Frequency of trichomonads as coinfecting agents in Pneumocystis pneumonia

OBJECTIVE: To investigate the incidence of the association of Trichomonas and Pneumocystis in the lung. STUDY DESIGN: Sixty-six bronchoalveolar lavage fluid (BALF) samples from immunocompromised patients with pneumocystosis were retrospectively examined microscopically. RESULTS: Trichomonads were found as coinfecting agents in 60% of BALF samples. The frequency and abundance of trichomonads was increased, up to 100%, in cases rich in Pneumocystis. CONCLUSION: The data suggest that pulmonary Trichomonas infection occurs frequently in the course of Pneumocystis pneumonia. The role of trichomonads in causing alveolar damage during Pneumocystis pneumonia is hypothetical.     

Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences

The presence of Pneumocystis organisms was detected by nested-PCR at mitochondrial large subunit (mtLSU) rRNA gene in 23 respiratory samples from Asian macaques representing two species: Macaca mulatta and M. fascicularis. A very high level of sequence heterogeneity was detected with 18 original sequence types. Two genetic groups of Pneumocystis could be distinguished from the samples. Within each group, the extent of genetic divergence was low (2.5+/-1.4% in group 1 and 2.3+/-1.7% in group 2). Genetic divergences were systematically higher when macaque-derived sequence types were compared with Pneumocystis mtLSU sequences from other primate species (from 5.3+/-2.7% to 19.3+/-3.0%). The two macaque-derived groups may be considered as distinct Pneumocystis species. Surprisingly, these Pneumocystis species were recovered from both M. mulatta and M. fascicularis suggesting that host-species restriction may not systematically occur in the genus Pneumocystis. Alternatively, these observations question about the species concept in macaques.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.     

Pneumocystis carinii and Pneumocystis wakefieldiae in Wild Rattus norvegicus Trapped in Thailand

ABSTRACT. This work reports for the first time the presence of two Pneumocystis species in wild Rattus norvegicus specimens from Thailand. Pneumocystis DNA was detected in 57.7% (15/26) wild rats without apparent association with typical pneumocystosis. Pneumocystis carinii was found alone in five rats (19.2%), Pneumocystis wakefieldiae was detected alone in six rats (23.1%), and two rats were infected by both species (7.7%). In addition, a new P. wakefieldiae variant sequence has been identified in three wild R. norvegicus specimens caught in the same geographical area. The high frequency of Pneumocystis in wild rats documented in this study and the apparent scarcity of severe pneumocystosis were consistent with an efficient circulation of rat Pneumocystis species in ecosystems.     

An evaluation of the occurrence of pneumocystis infection through examination of pharyngeal and oral swabs using the nested PCR method

A method of collecting pharyngeal and oral swabs from humans and laboratory rats, to be examined later for Pneumocystis infection with simple and nested PCR, was developed. The swabs were obtained from 15 HIV-infected patients, including 5 suffering from PCP, and from 10 immunocompetent children aged 2 to 6 years. Furthermore, the swabs were taken from 30 healthy laboratory rats and 23 animals subjected to immunosuppressive treatment. DNA of Pneumocystis was detected in all the examined rats with nPCR method, but only in 47% of healthy animals when simple PCR was used. Nested PCR examination of swabs collected from human subjects revealed the infection with Pneumocystis in all HIV-infected patients with PCP, and in 8 out of 10 symptomless carriers of Pneumocystis; moreover, the examination was positive in 2 out of 10 immunocompetent children. It was concluded, that noninvasive method of collecting pharyngeal and oral swabs in conjunction with very sensitive method of amplification DNA by nPCR is suitable for measuring the prevalence of Pneumocystis infection in the populations of humans and laboratory animals. The developed method offers a possibility of safe diagnosis of pneumocystosis in patients whose clinical status precludes collection of BAL through bronchoscopy.     

Host-microbe interplay in persistent Staphylococcus aureus nasal carriage in HIV patients

It has been shown that persistent Staphylococcus aureus nasal carriage results in increased bacterial dispersal and a higher risk of infection compared to non-or-intermittent S. aureus carriage. Although many studies investigated S. aureus nasal carriage in HIV patients, none compared persistent carriage to non-persistent carriage nor were studies performed in the HAART era. We investigated the host-microbe interplay of persistent S. aureus nasal carriage in HIV-infected patients by studying host determinants of persistent carriage as well as the genetic structure of S. aureus strains isolated. We compared this genetic structure with the previously determined population structure of S. aureus isolates obtained from healthy individuals. Between February 2004 and June 2005 all HIV patients visiting the outpatient department of Erasmus MC (Rotterdam, The Netherlands) were asked to participate in this study. Participants were interviewed and screened for persistent S. aureus carriage using two semi-quantitative nasal swab cultures. For 443 patients two cultures were available, 131 (29.6%) were persistent carriers, which is significantly higher as compared to healthy individuals from the same geographic region (17.6%; P<0.0001). Male sex (odds ratio [OR], 2.22; 95% confidence interval [CI], 1.32-3.73), current smoking (OR, 0.58; 95% CI, 0.38-0.90), Pneumocystis jiroveci pneumonia (PCP) prophylaxis (OR, 0.39; 95% CI, 0.16-0.97) and antiretroviral therapy (OR, 0.61; 95% CI, 0.38-0.98) were independent determinants of persistent carriage. Only two strains were mecA positive (1.2%) and no PVL positive strains were detected. The population structure of S. aureus strains isolated from HIV patients appeared to be strongly overlapping with that of S. aureus isolates from healthy individuals.     

Population structure of rat-derived Pneumocystis carinii in Danish wild rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Structure of Major Surface Determinants and DNA Diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which, causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii , we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105–120 kd. It includes 6 isoelcclric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunorcactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

Diagnosis,Burden and Mortality of Pneumocystis jirovecii Pneumonia in Venezuela

Current Fungal Infection Reports - The aim of this work is to contribute to the knowledge of diagnosis, burden, and mortality of pneumocystosis or Pneumocystis jirovecii pneumonia (PCP) in...     

Healthy skin of many animal species harbors papillomaviruses which are closely related to their human counterparts

Papillomaviruses associated with clinical symptoms have been found in many vertebrate species. In this study, we have used an L1 gene consensus PCR test designed to detect a broad spectrum of human skin papillomaviruses to analyze swab samples from healthy skin of 111 animals belonging to 19 vertebrate species. In eight of the species, papillomavirus DNA was found with the following prevalences: chimpanzees, 9 of 11 samples positive; gorillas, 3 of 4; long-tailed macaques, 14 of 16; spider monkeys, 2 of 2; ruffed lemurs, 1 of 2; cows, 6 of 10; European elks, 4 of 4; aurochs, 1 of 1. In total, 53 new putative animal papillomavirus types were found. The results show that skin papillomaviruses can be detected in healthy skin from many different animal species and are sufficiently related genetically to their human counterparts to be identified by a human skin papillomavirus primer set (FAP59 and FAP64).     

Epidemiology of Pneumocystis jirovecii Pneumonia in Venezuela

Current Fungal Infection Reports - The aim of this work is to contribute to the knowledge of the epidemiology of pneumocystosis or Pneumocystis jirovecii pneumonia (PCP) in Venezuela, by an updated...     

Structure of major surface determinants and DNA diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105-120 kd. It includes 6 isoelectric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunoreactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

Pneumocystis jirovecii pneumonia in developing countries

Pneumocystis pneumonia (PcP) is a serious fungal infection among immunocompromised patients. In developed countries, the epidemiology and clinical spectrum of PcP have been clearly defined and well documented. However, in most developing countries, relatively little is known about the prevalence of pneumocystosis. Several articles covering African, Asian and American countries were reviewed in the present study. PcP was identified as a frequent opportunistic infection in AIDS patients from different geographic regions. A trend to an increasing rate of PcP was apparent in developing countries from 2002 to 2010.     

Update on Diagnosis of Pneumocystis Pulmonary Infections

Pneumocystis jirovecii is a widespread fungal colonizer of the human lung. Proliferation of the pathogen in the alveoli is controlled by the immune system in healthy individuals. When the immune system is impaired, pneumocystosis can emerge, resulting in a pulmonary infection. Formerly, the disease occurred mainly in acquired immune deficiency syndrome (AIDS) patients, accompanied by a high mortality. Now it is increasingly seen in patients with immunosuppressive treatment. Traditionally, laboratory diagnosis is based on the microscopic detection of cysts and trophic forms of P. jirovecii in respiratory samples. Quantitative PCR-based methods will revolutionize laboratory diagnosis. However, cutoffs have to be established to discriminate between colonization (clinically irrelevant) and infection. Furthermore, the data on the serological detection of (1→3)-ß-D-glucan to diagnose or exclude pneumocystosis is promising.     

Effect of sulfamethoxazole-trimethoprim and sulfadoxine-primethamine against fatal pneumocystosis in SCID mice.

The effect of sulfamethoxazole-trimethoprim (S-T) and sulfadoxine-primethamine (S-P) against Pneumocystis carinii (Pc) were examined in severe combined immunodeficiency (SCID) mice which are known to be susceptible to Pc. These animals develop fatal pneumocystosis without treatment with any immunosuppressant. The results suggested that S-T and S-P were effective against both trophic and cystic forms of Pc in SCID mice.     

Population Structure of Rat-Derived Pneumocystis carinii in Danish Wild Rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Pneumocystis carinii in corticosteroid-treated voles: a comparison of three different staining methods.

Pneumocystis carinii (PC) is an opportunistic pathogen which causes clinical disease in immunocompromised hosts. Three different staining protocols were employed to detect this organism in lung samples of corticosteroid treated voles in order to discover a suitable method for large-scale screening. The procedures employed were: Grocotts methenamine silver (GMS)-stained paraffin sections, toluidine blue O-stained impression smears, and methenamine-silver-stained frozen sections. GMS-stained paraffin sections were relatively easy to interpret and gave more positive results than the other methods. It seemed to be the satisfactory method for large-scale population analyses. An unexpected result was that methylprednisolone treatment did not induce in voles a similarly fatal pneumocystosis infection as occurred in rats. All infections found in voles were mild. This might be due to species-dependent differences in metabolizing methylprednisolone.     

Cost-effective analysis of genotyping using oral cells in the geriatric population

We evaluated the cost-effectiveness of using buccal swab brushes in comparison with blood samples for obtaining DNA for large epidemiological studies of the elderly population. The data reported here are from the third phase of the Integral Study of Depression among the Elderly in Mexico City's Mexican Institute of Social Security, conducted in 2007. The total cost of the two procedures was determined. The measurement of effectiveness was the quality and quantity of DNA measured in ng/μL and the use of this DNA for the determination of apolipoprotein E (APO E) polymorphism by PCR. Similar rates of amplification were obtained with the two techniques. The cost of the buccal swab brushes, including sample collection and DNA extraction, was US$16.63, compared to the cost per blood sample of US$23.35. Using the buccal swab, the savings was US$6.72 per patient (P < 0.05). The effectiveness was similar. Quantity and quality of DNA obtained were similar for the oral and blood procedures, demonstrating that the swab brush technique offers a feasible alternative for large-scale epidemiological studies.     

Methods for Identifying Neisseria meningitidis Carriers: A Multi-Center Study in the African Meningitis Belt

Objective

Detection of meningococcal carriers is key to understanding the epidemiology of Neisseria meningitidis, yet no gold standard has been established. Here, we directly compare two methods for collecting pharyngeal swabs to identify meningococcal carriers.

Methods

We conducted cross-sectional surveys of schoolchildren at multiple sites in Africa to compare swabbing the posterior pharynx behind the uvula (U) to swabbing the posterior pharynx behind the uvula plus one tonsil (T). Swabs were cultured immediately and analyzed using molecular methods.

Results

One thousand and six paired swab samples collected from schoolchildren in four countries were analyzed. Prevalence of meningococcal carriage was 6.9% (95% CI: 5.4-8.6%) based on the results from both swabs, but the observed prevalence was lower based on one swab type alone. Prevalence based on the T swab or the U swab alone was similar (5.2% (95% CI: 3.8-6.7%) versus 4.9% (95% CI: 3.6-6.4%) respectively (p=0.6)). The concordance between the two methods was 96.3% and the kappa was 0.61 (95% CI: 0.50-0.73), indicating good agreement.

Conclusions

These two commonly used methods for collecting pharyngeal swabs provide consistent estimates of the prevalence of carriage, but both methods misclassified carriers to some degree, leading to underestimates of the prevalence.     

Antibodies to the infective agents of opportunistic infections in blood of patients with hemoblastosis complicated with pneumonia

The examination of 112 hematological patients with diagnosed acute and chronic leucosis, lymphoma, myeloma, anemia, melanoma and other diseases revealed not a single subject among these examinees in whom no markers of opportunistic infections were detected. Low titers of antibodies to Pneumocystis carinii, cytomegalovirus (CMV), Epstein-Barr virus (EBV) were noted in 42%, 46.4% and 40.2% of examinees, respectively. Markers of acute diseases, such as class IgM, IgG antibodies in high titers, as well as P.carinii, CMV, EBV antigens, were detected in 37.5%, 30.4% and 22.3% of patients of a hematological hospital. In the group of comparison (donors) these figures were, respectively, 15.3%, 2.4% and 6.9%. The signs of monoinfection were detected in 11.6% (pneumocystosis), in 10.7% (CMV infection) and in 14.3% (EBV infection), while the markers of two infections, EBV infection and pneumocystosis, were detected in 9.8%, EBV and CMV infections in 11.6%, pneumocystosis and CMV infection in 14.3%; mixed contamination with all three infective agents was detected in 12.5% of the patients.