Kazuhiro Iwasaku

Is Neisseria gonorrhoeae Initiating a Future Era of Untreatable Gonorrhea?: Detailed Characterization of the First Strain with High-Level Resistance to Ceftriaxone

ABSTRACT Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants (penA, mtrR, penB, ponA, and pilQ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary.

Ceftriaxone-Resistant Neisseria gonorrhoeae, Japan

To the Editor: Spread of multidrug-resistant Neisseria gonorrhoeae is a major public health concern. Effective antimicrobial therapy is a key element in gonorrhea control. However, N. gonorrhoeae has developed resistance to multiple classes of antimicrobial drugs, including β-lactams, tetracyclines, and fluoroquinolones (1–3). Even an extended-spectrum oral cephalosporin-resistant, cefixime-resistant N. gonorrhoeae has emerged, and cefixime has now been withdrawn from use in Japan. Best practice treatment is limited to injectable extended-spectrum cephalosporins, such as ceftriaxone and spectinomycin. The emergence of ceftriaxone-resistant N. gonorrhoeae threatens effective disease control. We identified a novel ceftriaxone-resistant N. gonorrhoeae isolated from a 31-year-old female commercial sex worker; MIC of ceftriaxone for this isolate was high (2 µg/mL). The woman visited a clinic in Kyoto for a routine examination for sexually transmitted infections in January 2009. Although she had no obvious symptoms or signs, a throat sample collected on her first visit yielded a positive result for N. gonorrhoeae by the strand displacement amplification test (ProbeTec ET, Becton Dickinson, Franklin Lakes, NJ, USA), but a vaginal sample taken at the same time was negative. After 2 weeks, another throat sample was positive for N. gonorrhoeae when cultured on Thayer-Martin medium, and the patient subsequently received 1 g ceftriaxone intravenously. Her pharyngeal sample was also N. gonorrhoeae positive by strand displacement amplification test on the third visit 2 weeks later, and further ceftriaxone treatment was prescribed. However, a culture for test of cure was not conducted because reinfection was considered. A negative result was finally obtained in April 2009. The culture showed positive reactions in oxidase and catalase tests. Gram staining showed gram-negative diplococci. The ID-test HN-20 Rapid system (Nissui, Tokyo, Japan) classified the bacterium as N. gonorrhoeae. Susceptibility was determined by the agar dilution method (4). For this strain, named H041, MIC of ceftriaxone was high (2 µg/mL), and the strain was highly resistant to penicillin G (4 µg/mL), cefixime (8 µg/mL), and levofloxacin (32 µg/mL). However, it demonstrated susceptibility to spectinomycin (16 µg/mL) and reduced susceptibility to azithromycin (0.5 µg/mL). To characterize the ceftriaxone-resistant N. gonorrhoeae H041, multilocus sequence typing characterized the strain as ST7363 (5), which is the predominant sequence type (ST) among cefixime-resistant clones (6). N. gonorrhoea multiantigen sequence typing (NG-MAST) was also performed (7). The NG-MAST strategy uses 2 genes, por and tbpB, for porin and a transferrin-binding protein, respectively. NG-MAST indicated that the strain H041 was ST4220 and contained the por2594 allele and the tbpB10 allele. NG-MAST 4220 is a novel ST. However, the tbpB10 allele is the most frequently observed allele (76.5%) among multilocus sequence typing-ST7363 N. gonorrhoeae strains (n = 81) (M. Ohnishi, unpub. data). Molecular typing suggested that the novel ceftriaxone-resistant N. gonorrhoeae, H041, is closely related to the ST7363 cefixime-resistant N. gonorrhoeae. Therefore, we compared SpeI-digested genomic DNA banding patterns of strain H041 with those of other N. gonorrhoeae strains by using pulsed-field gel electrophoresis as described (8). Four ST7363 strains, including N. gonorrhoeae H041, and 4 ST1901 strains (another major ST among cefixime-resistant N. gonorrhoeae strains) (6) were analyzed. The banding pattern of SpeI digested H041 genomic DNA was similar to that of other ST7363 strains and indistinguishable from that of cefixime-resistant but ceftriaxone-susceptible NG0207 (Figure). Figure Pulsed-field gel electrophoresis patterns of ceftriaxone-resistant Neisseria gonorrhoeae strain H041 and other multilocus sequence typing (MLST) ST7363 and ST1901 strains. SpeI-digested genomic DNA from ceftriaxone-resistant N. gonorrhoeae H041, 3 of ... We describe the emergence of ceftriaxone-resistant N. gonorrhoeae, isolated from a pharyngeal specimen from a female commercial sex worker. At 2 µg/mL, the MIC was 4-fold higher than that of the previously reported ceftriaxone nonsusceptible strain (9). Our susceptibility testing suggests that only azithromycin and spectinomycin are effective drugs for treating this strain. In this case, eradication was successful, although N. gonorrhoeae colonization of the pharynx may just be tempory because the pharynx is not an ideal site for N. gonorrhoeae growth. From the routine examinations of commercial sex workers during January–March 2009, 40 N. gonorrhoeae were isolated in the clinic, but no other ceftriaxone-resistant strains were isolated. There is no evidence of dissemination of this strain in Kyoto. Three independent molecular subtyping methods indicated that the ceftriaxone-resistant H041 strain was N. gonorrhoeae, and it might originate from an ST7363 cefixime-resistant N. gonorrhoeae clone. There are several possible mechanisms for the acquisition of resistance, including formation of a new mosaic type penA allele as penA-X cefixime resistance and acquisition of an extended-spectrum β-lactamase gene. The H041 strain did not produce β-lactamase in a nitrocephin test. Further molecular analysis is needed to elucidate the precise mechanism of the ceftriaxone resistance of the H041 strain. The emergence of ceftriaxone-resistant N. gonorrhoeae raises concerns for controlling gonorrhea because ceftriaxone is widely recommended and the first-line treatment for gonorrhea around the world. N. gonorrhoeae has a potential to gain an extraordinarily high MIC to ceftriaxone. Surveillance for ceftriaxone-resistant N. gonorrhoeae should be strengthened.

Serum and urinary estrone sulfate during the menstrual cycle, measured by a direct radioimmunoassay, and fate of exogenously injected estrone sulfate

Serum and early-morning urinary levels of estrone sulfate during the menstrual cycle were measured by a direct radioimmunoassay without hydrolysis. These levels were high and showed prominent peaks [serum, 2.67 +/- 0.37 ng/ml (mean +/- SE); urine, 5.82 +/- 2.3 micrograms/l] around the day of the preovulatory estradiol-17 beta peak, and increased again during the luteal phase. Following intravenous injection of estrone sulfate, serum estrone sulfate, estrone and estradiol-17 beta were measured. The conversion of estrone sulfate to estrone and/or estradiol-17 beta was very small during their transit in the general circulation.

Estrogen-related receptor α expression and function are associated with vascular endothelial growth factor in human cervical cancer.

Introduction: Estrogen-related receptor &agr; (ERR&agr;), one of orphan nuclear receptors with an unknown ligand, is expressed in various types of cancer. Increased ERR&agr; levels are associated with a higher risk of recurrence and poor clinical outcome in breast cancer, suggesting that ERR&agr; could be a negative prognostic factor. Recently, it has been suggested that vascular endothelial growth factor (VEGF) could be one of the transcriptional targets of ERR&agr; in breast cancer. Here, we examined the expression of ERR&agr; and the association of ERR&agr; with VEGF in uterine cervical cancer cells and tissues. Methods: We evaluated the expression of ERR&agr; and VEGF by immunohistologic analysis using specimens from 40 patients with invasive cervical cancer. We also evaluated the VEGF promoter activity of ERR&agr; in cervical cancer cell lines by transfection and luciferase assay. We overexpressed or knocked down ERR&agr; and examined VEGF expression by real-time polymerase chain reaction. Finally, cell proliferation assay was performed to examine whether ERR&agr; affects tumor growth in cervical cancer. Results: Immunohistologic analysis demonstrated that ERR&agr; expression in cervical cancer tissues was higher than that in noncancerous tissues and that there was a positive association between ERR&agr; and VEGF expression in cancer tissues (P < 0.05). We showed that ERR&agr; stimulated the VEGF promoter activity in cervical cancer cell lines. We further showed the overexpression and knockdown of ERR&agr;-regulated VEGF expression level by real-time polymerase chain reaction. Moreover, we showed that ERR&agr; and VEGF knockdown by small interfering RNA or an inverse agonist of ERR&agr;, XCT 790, could suppress cell growth compared with control cells in cervical cancer. Conclusions: We have provided compelling evidence that ERR&agr; affects VEGF expression and tumor growth in cervical cancer. These results justify further investigation into the use of ERR&agr; as a therapeutic target for patients with uterine cervical cancer.

Premature delivery due to intrauterine Candida infection that caused neonatal congenital cutaneous candidiasis: A case report

Congenital cutaneous candidiasis is a very rare disease with less than 100 cases published in the medical literature. Neonates having this disease present with systemic skin lesions caused by intrauterine Candida infections. We present a case of threatened premature delivery due to Candida chorioamnionitis, which caused both maternal postpartum endometritis and neonatal congenital cutaneous candidiasis. A 34‐year‐old woman who was admitted for fetal membrane bulging at 20 weeks of gestation underwent McDonald cervical cerclage. We diagnosed threatened premature delivery due to intrauterine infection; therefore, we terminated the gestation by cesarean section at 24 weeks of gestation. Fungi‐like yeast was detected in infantile gastric juice. Histopathological findings of the placenta revealed that Candida albicans mycelium invaded the placenta, chorioamniotic membrane and umbilical cord.

Hematoma and abscess formation caused by Mycoplasma hominis following cesarean section

Mycoplasma species cannot be identified by routine bacteriological culture methods and are resistant to common antimicrobial agents. Mycoplasma hominis usually colonizes the lower urogenital tract and causes pyelonephritis, pelvic inflammatory disease, chorioamnionitis, rupture of fetal membranes, preterm labor, postpartum fever, postabortal fever, and neonatal infection. This organism is highly prevalent in cervicovaginal cultures of sexually active women. M. hominis, M. genitalis, Ureaplasma urealyticum, and U. parvum may invade and infect placental and fetal tissues, leading to adverse pregnancy outcomes. M. hominis occasionally causes nongenitourinary infection of the blood, wounds, central nervous system, joints, or respiratory tract. We present a case of a 27-year-old woman who developed abdominal wound hematoma and abscess after cesarean section. The wound was drained, but her high fever persisted, in spite of antibiotic treatment using flomoxef sodium and imipenem·cilastatin sodium. Because the exudate exhibited M. hominis growth in an anaerobic environment, we administered the quinolone ciprofloxacin. This therapy resolved her fever, and her white blood cell count and C-reactive protein level diminished to the normal ranges. To our knowledge, there are four published articles regarding the isolation of M. hominis from postcesarean incisions. Based on the current study and the literature, infection by this pathogen may cause hematoma formation with or without abscess after cesarean section or in immunosuppressed postoperative patients. In such cases, physicians may need to suspect Mycoplasma infection and initiate appropriate antibacterial treatment as soon as possible in order to avoid persistent fever.

Estrogen-related receptor-γ regulates estrogen receptor-α responsiveness in uterine endometrial cancer.

Objective Estrogen-related receptors (ERRs) are orphan nuclear receptors that modulate the estrogen receptor (ER)-mediated pathway and play roles in the regulation of breast and prostate cancer cell growth. However, the significance of the localization and the function of ERRs in uterine endometrial cancer remain unclear. We aimed to measure the expression of ERR&ggr; and determine its association with the ER-mediated pathway in human endometrial cancer. Methods Proliferation, luciferase, and quantitative polymerase chain reaction assays were performed in ER&agr;-positive (Ishikawa) and ER&agr;-negative (HEC1A) endometrial cancer cell lines. The association between ERR&ggr; and ER&agr; expressions was determined by immunohistochemical analysis in uterine endometrial cancer tissues. Results Estrogen-induced estrogen response element transcriptional activity was repressed by ERR&ggr; in ER&agr;-positive cells but was stimulated by ERR&ggr; in ER&agr;-negative cells. The stable overexpression of ERR&ggr; regulated the in vitro cell growth in the ER&agr;-positive and ER&agr;-negative endometrial cancer cell lines. A selective ERR&ggr; agonist, DY131, inhibited the growth of the ER&agr;-positive endometrial cancer cells but promoted that of the ER&agr;-negative cancer cells. Furthermore, we found that ERR&ggr; is expressed in the nuclei of human uterine endometrial cancer tissues. Estrogen-related receptor &ggr; was not associated with pathological parameters such as the International Federation of Gynecology and Obstetrics stage and histological type. The uterine endometrial cancer tissues with ERR&ggr;-positive/ER&agr;-negative status may have a significantly poor prognosis. Conclusions The relationship between ERR&ggr; and ER&agr; status could be a predictive marker for the treatment of uterine endometrial cancer, which provides an impetus for the identification of ligands for nuclear orphan receptor ERR&ggr;.

Prenatal Diagnosis of Agnathia-Otocephaly Using Sonography and Magnetic Resonance Imaging

Agnathia-otocephaly is a rare lethal malformation characterized by micrognathia/agnathia, microglossia/aglossia, microstomia, and several types of abnormally positioned ears. Most cases of agnathia-otocephaly are sporadic; however, our case was an extremely rare case of agnathiaotocephaly following a sibling case of micrognathia. In this case, 2-dimensional (2D) sonography, magnetic resonance imaging (MRI), and subsequent 3-dimensional (3D) sonography led us to a precise prenatal diagnosis of agnathia-otocephaly. The patient was a 40-year-old woman, gravida 3, para 3. She had histories of left anophthalmia, left sensorineural hearing loss, and infertility. Her husband had a history of right amblyopia. However, the cause had not been clear. Her first pregnancy resulted in dichorionic triamniotic triplets; 2 fetuses died in utero at 15 weeks’ gestation. A live female neonate was delivered vaginally at 38 weeks (weight, 2322 g; Apgar scores, 9 and 10 at 1 and 5 minutes, respectively); the 2 dead fetuses were delivered as fetuses papyraceus simultaneously. The female child was healthy and in good condition for 6 years after birth. In her second pregnancy, MRI findings at 31 weeks revealed a male fetus with mandibular hypoplasia. The neonate was delivered vaginally at 37 weeks (weight, 2287 g; Apgar scores, 1 and 1 at 1 and 5 minutes) and died of airway obstruction shortly after birth. The autopsy showed that the neonate had micrognathia without other abnormalities (Figure 1). In her third pregnancy, the amniotic fluid was abundant (amniotic fluid index, 26 cm) at 25 weeks. We collected the amniotic fluid to reduce the volume and to perform a karyotyping analysis. The results revealed a normal 46,XY karyotype. Two-dimensional sonography showed micrognathia, orbital hypotelorism, downward displacement of the orbits, proboscis, low-set ears, and a normal brain. Other minor abnormalities were not detected. Magnetic resonance imaging confirmed the 2D sonographic findings. At this time, we thought about the possibility of agnathia-otocephaly. Subsequent 3D sonography showed a total 3D craniofacial construction and extremely low-set ears adjacent to each other medially, which confirmed our diagnosis (Figure 2, A and B). We informed the parents of the diagnosis of agnathiaotocephaly, the poor prognosis, and the fact that all survivors require support to maintain airway patency, such as tracheostomy or ex utero intrapartum treatment. However, the parents did not agree to those treatments. A male neonate was delivered vaginally at 38 weeks (weight, 2454 g; Apgar scores, 1 and 1 at 1 and 5 minutes) and died of total airway obstruction shortly after birth. Externally, the neonate had agnathia, extremely low-set ears adjacent to each other medially, a proboscis with 2 nostrils, a small mouth cavity, and downward-slanting palpebral fissures. The subsequent internal autopsy revealed bilateral auditory meatus atresia, a hypoplastic palate, a rudimentary tongue, a small trachea-oropharynx connection, 2 nostrils connecting with an extremely small blind-ending duct, orbital hypotelorism, and normal-size orbits located laterally and caudally to the proboscis. The brain and other organs were normal. The skeletal structure showed total mandibular agenesis and a rudimentary maxillary bone (Figure 2C). Clinical Letters

Chlamydial Proctitis in Patients with Chlamydial Cervicitis

Aims: We investigated the status of chlamydial proctitis, detected using a transcription-mediated amplification (TMA) method, in rectal mucosal swab samples from patients with chlamydial cervicitis. Methodology: Patients with chlamydial cervicitis were interviewed, and rectal mucosal swab samples were collected for TMA. If the patient agreed, colonoscopy was also conducted. Chlamydial proctitis was treated with a single dose of oral azithromycin (2000 mg). Three weeks after treatment, additional samples from the cervix and rectal mucosa were subjected to TMA, and follow-up colonoscopy was performed. Results: Among the 59 patients, 4 had diarrhea and 3 had melena; only 1 patient had practiced Original Research Article Iwasaku et al.; ISRR, 3(1): 8-13, 2015; Article no.ISRR.2015.002 9 anal sex. The rectal mucosal TMA test was positive in 43 (72.9%) cases. After treatment, TMA tests of the cervix and rectal mucosa were negative in all patients and in 26 (86.7%) of 30 patients, respectively. Conclusion: The clearance rate of chlamydial infection of the rectal mucosa was not 100% and the cervical samples became negative in all cases following treatment in this study. Further studies may be needed to determine the optimal indicator for evaluating patient treatment responses and to reliably clear the infection with an alternate drug or dosing regimen.

Efficacy and safety of intravenous azithromycin followed by oral azithromycin for the treatment of acute pelvic inflammatory disease and perihepatitis in Japanese women

UNLABELLED Pelvic inflammatory disease (PID) is mainly caused by ascending infection from the vaginal flora including the sexually transmitted organisms, Neisseria gonorrhoeae and Chlamydia trachomatis, and lower genital tract endogenous anaerobes, leading to serious consequences including infertility and ectopic pregnancy. To evaluate the efficacy and safety of azithromycin in the treatment of PID that requires initial intravenous therapy, we conducted a multicenter, unblinded, non-comparative phase 3 trial. Intravenous azithromycin (500 mg, once daily) for 1 or 2 days followed by oral azithromycin (250 mg once daily) to complete a total of 7 days treatment was administered to 60 Japanese women with acute PID. The clinical and bacteriological responses were assessed at the end of treatment, and on Days 15 and 29. The most commonly detected baseline causative pathogens were C. trachomatis (12 strains), Prevotella bivia (10 strains), Streptococcus agalactiae (7 strains), N. gonorrhoeae and Peptostreptococcus anaerobius (6 strains each). The clinical success rate on Day 15 was 94.1% (48/51 subjects including perihepatitis). The clinical efficacy and bacterial eradication rates against C. trachomatis and N. gonorrhoeae (including 2 quinolone-resistant strains) were both 100%. Common treatment-related adverse events were diarrhoea, injection site pain, and nausea. All adverse events were mild or moderate in severity. Azithromycin intravenous-to-oral switch therapy demonstrated excellent clinical and bacteriological effects for PID caused by various etiologic agents including quinolone-resistant strains and strains with low susceptibility to azithromycin at in vitro testing. The therapy was well tolerated in the treatment of PID in Japanese women. REGISTRATION NUMBER NCT00871494.