence interval = 0.99-12.09). CONCLUSION: Most cases of Pneumocystis colonization were detected in those patients where renal transplantion had taken place more than 2 years previously. As most PcP cases occur within the first 2 years of transplantation, colonization does not seem to play a role in the development of acute PcP in this period. Though Pneumocystis pneumonia is likely to be a newly acquired infection in the first 2 years after transplantation, colonized patients remain a potential source of transmission of Pneumocystis jirovecii.

Imbalance of pro- and antifibrogenic genes and bile duct injury in murine Schistosoma mansoni infection-induced liver fibrosis.

OBJECTIVES: The murine model of Schistosoma mansoni infection is characterized by strong fibrosis and little hepatocellular injury. The objective of this study was to evaluate the potential link between hepatic schistosomiasis and bile duct injury in relation to the expression of profibrotic cytokines and fibrosis-related genes. METHODS: Hepatic schistosomiasis was induced via percutaneous infection of mice with 50 S. mansoni cercariae. Markers of fibrosis including matrixmetalloproteinases (MMPs) and tissue-inhibitors of metalloproteinases (TIMPs), as well as markers of bile duct injury (keratin-19, VCAM-1) were studied during 24 weeks after infection by RT-PCR and immunohistochemistry. RESULTS: Liver biochemistry revealed no differences in serum transaminase and alkaline phosphatase levels in infected and uninfected mice. Total liver hydroxyproline content was increased 5-fold (P < 0.05) after infection. Gene expression analysis revealed MMP-2 (12-fold, P < 0.05) and TIMP-1 (48-fold, P < 0.05) up-regulation after infection. The balance of MMP and TIMP was shifted towards TIMP. Bile ducts were engulfed by adjacent granulomas resulting in ductular proliferation (keratin-19). VCAM-1 expression and inflammatory infiltrates were reduced. CONCLUSIONS: This study demonstrates that schistosomiasis is associated with (i) an imbalance of MMP-2 and TIMP-1 as key players of fibrogenesis and (ii) with secondary bile duct alterations leading to ductular proliferation possibly contributing to fibrosis.