Pathogenicity of Tritrichomonas mobilensis: subcutaneous inoculation in mice

The standard "subcutaneous mouse assay" was used to investigate the inherent pathogenicity of Tritrichomonas mobilensis, an intestinal parasite of squirrel monkeys. C57B1/6 mice given subcutaneous bilateral inocula of T. mobilensis died by day 4 postinoculation with lesions too small to be measured. Control mice similarly inoculated with pathogenic and nonpathogenic strains of Trichomonas vaginalis survived the challenge and produced lesions on day 6 with mean volumes in agreement with previous reports. CD1 mice similarly inoculated with standard and double doses of trichomonads (T. mobilensis) again produced small lesions. CD1 mice inoculated at double dosage were moribund or dead on days 5 and 6, respectively, postinoculation. Necropsies were performed on dead and sacrificed mice. Tissues were obtained from internal organs for histology and culture. Unexpectedly, trichomonads were cultured from liver and lung of C57B1/6 mice at the standard level of inoculation and liver, lung, and spleen of CD1 mice at the higher level of inoculation. Although trichomonads are normally considered surface-dwelling noninvasive organisms, the penetration of trichomonads to deep tissues is not without precedent. Tritrichomonas foetus and Trichomonas gallinae are known to invade tissues of their respective hosts. Trichomonas vaginalis has been demonstrated in subepithelial areas of both the prostate gland and cervix of humans. The ability of several species of trichomonads to invade tissues and/or migrate to other sites in their hosts suggests a need for revision of the concept of trichomonads as strictly lumen or surface-dwelling parasites.     

Electron microscope observations on the interaction of Mycoplasma fermentans with Trichomonas vaginalis

Cultures of Trichomonas vaginalis were found to be contaminated with Mycoplasma fermentans. By means of electron microscopy the interaction between the prokaryotic organisms and the trichomonads was examined. Cells of M. fermentans were observed in the medium; some of them were attached to the surface of the trichomonads and others were observed in membrane-bounded vacuoles of trichomonads. They were also present in the ground substance of the cytoplasm. The mycoplasmas divided by binary fission like other prokaryotes. The most obvious change occurring in the infected trichomonad cells was an increase in number of vacuoles containing mycoplasmas.     

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.     

Passive Immunization of Pigeons Against Trichomoniasis

SYNOPSIS Nonimmune homing pigeons Columba livia were infected with the Jones' Barn strain of Trichomonas gallinae and subsequently transfused with plasma from acute or chronically infected pigeons harboring one of 3 different strains of T. gallinae. The transfusions were either a single 2 ml dose given one day after inoculation or three 1 ml doses given 0, 5, and 10 days after inoculation. Plasma from pigeons harboring any of the 3 strains was capable of passively immunizing nonimmune birds. All birds which were immunized with plasma from infected pigeons survived until killed at the end of the test period and no visceral lesions were found on necropsy but trichomonads were present in the oropharynx. All controls (untreated or transfused with normal plasma) died of visceral trichomoniasis.
Immune plasma produced some lysis of trichomonads in vitro, and inhibition of motility and vacuolization occurred in some of the non-lysed organisms. The overall lytic activity in vitro affected less than 10% of the suspended trichomonads.     

A simple micropore system for experimental studies on trichomonad parasites

A simple leak-free micropore chamber containing protozoan parasite species was implanted subcutaneously on the back of hamsters and evaluated for viability and multiplication of protozoan parasites. Trophozoites of defined strains of Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Tritrichomonas foetus were used; their survival and multiplication in the chambers formed the basis of evaluation. Entamoeba histolytica and G. lamblia did not survive more than 6 hr and succumbed due to cellular adhesion. Trichomonas vaginalis and T. foetus survived 3 and 6 days and multiplied a maximum of 3.6 and 26 times, respectively. This indicated that exchange of body fluids and cells needed for the survival and multiplication of trichomonads readily occurs. This preliminary observation showed that micropore chambers may be useful for chemotherapeutic and immunological studies on trichomonads in ectopic sites.     

Ultrastructural changes of Trichomonas vaginalis prior and after cryopreservation

Fine structural changes of Trichomonas vaginalis are described prior and after the freezing process in liquid nitrogen. Dimethyl sulfoxide (DMSO) used as the cryoprotectant caused distinct alterations of the cytoplasm when trichomonads were equilibrated with 5% DMSO under various experimental conditions. Changes were bubble-like protrusions, fissuration and/or vacuolation of the cytoplasm, doubling and removal or/and rupture of the cell membrane. Apart from these findings cryopreservation caused marked alterations on the hydrogenosomes, such as condensation and flocculence of the usually homogeneous contents; in addition numerous hydrogenosomes fused while loosing membrane at the site of fusion. However, several parasites revealed normal hydrogenosomes after the freezing process. It is assumed that these organisms survived freezing and thawing as demonstrated by successful cultivation of recovered trichomonads.     

Investigation of the significance of Oil Red O‐positive macrophage excess in bronchoalveolar lavage fluid during HIV infection

L. Lacoste‐Collin, G. Martin‐Blondel, C. Basset‐Léobon, V. Lauwers‐Cancès, D. d’Aure, J. Aziza, A. Berry, B Marchou, M.B. Delisle and M. Courtade‐Saïdi Investigation of the significance of Oil Red O‐positive macrophage excess in bronchoalveolar lavage fluid during HIV infection Objective: To assess the significance of increased levels of Oil Red O‐positive macrophages (ORO‐PM) in bronchoalveolar lavage fluids (BALFs) from HIV‐positive patients. Methods: Cytological data for seventy BALF samples from 66 consecutive HIV‐infected patients were analysed according to antiretroviral therapy regimen, presence of Pneumocystis jiroveci infection, blood CD4⁺T cell count, HIV‐1 viral load and plasma lipid levels. Non‐parametric tests were used to compare the values between groups. Results: The percentages of ORO‐PM were high in this group: 40% [6–80] (median [interquartile range]). They were positively correlated with the BALF total cell count, 21% [5–48.5] for <300 cells/mm³ and 60% [26.5–80] for >300 cells/mm³ (P < 0.01) but inversely correlated with the percentage of BALF lymphocytes, 50% [20–80] for <15% lymphocytes and 11.5% [2–47] for ≥15% lymphocytes (P < 0.01). Antiretroviral therapy with or without protease inhibitors, plasma lipid levels, HIV‐1 viral load, blood CD4⁺T cell count or presence of a Pneumocystis jiroveci infection were not correlated with the ORO‐PM status. Conclusion: Significantly increased numbers of ORO‐PM were correlated with high total cell counts and low lymphocyte counts in BALF, irrespective of disease activity or treatment. Extended work on a larger series of patients needs to be conducted.     

Axenic Cultivation of Trichomonas tenax, the Oral Flagellate of Man I. Establishment of Cultures

SYNOPSIS. A technique for the axenic cultivation of Trichomonas tenax , the oral flagellate of man, is presented. The medium employed consists of a nutrient broth supplemented with horse serum and a cell-free extract of chick embryo. Three strains established in this medium have been maintained for 16, 23 and 48 months respectively.
All the cultures were initiated with trichomonads grown in association with Trypanosoma cruzi. Attempts to establish axenic cultures with trichomonads obtained from xenic cultures containing a bacterial flora of unknown composition met with failure. This suggests that the successful cutcome of the process of axenization is to a certain extent dependent upon the type of organism(s) with which T. tenax is associated in culture. Furthermore, these findings may serve to explain earlier failures since not only were all of these attempts made with media lacking tissue extract supplements but all were made using bacteria-trichomonad cultures as a source of trichomonads.     

Comparative study of ferredoxin-linked and oxygen-metabolizing enzymes of trichomonads

1. The activities of pyruvate:methyl viologen oxidoreductase (EC 1.2.7.1), hydrogenase (EC 1.18.99.1), NADH:methyl viologen oxidoreductase (EC 1.6.99.3), NADPH:methyl viologen oxidoreductase (EC 1.6.99.1), NADH oxidase (EC 1.6.99.3) and NADPH oxidase (EC 1.6.99.1) were determined for Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum. 2. The three trichomonad species were found to differ significantly, especially with respect to NADH oxidase and NADH:methyl viologen oxidoreductase activities. 3. The species differences in ferredoxin-linked and oxygen-metabolising enzymes may be related to the ways in which the trichomonads are adapted for growth in their respective hosts.     

Reactive type II pneumocytes in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the prevalence of reactive type II pneumocytes (RPII) in bronchoalveolar lavage (BAL) fluid samples obtained from patients with various pulmonary disorders. STUDY DESIGN: Consecutive BAL fluid samples were screened for the presence of RPII on May-Grünwald-Giemsa-stained cytocentrifuge preparations. BAL fluid samples with and without RPII were compared with regard to prevalence, associated clinical diagnoses and cytologic findings. RESULTS: RPII were generally large cells with a high nuclear:cytoplasmic ratio and deeply blue-stained, vacuolated cytoplasm. Most RPII occurred in cohesive cell groups, and the vacuoles tended to be confluent. Cytologic findings associated with RPII were foamy alveolar macrophages, activated lymphocytes and plasma cells. RPII were present in 94 (21.7%) of 433 included BAL fluid samples. The highest prevalences were noted in patients with systemic inflammatory response syndrome and alveolar hemorrhage. In addition, RPII tended to occur more frequently in ventilator-associated pneumonia, Pneumocystis carinii pneumonia, extrinsic allergic alveolitis and drug-induced pulmonary disorders. In contrast, RPII were not observed in BAL fluid samples obtained from patients with sarcoidosis. CONCLUSION: RPII were prevalent in about 20% of BAL fluid specimens. They were associated mainly with conditions of acute lung injury and not observed in sarcoidosis.     

Effect of iron on the virulence of Trichomonas vaginalis

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T. vaginalis is reduced.     

Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli

Pneumocystis pneumonia or PCP is caused by Pneumocystis jirovecii, an obligate parasite of the human lung. In this study P. jirovecii genomic sequence encoding FAS, a trifunctional protein including dihydroneopterin aldolase (DHNA), hydroxymethyldihydropterin pyrophosphokinase (PPPK) and dihydropteroate synthase (DHPS) were identified by PCR amplification from fixed broncheolar lavage samples from patients having Pneumocystis pneumonia. The P. jirovecii trifunctional DHNA-PPPK-DHPS genes (PjFAS) showed a high degree of conservation with the rat Pneumocystis carinii and P. carinii f. sp. macaca sequences. To test the functionality of the PjFAS sequences introns were removed followed by cloning and expression of PjFAS sequences in a DHPS-disrupted Escherichia coli strain. Complementation depended on the presence of N-terminal FAS sequences in addition to a glutathione S- transferase tag to the N-terminus of PjFAS. Functional complementation allowed evaluation of DHPS mutations implicated with sulfa drug resistance.     

Diagnostic utility of p16 immunocytochemistry for Trichomonas in urine cytology

We present a case in which p16 immunocytochemistry helped establish the diagnosis of Trichomonas in urine from a male patient. Based on this finding, we recommend p16 immunocytochemistry as a diagnostic tool for unexpected patients or specimen types in which potential trichomonads are identified following routine cytologic evaluation.     

Trichomonas vaginalis: reactive oxygen species mediates caspase-3 dependent apoptosis of human neutrophils

There are many neutrophils in the vaginal discharge from women infected with Trichomonas vaginalis. The aim of our study was to determine whether human neutrophil apoptosis may be regulated by reactive oxygen species (ROS) in response to trichomonads infection. Incubation of human neutrophils with live trichomonads caused marked receptor shedding of CD16, decrease of mitochondrial membrane potential (MMP) and caspase-3 activation in human neutrophils. These proapoptotic effects of T. vaginalis on neutrophils were inhibited by pretreatment of neutrophils with an inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI), suggesting an important role of intracellular ROS accumulation in T. vaginalis-triggered apoptosis. Indeed, large amounts of ROS levels were detected in neutrophils incubated with live trichomonads, and were also effectively inhibited by DPI. However, pan-caspase inhibitor z-VAD-fmk or caspase-3 inhibitor z-DEVD-fmk did not affect T. vaginalis-induced ROS generation in neutrophils. These results suggest that ROS-dependent caspase-3 activation plays an important role in apoptosis of human neutrophils induced by T. vaginalis.     

Bacteriological Assessment of Healthcare-Associated Pneumonia Using a Clone Library Analysis

Background

The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen of HCAP.

Methods

We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed.

Results

Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. Staphylococcus aureus was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method.

Conclusions

The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.     

Serum lactic dehydrogenase predicts mortality in patients with AIDS and Pneumocystis pneumonia.

Serum lactic dehydrogenase (LDH) activity was compared with mortality in patients with the acquired immunodeficiency syndrome (AIDS) and Pneumocystis carinii pneumonia during the first four days of admission to assess the test''s predictive value. In 30 admissions, 29 patients who survived an episode of Pneumocystis pneumonia had a mean LDH value of 385 IU, with five values greater than 520 IU. Eight with pneumonia who died had a mean value of 926 IU: all had values higher than 520 IU. The mean LDH values for 20 patients with AIDS (35 admissions) who survived and 4 who died of non-Pneumocystis disease were 240 IU and 350 IU, respectively; these patients were the control population. The positive and negative predictive values for survival using 520 IU as the threshold are 61% and 100%. Thus, LDH measurements in the first days of admission for P carinii pneumonia predict mortality and are useful in guiding future management.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

The interaction of Trichomonas vaginalis with epithelial cells, polymorphonuclear leucocytes and erythrocytes on vaginal smears: light microscopic observation

In this study, vaginal smears taken from 400 patients were examined cytologically using the Papanicolaou technique. Twenty of the 400 patients were detected as harbouring Trichomonas vaginalis. The interactions of T. vaginalis with epithelial cells, polymorphonuclear leucocytes (PMNLs) and erythrocytes were determined at light microscopic level. It was observed that T. vaginalis were juxtaposed to the epithelial cells and changed shape according to the contours of the epithelial cell revealing the cytopathic effect of trichomonads on epithelial cells. Trichomonads attached to PMNLs produced pseudopodia to phagocytose the cells. Occasionally an amoeboid shaped T. vaginalis organism was seen trailed by a row of PMNLs. This light microscopic study supports the production by trichomonads of a chemotactic factor for PMNLs. Phagocytosed erythrocytes were also seen in the cytoplasm of T. vaginalis, suggesting the need for the patient to be tested for anaemia.