Human polyomavirus 9 in immunocompromised patients in the University Hospital in Hradec Kralove, Czech Republic.

Human polyomaviruses such as JC polyomavirus and BK polyomavirus have long been well known pathogens of immunocompromised patients. Several new members of this viral family have been described during the last decade. Human polyomavirus 9 seems to be a novel pathogen of transplanted patients according to some studies. The aim of our study was to determine the presence of human polyomavirus 9 in patients after kidney or stem cell transplantation (SCT) at the University Hospital in Hradec Kralove, Czech Republic. Overall 100 patients, 65 after kidney transplantation and 35 after SCT, were included into the study. At least three follow-up samples from each patient were examined for human polyomavirus 9 DNA presentation with the two previously described in-house PCR protocols. Despite the frequent reactivation of human CMV (14.3% in kidney transplantation and 63.3% after SCT) or BK polyomavirus in our patient group, there was no positivity for human polyomavirus 9 either in blood samples or urine samples. One of the possible reasons for this discrepancy versus previous published studies could be a relatively low proportion of patients treated by induction therapy before kidney transplantation in our study cohort.

Amniotic fluid calreticulin in pregnancies complicated by the preterm prelabor rupture of membranes.

OBJECTIVE: This study aimed to determine the amniotic fluid calreticulin concentrations in women with the preterm prelabor rupture of membranes (PPROM) based on the microbial invasion of the amniotic cavity (MIAC), intraamniotic inflammation (IAI) and microbial-associated IAI. METHODS: One hundred sixty-eight women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis and were assayed for calreticulin concentrations by ELISA. IAI was defined as an amniotic fluid interleukin-6 concentration > 745 pg/ml. Microbial-associated IAI was defined as the presence of both MIAC and IAI. RESULT: Women with MIAC (with MIAC: median 54.4 ng/ml, versus without MIAC: median 32.6 ng/ml; p < 0.0001), IAI (with IAI: median 66.8 ng/ml, versus without IAI: median 33.0 ng/ml; p < 0.0001) and microbial-associated IAI (with microbial-associated IAI: median 82.5 ng/ml, versus without microbial-associated IAI: median 33.7 ng/ml; p < 0.0001) had higher concentrations of calreticulin than women without these complications. An amniotic fluid calreticulin concentration of 81.4 ng/ml was found to be the best cutoff point for identifying women with microbial-associated IAI. CONCLUSIONS: The presence of microbial-associated IAI is associated with increased amniotic fluid calreticulin concentrations. Calreticulin seems to be a promising marker for the early identification of PPROM complicated by microbial-associated IAI.

Umbilical cord blood markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes.

OBJECTIVE: To determine umbilical cord blood total antioxidant capacity (TAC), ferric reducing antioxidant power (FRAP), thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs) and markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) and their associations with microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA), funisitis and selected aspects of short-term neonatal morbidity. MATERIALS AND METHODS: One hundred and sixty-five women with singleton pregnancies complicated by PPROM were included in this study. Blood samples were obtained by venipuncture from the umbilical cord vein after the delivery of the newborn. The umbilical cord blood concentrations of TAC, FRAP, TBARS and AGEs were measured. RESULTS: The presence of MIAC, HCA and funisitis did not show differences in the umbilical cord blood TAC, FRAP, TBARS and AGEs concentrations. Positive correlations were found between the gestational age at sampling and umbilical cord blood TAC and AGEs concentrations (TAC: rho = 0.26; p = 0.001; AGEs: rho = 0.35; p < 0.0001). There was no association between umbilical cord blood TAC, FRAP, TBARS and AGEs concentrations and selected aspects of short-term neonatal morbidity. CONCLUSIONS: Oxidative stress is associated with PPROM, as indicated by the presence of markers tested in the umbilical cord blood; however, the evaluated oxidative stress markers are not influenced by the presence of MIAC and/or HCA, and funisitis or subsequent development of selected aspects of short-term neonatal morbidity.

Ureaplasma species and Mycoplasma hominis in cervical fluid of pregnancies complicated by preterm prelabor rupture of membranes.

OBJECTIVE: To evaluate Ureaplasma species and Mycoplasma hominis DNA in the cervical fluid and their association with microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA) in pregnancies complicated by preterm prelabor rupture of membranes (PPROM). STUDY DESIGN: A prospective study of 68 women with singleton pregnancies complicated by PPROM between 24(0/7) and 36(6/7) weeks was conducted. Cervical fluid and amniotic fluid were collected from all women at the time of admission. The Ureaplasma species and Mycoplasma hominis DNA in the cervical fluid were identified using specific real-time PCR. RESULTS: Ureaplasma species and Mycoplasma hominis DNA were identified in 59% (40/69) of the cervical fluid samples. Women with the presence of Ureaplasma species DNA with and without Mycoplasma hominis DNA in the cervical fluid had a higher rate of MIAC alone [35% (14/40) versus 11% (3/28); p = 0.02] and a higher rate of the presence of both MIAC and HCA [30% (12/40) versus 4% (1/28); p = 0.01] than women without Ureaplasma species and Mycoplasma hominis DNA in the cervical fluid. CONCLUSIONS: The presence of Ureaplasma species DNA with and without Mycoplasma hominis DNA in the cervical fluid is associated with a higher risk of MIAC or MIAC and HCA together in pregnancies complicated by PPROM.

Streptococcus agalactiae in pregnancies complicated by preterm prelabor rupture of membranes.

OBJECTIVE: The main aim of this study was to evaluate the presence of Streptococcus agalactiae (S. agalactiae) in the vagina and the amniotic fluid in pregnancies complicated by preterm prelabor rupture of membranes (PPROM). The next aim was to evaluate the incidence of S. agalactiae early onset sepsis in newborns from PPROM pregnancies, with respect to the presence of S. agalactiae in the vagina and the amniotic fluid. METHODS: Singleton gestations with PPROM between 24 + 0 and 36 + 6 were included. A vaginal swab was obtained, and amniocentesis was performed at admission. The presence of S. agalactiae in the vagina and in the amniotic fluid was assessed by culture and by real-time polymerase chain reaction, respectively. RESULTS: In total, 336 women were included. The presence of S. agalactiae in the vaginal and amniotic fluid was found in 9% (31/336) and 1% (3/336) of women. One woman had S. agalactiae in the amniotic fluid but was negative for the presence of S. agalactiae in the vaginal fluid. Early onset neonatal sepsis developed in one newborn from pregnancies complicated by the presence of S. agalactiae in the amniotic fluid. CONCLUSION: The presence of S. agalactiae in the vagina and amniotic fluid complicated approximately each 10th and each 100th PPROM pregnancy. Cultivation-negative findings of S. agalactiae in the vagina did not exclude the positivity of the amniotic fluid for S. agalactiae and the development of early onset sepsis in newborns.

Intraamniotic Inflammation in Women with Preterm Prelabor Rupture of Membranes.

OBJECTIVE: To characterize subgroups of preterm prelabor rupture of membranes (PPROM) and short-term neonatal outcomes based on the presence and absence of intraamniotic inflammation (IAI) and/or microbial invasion of the amniotic cavity (MIAC). METHODS: One hundred and sixty-six Caucasian women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis (n=166) and were assayed for interleukin-6 levels by a lateral flow immunoassay. The presence of Ureaplasma species, Mycoplasma hominis, Chlamydia trachomatis, and 16S rRNA was evaluated in the amniotic fluid. IAI was defined as amniotic fluid IL-6 values, measured by a point of care test, higher than 745 pg/mL. RESULTS: Microbial-associated IAI (IAI with MIAC) and sterile intraamniotic inflammation (IAI alone) were found in 21% and 4%, respectively, of women with PPROM. Women with microbial-associated IAI had higher microbial loads of Ureaplasma species in the amniotic fluid than women with MIAC alone. No differences in the short-term neonatal morbidity with respect to the presence of microbial-associated IAI, sterile IAI and MIAC alone were found after adjusting for the gestational age at delivery in women with PPROM. CONCLUSIONS: Microbial-associated but not sterile intraamniotic inflammation is common in Caucasian women with PPROM. The gestational age at delivery but not the presence of inflammation affects the short-term neonatal morbidity of newborns from PPROM pregnancies.

Cervical microbiota in women with preterm prelabor rupture of membranes.

OBJECTIVE: To analyze the cervical microbiota in women with preterm prelabor rupture of membranes (PPROM) by pyrosequencing and to document associations between cervical microbiota, cervical inflammatory response, microbial invasion of the amniotic cavity (MIAC), histological chorioamnionitis, and intraamniotic infection (IAI). STUDY DESIGN: Sixty-one women with singleton pregnancies complicated by PPROM were included in the study. Specimens of cervical and amniotic fluid were collected on admission. The cervical microbiota was assessed by 16S rRNA gene sequencing by pyrosequencing. Interleukin (IL)-6 concentration in the cervical fluid and amniotic fluid was measured by ELISA and lateral flow immunoassay, respectively. RESULTS: Four bacterial community state types [CST I (Lactobacillus crispatus dominated), CST III (Lactobacillus iners dominated), CST IV-A (non-Lactobacillus bacteria dominated), and CST IV-B (Gardnerella vaginalis and Sneathia sanguinegens dominated)] were observed in the cervical microbiota of women with PPROM. Cervical fluid IL-6 concentrations differed between CSTs (CST I = 145 pg/mL, CST III = 166 pg/mL, CST IV-A = 420 pg/mL, and CST IV-B = 322 pg/mL; p = 0.004). There were also differences in the rates of MIAC, of both MIAC and histological chorioamnionitis, and of IAI among CSTs. No difference in the rate of histological chorioamnionitis was found among CSTs. CONCLUSIONS: The cervical microbiota in PPROM women in this study was characterized by four CSTs. The presence of non-Lactobacillus CSTs was associated with a strong cervical inflammatory response and higher rates of MIAC, both MIAC and histological chorioamnionitis, and IAI representing a PPROM subtype with pronounced inflammation. CST I represents the dominant type of PPROM with a low rate of MIAC, IAI, and the combination of MIAC and histological chorioamnionitis.

[Human polyomaviruses]. / Lidské polyomaviry.

Polyomaviruses belong to a group of viruses that has recently attracted the attention of many research groups. During 35 years, JC and BK viruses, known pathogens in immunocompromised patients, seemed to be the only human polyomaviruses. But in 2007, two other polyomaviruses, WU and KI, were isolated whose pathogenicity is still a matter of discussion. A year later, another human polyomavirus, associated with Merkel cell carcinoma, was identified, and seven more were described by the end of 2014. Some of them were found to be related to various diseases, others seem to be a part of the normal skin and mucosal microbiome. The article summarizes basic information about all so far described human polyomaviruses.

[Determination of the naturally occurring Q80K mutation in the HCV NS3 protease gene]. / Vysetrení prilrozene variant Q80K v genu pro proteázu NS3 HCV.

OBJECTIVES: The aim was to introduce a diagnostic method for detecting variants of hepatitis C virus (HCV) with protease NS3 resistance primarily to simeprevir (Q80K mutation in HCV genotype 1a) and its subsequent use in routine practice. MATERIAL AND METHODS: The detection of HCV resistance-associated variants in the NS3 protease gene by sequence analysis was introduced in the molecular biology laboratory of University Hospital Hradec Kralove in 2015. The primers were designed by sequence analysis software Custom Primers - OligoPerfect™ Designer. The method was optimized for HCV genotype 1a. The search for variants was performed using two programs. RESULTS: A total of 16 patients with genotype 1a chronic hepatitis C have been examined since 2015. In five of them, the Q80K variant was detected. CONCLUSION: The development of resistance to antiviral therapy for chronic hepatitis C gained importance after the introduction of direct-acting antivirals. Given the relatively high prevalence of the Q80K mutation in HCV genotype 1a, it is crucial to confirm its presence or absence before the therapy is initiated. The reported method enables clear and early detection of the Q80K mutation.

Cryptococcus neoformans meningoencephalitis in a patient with polyarteritis nodosa.

Case of 59-year-old male with chronic obstructive pulmonary disease and a number of comorbidities, who has developed meningoencephalitis caused by Cryptococcus neoformans var. grubii with polyarteritis nodosa diagnosed during hospitalization, was presented. Before evidence of meningoencephalitis, the patient was being treated with ketoconazole and low doses of fluconazole (200 mg/day) for alleged candidiasis. The dosage was increased (800 mg/day) following laboratory diagnosis of C. neoformans based on positive latex agglutination test and biochemical identification of encapsulated yeast isolated from the blood and CSF. Later, the yeast identification was confirmed by sequencing analysis. Owing to inadequate clinical response, fluconazole therapy was switched to voriconazole (400 mg/day) and later to intravenous amphotericin B (1.0 mg/kg per day). Despite of a temporary stabilization and improvement, which correlated with decline of cryptococcal antigen titers (from 1:1024 to 1:8), after 6 weeks, the patient's underlying condition deteriorated due to severe pancolitis and serious nosocomial bacterial infections. The patient died of multiorgan failure several days later. Our case demonstrates a possible connection between the development of life-threatening cryptococcosis and an autoimmune vasculitis disease and emphasizes that the outcome of the management of cryptococcal meningoencephalitis is highly dependent on early diagnosis, adequate treatment, including dosage, and last but not least control of underlying disease and risk factors.

Bedside assessment of amniotic fluid interleukin-6 in preterm prelabor rupture of membranes.

OBJECTIVE: The objective of the study was to determine the diagnostic indices and predictive values by bedside assessment of amniotic fluid interleukin-6 (IL-6) concentration in the identification of microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA) in patients with preterm prelabor rupture of membranes. STUDY DESIGN: One hundred twenty-four women with singleton pregnancies were included in this study. The amniotic fluid was sampled by transabdominal amniocentesis at the time of admission. IL-6 concentrations were assessed with an immunoassay. RESULTS: The presence of MIAC, HCA, or the coexistence of both was associated with higher amniotic fluid concentrations of IL-6 in both a crude and adjusted analysis. The amniotic fluid concentration of IL-6 of 1000 pg/mL was determined to be the best cutoff value for the prediction of MIAC (sensitivity of 50%, specificity of 95%, positive predictive value of 82%, negative predictive value of 81%, and likelihood ratio of 8.4) or both MIAC and HCA (sensitivity of 60%, specificity of 94%, positive predictive value of 75%, negative predictive value of 88%, and likelihood ratio of 9.4). CONCLUSION: The bedside assessment of amniotic fluid IL-6 seems to be an easy, rapid, and inexpensive method for the prediction of MIAC or both MIAC and HCA in pregnancies complicated by preterm prelabor rupture of membranes.

Outbreak of fungal endophthalmitis due to Fusarium oxysporum following cataract surgery.

Outbreak of exogenous Fusarium endophthalmitis after cataract surgery was evaluated. Twenty patients developed postoperative endophthalmitis. In 19 eyes, pars plana vitrectomy (PPV) was performed, in 14 cases (74 %) with primary intraocular lens explantation. In one case, the PPV was not performed because of poor general condition of the patient. Symptoms of endophthalmitis (damaged vision, iritis, tyndallization in anterior chamber, hypopyon) occurred at intervals of 16-79 days (mean 31.3 days). Fungal etiology was documented in 12 eyes (60 %). Fusarium oxysporum was evidenced by culture and/or microscopy and confirmed by PCR and sequencing analysis. Eighteen (90 %) patients were treated with oral voriconazole (400 mg/day) for a period of 4-6 weeks. The final visual acuity was 6/15 in 1 case (5 %), 6/60 and worse in 17 eyes (85 %), and in 2 cases (10 %), enucleation had to be performed. Viscoelastic filling material was suggested the most likely source of infection. Endophthalmitis caused by Fusarium spp. are a potentially big threat for patients with serious impact on vision. Successful management of the infection is highly dependent on early diagnosis including species identification and antifungal susceptibility testing, and on aggressive and long-term treatment.

Microbial load of umbilical cord blood Ureaplasma species and Mycoplasma hominis in preterm prelabor rupture of membranes.

OBJECTIVE: To evaluate Ureaplasma species and M. hominis DNA in the umbilical cord blood and its correlation with its microbial load in the amniotic fluid, as a measure of microbial burden in fetal inflammatory response and neonatal outcome in pregnancies complicated by preterm prelabor rupture of membranes (pPROM). STUDY DESIGN: A retrospective study of 158 women with singleton pregnancies complicated by pPROM between 24(0/7) and 36(6/7) weeks was conducted. Amniotic fluid was obtained from all women by transabdominal amniocentesis, and umbilical cord blood was obtained by venipuncture from umbilical cords immediately after the delivery of the neonates. The Ureaplasma species and M. hominis DNA was quantitated using absolute quantification techniques. RESULT: Ureaplasma species and M. hominis DNA was identified in 9% of the umbilical cord blood samples. No correlation between the amniotic fluid and umbilical cord blood microbial load was observed. The presence of Ureaplasma species and M. hominis DNA in the umbilical cord blood had no impact on short-term neonatal morbidity. CONCLUSIONS: A high microbial load of genital mycoplasma Ureaplasma species DNA in the umbilical cord in pregnancies complicated by pPROM is not associated with a high fetal inflammatory response and is therefore not associated with serious neonatal morbidity.