Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

Higher temperatures reduce the number of Trypanosoma cruzi parasites in the vector Triatoma pallidipennis.

BACKGROUND: Relatively little is known about how pathogens transmitted by vector insects are affected by changing temperatures analogous to those occurring in the present global warming scenario. One expectation is that, like their ectothermic vectors, an increase in temperature could reduce their fitness. Here, we have investigated the effect of high temperatures on the abundance of Trypanosoma cruzi parasites during infection in the vector Triatoma pallidipennis. METHODS: We exposed T. pallidipennis nymphs to two strains (Morelos and Chilpancingo) of T. cruzi. Once infected, the fifth-instar bugs were distributed among three different temperature groups, i.e. 20, 30, and 34 °C, and the resulting parasites were counted when the bugs reached adulthood. RESULTS: The number of parasites increased linearly with time at 20 °C and, to a lesser extent, at 30 °C, especially in the Chilpancingo compared to the Morelos strain. Conversely, at 34 °C, the number of parasites of both strains decreased significantly compared to the other two temperatures. CONCLUSIONS: These results suggest negative effects on the abundance of T. cruzi in T. pallidipennis at high temperatures. This is the first evidence of the effect of high temperatures on a pathogenic agent transmitted by an insect vector in the context of global warming. Further tests should be done to determine whether this pattern occurs with other triatomine species and T. cruzi strains.

Rectal Swabs as an Alternative Sample Collection Method to Bulk Stool for the Real-Time PCR Detection of Giardia duodenalis.

Though bulk stool remains the gold standard specimen type for enteropathogen diagnosis, rectal swabs may offer comparable sensitivity with greater ease of collection for select pathogens. This study sought to evaluate the validity and reproducibility of rectal swabs as a sample collection method for the molecular diagnosis of Giardia duodenalis. Paired rectal swab and bulk stool samples were collected from 86 children ages 0-4 years living in southwest Niger, with duplicate samples collected among a subset of 50 children. Infection was detected using a previously validated real-time PCR diagnostic targeting the small subunit ribosomal RNA gene. Giardia duodenalis was detected in 65.5% (55/84) of bulk stool samples and 44.0% (37/84) of swab samples. The kappa evaluating test agreement was 0.81 (95% CI: 0.54-1.00) among duplicate stool samples (N = 49) and 0.75 (95% CI: 0.47-1.00) among duplicate rectal swabs (N = 48). Diagnostic sensitivity was 93% (95% CI: 84-98) by bulk stool and 63% (95% CI: 49-75) by rectal swabs. When restricting to the lowest three quartiles of bulk stool quantitation cycle values (an indication of relatively high parasite load), sensitivity by rectal swabs increased to 78.0% (95% CI: 64-89, P < 0.0001). These findings suggest that rectal swabs provide less sensitive and reproducible results than bulk stool for the real-time PCR diagnosis of G. duodenalis. However, their fair sensitivity for higher parasite loads suggests that swabs may be a useful tool for detecting higher burden infections when stool collection is excessively expensive or logistically challenging.

Evaluation of Anatomically Designed Flocked Rectal Swabs for Use with the BioFire FilmArray Gastrointestinal Panel for Detection of Enteric Pathogens in Children Admitted to Hospital with Severe Gastroenteritis.

Diagnosing diarrheal disease is difficult in part due to challenges in obtaining and transporting a bulk stool specimen, particularly in resource-limited settings. We compared the performance of flocked rectal swabs to that of traditional bulk stool samples for enteric pathogen detection using the BioFire FilmArray gastrointestinal panel in children admitted to four hospitals in Botswana with community onset severe gastroenteritis. Of the 117-matched flocked rectal swab/stool pairs, we found no significant difference in pathogen detection rates between the flocked rectal swab samples and traditional bulk stool sampling methods for any bacterial (168 versus 167, respectively), viral (94 versus 92, respectively), or protozoan (18 versus 18, respectively) targets. The combination of flocked rectal swab samples with FilmArray testing allows for the rapid diagnosis of infectious gastroenteritis, facilitating a test-and-treat approach for infections that are life-threatening in many resource-limited settings. The culture recovery rates for bacterial pathogens utilizing this approach need to be assessed.

Trypanosoma cruzi surface mucins are involved in the attachment to the Triatoma infestans rectal ampoule.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process. METHODS/PRINCIPLE FINDINGS: Here we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2-3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfß1-4[Galpß1-6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfß1-4[Galpß1-6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins' O-glycans on the former but not the latter parasite clone. CONCLUSION/SIGNIFICANCE: These results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies.

Evaluation of rectal swab use for the determination of enteric pathogens: a prospective study of diarrhoea in adults.

OBJECTIVES: A stool sample is the sample of choice for microbiological testing of enteric pathogens causing diarrhoea, but a rectal swab can be a more practical alternative. A prospective observational study was performed to evaluate the diagnostic performance of flocked rectal swab specimens using the syndromic molecular approach to determine the aetiology of diarrhoea in adults. METHODS: We compared the performance of rectal swabs with stool samples as the reference standard in determining viral, bacterial and protozoal pathogens using real-time multiplex PCR as well as standard stool culture. Paired samples of stool and rectal swab specimens were collected from 304 adult patients with diarrhoea, presented at the Department of Infectious Diseases, University Medical Centre Ljubljana, between June 2016 and August 2017. RESULTS: Overall sensitivity of rectal swab samples in the syndromic molecular approach was 83.2% (95% CI 77.2%-88.1%). Pathogen group-specific analysis of rectal swabs showed sensitivity of 65.6% (95% CI 52.7%-77.1%) for viruses and 57.1% (95% CI 28.9%-82.3%) for parasites. For bacteria, sensitivity was 86.5% (95% CI 79.5%-91.8%) when PCR was performed and 61.4% (95% CI 52.4%-69.9%) when culture for bacteria was performed. Mean threshold cycle (Ct) values for most pathogens were higher in rectal swab specimens than in stool specimens. CONCLUSIONS: Our results indicate that rectal swabs can be used in the diagnosis of diarrhoea in adults when stool specimens are not available or when rapid aetiological determination is needed. However, rectal swabs should be analysed using a molecular approach. The mean Ct value for most pathogens is higher in rectal swab specimens than in stool specimens.

Cosmocerca bengalensis sp. nov. (Nematoda: Cosmocercidae) in Hoplobatrachus tigerinus (Daudin, 1803) (Amphibia, Anura, Dicroglossidae) from West Bengal, India.

Cosmocerca bengalensis sp. nov. (Ascaridida, Cosmocercidae) recovered from the rectum of an Indian bullfrog, Hoplobatrachus tigerinus (Daudin, 1803), collected from Hetampur town in Birbhum district of West Bengal, India, is described and illustrated. This species is similar to C. acanthurum, C. banyulensis, C. cruzi, C. japonica, C. kalesari, C. microhylae, C. novaeguineae, C. ornata, C. paraguayensis, C. parva, C. podicipinus and C. travassosi in having 5 pairs of plectanes supporting preanal papillae but differs from these species by general morphometry, absence of somatic papillae in females, absence of gubernaculum and having only one pair of adanal papillae in males and one pair postanal papillae in females except C. microhylae. Present parasites differ from C. microhylae by absence of gubernaculum and general morphometry. Cosmocerca bengalensis sp. nov. represents the thirtheith species assigned to the genus, seventh from Oriental region and fifth species from India.

Balantidium grimi n. sp. (Ciliophora, Litostomatea), a new species inhabiting the rectum of the frog Quasipaa spinosa from Lishui, China.

Balantidium grimi n. sp. is described from the rectum of the frog Quasipaa spinosa (Amphibia, Dicroglossidae) from Lishui, Zhejiang Province, China. The new species is described by both light microscopy (LM) and scanning electron microscopy (SEM), and a molecular phylogenetic analysis is also presented. This species has unique morphological features in that the body shape is somewhat flattened and the vestibulum is "V"-shaped, occupying nearly 3/8 to 4/7 of the body length. Only one contractile vacuole, situated at the posterior body, was observed. The phylogenetic analysis based on SSU-rDNA indicates that B. grimi groups together with B. duodeni and B. entozoon. In addition, the genus Balantidium is clearly polyphyletic.

First detection of Echinococcus granulosus sensu stricto (G1) in dogs in central Sudan.

Eighty-four stray dogs shot as a part of a governmental rabies control program in two neighboring towns of central Sudan were examined for the presence of Echinococcus spp. and other intestinal helminths. Echinococcus worms were identified to species level by PCR and gene sequencing. For comparative reasons, rectal content of the necropsied dogs was examined for helminth eggs and subjected to copro-PCR for Echinococcus. At necropsy, 51.2% (43/84) of the dogs harbored Echinococcus canadensis (G6/7) worms with worm burdens ranging from 22,000 to 80,000. Dipylidiun caninum was found in 53.6% of the dogs. At coproscopy, taeniid eggs were found in 37 of the 43 dogs which were positive for Echinococcus at necropsy, but none in the 41 necropsy-negative dogs. In addition, 58% of the rectal samples contained eggs of Toxocara spp., 34.5% eggs of Trichuris spp. (34.5%), and 26% eggs of Ancylostoma caninum. Copro-PCR gave positive results for E. canadensis with 97.5% (39/40) of nonhibiting samples from the necropsy positive dogs; the one remaining dog tested positive for E. granulosus sensu stricto (G1), whose partial cox1 and nad1 sequences showed a 100% identity with various reference sequences of the G1 genotype. 100% of 38 non-inhibited samples taken from the necropsy-negative dogs were also negative in copro-PCR. This is the first study which combines prevalence and genetic identification of Echinococcus spp. in dogs of Sudan. Together with a recent report from cattle, it confirms the autochthonous presence, at low level, of E. granulosus sensu stricto in Central Sudan.

Clinical diagnostic value of viable Schistosoma japonicum eggs detected in host tissues.

BACKGROUND: Schistosomiasis, one of the neglected tropical diseases, is endemic in more than 70 countries. However, the clinical diagnosis of patients with a low degree of infection is an unsolved technical problem. In areas endemic for schistosomiasis japonica, proctoscopy detection of eggs has been one method used for clinical diagnosis. However, it is often a challenge to find typical live eggs and it is difficult to distinguish live eggs from large numbers of partially degraded and/or completely degraded eggs within colon biopsy tissue. To address this problem, we tested six different morphological and biochemical/molecular markers (ALP; morphological characteristics of egg; CalS (calcified substance); AOS (antioxidase); SDHG (succinic dehydrogenase) and SjR2 mRNA (retrotransposons 2 of S.japonicum genome mRNA)), including four new markers (CalS; AOS; SDHG and SjR2 mRNA.), to determine the viability of S. japonicum eggs deposited in human and mouse colon tissues. Our ultimate aim is to obtain a new method that is more sensitive, practical and accurate to clinically diagnose schistosomiasis. METHODS: Tissue samples were collected from mice at six different time points during S. japonicum infection with or without treatment with praziquantel (PZQ). Four new biochemical or molecular markers were used for the detection of egg viability from mouse liver and intestinal samples: CalS; AOS; SDHG and SjR2 mRNA. Subsequently, all markers were employed for the detection and analysis of eggs deposited in biopsy materials from patients with suspected schistosomiasis japonica for clinical evaluation. Microscopic examination of the egg morphology, worm burden in vivo and ALP (alkaline phosphatase) levels were used as a reference standard to evaluate the sensitivity and reliability of four new markers detecting egg viability. RESULTS: The results of the study showed that the morphology of S. japonicum eggs deposited in tissues of hosts with schistosomiasis, especially cases with chronic schistosomiasis, is complex and egg viability is difficult to judge morphologically, particularly eggs with a fuzzy structure or partially modified eggs. We found that the majority of the viable schistosome eggs determined by four new markers (CalS, AOS, SDHG and SjR2 mRNA) were morphologically difficult to identify. CONCLUSIONS: Among the markers, the most sensitive and specific method was the detection of SjR2 mRNA and the most simple, rapid and practical method was the detection of SDHG. Therefore, the detection of SDHG is the most practical for clinical application and its use could improve the accuracy in diagnosing active schistosome infection.

Expansion of Comprehensive Screening of Male Sexually Transmitted Infection Clinic Attendees with Mycoplasma genitalium and Trichomonas vaginalis Molecular Assessment: a Retrospective Analysis.

Of 1,493 encounters of males at a sexually transmitted infection (STI) clinic in a community with a high prevalence of STI, Chlamydia trachomatis was detected in 8.7% and Neisseria gonorrhoeae was detected in 6.6%. Additional Trichomonas vaginalis and Mycoplasma genitalium screening found 17.4% and 23.9% of the encounters, respectively, to be positive for STI. STI agents were detected in 13.7% of urine specimens; addition of pharyngeal and rectal collections to the analysis resulted in detection of STI agents in 19.0% and 23.9% of encounters, respectively. A total of 101 (23.8%) encounters of identified STI involved sole detection of M. genitalium Expansion of the STI analyte panel (including M. genitalium) and additional specimen source sampling within a comprehensive STI screening program increase identification of male STI carriers.

Evaluation of Presto(plus) assay and LightMix kit Trichomonas vaginalis assay for detection of Trichomonas vaginalis in dry vaginal swabs.

This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05).

Clinicopathological characteristics and prognosis of schistosomal colorectal cancer.

AIM: The aim of the present study was to investigate the clinicopathological characteristics and prognostic factors of schistosomal colorectal cancer. METHOD: A total of 74 consecutive schistosomal colorectal cancer patients who underwent curative surgery from July 2009 to July 2012 were included in this study. The clinical and pathological characteristics of all 74 patients were analysed and univariate and multivariate analyses were performed. This study demonstrated positive correlations between the site of deposition of schistosomal eggs and certain essential variables. RESULTS: Depositional site of schistosome eggs, carcinoembryonic antigen (CEA) level and the pathological N and T stages were statistically significantly correlated with overall survival (OS). The pathological T stage and the CEA level were independent prognostic factors for OS. The site of deposition of schistosome eggs was positively correlated with the T and N stages, tumour size, the CEA level and the resection margins. CONCLUSIONS: Schistosome eggs might be associated with tumorigenesis. The site of deposition of schistosome eggs was statistically significantly correlated with OS but it was not an independent prognostic factor for OS. It was, however, correlated with the depth of the tumour. The presence of schistosoma eggs at the margin did not affect the patient's prognosis or anastomotic healing. The existing standard surgical approach was equally applicable to schistosomal colorectal cancer. It was not necessary to expand the scope of surgical resection.

On a new species of Cosmocerca (Nematoda: Cosmocercidae) from Microhyla rubra (Anura: Microhylidae) from West Bengal, India.

Cosmocerca microhylae sp. nov., recovered from the rectum of a red narrow-mouthed frog, Microhyla rubra (Jerdon, 1854) (Anura: Microhylidae), collected from Bolpur in the Birbhum district of West Bengal, India, is described and figured. This species is similar to C. acanthurum, C. banyulensis, C. cruzi, C. japonica, C. kalesari, C. novaeguineae, C. ornata, C. paraguayensis, C. parva, C. podicipinus and C. travassosi in having 5 pairs of plectanes supporting preanal papillae but differs from these species by smaller size, absence of somatic papillae in females and having only one pair of adanal papillae in males and one pair postanal papillae in females. Cosmocerca microhylae sp. nov. represents 27th species assigned to the genus, and 4th species from India.

Aplectana dubrajpuri sp. nov. (Nematoda: Cosmocercidae) in Hoplobatrachus tigerinus (Anura: Dicroglossidae) from Dubrajpur, Birbhum, West Bengal, India.

Aplectana dubrajpuri sp. nov., recovered from the rectum of Indian bull frog, Hoplobatrachus tigerinus, collected from Dubrajpur in the Birbhum district of West Bengal, India, is described and illustrated. This species is characterised by absence of gubernaculum and differs from other species of Aplectana which lack a gubernaculum (viz. A. akhrami, A. artigasi, A. chilensis, A. crossodactyli, A. crucifer, A. delirae, A. meridionalis, A. papillifera, A. praeputialis, A. tarija and A. vercammeni) by smaller size of males and females, absence of somatic papillae in females and number and distribution of caudal papillae in males which include 3 pairs precloacal, 1 pair adcloacal, 14 pairs postcloacal and a single unpaired small papilla on the upper lip of cloaca. Aplectana dubrajpuri sp. nov. represents 51st species assigned to the genus and 3rd from India.