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string | predicted_class
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|---|---|---|
Plus signs (+) indicate ticks infected with R. rhipicephali and slashes (∕) indicate ticks infected with R. philipii 364D. Pearson product moment correlation; R = − 0.44, P < 0.01. (A) Mission Trails; (B) Lopez Canyon; (C) Escondido Canyon; (D) Peñasquitos Canyon.
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| 85.75 |
Amplification of vertebrate cytochrome b gene was attempted to determine the origin of the ticks’ host blood meals; however, no cytochrome b was amplified from the ticks. This may have been due to ticks being captured before feeding as they were questing for a blood meal. SourceTracker analysis revealed that 31% of ticks had microbiomes that were between 1.1 and 27.4% similar to dog skin microbiomes (Table S2). Ticks negative for R. philipii or R. rhipicephali were more likely to have microbiomes similar to dog skin than ticks that were infected with R. philipii or R. rhipicephali (Generalized Linear Model; P = 0.023). Sphingomonadaceae, Oxalobacteraceae, and Comamonadaceae were the most abundant families of bacteria shared between tick and dog skin microbiomes. The tick microbiomes were less than 1% similar to microbiomes of the skins of fish, iguana, pigeon, rat, and human, as well as human oral, plant and soil microbiomes (Table S2).
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| 100.0 |
D. occidentalis is one of the most common tick species found in San Diego and is a vector of human pathogens including Francisella tularensis and Rickettsia philipii. This is the first study of the microbiome of D. occidentalis ticks using NGS technologies to examine pathogen interference within the tick microbiome. Although Francisella tularensis has been detected previously in ticks in San Diego (Kugeler et al., 2005), none of the ticks harbored this bacterium or genera of other recognized zoonotic tick-borne pathogens such as Borrelia, Anaplasma, Ehrlichia, Babesia or Bartonella; however, a low percentage of the ticks were infected with spotted fever group Rickettsia: 2.5% with R. philipii and 8.2% with R. rhipicephali. This is a slightly lower prevalence of R. philipii than surveys of ticks performed in Orange, Riverside, Los Angeles, Santa Barbara and Ventura counties north of San Diego, that reported an overall 7.5% prevalence of R. philipii (Wikswo et al., 2008) but is within the range of R. philipii prevalence reported from northern California of 0.4–5.1% (Lane et al., 1981; Philip, Lane & Casper, 1981). Similar to other tick species, the microbiome of D. occidentalis was dominated by Proteobacteria, primarily Rickettsia or Francisella, with lesser amounts of Sphingomonas, Methylobacterium and Hymenobacter (Bacteroidetes). These last three genera are all decomposer microbes found in the soil and except for Hymenobacter, have been detected in other tick microbiome studies. Even though the ticks were washed multiple times before DNA extraction, the possibility that some of these represent surface bacteria cannot be completely excluded. Although not performed in this study, removal of any OTUs detected by sequencing a sterile water negative control would also improve the sequence quality of future analyses. However, it is worth noting that members of the genus Sphingomonas were also found in two different studies of Ixodes tickes (Van Treuren et al., 2015), including one that studied larvae, suggesting that this genus may be arthropod-associated.
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| 99.94 |
Although 58 of the ticks were negative for SFGR by real-time PCR of the rompA gene and IGR, all of the ticks contained OTUs whose partial 16S rRNA gene segments aligned with SFGR in GenBank. The cause of this discrepancy may be due to the increased sensitivity of the Illumina sequencing platform compared to real-time PCR of rompA and IGR sequences and/or the presence of other Rickettsia spp. with highly conserved 16S rRNA genes but that lack rompA and IGR sequences complementary to the PCR primers used. Analysis of other genes would be required to resolve them at the species level (Eremeeva, Yu & Raoult, 1994; Regnery, Spruil & Plikaytis, 1991). Additional data support that more than two different Rickettsia species were present within the tick population tested. R. rhipicephali was detected by real-time PCR of the rompA gene and/or IGR in ticks that had OTU 837189 counts greater than 5900/tick, except for two ticks, T14-0667 and T14-0769 that had high OTU 837189 counts of 73,527 and 53,714, respectively, but were negative for R. rhipicephali. Similarly, R. philipii was detected in ticks with OTU 553807 counts ranging from 11 to 2,158, except for one sample, T14-0667, that had 884 counts of OTU 553807 yet was negative for R. philipii by real-time PCR of rompA gene and IGR. These findings are consistent with the presence of species of Rickettsia different from R. rhipicephali and R. philipii that could not be discriminated by the partial 16S rRNA gene or rompA and IGR sequences. The two most abundant Francisella OTUs, 840032 and 399541, accounted for over 90% of all Francisella OTUs and were 100% identical to Francisella-like endosymbionts (FLE) of D. occidentalis (GenBank accession numbers AY805304 and AY375402 for OTU 840032, and KU355875 for OTU 399541). Taken as a whole, these results are consistent with tick co-infection with a mixture of Rickettsias and FLEs.
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| 100.0 |
The number of unique OTUs detected in D. variabilis was 6.4 times higher than found in a study of Ixodes ticks, although, sequence depth was approximately two times greater in our study and, as noted, the vast majority of the OTUs occurred at very low frequencies (Van Treuren et al., 2015). OTUs that occurred only once in the data were removed. However, a presence threshold (i.e., requiring OTUs to be present in more than one tick) was not applied so that rare species that contributed to microbiome differences between locations would not be filtered out. This resulted in a large number of rare OTUs analyzed in our data, although, the interesting inverse relationship of the dominant endosymbiont species was not affected.
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| 100.0 |
The low frequency OTUs found in the study could have partially been the result of contamination.Although negative water controls did not yield visible products when subjected to 16S rRNA PCR, these controls were not subjected to NGS sequencing. SourceTracker analysis did not find evidence of skin contaminants commonly found in reagents or samples (Hewitt et al., 2013). SourceTracker was originally designed to analyze laboratory and built environment contamination and skin bacteria are the most common contaminants in these environments (Kelley & Gilbert, 2013), suggesting that investigator introduced contamination was not a major contributor to the low frequency OTU numbers. Nonetheless, in future studies would be much improved by the incorporating negative water extraction controls carried though to NGS. Future studies would also be significantly improved by following a more rigorous contamination protocol procedure such as that outlined by Kozich et al. (2013): https://github.com/SchlossLab/MiSeq_WetLab_SOP/blob/master/MiSeq_WetLab_SOP_v4.md.
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| 100.0 |
Similar to Ixodes scapularis and Amblyomma americanum ticks, female D. occidentalis ticks harbored a less diverse array of bacteria than males (Fig. 2) (Ponnusamy et al., 2014; Van Treuren et al., 2015). Endosymbionts belonging to Rickettsia, Coxiella, Francisella and Arsenophous genera have been found in different tick species and are thought to interfere with and partially exclude other bacteria and pathogenic forms of closely related organisms from transovarial transmission leading to lower alpha diversity in female ticks than males (Burgdorfer & Brinton, 1975; Macaluso et al., 2002; Niebylski, Peacock & Schwan, 1999; Noda, Munderloh & Kurtti, 1997; Reinhardt, Aeschlimann & Hecker, 1972; Telford III, 2009). In D. occidentalis, a higher percentage of Rickettsia and Francisella in the microbiomes of female ticks than male ticks may have similarly decreased species richness in female ticks compared to males (Fig. 2).
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| 99.94 |
The beta diversity of the endosymbionts and non-endosymbionts differed with respect to location. Although non-endosymbionts demonstrated a small association with location, geographic association was not observed by the Rickettsia and Francisella endosymbionts. In addition, Procrustes analysis results demonstrated that Rickettsia and FLE beta diversities had different relationships to geographical locations than the other microbiome components, illustrating that different factors shape Rickettsia and FLE components of the D. occidentalis microbiome than other non-endosymbiont microbiome members. One factor that appeared to contribute to the geographical differences in the non-endosymbiont microbiome was isolation by distance. Geographical differences in bacterial community composition in the same hematophagous insect species has been seen in fleas and ticks, however, the causes are not completely known (Jones, Knight & Martin, 2010; Van Treuren et al., 2015). Differential geographic localization of Nevskia, Curtobacterium and Sphingomonas, genera that are associated with environmental sources such as the air-water interface (Nevskia) and soils (Curtobacterium and Sphingomonas), may be the result of differences in soil microbial ecology at each location (Van Treuren et al., 2015). Alternatively, non-endosymbiont microbiome differences could be the result of stochastic or different populations of ticks at each location. In contrast, the dependency of Rickettsia and Francisella endosymbionts on their D. occidentalis host may have restricted the degree of variation that population separation could impart upon these endosymbionts (Budachetri et al., 2014).
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| 99.94 |
One of the primary hypotheses of this study was to determine if negative associations between bacteria, suggestive of interference, occurred within ticks especially with respect to pathogens. Indeed, a strong inverse relationship was observed between Rickettsia and FLE infection (Fig. 6) and a Random Forests supervised learning model successfully predicted the absence of SFGR within the ticks. Not surprisingly, FLE OTU 840032 contributed most to the model. FLE and different uncategorized Rickettsia co-infection in ticks has been previously observed but not enumerated (Niebylski et al., 1997; Scoles, 2004) and partial interference between co-infection by different Rickettsia species has been demonstrated (Burgdorfer, Hayes & Mavros, 1981; Macaluso et al., 2002). Although the quantitative 16S rRNA gene sequence data of FLE and Rickettsia co-infection in this study do not directly measure interference between the organisms, they are consistent with interference between FLE and Rickettsias and require further experimental studies for confirmation.
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| 100.0 |
The mechanisms by which Rickettsia and Francisella interfere with each other in co-infections are not known and there can be many kinds of interaction, direct or indirect, between microbes and the tick host which can be influenced by the infection behavior of the different bacterial species. Although the localization of R. rhipicephali and R. philipii within ticks has not been determined, FLEs have been found in female tick reproductive tissues and hemolymph (Goethert & Telford, 2005; Scoles, 2004). In addition, non-Francisella bacteria were also associated with low Rickettsia to Francisella ratios. Planococcaceae and Geobacillus were associated with greater abundance of Francisella relative to Rickettsia within the ticks. Although blood meals of the ticks could not be detected by amplification of vertebrate cytochrome b gene from the ticks, 31% of the tick microbiomes had microbiome components similar to canine skin which may suggest the source of a prior blood meal if they incorporated some of the skin flora into their own microbiome as has been shown with host blood microbiomes following feeding (Zhang et al., 2014). Use of SourceTracker for comparison of tick and skin microbiomes is a novel approach and, interestingly, generalized linear models showed that ticks with canine skin microbiome components were less likely to be infected with Ricketssia which is consistent with R. rhipicephali and R. philipii being endosymbionts without a canine host.
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| 99.94 |
The results of this study suggest that FLE and Rickettsia endosymbionts partially exclude each other in co-infections of the same D. occidentalis tick. Although interference between Rickettsia co-infections has been known for many years, this is the first study that suggests possible exclusion between different endosymbiont genera in ticks. Whether FLEs can be shown to inhibit Rickettsia co-infection in the laboratory, the mechanisms for their interaction and whether they could be propagated through a tick population as a means to render ticks unable to vector pathogenic Rickettsia are intriguing prospects that warrant further investigation. In addition to more mechanistic investigations, future research should also apply approaches such as shotgun metagenomics that can be used to assemble long sequence reads and even complete genomes from whole microbiome samples for more accurate species and strain identification.
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| 100.0 |
Ovarian cancer is the most lethal gynaecological cancer and the sixth most common cause of cancer related death among Western women . Although ovarian cancers represent only 30% of cancers of the female genital tract, they are responsible for half of the deaths . The disproportionately high mortality rate is attributed to the late presentation of the disease. Despite advances in surgery and chemotherapies, no substantial improvement in ovarian cancer survival has been observed over the last two decades . A greater understanding of the mechanisms involved in the progression of ovarian cancer will aid in the discovery of novel molecular prognostic indicators as well as new therapeutic targets. To increase our understanding of the molecular mechanisms involved in ovarian cancer progression and to identify novel therapeutic targets we recently studied the interaction of ovarian cancer and peritoneal cells [3–5]. Keratins K5 and K6c were amongst the proteins that were identified in the ovarian cancer peritoneal cell co-culture secretome by MALDI-TOF/TOF mass spectrometry .
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| 99.94 |
Keratins are intermediate filament proteins responsible for structural integrity of epithelial cells and play an important role in epithelial cell protection. They also play roles in cell polarization, cell size regulation, protein translation and organelle positioning . Fifty four functional keratin proteins have been identified in human epithelial cells including 28 type I (acidic forms, K9-K28) and 26 type II (basic forms, K1-K8 and K71-K74) proteins [7, 8]. They contain a central rod of ~310 amino acids with a helical conformation flanked by non-helical head and tail domains of variable length . A characteristic feature of keratin proteins is their pairing with other keratin proteins. They form obligate heterodimers between a type I keratin and a type II keratin via their rod domains and the resulting heterodimers and tetramers form the basic building units of the keratin filaments [7, 8].
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| 99.9 |
K5 (encoded by gene KRT5) is a high molecular weight (predicted 62.6 kDa), basic type keratin expressed in the basal, intermediate, and superficial layers of stratified epithelia as well as transitional epithelia and complex epithelium . It is most often complexed with K14 . K5 positive cells have been identified in both luminal and basal epithelium of the normal breast and K5 has been implicated as a stem cell marker [10, 11]. A recent study has highlighted that K5 expressing basal cells in the healthy and regenerating urothelium are self-renewing and unipotent .
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| 100.0 |
K6 protein is also a high molecular weight (predicted 60.3 kDa), basic type keratin known to be expressed by proliferating squamous epithelia and usually complexes with K16 . Three isoforms of K6 exist (K6a, K6b, and K6c) which are encoded by three distinct genes: KRT6A, KRT6B, and KRT6C [13, 14]. K6a is the most abundant, representing about 77% of all forms found in epithelia and shares at least 97.6% amino acid identity with other K6 proteins. K6a has been detected in subpopulations of luminal and ductal myoepithelial cells in human mammary glands . A high proliferative population of K6a positive cells has also been described in the prostate gland . There have been only a few studies which have investigated the expression of the K6c isoform in human tissues as until recently there was a lack of isoform-specific gene probes and antibodies.
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| 99.94 |
Monoclonal antibodies to K5 and K5/6 have been used to identify basal-like triple negative breast cancers [17, 18] and high K5/6 expression was found to be associated with an increased risk of breast cancer relapse and death [17, 19, 20]. Focal K5/6 expression has also been described in adenocarcinomas of the endometrium, pancreas and ovary [21, 22]. In addition, K5+ subpopulation of cells have been identified in ER+ PR+ luminal breast cancers [23, 24] and are increased in patients whose luminal breast cancers develop resistance to endocrine treatment and chemotherapy [25, 26].
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review
| 98.25 |
Whilst other keratins have been shown to have diagnostic or prognostic utility in ovarian cancer [27–30], limited studies to date have examined K5 and K6 expression in this malignancy. We therefore investigated the prognostic significance of KRT5 and KRT6 mRNA expression in publically available serous ovarian cancer data sets . Additionally, monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and cohorts of high grade serous ovarian carcinomas at surgery and after neoadjuvant chemotherapy. Furthermore, KRT5 and KRT6C mRNA expression was assessed in chemotherapy sensitive and chemotherapy resistant primary serous ovarian cancer cells derived from patient ascites. We also evaluated whether K5+ cells are increased in serous ovarian cancer patients following chemotherapy treatment. To our knowledge, this is the first study to investigate the relationships between KRT5 mRNA, KRT6 mRNA, K5/6, and K5 protein expression with serous ovarian cancer patient outcome.
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| 99.94 |
Using qRT-PCR KRT5 is expressed by metastatic OVCAR-5, OV-90, and SKOV-3 ovarian cancer cells, as well as by poorly metastatic OVCAR-3 cells but not the peritoneal cell line, LP-9 (Figure 1A). KRT6C was expressed by all ovarian cancer cell lines as well as LP-9 cells (Figure 1B). The K5/6 antibody detected bands at ~52 kDa in all cell line extracts and faint bands at ~56 kDa in protein extracts from OVCAR-5, OV-90 and SKOV-3 cells (Figure 1C). Using human ovarian cancer tissue extracts shown to express high and low K5/6 and K5 positivity (see inserts in Figure 1C and 1D) and antibodies to only K5, we confirmed that the 56 kDa and 52 kDa bands were K5 and K6, respectively. Two bands at ~52 kDa and ~56 kDa were observed with the K5/6 antibody in the ovarian cancer tissue extracts (Figure 1C), however the K5 antibody (Abcam) only detected a single band at ~56 kDa in the ovarian cancer tissue (Figure 1D).
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| 100.0 |
(A) KRT5 mRNA expression in ovarian cancer cell lines and LP-9 cells. (B) KRT6C mRNA expression in ovarian cancer cell lines and LP-9 cells. Relative expression was normalized to the house keeping gene β-actin using the 2-ΔΔCT method with the same calibrator (OVCAR-3). Data is expressed as the mean fold change ± SEM from 4-11 individual RNA samples obtained from 2-4 independent experiments. (C) Equal amounts protein for the cell lines (40 μg) and ovarian cancer tissue extracts (5 μg) were run on a 4-20% SDS-PAGE gel and immunoblotted with mouse monoclonal K5/6 antibody (1/200, clone D5/16 B4, Dako). K5/6 antibody detected K6 bands at ~52 kDa in all cell line extracts and faint K5 bands at ~56 kDa in cell extracts from OVCAR-5, OV-90 and SKOV-3 cells. Both K5 and K6 were detected in ovarian cancer tissue extracts. White asterisks indicate faint K5 bands detected with K5/6 antibody. (D) Western blotting with rabbit monoclonal K5 antibody (1/5000, clone EPR1600Y, Abcam) antibody using the same protein samples from (C) confirmed K5 expression in ovarian cancer tissue extracts. A mouse monoclonal antibody to β-actin (1/10,000, clone AC-15, Sigma Aldrich A3854) was used as a loading control. Ovarian cancer tissues sections with high and low K5/6 (C) or K5 (D) immunostaining were used as positive controls for the western blots (scale bar = 50 μm).
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| 100.0 |
K5/6 immunostaining was abundant in the skin epidermis (Figure 2A) but little or no staining was observed in the ovarian surface epithelium (OSE) of normal ovaries (8/8, Figure 2B, Table 1A). High K5/6 immunostaining (score 2 or 3) was present in 25% (2/8) of the benign serous cystadenoma (Figure 2C, Table 1A), 60% (6/10) high grade serous borderline tumors (Figure 2D, Table 1A) and 29.9% (35/117) of the serous ovarian cancer cases (Table 1A). Sixteen percent (19/117) of the serous ovarian cancer tissues were negative for K5/6. Examples of low (score = 1) and high (score = 3) K5/6 immunostaining in serous ovarian cancer tissues are shown in Figure 2E and 2F, respectively. K5/6 immunostaining was increased in serous borderline tumors and serous carcinomas compared to normal ovaries (P = 0.006, Chi-Square test, Table 1A). Similar staining patterns were observed with a monoclonal antibody which detects only K5 (Table 1C), (Supplementary Figure 1). However, a higher proportion of serous carcinomas (66%, 70/106) had high K5 immunostaining compared to K5/6 immunostaining. Neither K5/6 nor K5 immunostaining were associated with patient age, FIGO stage, tumor grade or the presence of residual disease (Supplementary Table 1).
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| 99.94 |
K5/6 immunohistochemistry using mouse monoclonal K5/6 antibody (1/50, clone D5/16 B4, DAKO) using Tris buffer (pH 9.0) microwave antigen retrieval. Human skin (A), normal ovary (B), benign serous cystadenoma (C), serous borderline tumor (D), serous carcinoma with low K5/6 immunostaining (E) serous carcinoma with high K5/6 immunostaining (F). K5/6 immunostaining in tissues obtained from the same patient at diagnosis (G) and following treatment carboplatin and paclitaxel (H). Scale bar = 100 μm. All images are same magnification.
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| 60.1 |
Using the publically available Kaplan-Meir online plotter tool which incorporates gene expression data from 13 ovarian cancer sets including the TCGA dataset , high KRT5 expression was associated with reduced progression-free survival (PFS, HR 1.38; 95% CI 1.16–1.64, P < 0.0001, Figure 3A) and overall survival (OS, HR 1.28 95%; CI 1.05–1.56, P = 0.013, Figure 3B). High KRT6 expression was also associated with reduced PFS (HR 1.26; 95% CI 1.07–1.47, P = 0.005, Figure 3C) but not OS (Figure 3D). No statistical correlation was found between KRT5 or KRT6 expression with patient age at diagnosis, tumor stage, tumor grade, or size of residual tumor after cytoreductive surgery in the TCGA dataset (data not shown).
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| 99.94 |
(A) Progression-free survival curve in stage III/IV serous ovarian cancers patients with low or high levels of KRT5 (Affymetrix probe set 201820_at). (B) Overall survival curve in stage III/IV serous ovarian cancers patients with low or high levels of KRT5 (Affymetrix probeset 201820_at). (C) Progression-free survival curve in stage III/IV serous ovarian cancers patients with low or high levels of KRT6 (Affymetrix probeset 209126_x_at, which detects all KRT6 isoforms). (D) Overall survival curve in stage III/IV serous ovarian cancers patients with low or high levels of KRT6 (Affymetrix probeset 209126_x_at, which detects all KRT6 isoforms).
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| 99.94 |
We confirmed in a cohort of advanced stage serous ovarian cancers that patients with high K5/6 or high K5 immunostaining (≥ 10.0%) had a significantly reduced PFS compared to patients with low K5/6 or low K5 positivity (< 10.0%, Figure 4A, 4C). The 24 months PFS rate was 34.4% in the group of patients with K5/6 positivity < 10% and only 10.5% in the group of patients with K5/6 positivity ≥ 10%. The 24 months PFS rate was 48.5% in the group of patients with K5 positivity < 10% and only 27.7% in the group of patients with K5 positivity ≥ 10%. Neither K5/6 nor K5 immunostaining was associated with OS (Figure 4B, 4D). Cox regression analysis also indicated that patients with high K5/6 positivity (≥ 10%) had a 1.78 fold increased risk of disease relapse (95% CI; 1.01–2.66, P = 0.017, Table 2). High K5 positivity was associated with a 1.90 fold increased risk of disease relapse (95% CI; 1.12–3.19, P = 0.017, Table 2). Other clinical and pathological parameters including patient age, clinical stage, tumor grade, and the presence of residual disease were not associated with PFS or OS in this advanced stage serous ovarian cancer cohort (Table 2).
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| 99.94 |
(A) Progression-free survival curve in stage III/IV serous ovarian cancers patients with high K5/6 immunostaining (≥ 10%, n = 30) and low K5/6 immunostaining (< 10%, n = 73, P = 0.014, log rank test). (B) Overall survival curve in stage III/IV serous ovarian cancers patients with high K5/6 immunostaining (> 10%, n = 33) and low K5/6 immunostaining (< 10%, n = 80, P = 0.673, log rank test). (C) Progression-free survival curve in stage III/IV serous ovarian cancers patients with high K5 immunostaining (≥ 10%, n = 63) and low K5 immunostaining (< 10%, n = 31, P = 0.015, log rank test). (D) Overall survival curve in stage III/IV serous ovarian cancers patients with high K5 immunostaining (≥ 10%, n = 69) and low K5 immunostaining (< 10%, n = 18, P = 0.144, log rank test).
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| 100.0 |
K5/6 was increased in serous ovarian cancer tissues following chemotherapy compared with chemonaïve tissues (P < 0.0001, Table 1B). Increased K5/6 immunostaining is evident in the images in Figure 2G and 2H that are an example of matching tissues from the same patient before and after chemotherapy treatment, respectively. Similar results were observed with the K5 monoclonal antibody (Table 1C, Supplementary Figure 1G and 1H). These findings are supported by mRNA expression studies in chemosensitive and chemoresistant primary ovarian cancer cells. KRT5 but not KRT6C mRNA expression was increased in primary cells derived from patients’ ascites with recurrent chemoresistant disease (n = 10) compared with primary cells from patients who responded to the chemotherapy treatment (n = 9; Figure 5A and 5B, P = 0.0006, Mann Whitney U test). KRT5 (Figure 5A), KRT6C mRNA (Figure 5B) and K5 protein (Figure 5C and 5D) were increased in OVCAR-5 cells made resistant to carboplatin (OVCAR-5-CBPR) compared with the parental OVCAR-5 cells by immunofluoresence. Furthermore, treatment with an IC50 dose of carboplatin significantly increased the number of K5+ cells in 4 serous ovarian cancer cell lines (OVCAR-5, OVCAR-3, OAW28 and COV362) (Figure 5C and 5D).
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| 100.0 |
KRT5 (A) and KRT6C (B) expression in chemotherapy resistant primary serous ovarian cancer cells (n = 10) compared to chemotherapy sensitive cells (n = 9) and OVCAR5 CBPR made resistant to carboplatin (CBP). KRT5 but not KRT6C was significantly increased in chemotherapy resistant cells (n = 10) compared to chemotherapy sensitive cells (n = 9, *P = 0.0006, Mann Whitney U test). Both KRT5 (*P < 0.0001, Mann Whitney U test) and KRT6C (*P = 0.0004, Mann Whitney U test) were significantly increased in CBP resistant OVCAR-5 CBPR cells compared to parental OVCAR-5 cells. Data for the primary cells is expressed as the mean fold change from 3-6 RNA samples from 2 independent experiments. Data for OVCAR5 cells is expressed as the mean fold change ± SEM from 10 individual RNA samples from 3 independent experiments. (C) K5 immunocytochemistry in serous ovarian cancer cells (OVCAR-5, OAW28, OVCAR-3 & COV362) ± 48 hr treatment with CBP IC50 and in parental OVCAR5 and CBP resistant OVCAR5 cells (OVCAR5 CBPR). (D) K5+ cells are increased following 48 hr treatment with an IC50 dose of CBP and the development of chemoresistance. *P < 0.05 (unpaired student t test), data is expressed as mean % positive cells ± SEM from 3-4 independent experiments.
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| 100.0 |
High grade serous ovarian carcinomas account for nearly 70% of ovarian malignancies. They are characterized by high initial chemosensitivity to platinum based therapies, however 75% of patients relapse after treatment and subsequently become chemotherapy resistant . The development of more effective molecularly targeted therapies to improve survival is urgently required. In this study we show that 1) KRT5 and KRT6C are expressed by ovarian cancer cell lines, 2) KRT5 expression levels predict reduced PFS and OS for serous ovarian cancer patients, 3) KRT6 expression levels predict reduced PFS but not OS for serous ovarian cancer patients, 4) Both high K5/6 or high K5 positivity in serous ovarian cancers can predict reduced PFS but not OS, 5) K5/6 and K5 immunostaining is increased in serous ovarian cancers following neoadjuvant chemotherapy, 6) KRT5 expression levels but not KRT6C are increased in serous primary cells derived from patients’ ascites with chemoresistant disease and 7) K5 protein expression is increased in serous ovarian cancer cell lines following carboplatin treatment. Our findings indicate that K5 expression could be used to predict serous ovarian cancer prognosis and may be used to identify cancer cells that are resistant to chemotherapy.
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| 99.94 |
K5 is usually detected using a K5/6 combination monoclonal antibody (clones D5/16B4) as it is closely related to K6. Co-expression of K5 and K6 has been reported in a number of different types of neoplasms including basal cell carcinoma , prostate cancer [34, 35], ductal breast carcinoma [36–38], mesothelioma [39, 40], lung carcinomas , melanoma, basal cell carcinoma, and salivary gland tumors . K5/6 overexpression is associated with poor prognosis of basal-like breast cancers [17, 18, 42] and was found to be an independent indicator of recurrence-free survival and/or OS in breast cancer [17, 19, 20, 43]. The K5/6 monoclonal antibody has been used for the diagnosis of poorly differentiated squamous carcinomas and undifferentiated nasopharyngeal carcinomas [21, 22]. It can distinguish between small cell lung carcinomas which are K5 negative and malignant mesothelioma which are K5 positive [21, 22]. K5/6 has superior sensitivity and reliability in differentiating between benign and malignant prostate glands when compared with K903 (high molecular weight keratins); and it has been used successfully in a five antibody panel (which also targets TRIM29, CEACAM5, SLC7A5, MUC1) to better classify the subtypes of lung carcinoma . The expression of K5/6 together with p63 has also been used to differentiate between adenosquamous carcinomas and adenocarcinomas in pleural effusion samples [45, 46]. Recently, K5 positive basal cells have also been identified as progenitors of bladder cancers .
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review
| 99.8 |
Several studies have investigated the expression of K5/6 in ovarian cancer but to date K5 or K6 expression has not been linked with ovarian cancer outcome. The incidence of K5/6 positivity (29.9%, Table 1B) in our study was similar to that observed in previous ovarian cancer studies which ranged from 25% to 55.4% [22, 48, 49]. However we observed a higher proportion of serous carcinomas (66%, Table 1C) with high K5 immunostaining which is comparable to a recent study reporting 50% K5 positivity in serous ovarian carcinomas . Our finding is in agreement with the study by Bhargava et al, 2008 who found that a monoclonal antibody to only K5 (clone XM26) was more sensitive than the K5/6 monoclonal antibodies (clones D5/16B4) in identifying basal-like breast carcinomas and reported a sensitivity of 97% for K5 but only 59% for K5/6 . The K5 antibody (clone EPR1600Y) used in our study is raised to a synthetic peptide in the head domain of keratin 5 whilst the K5/6 antibody clones were raised against purified keratin proteins. It has been suggested that the lower sensitivity of the K5/6 antibodies by immunohistochemistry may be caused by an interference between each other's antigenic binding sites by steric hindrance .
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study
| 100.0 |
Gene expression studies have previously identified only KRT5 mRNA and not KRT6 isoforms in normal breast and basal-like breast cancer in humans [11, 21, 51]. Consequently in normal breast tissues and cancer, the K5/6 antibody is thought to target only K5 . We found LP-9 peritoneal cells to express KRT6C but not KRT5. We confirmed that K6 protein is expressed in ovarian cancer cell lines and LP-9 cells but only faint K5 bands could be detected with K5/6 in OVCAR-5, OV-90 and SKOV-3 cell extracts. The low expression of K5 protein in ovarian cancer cell lines was confirmed by immunofluorescence as only 10–15% of ovarian cancer cells had detectable K5 protein without carboplatin treatment (see Figure 5C and 5D). The observed molecular weights of K5 (56 kDa) and K6 (52 kDa) were close to the predicted molecular weight of K5 (62 kDa) and K6 (60 kDa), respectively, and consistent with previous studies that have observed K5 at 56 kDa in rat liver cancer and K6 at 50 kDa in bladder cancer .
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study
| 100.0 |
K5/K14 form the main keratins in keratinocytes of stratified squamous epithelia of the epidermis as well as mucosal non-keratinizing stratified squamous epithelia . K5 is strongly expressed in the undifferentiated basal cell layer which contains stem cells and is reduced in the differentiating suprabasal cell layers . Our immunostaining in human skin using the K5/6 and K5 antibodies concur with this finding. Recent studies have reported that K5/K14 modulates cell proliferation and cell differentiation in the stratified epithelia via the P13K/Akt pathway and K5/K14 negatively regulates cell differentiation via the Notch 1 signaling pathway . Consequently K5/K14 is thought to play an important role in the maintenance of cell proliferation in the basal layer of stratified epithelia. It is likely that K5 regulates similar pathways in serous ovarian cancer cells.
|
study
| 100.0 |
Greater than 50% of ER+PR+ tumors contain ER-PR-K5+ subpopulations and K5+ cells are increased in ER+ breast tumors following treatment with neoadjuvant endocrine therapy . ER-PR-K5+ luminal breast cancer cell populations, termed ‘luminobasal’ cells exhibiting enhanced progenitor properties can be induced by progestins, glucocorticoids, as well as mineralocorticoids [26, 56–58] and blocked by anti-progestins and prolactin . Interestingly, these K5+ breast cancer cells were found to be less sensitive to 5-fluorouracil and docetaxel in in vitro culture and exhibited reduced apoptosis . A recent study investigated metastasis formation in ovariectomized mice injected with luminal breast cancer cell lines and assessed the metastatic process following treatment with estradiol or estradiol + progestin . The untreated ovariectomized mice were metastasis-free until they were supplemented with estradiol or estradiol + progestin. Unlike the parental cells that were predominately ER+PR+K5- the metastases formed following estradiol or estradiol + progestin contained significantly increased proportions of ER-PR-K5+ cells (6–30%). This finding may have important implications for women on hormonal contraception or replacement therapy who may harbor dormant K5+ micrometastases. It has also been suggested that basal-like breast cancers in BRCA1 deficient women may potentially arise from K5+ luminal progenitors . Compounds that can effectively target these K5+ cells have the potential to improve the outcome of luminal breast cancers and basal-like breast cancers. Targeting K5+ cells may also be effective in reducing recurrence in patients with serous ovarian carcinoma. Indeed many similarities have been observed between basal-like breast cancers and serous ovarian carcinoma .
|
study
| 96.94 |
A recent study by Corr et al (2015) demonstrating that K5+ ovarian cancer cells were more resistant to cisplatin-induced apoptosis than K5- cells has suggested that K5 is a marker of a chemoresistant subpopulation of ovarian cancer cells . Their observation that the number of K5+ cells increased following cisplatin treatment agrees with the carboplatin data presented in our study. We additionally showed that K5 and K5/6 immunostaining is significantly increased following neoadjuvant chemotherapy treatment and that KRT5 mRNA is increased in chemoresistant compared to chemosensitive serous primary ovarian cancer. These findings support the notion that K5 plays an important role in the development chemotherapy resistance.
|
study
| 100.0 |
In conclusion, this study found for the first time that serous ovarian carcinomas with increased KRT5 and KRT6 mRNA expression, as well as increased K5 or K5/6 immunostaining have an increased risk of disease relapse. K5/6 and K5 expression may therefore be used for predicting the prognosis of serous ovarian cancer patients and to aid patient management. In addition our findings that K5 is increased following carboplatin treatment and in chemotherapy resistance cells suggest that K5 could also be used to identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may prevent recurrence and chemotherapy resistance in serous ovarian cancer patients.
|
study
| 99.94 |
The human ovarian cancer cell lines OVCAR-3, SKOV-3, and OV-90 were purchased from American Type Culture Collection (ATCC, VA, USA). OVCAR-5 cells were obtained from Dr Thomas Hamilton (Fox Chase Cancer Center, PA, USA) and the peritoneal cells, LP-9 were purchased from Coriell Cell Repositories (NJ, USA). COV362 and OAW28 were purchased from the European Collection of Cell culture (ECCC). OV-90, OVCAR-3, SKOV-3 and OVCAR-5 cell lines were grown in RPMI 1640 media (cat no. R8758, Sigma Adrich, St Louis, USA) whilst COV362 and OAW28 were grown in DMEM media (cat no. 10567-022, Gibco, Life Technologies, Mulgrave, Vic, Australia). All cell lines are cultured with 10% fetal bovine serum (Sigma Aldrich) and maintained at 37°C in an environment of 5% CO2. OVCAR-5 cells were made resistant to carboplatin (OVCAR-5 CBPR) following treatment with 8 cycles of carboplatin (CBP, 50 μM, Hospira Australia Pty, Ltd). The OVCAR-5 CBPR cells exhibited an IC50 (273 μM) to carboplatin that was nearly 3-fold higher than that for the parental OVCAR-5 cells (99 μM) (data not shown)
|
study
| 100.0 |
Primary ovarian cancer cells were derived from ascites collected from serous ovarian cancer patients after informed consent and with approval of the Royal Adelaide Hospital Human Ethics Committee as described previously . All primary cells were grown in Advanced RPMI 1640 medium (cat no 12633-020) supplemented with 4 mM L-glutamine, 10% FBS (Sigma Aldrich, St Louis, MO, USA) and antibiotics (100 U penicillin G, 100 μg/ml streptomycin sulfate and 100 μg/ml amphotericin B, Sigma Aldrich). Methods were carried out in accordance with the approved guidelines. The clinicopathological characteristics of the patients whose ascites was used to isolate the primary cells are shown in Supplementary Table 2.
|
study
| 100.0 |
Cells were plated at 5,000 cells in 96 well plates and cultured until confluence for 72–96 hr. Total RNA was isolated and reverse transcribed using the TaqMan® Gene expression Cells-to-CT™ kit (Applied Biosystems, Mulgrave, Victoria, Australia), as per the manufacturer's instructions. Briefly, lysis solution with DNAse was added to each well and incubated for 5 min at room temperature. Stop solution was then added to each well and mixed. The lysate (10 μl) was added to a 40 μl reverse transcription master mix and reverse transcribed for 1 hr. Resultant cDNA was stored as 50 μl aliquots at −20°C for qRT-PCR analysis. qRT-PCR reactions were performed on triplicate samples using TaqMan® primer sets for KRT5 (Hs00361185_m1), KRT6C (Hs00752476_s1) using the Quantstudio 12K Flex Real Time PCR System (Applied Biosystems). Briefly, PCR reactions were made up to 10 μl and contained TaqMan® Gene Expression Master Mix (2×), primers for the gene of interest, nuclease free water, and the sample cDNA. PCR cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min (with 40 cycles following of 95°C for 15 sec), and 60°C for 1 min. CT values were normalised to the house keeping gene β-actin (Human ACTB 4333762, Applied Biosystems) and calibrator using the 2−ΔΔCT method.
|
study
| 99.94 |
OVCAR-5, OVCAR-3, OV-90, SKOV-3, and LP-9 cells a were grown to 80% confluence in 75 cm2 flasks (Corning, Sigma Aldrich) and cell extracts were collected. Cells were dislodged using a cell scraper and resuspended in 200 μl of RIPA buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M sodium chloride, 50 mM Tris- HCL and 1 mM EDTA, pH 8.0 with protease inhibitor) and spun at 7000 rpm (Eppendorf 5424 centrifuge) for 10 min and stored at −20°C. Equal amounts of protein were electrophoresed and transferred to PVDF membranes (GE Healthcare, Little Chalfont, England) as described previously . Proteins bands were detected with mouse monoclonal K5/6 antibody (1/200, clone D5/16 B4, Dako, Glostrup, Denmark) or K5 rabbit monoclonal antibody (1/5000, clone EPR1600Y, Abcam Ab75869, Melbourne, Vic, Australia) with anti-mouse or anti-rabbit IgG peroxidase-conjugated secondary antibodies, enhanced chemiluminescence, and autoradiography as described previously . β-actin mouse monoclonal antibody (1/10,000, clone AC-15, Sigma Aldrich A3854) was used as a loading control. Ovarian cancer tissue extracts prepared in RIPA buffer with high K5/6 and K5 immunostaining used as positive controls for the western blots.
|
study
| 100.0 |
Tissue sections were obtained from formalin fixed paraffin embedded blocks from normal ovaries (n = 8), benign serous tumors (n = 8), serous borderline tumors (n = 10), and primary advanced stage (FIGO stage III/IV) serous ovarian cancers (n = 126). The cancer tissues were assembled into tissue microarrays (TMAs, 1 mm triplicate cores) from archived tissue (cancer areas identified by pathologist, AR) obtained from serous ovarian cancer patients diagnosed between 1988 and 2012. An additional 21 tissues were obtained from patients with high grade serous cancer after they had received neoadjuvent chemotherapy. Approval was obtained from the Royal Adelaide Hospital Human Ethics Committee and methods were carried out in accordance with the approved guidelines. Detailed pathological and clinical characteristics of the patient samples are summarized in Supplementary Table 3.
|
study
| 99.94 |
The Kaplan-Meier plotter tool (http://kmplot.com/analysis/) was used to generate survival curves combining KRT5 (Affymetrix probe 201820_at) and KRT6 (Affymetrix probe 209126_x_at detects all KRT6 isoforms) mRNA data from 13 public ovarian cancer datasets . The Kaplan-Meier analysis was performed on the 2015 version database (n = 1648) and patients were split by the best cut-off selected by the online plotter tool . PFS and OS data was available for 922 and 956 stage III/IV serous ovarian cancer patients, respectively. cBioPortal (http://www.cbioportal.org/) was used to assess correlations between KRT5 and KRT6 expression levels with clinicopathological parameters features in the TCGA 2011 dataset [62, 63].
|
study
| 100.0 |
Immunohistochemistry was performed as previously described . Briefly, tissue sections (5 μm) underwent microwave antigen retrieval for 10 minutes at 100°C in a steam microwave (Sixth Sense, Whirlpool, VIC, Australia) in 10 mM Tris buffer, 1 mM EDTA (pH 9.0). Sections were incubated overnight with mouse monoclonal antibody which detects both K5 and K6 (1/50, clone D5/16 B4, Dako) or K5 rabbit monoclonal antibody (1/400, clone EPR1600Y, Abcam Ab75869), in blocking buffer (5% normal goat serum) at 4°C. Visualization of immunoreactivity was achieved using biotinylated anti-mouse or anti-rabbit immunoglobulins streptavidin-peroxidase conjugate, diaminobenzidine substrate as described previously . Human skin was used as a positive control and negative controls included tissues incubated with no primary antibody or with mouse or rabbit immunoglobulins. Slides were digitally scanned using the NanoZoomer Digital Pathology System (Hamamatsu Photonics, SZK, Japan) and images were collected using NDP view imaging software (NDP scan software v2.2, Hamamatsu Photonics). K5/6 or K5 expression was quantified using a visual grading system based on the extent of staining. Percentage of positive tumor cells was graded on a scale from 0–3; 0 = none, 1 = 1–9 %, 2 = 10–50%, 3 = > 50% by two independent assessors used previously for breast cancer [65, 66]. High K5/6 or K5 immunostaining was defined as ≥ 10% positivity and < 10% was defined as low K5/6 or K5 immunostaining.
|
study
| 100.0 |
Ovarian cancer cells (2 × 104 cells/well) were plated in 8 well tissue culture chamber slides (Nunclon™ Lab-Tek II Chamber slide, RS Glass Slide, Naperville, IL) in 500 μl 10% FBS RPMI for 24 h and treated for 48 h with IC50 concentration of CBP or control media. CBP IC50 for serous ovarian cancer cell lines were previously determined to be ~100 μM for OVCAR-5, OVCAR-3 and OAW28 cells and 500 μM for COV362 cells. Cells were fixed with cold 100% methanol (5 min) and cold 100% acetone (3 min), washed with PBS and blocked with 5% goat serum and incubated overnight with rabbit monoclonal K5 (1/50, clone EPR1600Y, Abcam Ab75869). K5 was visualized with goat anti-rabbit Alexa Fluor® 488 (1/200, 1hr at RT, catalogue no. A11034, Molecular Probes, Life Technologies) and slides were mounted with Prolong Gold Antifade Mountant with Dapi (catalogue no. P36941, Molecular Probes, Life Technologies). Cells were viewed with an epifluorescence microscope (BX50, Olympus Australia) and imaged using a 40× objective and a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Negative controls included rabbit immunoglobulin or no primary antibody. The percentage of K5+ cells in controls and following carboplatin treatment were evaluated in 5 high power images (~100–200 cells).
|
study
| 100.0 |
All statistical analyses were performed using SPSS for Windows software (Version 21.0, SPSS Inc., Chicago, IL, USA). Chi-squared test was performed to determine the correlation of K5/6 immunostaining in ovarian tumor tissues with clinical and pathological parameters. The Mann Whitney U test was used to assess differences between KRT5 and KRT6C expression in the chemotherapy sensitive and chemotherapy resistant primary serous ovarian cancer cells and the parental OVCAR-5 and carboplatin resistant OVCAR-5 CBPR cells. The one way ANOVA with the Dunnet C Post hoc test was used to assess differences between Z scores for KRT5 and KRT6 expression and clinical parameters as data was normally distributed. Kaplan-Meier and univariate Cox Regression analyses were performed to assess the association of K5/6 expression in the advanced stage ovarian cancer TMA cohort with PFS and OS. Relapse or death due to ovarian cancer was used as the endpoint to determine whether KRT5, or KRT6C expression and K5/6 positivity was associated with PFS or OS. Statistical significance was accepted at P < 0.05.
|
study
| 100.0 |
The incidence rate of tuberculosis (TB) is very high in China, and data from Centers for Disease Control in recent years have shown a growing number of patients with multidrug-resistant TB and refractory TB. Treatment for patients with severe TB infection has drawn widespread attention. Further, the diagnosis of fever of unknown origin (FUO), which is commonly caused by infectious diseases, autoimmune diseases, and malignant tumors, has become a problem that clinicians often face in large general hospitals. With a diverse range of clinical manifestations, TB is a common cause of FUO.
|
other
| 99.9 |
Hemophagocytic syndrome, also known as hemophagocytic lymphohistiocytosis (HLH), is associated with cytokine storm and inflammation, which seriously impact quality of life. HLH is rare, with a lower incidence in adults than in children, and usually presents secondary to cancer, infections, autoimmune, and other diseases. Among infectious diseases, secondary or acquired HLH is commonly associated with viral infections, such as Epstein-Barr virus and cytomegalovirus, and also bacterial infections; secondary HLH can also be caused by TB-HLH.
|
review
| 99.9 |
Hemophagocytic lymphohistiocytosis is a severe complication of TB infection and has a high mortality. For patients with FUO, HLH-related clinical manifestations sometimes present before the final diagnosis of TB. Existing literature on TB-HLH in China and abroad are limited to case reports. Thus, it is necessary to understand the characteristics of secondary TB-HLH and its prognosis to achieve early recognition and treatment.
|
review
| 99.75 |
Using the medical record system of Peking Union Medical College Hospital (PUMCH), a total of 371 patients who were admitted to our hospital with FUO and finally diagnosed with TB in the past 10 years (from January 2006 to December 2015) were analyzed (including 3 categories: patients with positive results using acid-fast bacilli-associated etiological examination, patients whose histopathological findings were inconsistent with TB, and patients who obtained a good outcome with clinical diagnosis and diagnostic antitubercular therapy [ATT]). Among these, a total of 8 cases were diagnosed as TB-HLH. Within the same period, a total of 294 patients diagnosed with HLH had been discharged from the hospital, and a total of 227 cases were verified to satisfy the HLH-2004 diagnostic criteria with complete information available for analysis.
|
study
| 99.94 |
We verified patient diagnoses and retrospectively analyzed clinical manifestations, physical signs, laboratory examinations, and treatments. These analyses were completed in combination with literature review and collection of follow-up diagnosis and treatment information for analysis; follow-up was accomplished by means of outpatient contact, telephone, and the use of a mobile phone app. FUO was defined as: fever that lasted ≥3 weeks; temperature >38.3°C ≥2 times; and no diagnosis even after evaluation of complete disease history, physical examination, and routine laboratory tests for ≥1 week.
|
study
| 87.1 |
SPSS 18.0 (SPSS Inc., Chicago, IL) was used for all statistical analysis. Categorical data are presented as numbers. Statistical comparisons between experimental groups were analyzed using Fisher exact test, and a 2-tailed P < 0.05 was considered statistically significant.
|
other
| 99.9 |
All human and animal studies have been approved by the Ethics Committee of Peking Union Medical College Hospital and have therefore been performed in accordance with the ethical standards specified in the 1964 Declaration of Helsinki and its later amendments. All study participants provided their written informed consent before inclusion in the study.
|
other
| 99.94 |
Among patients admitted to our hospital with FUO, who were finally diagnosed with TB, the ratio of male to female was 1.14:1. The average age was 42.1 ± 17.5 (mean ± standard deviation [SD]) years (range 18–82 years). Among these patients, HLH incidence rate was 2.16%, with male-to-female ratio of 1:3 and an average age of 46.6 ± 19.3 years (range 23–78 years). There was a higher proportion of females among the TB-HLH patients than the proportion of females among TB patients (P = 0.001).
|
study
| 99.94 |
Among the 227 HLH patients, there were 91 infection-associated HLH patients. Detailed information is shown in Table 3. In total, 8 patients were diagnosed with TB-HLH, accounting for 3.52% of HLH cases. The 8 patients were identified and screened for other possible causes of HLH during the course of disease evolution, and no positive results were found.
|
study
| 99.94 |
The 8 patients diagnosed with TB-HLH had no underlying diseases (Table 4). Overall, the disease had no specific clinical manifestations or symptoms, and the patients manifested a fever with no specific features, such as irregular fever and continued fever. Half of the patients (4 cases) presented with skin rash, which presented in different forms. The incidence rates of other positive symptoms including lymphadenectasis, splenomegaly, serous effusion, icterus, bleeding tendency, hepatomegaly, nervous system symptoms (including lethargy, delirium), and arthralgia were 87.5%, 75%, 75% (pericardial effusion 37.5%, pleurisy 25%, and ascites and pelvic fluid 12.5%), 50%, 50%, 37.5%, 37.5%, and 25%, respectively.
|
study
| 97.25 |
Regarding laboratory examinations, all patients had abnormalities in liver function, hemogram findings such as moderate and severe anemia, and a reduced number of blood platelets. Other findings associated with blood coagulation included significantly reduced fibrinogen levels, significantly elevated serum ferritin levels, hyperplasia, and histiocytic hemophagocytosis in the bone marrow. Apart from this, the patients showed increased levels of inflammatory markers such as erythrocyte sedimentation rate and C-reactive protein/high-sensitivity C-reactive protein. In addition, 7 patients had a reduced leukocyte or neutrophil counts, 3 of whom exhibited granulocytosis. Six patients showed diminished natural killer (NK) cell activity and reduced numbers of NK cells. Three patients underwent a serum CD25 level examination, and CD25 was elevated in all patients. Four patients underwent serum β2-microglobulin examination, and the level was elevated in 3 patients, whereas it was normal in 1 patient.
|
clinical case
| 99.44 |
All 8 TB-HLH patients underwent necessary infectious disease screening, and all of them presented etiological evidence of TB infection. In 2 cases, the rapid mycobacteria cultures using blood samples showed positive acid-fast stain results. In 1 case, the acid-fast stain of a sputum smear (bronchoscopy) was positive, whereas in 3 cases, positive acid-fast stain sputum using the polymerase chain reaction technique was found (the sputum sample was obtained via bronchoscopy in 1 case). In 1 case, the ascites tested were positive, and in another case, the cerebrospinal fluid was positive, both using the acid-fast stain method. Five of the patients received a T.SPOT TB examination with blood samples, and the results were all 0. For the remaining 3 patients, inspection was not available when they were in the hospital. Moreover, to evaluate the possibility of HLH secondary to cancers, 3 patients underwent positron emission tomography/computed tomography examination, and no clear signs of malignant diseases were found.
|
clinical case
| 98.8 |
Imaging examination revealed varied results: pleural effusion (5 cases), pulmonary infiltrates (4 cases), multiple lung nodules (2 cases), thick-walled cavity (1 case), sign of budding (1 case), ascites (1 case), pericardial effusion (a trace amount, 3 cases), mediastinal lymphadenectasis (4 cases), and retroperitoneal lymphadenectasis (2 cases).
|
clinical case
| 99.0 |
The HLH-2004 and H-score scoring systems were highly consistent in diagnosing patients with HLH in this study. All 8 TB-HLH patients met the criteria of the 2 diagnostic systems. The mean disease course of all patients diagnosed with HLH was 7.625 months. It is worth noting that HLH was diagnosed before TB in 5 patients. The 8 patients with secondary HLH were diagnosed with pulmonary TB (4 cases), miliary TB (2 cases), abdominal TB (1 cases), and tuberculous meningitis (1 case). Regarding complications, 1 patient suffered a pulmonary bacterial infection during the course of treatment, and another patient was infected with cytomegalovirus after diagnosis (CMV-DNA 1900 copies/mL) that resolved after 1 week with antiviral therapy.
|
clinical case
| 67.3 |
Due to rapid development of the disease, 2 patients refused further laboratory tests and treatments, were discharged from PUMCH, and returned home. They did not receive any treatments for HLH (including glucocorticoid and immunosuppressive agents). However, all 8 patients were given ATT (3 patients received treatment only with 2 first-line anti-TB drugs due to severe liver dysfunction, whereas the rest received treatment with 4 first-line anti-TB drugs). Four patients with respiratory failure underwent tracheal intubation and breathed with the help of a ventilator. Two patients experienced septic shock. Six patients received a sufficient amount of glucocorticoid therapy based on the HLH-2004 program, including 1 who received etoposide (VP16) and another who received cyclosporine A (CsA). For supportive treatment, 3 patients received intravenous immunoglobulin therapy, 7 patients received transfusions of red blood cells with fresh frozen plasma, and 5 patients received platelet transfusions for symptomatic therapy.
|
clinical case
| 99.9 |
Among the 8 patients with TB-HLH, 3 were admitted to our hospital from the emergency room. All 8 patients were in critical condition during their stay in the hospital. Three patients died during hospitalization, another 3 gave up further treatment and died soon after discharge, 1 patient improved during hospitalization, and 1 improved after receiving treatment in a TB hospital. Currently, the 2 patients who remained relatively stable are no longer receiving ATT (adhere to ATT at least 1 year) or glucocorticoid therapy (adhere to glucocorticoid at least glucocorticoid 8 weeks according to HLH-2004).
|
clinical case
| 99.9 |
China has the second highest incidence rate of TB infections in the world. Therefore, the Chinese government has instituted specific health policies and created specialized TB hospitals to treat confirmed patients with TB. However, TB is known as “a great mimicker” due to its diverse range of clinical manifestations, which make early diagnosis difficult and may even result in misdiagnosis. In large first-class hospitals in China, TB patients are admitted to the hospital mostly due to FUO, and after undergoing abundant and differential diagnosis, they are finally diagnosed with TB. Therefore, the diagnosis of TB that presents as FUO is quite difficult. According to clinical data published by our hospital, TB infection ranks first in the discharge diagnoses of patients admitted with FUO. Meanwhile, in recent years, a growing number of patients have been infected with drug-resistant TB, refractory TB, and severe TB; these patients have to seek medical care in large general hospitals because of difficulties in diagnosis and treatment. TB-HLH is a type of severe TB. Our retrospective case analysis has demonstrated that all TB-HLH patients in our study were admitted to the hospital with FUO, which is undoubtedly a challenge for clinicians. TB-HLH was first reported in the 1980s, and there has been a growing amount of literature on TB-HLH in recent years. Nevertheless, these reports both from China and abroad, are single case reports. The current report is the largest single-center case summary in the world thus far.
|
clinical case
| 99.7 |
The pathophysiology of HLH involves excessive immune activation and cytokine storm, but the underlying mechanism of TB-HLH remains unclear, although TB infection disrupts immune homeostasis leading to secondary HLH. In patients with TB, the levels of interferon-γ, tumor necrosis factor-α, and granulocyte/monocyte colony-stimulating factor are higher than those in a healthy population. Moreover, in HLH patients with cytokine storm, the increased levels of cytokines mentioned above along with macrophage colony-stimulating factor and interleukin (IL) family cytokines, including IL-1, IL-6, and IL-18 among others, are important markers. Specifically, Mycobacterium tuberculosis could act as an obligate intracellular pathogen after phagocytosis by phagocytic cells to induce TH1-mediated cytotoxicity, activating macrophages and NK cells, further releasing a large quantity of cytokines and chemokines, leading to TB and HLH-related symptoms. Meanwhile, M tuberculosis can induce migration of monocytes and macrophages to regional lymph nodes through a mechanism mediated by IL-12 and IL-15, leading to antigen-specific T-cell expansion, stimulation of persistent cytokine release, followed by subsequent activation and proliferation of macrophages.[10–13]
|
review
| 98.75 |
According to international literature, the mortality of patients diagnosed with TB-HLH who did not receive ATT is reported to be 100%, whereas ATT in combination with immunotherapy can reduce the mortality rate by 40% to 60%. Due to the long-term characteristics and efficacy of ATT, the utility of sufficient immunotherapy at an early stage of treatment is important to suppress cytokine storm in patients with HLH. Patients who received immunotherapy also experienced significantly higher overall survival rates than patients who did not receive ATT.[1,9,14–16]
|
review
| 99.6 |
Meanwhile, a few scholars believe that early and effective ATT is the key to preventing HLH in TB patients, and is the cornerstone of TB-HLH treatment. Increased knowledge of TB infection, early diagnosis, and early treatment can, to a certain extent, prevent the occurrence of HLH. The mean disease course for patients was 7.6 months in our study. Specifically, there were 6 cases that developed within 6 months of initial presentation, and the disease progressed rapidly before and after admission to our hospital. These patients did not receive ATT previously. Thus, it is inadequate to assess whether effective TB treatment can prevent HLH.
|
study
| 99.94 |
The inclusion criteria were strict in this study. The 8 TB-HLH patients were admitted to the hospital with FUO and with clear evidence of TB. Cases with other secondary HLH were excluded. All of these patients received ATT, and 6 of them received specific treatment for HLH. However, the mortality rate was significantly higher than that for other TB-HLH cases reported domestically and abroad. The reason might be related to their FUO background. In our country, patients diagnosed with TB usually go to specialized TB hospitals for treatment. Our hospital is a center for diagnosis and treatment of rare and tough diseases nationwide. These patients were hospitalized with FUO, representing patients who received more than 3 weeks of treatment before admission with unclear diagnosis, suggesting that this group of patients had atypical clinical manifestations, symptoms, and severe organ damage.
|
study
| 99.94 |
A previous study has shown that, among patients with FUO who were finally diagnosed with TB, those with bloody disseminated TB and tuberculous meningitis accounted for 4% and 6% of the cases, respectively. Most patients with TB and HLH reported previously have had underlying diseases, such as chronic renal failure, cancer, diabetes, acquired immune deficiency syndrome, and renal failure. Though this group of patients did not have underlying diseases, they were in poor condition. A high proportion of disseminated pulmonary TB and tuberculous meningitis was also present. In the blood system, the levels of hemoglobin and platelets decreased in the patients with TB-HLH in our study, and coagulation abnormalities were more severe than those commonly observed in patients with TB. Among the 8 patients with TB-HLH, 5 patients eventually developed respiratory failure, and none of them had renal failure.
|
study
| 100.0 |
The typical imaging features of disseminated pulmonary TB include miliary nodule shadows scattered throughout the 2 lungs, and the nodules were usually easy to recognize. However, secondary pulmonary TB typically occurred in the posterior segment of the apical superior lobe and dorsal lower lobe, and it developed more often on the right than on the left. The lung imaging features of this group of patients with disseminated pulmonary TB were atypical, and final diagnoses were based on positive blood cultures. Secondary TB lesions were mainly diffused in both lungs and involved the lower lobe. The predominant morphologies of pulmonary lesions were effusion shadows, nodule shadows, and empty shadows. There was 1 case in our study with diffused interstitial changes in both lungs. In summary, this group of patients had many atypical clinical TB symptoms. Their fever was not low-grade in the late afternoon, which is commonly seen in TB, and the imaging features demonstrated atypical manifestations of TB, presenting rare serious complications mentioned above. These atypical symptoms undoubtedly interfered with the clinicians’ abilities to correctly diagnose TB, but they also helped raise awareness of atypical TB. Cases like these provide a reminder that screening of atypical lesions should not be ignored, such as those in the lower lobe of lungs and those with morphology atypical of TB. Some researchers have suggested that routine tracheal tube brushing or bronchoalveolar lavage fluid inspection for patients suspected to be infected with TB, who have no phlegm or produce negative results in a sputum smear test, can effectively improve the rate of discovery. Yet, for severe TB patients with secondary HLH, low platelet counts and poor coagulation functions might not be able to tolerate invasive inspection and operation for the final diagnosis of TB. Therefore, if suspected, sputum examination and blood culture for TB should be carried out repeatedly, and diagnostic anti-TB treatment should be given actively to those who have no contraindication to ATT.
|
clinical case
| 96.56 |
Most previously reported studies suggest that clinicians consider a diagnosis of HLH when they encounter patients with TB who have reduced levels of blood cells, hepatosplenomegaly, and coagulation abnormalities. However, in our study, the majority of HLH manifestations appeared before the diagnosis of TB or acted as a portion of the inflammatory storm that occurred in patients with acute and fulminant TB.
|
study
| 100.0 |
We noticed an emergence of HLH manifestations in patients with FUO, and thus, the doctors maybe cautioned that screening for TB should be considered during diagnosis. Given the high mortality rate in this group of patients who did not display specific clinical manifestations, careful consideration by clinicians is necessary to achieve early diagnosis, and early intervention may help improve the prognosis.
|
clinical case
| 98.2 |
Trichomonas vaginalis is a flagellated protozoan parasite that infects the human urogenital tract, causing the most common non-viral, sexually transmitted infectious disease worldwide . The prevalence of T. vaginalis among women in sub-Saharan Africa is 11.5% . In about half of the infected women, T. vaginalis causes a malodorous vaginal discharge, vulval irritation and inflammation, and a ‘strawberry cervix’ characterized by punctate hemorrhagic lesions . Men typically remain asymptomatic, but can suffer from urethral discharge, dysuria, urethritis, epididymitis and prostatitis [2, 3]. Trichomonas vaginalis has been associated with adverse pregnancy outcomes, such as preterm birth and premature rupture of membranes [4–6], increased shedding and acquisition of the human immunodeficiency virus (HIV) [7, 8], hence contributing to the HIV pandemic.
|
review
| 99.9 |
The global prevalence of T. vaginalis and the health sequelae associated with it have necessitated the need to understand its genetic make-up. Trichomonas vaginalis is a complex pathogen, with a genome size of ~160 megabases, two-thirds of which is composed of repeats and transposable elements . Some strains of T. vaginalis can harbor up to four types of Trichomonas vaginalis viruses (TVVs) . TVVs are members of the family Totiviridae under the distinct genus Trichomonasvirus . Carriage of TVVs has been suggested to upregulate pro-inflammatory host responses and T. vaginalis immunogenic protein P270 and is also associated with differential qualitative and quantitative expression of cysteine proteinases . Thus, since TVVs induce various phenotypic changes that may impact T. vaginalis virulence , determining carriage of TVVs seems to be essential in the characterization of T. vaginalis infection. However, no method has been adapted as a standard clinical diagnostic test for TVVs .
|
review
| 98.4 |
Better understanding the diversity of T. vaginalis and geographical distribution of various genotypes may improve our knowledge regarding the epidemiology of this infectious agent and contribute to vaccine development efforts. At present, none of the described techniques have been recognized as the “gold standard” for genotyping of T. vaginalis isolates [16–23].
|
study
| 99.94 |
In this study, we opted to sequence the T. vaginalis actin gene to better understand the genetic diversity of T. vaginalis. Actin is a ubiquitous well conserved structural protein in all eukaryotic cells and has been used to clarify the molecular phylogeny of protists, plants, animals and fungi .
|
study
| 100.0 |
The data produced by sequencing are unambiguous, reproducible and portable, thus offering the advantage that public databases can be constructed. In Kenya, screening for T. vaginalis is not routinely done. However, recent studies have indicated that there is high prevalence of T. vaginalis among different groups in Kenya [26–29]. Despite this, no typing has been carried out. In this study, we genotyped T. vaginalis isolated from pregnant women attending ANC in Kilifi, Kenya.
|
study
| 100.0 |
From July through to September 2015, we conducted a cross-sectional study at the antenatal care clinic of Kilifi County Hospital, Kenya. The main aim of that study was to describe the prevalence and predictors of curable sexually transmitted infections (STIs) among pregnant women attending to the antenatal care clinic . Women were eligible if they met the following criteria: age 18–45 years, gestation ≥ 14 weeks, resident of the Kilifi Health and Demographic Surveillance area, willingness to undergo free STI and bacterial vaginosis screening procedures, and able and willing to give written informed consent . The current study presents a secondary objective of the above-mentioned study, namely to perform typing of T. vaginalis clinical isolates from this study population.
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study
| 100.0 |
A total of 349 pregnant women were included in the study. A nurse collected vaginal secretions from the vaginal introitus using a sterile cotton swab. The vaginal swab was inoculated at the clinic in the upper-chamber of an InPouch system (BioMed Diagnostics, White City, Oregon, USA). The inoculated InPouch was transferred to the laboratory within 15 min for direct microscopy of the upper chamber, after which it was merged with the lower chamber and incubated at 37 ± 1 °C under aerobic conditions. Daily microscopic observation of the InPouch system was performed and media with motile trichomonads within 5 days of culture were considered positive for T. vaginalis. Two ml of the contents of each InPouch system positive for T. vaginalis were transferred into a 2.0 ml Eppendorf tube and stored at -80 °C until shipment to the Laboratory of Bacteriology Research (Ghent University, Belgium) using shipping boxes filled with dry ice (-78.5 °C). The samples were stored at -80 °C until molecular analysis was performed.
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study
| 100.0 |
Women found to be positive for T. vaginalis using direct microscopy, i.e. microscopy of the upper chamber, were treated on the same day, while women who were negative for T. vaginalis using direct microscopy but positive on culture were contacted to return to the clinic for treatment immediately after the culture turned positive. Partners of women treated for T. vaginalis infection received presumptive treatment.
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other
| 91.7 |
Frozen cultures were first thawed at room temperature for 30 min, after which the tubes were vortexed for 30 s to ensure that a homogeneous mix was achieved before starting the nucleic acid extraction. Total nucleic acid extraction was performed using the NucliSENS® easyMAG® (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions (generic protocol 2.0.1).
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other
| 65.5 |
The actin gene was amplified using the outer primers previously used in a nested polymerase chain reaction (PCR), i.e. primers Tv8S (5′-TCT GGA ATG GCT GAA GAA GAC G-3′) and Tv9R (5′-CAG GGT ACA TCG TAT TGG TC-3′) , with the following thermocycling conditions: 5 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 55 °C and 3 min at 72 °C, followed by 7 min at 72 °C. This was performed on the ABI Veriti thermocycler platform (ThermoFisher Scientific, Waltham, Massachusetts, USA).
|
study
| 95.4 |
PCR amplification products were visualized under UV light after electrophoresis on 1% agarose gels in Tris-acetate-EDTA buffer pH 8.5 (30 min at 10 V/cm) and staining with ethidium bromide (0.5 mg⁄ l; Sigma, Bornem, Belgium). The size of the amplified products was assessed by comparison with a commercial weight marker, Smart Ladder (Eurogentec, Liege, Belgium).
|
study
| 99.9 |
Trichomonas vaginalis actin sequences were edited using Chromas Lite 2.01 (http://technelysium.com.au/wp/). Furthermore, we identified the T. vaginalis actin genotypes amongst our isolates by means of in silico RFLP-analysis Webcutter version 2.0; http://rna.lundberg.gu.se/cutter2/with the three restriction enzymes (HindII, MseI and RsaI) used by Crucitti et al. . The in silico analysis was done on our clinical isolates and on retrieved sequences representing the genotypes that have been proposed. To identify if there were additional T. vaginalis actin genotypes not captured by the previous proposed scheme, in silico RFLP was performed on actin sequences retrieved from the GenBank whose actin genotype is yet to be documented. The sequences were of at least 1100 bp in length (Additional file 1: Table S1).
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study
| 100.0 |
To compare results obtained in the RFLP analysis, and to determine the genetic variation among the identified genotypes, we performed phylogenetic analysis of the sequences. Sequences obtained in our study were aligned using MEGA software and compared with analogous sequences representative of known T. vaginalis actin genotypes identified in from our isolates.
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study
| 100.0 |
Evolutionary distances were calculated by Kimura’s two-parameter model (Kimura, 1980) and a phylogenetic tree was generated using the Maximum Likelihood method using the MEGA software (version 7.0) . Finally, confidence levels were estimated using bootstrap resampling on 1000 randomly selected pseudoreplicates.
|
study
| 99.94 |
Synthesis of cDNA was performed on the nucleic acid extract using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Gent, Belgium). The cDNA was used to amplify the four known TVVs by means of previously described type specific primers: TVV1F2875 (5′-ATT AGC GGT GTT TGT GAT GCA-3′) and TVV1R3443 (5′-CTA TCT TGC CAT CCT GAC TC-3′), TVV2F1401 (5′-ATT AGC GGT GTT TGT GAT GCA-3′) and TVV2R1953b (5′-GGT TCG TGG AAG CGG TTG ATG A-3′), TVV3F1474 (5′-CTA CCA AGA AGG AGG CTT GA-3′) and TVV3R2025b (5′-GGT TCG TGG AAG CGG TTG ATG A-3′), and TVV4F1338 (5′-ATG CCA GTT GCT TTC CG-3′) and TVV4R1834 (5′-TTC CCC AAT AGT TAT CAG-3′) . The amplification conditions were: 5 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 50 °C and 45 s at 72 °C, followed by 7 min at 72 °C. The PCR products were visualized by electrophoresis as described above.
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study
| 99.94 |
The socio-demographic and clinical data of the participants were entered in the REDCapTM electronic data capture tool, version 6.5.0 (Vanderbilt University, Nashville, Tennessee). Prevalence of T. vaginalis was expressed as a percentage with exact 95% binomial confidence intervals (CIs). Univariable logistic regression was used to determine associations of T. vaginalis presence with socio-demographic data, hygienic data, sexual behavior, and vaginal signs & symptoms. Variables significant at P ≤ 0.1 in bivariable analysis were entered into a multivariable model to identify independent associations. Crude odds ratios (COR) and adjusted odds ratios (AOR) were calculated. These statistical analyses were done using STATA, version 13.1 (Stata-Corp, College Station, Texas).
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study
| 100.0 |
The median age and gestation age including the interquartile range (IQR) for the participants was 27 (22–31) years and 25 (20–30) weeks, respectively. The majority of the participants were married (93.6%) and Christian (72.3%). Approximately a quarter of the participants had received secondary school education and above. The median age and IQR of sexual debut was 18.6 (16–20) years, although 17.4% of the participants were not sure or did not respond to this question. Seventy-three percent of the participants had given birth before and the number of children ranged between 1 and 10. Sixty-five percent of the participants had previously experienced signs/symptoms associated with reproductive tract infection and about one third had ever received syndromic treatment for genital signs or symptoms of infection. The socio-demographic characteristics of the participants are summarized in Table 1.Table 1Univariable and multivariable analysis of characteristics of pregnant women with T. vaginalis attending antenatal care clinic at Kilifi County Hospital, KenyaVariable N = 349Percentage with T. vaginalis Crude OR (95% CI) P-valueDemographic characteristics Age group (years) 18–241326.81.1 (0.4–2.5)0.894 ≥ 252176.5ReligionChristian2485.7 Muslim545.71.0 (0.3–3.5)0.979 Other/None4712.82.4 (0.9–6.7)0.083a Education None637.9 Primary2007.00.9 (0.3–2.5)0.802 Secondary/Tertiary864.70.6 (0.1–2.1)0.411Marital status Single254.0 Married2246.81.7 (0.2–13.5)0.593Residency Living with partner2555.5 Not living with a partner949.61.8 (0.8–4.4)0.178Employment status Employed/self-employed1995.0 Unemployed1508.71.8 (0.8–4.2)0.180Parity 0946.41.1 (0.3–3.5)0.843 1–21337.51.3 (0.5–3.6)0.570 3+1225.7Gestational age (weeks)c 14–251835.5 ≥ 261657.91.5 (0.6–3.4)0.382Hygiene characteristics Toilet type Flushing toilet1177.7 Pit latrine1975.10.6 (0.3–1.6)0.350 Bush/Other3511.41.5 (0.4–5.4)0.491Mode of cleaning after visiting the toilet Tissue paper/Other solid materials1062.8 Water2438.23.1 (0.9–10.5)0.074a Behavioral characteristics Sexual debut age (years) ≤ 171068.51.8 (0.7–4.7)0.231 ≥ 181834.9 Do not know/No response606.71.5 (0.4–5.0)0.523Number of lifetime sex partnersc 11583.8 2+1899.02.5 (1.0–6.5)0.060a Polygamous partner No3036.91.6 (0.4–7.2)0.515 Yes464.4Alcohol consumption ever No2626.50.9 (0.4–2.5)0.894 Yes876.9Tobacco use No3326.61.1 (0.1–8.9)0.904 Yes175.9Other drugs/substance use ever No3326.61.1 (0.1–8.9)0.904 Yes175.9Sexually transmitted infections/reproductive tract infections HIV Negative3246.2 Positive2512.02.1 (0.6–7.5)0.268Bacterial vaginosisd Negative2936.5 Positive537.61.1 (0.4–3.6)0.775Clinical signs and symptoms of STI Previous history of vaginal discharge No1225.6 Yes2277.11.2 (0.5–3.1)0.638Previous syndromic treatment of genital infection No2296.6 Yes1206.71.0 (0.4–2.5)0.967Current vaginal discharge (self-reported)c No963.1 Yes2527.92.7 (0.8–9.2)0.119Abnormal vaginal discharge foul smell/color (observed)c No2715.9 Yes779.11.6 (0.6–4.0)0.324Dysuriac No2636.1 Yes858.21.4 (0.5–3.5)0.489Dyspareuniac No2454.9 Yes10310.72.3 (1.0–5.4)0.053Vaginal itchingc No2316.5 Yes1176.81.1 (0.4–2.6)0.903Lower abdominal painc No1698.3 Yes1795.00.6 (0.2–1.4)0.226Genital wartsc No3416.2 Yes728.66.1 (1.1–33.3)0.037a Genital ulcer (observed)c No3385.6 Yes1040.011.2 (2.9–43.1) < 0.001b Vaginitisc No3346.6 Yes147.11.1 (0.1–8.7)0.935Symptomaticc No1456.9 Yes2036.40.9 (0.4–2.2)0.855 aSignificant in univariable analysis bSignificant on multivariable association; symptomatic (any or a combination of the three symptoms, i.e. genital ulcer, lower abdominal pain or abnormal vaginal discharge) cMissing data/some participants did not respond to this question(s) dBacterial vaginosis results for three participants were not available due to poor slides
|
study
| 99.94 |
A total of 23/349 (6.6%, 95% CI: 4.2–9.7) women were culture-positive for T. vaginalis, and 34.8% of these cases were positive by direct microscopy prior to further incubation. Based on symptoms routinely used in syndromic management of STIs (i.e. genital ulcer, lower abdominal pain or abnormal vaginal discharge), 43.5% of the 23 women with T. vaginalis infection were asymptomatic. The most commonly self-reported symptoms amongst all participants included vaginal discharge (72.4%). However, during collection of specimens by the study nurse, only 22.1% of the participants had an abnormal discharge (defined as excess discharge/foul smelling discharge/colored discharge) upon examination. A total of 51.4% of the women reported having lower abdominal pain, genital ulcers were observed in 2.9% of the women. Dyspareunia, genital warts and genital ulcers were the only clinical signs or symptoms significantly associated with T. vaginalis infection (Chi-square test: χ 2 = 3.93, df = 1, P < 0.048; χ 2 = 5.58, df = 1, P < 0.018; and χ 2 = 18.60, df = 1, P < 0.05, respectively).
|
study
| 100.0 |
Univariable analysis indicated that T. vaginalis infection was more common among participants who were traditionalist or reported having no religion compared to participants who were Christians or Muslims, used water to clean themselves after visiting the toilet compared to those who used tissue paper or other solid materials, reported having ≥ 2 lifetime sexual partners, reported dyspareunia, had genital warts, and/or had a genital ulcer (Table 1). In multivariable analysis, the only independent predictor associated with T. vaginalis was having a genital ulcer (AOR = 7.6, 95% CI: 1.4–42.3).
|
study
| 100.0 |
The actin gene target could be amplified from 21 of the 23 T. vaginalis isolates. All 21 amplicons had the expected length of approximately 1100 bp. The two remaining isolates could only be amplified by a higher primer concentration of 0.5 μM, instead of 0.3 μM. However, one of these clinical isolates did not yield an interpretable sequence and thus only sequences from 22 clinical isolates were utilized in the typing. Five different actin types (E, G, I, N and P) were identified according to the position and the number of cleavage sites, following the scheme proposed by Crucitti et al. (Table 2). The most prevalent actin genotype was E, representing 50.0% of the isolates. The other genotypes were, in order of descending frequency, N (27.3%), G (13.6%) and I and P (each 4.5%).Table 2Number and position of restriction sites using HindII, MseI and RsaI restriction enzymesGenotypeNo. of isolates HindII MseI RsaI213273699185314518103190426878994E11××××××××G3×××××××I1××××××××N6×××××××××P1×××××××××× indicates the presence of a restriction cut site
|
study
| 100.0 |
Multiple sequence analysis to compare polymorphic sites found on our actin sequences, and those retrieved from the GenBank, revealed a total of 33 single nucleotide differences in the open reading frame of the actin gene. Three of these single nucleotide polymorphisms were exclusively found in actin sequences from our study (Additional file 2). The nucleotide sequences obtained of the actin gene for all the 22 isolates were submitted in GenBank under accession numbers: (MF350322–MF350343). The phylogenetic analysis (Fig. 1) showed that actin genotype E clustered with a bootstrap value of 99. Lower bootstrap values were observed for actin genotypes N, G, I and P.Fig. 1Phylogenetic tree for actin gene nuclotide sequences of T. vaginalis. The tree was generated using Maximum Likelihood method. Bootstrap test for 1000 replicates. Sequences from Kilifi isolates (n = 22) have accession numbers with the prefix “MF”, all other sequences were retreived from GenBank and are representative of the five actin genotypes. Scale-bar: 0.001 (1 substitution per 1000 nucleotides)
|
study
| 100.0 |
Phylogenetic tree for actin gene nuclotide sequences of T. vaginalis. The tree was generated using Maximum Likelihood method. Bootstrap test for 1000 replicates. Sequences from Kilifi isolates (n = 22) have accession numbers with the prefix “MF”, all other sequences were retreived from GenBank and are representative of the five actin genotypes. Scale-bar: 0.001 (1 substitution per 1000 nucleotides)
|
study
| 99.94 |
TVVs were present in 43.5% (95% CI: 23.2–65.5) (10/23) of T. vaginalis isolates. Trichomonas vaginalis virus type 1 (TVV1) was the most prevalent (39.1%), followed by TVV2 (26.1%), TVV3 (17.4%) and TVV4 (13.0%). Nine out of 10 T. vaginalis isolates with TVVs harbored more than one type of TVV (Table 3). TVV1 was present in all virus-positive T. vaginalis isolates but one (TV207). All 11 genotype E isolates were virus-negative.Table 3Genotypes of Trichomonas vaginalis and carriage of T. vaginalis viruses, in relation to symptoms among 23 T. vaginalis isolates in Kilifi, KenyaSample IDTVV1TVV2TVV3TVV4GenotypeSymptomatica TV279+–+–*+TV022––––E+TV042––––E+TV050––––E–TV066––––E+TV075––––E+TV176––––E–TV188––––E–TV203––––E+TV224––––E–TV299––––E+TV323––––E–TV185+–++G–TV207–+––G+TV238+++–G+TV307++––I–TV116++––N–TV131++––N+TV156+––+N–TV190+++–N+TV210––––N+TV234+––+N–TV140––––P+ Abbreviations: TVV1 Trichomonas vaginalis virus type 1, TVV2 T. vaginalis virus type 2, TVV3 T. vaginalis virus type 3, TVV4 T. vaginalis virus type 4 Code: *Not typed; + present; − absent aSymptomatic: any or a combination of the three symptoms, i.e. genital ulcer, lower abdominal pain and/or abnormal vaginal discharge
|
study
| 100.0 |
Finally, the distribution of symptomatic and asymptomatic cases was not linked to any particular T. vaginalis actin genotype. Similarly, presence or absence of TVVs did not appear to have an influence as to whether a patient was symptomatic or asymptomatic.
|
study
| 100.0 |
To our knowledge, this is the first study determining T. vaginalis genotypes and co-occurrence of T. vaginalis viruses in Kenya. We sequenced the actin gene for 22 isolates and identified five types of T. vaginalis by in silico RFLP-analysis of the amplified actin gene. We found notable genetic diversity by full actin gene sequence analysis among T. vaginalis isolates in Kilifi, as well as those retrieved from GenBank. Prevalence of T. vaginalis in this study (6.6% in 349 pregnant women) was high but, however, fell short of the prevalence of T. vaginalis among women aged 15–49 years in the World Health Organization Africa region in 2012, which was estimated to be 11.5% (95% CI: 9.0–14.6) . This is suggestive of lower rates of T. vaginalis among the general population of women in Kilifi, Kenya compared to other African countries .
|
study
| 100.0 |
Although T. vaginalis does not traditionally present with genital ulcers , multivariable analysis showed that genital ulcers were the only predictor of an infection with T. vaginalis in our study. Ulcers could not be due to syphilis, which was not diagnosed in any of the women with T. vaginalis, but we did not test for the Herpes simplex virus, which is also associated with genital ulcers. The association of genital ulcer with T. vaginalis is not unique to our study as it has been reported amongst female sex workers in China .
|
study
| 100.0 |
Nucleotide sequence analysis of actin sequences showed 33 polymorphic sites, three of which caused amino acid substitution. Two of these amino acid substitutions have been previously reported to occur in genotypes G, N, I and P, in which nucleotide 371 substituted alanine for valine, and nucleotide 904 substituted lysine for glutamine [20, 34]. A unique polymorphism leading to an amino acid substitution, in which nucleotide 892 substituted threonine for serine, was observed to be exclusively present on a GenBank sequence (accession number XM_001301892). As such, in silico genotyping of isolates provides an opportunity to distinguish closely related isolates based on these polymorphic sites and to further identify such polymorphic sites.
|
study
| 100.0 |
Our phylogenetic analysis confirms RFLP as a good typing method, as the results from this method were in agreement with phylogenetic analysis. Phylogenetic analysis and detection of carriage of TVVs, revealed that none of the isolates of the most prevalent actin genotype E harbored a TVV. Furthermore, phylogenetic analysis indicated that genotype E formed a distinct phylogenetic lineage, suggesting clonal stability of this genotype .
|
study
| 100.0 |
The high prevalence (43.5%) of TVVs found in this study is comparable to a prevalence of 55% (95% CI: 38.4–70.7) among Cuban isolates . Two recent studies have reported lower carriage of TVV, 18.7% (95% CI: 11.5–28.0) in the Philippines and 17.3% (95% CI: 7.8–31.4) among Iranian isolates , although the latter study only determined the presence of TVV1. However, higher prevalence rates of TVV have been reported as well, 81.9% (95% CI: 71.1–90.0) in South Africa , and 75.0% (95% CI: 55.1–89.3) in Baltimore City, Maryland . The presence of TVV, in addition to metronidazole susceptibility, has been found to differ significantly between T. vaginalis isolates genotyped by a panel of 21 microsatellites and six single-copy genes of T. vaginalis, which classifies T. vaginalis into two types: type 1 and type 2 . Type 2 is characteristically free of TVV and resistant to metronidazole . Metronidazole susceptibility in relation to actin genotypes is yet to be determined.
|
study
| 100.0 |
Fifty-seven percent of isolates in our study did not harbor TVV, suggesting that they might be of type 2. Our study might have been biased towards type 2; 63.6% were recovered from patients who were diagnosed by culture, after direct microscopy had been determined to be negative, suggesting that the parasite load in the patient was low. Conrad et al. observed that type 1 parasites are often diagnosed by direct microscopy and suggested that this may be indicative of higher parasite load in type 1 which harbor TVVs. Additional studies, sampling a more diverse population and other regions in Kenya, are needed to confirm the population type and distribution of T. vaginalis in the country.
|
study
| 100.0 |
A total of 22 TVVs were identified in 10 T. vaginalis cultures, with multiple TVVs detected in nine cultures (Table 2). The higher prevalence of T. vaginalis cultures with either TVV1 or TVV2 than with TVV3 or TVV4 is consistent with previous reports [10, 37]. In these publications, concurrent TVV infection, with at least two or 3 TVVs, was recorded in six and three T. vaginalis sample cultures, respectively. Although we identified single actin genotypes in all T. vaginalis cultures, which is indicative for the presence of only 1 T. vaginalis strain per culture, we cannot rule out that the presence of the multiple TVVs may be the result of a mixture of T. vaginalis strains, each infected with a different TVV. Therefore, our data does not necessarily indicate concurrent infection of TVVs in a single TV strain. The lytic cycle of TVVs is yet to be described, and attempts to infect uninfected isolates have been unsuccessful . Therefore, it is plausible that the virus may solely be acquired through vertical transmission, making its presence an important genetic marker .
|
study
| 100.0 |
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