Molecular detection of Mycoplasma genitalium in endocervical swabs and associated rates of macrolide and fluoroquinolone resistance in Hong Kong.

INTRODUCTION: There is a global trend of increasing macrolide and fluoroquinolone resistance in Mycoplasma genitalium (MG), such that international guidelines recommend molecular detection of resistance if a patient has MG-positive test results. Tests for MG are not routinely performed in Hong Kong. This study examined the detection of MG in endocervical swabs and the associated macrolide and fluoroquinolone resistance rates. METHODS: Endocervical swabs received from two sexual health clinics in Hong Kong for routine assessments of Chlamydia trachomatis and Neisseria gonorrhoeae were also subjected to detection of MG. All MG-positive samples were tested for resistance-mediating mutations in 23S rRNA, parC, and gyrA genes. Laboratory records and past results for each patient were analysed. RESULTS: In total, endocervical swabs from 285 patients were included in this study. Mycoplasma genitalium was detected in swabs from 21 patients (7.4%) by real-time polymerase chain reaction with a commercial kit. Among MG-positive samples which were successfully analysed further, macrolide resistance-mediating mutations in 23S rRNA were found in 42.1% (8/19); fluoroquinolone resistance-related mutations in parC and gyrA were found in 65% (13/20) and 0% (0/20), respectively. All macrolide-resistant MG strains were also fluoroquinolone-resistant (42.1%, 8/19). No assessed factors were associated with the detection of MG or resistance-related mutations. CONCLUSION: In Hong Kong, MG was detected in endocervical swabs from 7.4% of patients in sexual health clinics, with high rates of macrolide and fluoroquinolone resistance. These findings warrant careful review of testing, clinical correlation, and treatment strategies for MG in the context of increasing antibiotic resistance.

Emergent genital infection by Leptotrichia trevisanii.

We report the first case of an association between Leptotrichia trevisanii and an episode of pelvic inflammatory disease (PID) and the second case of the isolation of this infection in the cervical canal. A 45-yr-old woman was admitted to our emergency department with clinical and radiological signs and symptoms compatible with an episode of PID. She was hospitalized for intravenous antibiotic control and treatment and the subsequent surgical drainage of abscesses. Cultures were taken throughout the process, but only cultures from cervical canal exudate were positive, with the growth of L. trevisanii species. It appears important to carry out a complete microbiological screening, not limited to conventional agents, on adequate clinical samples to detect possible infectious agents that may be missed in these cases.

Uterine cervical ulceration caused by group C streptococcal infection during pregnancy: A case report.

Causes of cervical ulceration include infection, collagen disease, malignant tumors and external stimuli. Cervical ulceration during pregnancy is rare. We present a case of cervical ulceration caused by group C streptococcal infection during pregnancy. A 36-year-old woman (gravida 1, para 0) complained of metrorrhagia, and a circular cervical ulcer of about 1.5 cm in diameter was detected on her cervix at 37 weeks' gestation. A biopsy and a cultivation test of the ulcer were performed, and pathological diagnosis was made as suppurative inflammation, and group C streptococcal infection was detected by the cultivation test. The ulcer had expanded to about 3 cm in diameter at the onset of labor at 40 weeks' gestation. An emergency cesarean section was performed because of failed induction of labor, and she was delivered of a male baby. The ulcer became gradually smaller after delivery, and completely disappeared on the 35th day after delivery.

HPV/Chlamydia trachomatis co-infection: metagenomic analysis of cervical microbiota in asymptomatic women.

HPV and Chlamydia trachomatis are the most common causes of sexually transmitted diseases worldwide. Most infections are asymptomatic and left untreated lead to severe reproductive tract sequelae such as cervical cancer and infertility. Interestingly, C. trachomatis may also increase the susceptibility to HPV infection as well as contribute to viral persistence. Recently, a growing body of evidence has suggested that the composition of the cervico-vaginal microbiota plays a key role in the susceptibility and outcome of genital infections caused by several pathogens, including HPV and C. trachomatis. The aim of our study was to undertake a metagenomic analysis of sequenced 16s rRNA gene amplicons to characterize the cervical microbiota from asymptomatic women with HPV/C. trachomatis co-infection. The composition of the cervical microbiota from HPV-positive or C. trachomatis-positive women was also analysed. The main finding of our study showed that the cervical microbiota in HPV/C. trachomatis co-infected women had a higher microbial diversity than the cervical microbiota in healthy controls (p<0.05). In addition, Aerococcus christensenii was associated with C. trachomatis infection. In conclusion, the increased cervical microbial diversity observed in HPV/C. trachomatis co-infected women and the detection of potential microbiological biomarkers of C. trachomatis infection will open the way to innovative approaches that may be helpful to identify women at risk of co-infection.

Co-infection of sexually transmitted pathogens and Human Papillomavirus in cervical samples of women of Brazil.

BACKGROUND: Some sexually transmitted infectious agents, such as Chlamydia trachomatis and Herpes simplex, cause local inflammation, and could contribute to Human Papillomavirus (HPV) and cervical lesion progression. Thus, the aim of this study was to determine any association between the presence of microorganisms of gynecological importance, sexual behavior, clinical and demographical variables to the development and progress of cervical lesions. METHODS: One hundred and thirty-two women between 14 and 78 years and living at Vitória da Conquista, Bahia, Brazil, were included (62 individuals with cervical lesions and 70 without lesions). They answered a questionnaire to provide data for a socioeconomic and sexual activity profile. Samples of cervical swabs were collected and analyzed by PCR to detect genital microorganisms and HPV. Quantitative PCR was used to detect and quantify Ureaplasma urealyticum and Ureaplasma parvum. Univariate and multiple logistic regression were performed to measure the association with the cervical lesions, and an odds ratio (OR) with 95% confidence intervals (95%CI) were calculated. The Mann-Whitney U test was also used to compare the microorganism load in the case and control groups. The significance level was 5% in all hypotheses tested. RESULTS: Cervical lesions were associated with: women in a stable sexual relationship (OR = 14.21, 95%CI = 3.67-55.018), positive PCR for HPV (OR = 16.81, 95%CI = 4.19-67.42), Trichomonas vaginalis (OR = 8.566, 95%CI = 2.04-35.94) and Gardnerella vaginalis (OR = 6.13, 95%CI = 1.53-24.61), adjusted by age and qPCR for U. parvum. U. parvum load showed a statistical difference between the case and control groups (p-value = 0.002). CONCLUSION: Variables such as stable relationship, HPV, T. vaginalis, G. vaginalis were associated with cervical lesions in epidemiological studies. U. parvum load was higher in woman with cervical lesions compared with women without lesions. Additional studies are needed to better understand the role of these factors in cervical lesion development.

[Pilot tests using molecular diagnostic assay cervicovaginal infection during pregnancy]. / Ensayo piloto del uso de pruebas moleculares para el diagnóstico de infecciones cervicovaginales en pacientes embarazadas.

Background: The prevalence of cervicovaginal infections during pregnancy has been associated with adverse perinatal outcomes however, the actual approach used for diagnosis is not effective. The aim of this study was to compare the diagnosis of vaginal infections in pregnant women using clinical, molecular diagnostic and traditional microbiological culture in a pilot study, to determine the prevalence and association with the development of preterm labor. Materials and methods: We performed a nested cross-sectional study composed by 54 women in a cohort of pregnant women in Mexico City. Cervicovaginal infections were evaluated by clinical methods, microbiology culture and a commercially available molecular biology test. Results: Prevalence of cervicovaginal infections during pregnancy was estimated between 28% and 50% according to methodologies. Considering the clinical diagnosis of preterm labor as the gold standard, all diagnostic tests were poor as predictors of preterm labor. Conclusion: Traditional approaches to establish the significance of cervicovaginal infection in pregnancy are exhausted, so be sought new ways to understand this complex relationship. Meanwhile it is recommended to continue to use traditional methods to identify infections during pregnancy in both knowledge of new methods aimed at understanding these relationships are sophisticated.

[FREQUENCY OF PRETERM BIRTH IN WOMEN WITH CERVICAL VAGINAL INFECTIONS.]

Premature birth (PB) occurs in 5% to 18% of births in the world. Children born prematurely are at increased risk of death, major neonatal complications, long-term adverse consequences for health and development. The cause of spontaneous preterm birth is often unknown, but it is believed that intrauterine infection is implicated in 40% of cases. The most likely route to intrauterine infection is ascending infection of the genital tract. Infection of the genital tract is more common in women with spontaneous preterm births in lower ge. tational age. s performed a retrospective research of 70 pregnant women and their newborns. In the group with preterm birth was reported high percentage of CVI - 37.14%, while in the control group this percentage is 17.14%. The results showed the increase in the incidence of CVI group of PB more than twice. There is a need an effective prevention and treatment of CVI during pregnancy. The timely detection of deviations from normal vaginal microflora and the correct therapeutic approach would reduce the risks of PB and infectious complications in newborns.

Autoimmune neutropenia of infancy with recurrent urinary tract infections: a case report.

Autoimmune neutropenia of infancy is characterized by minor intercurrent infections despite severe neutropenia; severe bacterial infections are uncommon. An infant developed recurrent urinary tract infections at 9 and 11 months of age. The identified uropathogens were Escherichia coli and Enterococcus faecalis, respectively. Empirical treatment with carbapenems, as broad-spectrum antibiotics, promptly resolved the infection without sequelae. Febrile neutropenic children with cancer and autoimmune neutropenia can develop urinary tract infections; therefore, in such infants, urine culture should be obtained through catheterization. In febrile neutropenic infants with no apparent fever source, cephalosporin monotherapy should not be selected empirically because Enterococci can be the involved pathogens.

Microbiological Infections in Women With Cervical Cytological Reports of Atypical Squamous Cells of Undetermined Significance.

OBJECTIVE: To study the role of cervicovaginal infections in women with cytological reports of atypical squamous cells of undetermined significance (ASC-US). MATERIALS AND METHODS: The study included 220 women admitted to the Clinic of Microscopy, Cervicovaginal and Vulvar Pathology of the Department of Gynecology and Obstetrics of the Tor Vergata University Hospital, Rome, Italy, enrolled between October 2012 and July 2013. RESULTS: Among the enrolled women, 105 women (47.7%) had ASC-US cytology, whereas 115 women (52.3%) had negative cytology. Microscopy showed infections more frequently in women with ASC-US than in those with negative cytology: 70.5% (74/105) vs 36% (41/115); p < .001. Cocci were present in 73.3% (77/105) of the women with ASC-US and in 43.5% (50/115) of those with negative cytology; p < .001. According to Ison score, 84% (88/105) of ASC-US was grade 0 vs 22% (25/115) of negative cytology, p < .001. Human papillomavirus was detected in 35% of the women with ASC-US. A statistically significant correlation between high pH and vaginal infections was found in women aged 20 to 29 (p = .003) and those 50 years or older in both cytological report groups; p < .001. CONCLUSIONS: Cervicovaginal infections are associated with a cytological report of ASC-US. Direct microscopy of vaginal specimens allowing immediate evaluation of the vaginal microflora and infectious agents may be a useful tool in managing women with cytological reports of ASC-US.

[Clinical efficacy of fluconazole, tinidazole and clindamycin vs fluconazole, tinidazole and azithromycin in the treatment of mixed cervical-vaginal infections, included those caused by Mycoplasma and Chlamydia trachomatis]. / Eficacia clínica de fluconazol, tinidazol y clindamicina vs fluconazol, tinidazol y azitromicina en el tratamiento de infecciones cérvico- vaginales mixtas, incluidas las causadas por Mycoplasma y Chlamydia trachomatis.

BACKGROUND: In the United States 19 million people acquire a sexually transmitted disease every year. Sexually transmitted diseases impact in gynecological terms because they may cause sterility, infertility and ectopic pregnancy. OBJECTIVE: To compare the effectiveness of two combinations of three oral antimicrobial drugs in the treatment of mixed cervical-vaginal infections, included those caused by Mycoplasma and Chlamydia trachomatis. MATERIAL AND METHOD: Aclinical, random, comparative, double-blind study included 50 patients assisting to infectology consult with diagnosis of mixed cervical-vaginal infection. Patients were divided into two groups: Group A (n = 25): fluconazole 37.5 mg, tinidazole 500 mg and azithromycin 250 mg; group B (n = 25): fluconazole 37.5 mg, tinidazole 500 mg and clindamycin 312.5 mg. Patients of both groups received two tablets twice p.o. for one day. Cultures were performed to corroborate the diagnosis and then to demonstrate effectiveness of the schemes studied. For the analysis of the data we used measures of central tendency, dispersion and inferential statistics for comparison of proportions by c2 and Fisher's exact tests with a significance level of p < 0.05. RESULTS: All patient got clinical cure; however, regarding the microbiologic eradication a positive case was identified in group A, requiring rescue treatment. The compliance in both groups was of 100%. In both groups, statistical analysis did not show significant differences. Three patients in group A had mild adverse effects. Patients mean age was 33.4 +/- 5.3 years. CONCLUSIONS: Both treatments showed similar effectiveness against mixed cervical-vaginal infections. Microbiological efficacy was of 96% and 100% in group A and B, respectively, besides, scheme of group B was better tolerated.

A genome-wide profiling of the humoral immune response to Chlamydia trachomatis infection reveals vaccine candidate antigens expressed in humans.

A whole genome scale proteome array consisting of 908 open reading frames encoded in Chlamydia trachomatis genome and plasmid was used to profile anti-chlamydial Ab responses. A total of 719 chlamydial proteins was recognized by one or more antisera from 99 women urogenitally infected with C. trachomatis. Revealing such a large C. trachomatis ANTIGENome in humans might partially be attributed to the significantly improved detection sensitivity of the whole genome scale proteome array assay because both linear and conformation-dependent Abs were detected by the array assay. Twenty-seven of the 719 Ags were recognized by >or=50% antisera, thus designated as immunodominant Ags. Comparison of Ag profiles recognized by live chlamydial organism-infected versus dead organism-immunized hosts led to the identification of infection-dependent or in vivo expressed Ags. The infection-dependent Ags induced Abs only in live organism-infected, but not in dead organism-immunized hosts. Many of these Ags were highly expressed during replication, but only minimally packaged into the infectious elementary bodies. Because inactivated whole chlamydial organism-based vaccines failed to induce protection in humans, identification of the infection-dependent or in vivo expressed immunodominant Ags in humans should greatly facilitate the selection of promising chlamydial subunit vaccine candidates for further evaluation. This approach may also be applicable to other pathogens.

Risk factors for Mycoplasma genitalium infection among female sex workers: a cross-sectional study in two cities in southwest China.

BACKGROUND: Mycoplasma genitalium (MG) is one of the common causes of non-gonococcal urethritis (NGU) in men and is associated with cervicitis, endometritis, and pelvic inflammatory diseases (PID) in women. The prevalence of MG infection has been reported to be high among female sex workers (FSWs) in many countries, but limited information is known among this population in China. METHODS: From July to September 2009, venue-based FSWs were recruited in two cities (Wuzhou and Hezhou) of Guangxi Autonomous Region in southwest China. Information of socio-demographic and behavioral characteristics was collected by a questionnaire-based interview. Cervical specimens were obtained for detection of MG using a real-time polymerase chain reaction (PCR) assay targeting mgpA gene. RESULTS: The overall prevalence of MG infection among 810 FSWs was 13.2% (95% CI = 10.87%-15.52%). MG infection was significantly associated with less education (adjusted odds ratio (AOR) = 2.36, 95% CI = 1.15-4.87) consisting of junior high school or below, being single (AOR = 2.27, 95% CI = 1.42-3.62), migrant background (AOR = 2.03, 95% CI = 1.29-3.20), and absence of any STI symptoms in the previous year (AOR = 1.66, 95% CI = 1.09-2.52). CONCLUSIONS: MG infection was prevalent among FSWs in the study areas. This pattern of infection suggests that an increasing attention should be paid to MG screening and treatment in this high risk population.

Correlation between Lactobacillus and expression of E-cadherin, ß-catenin, N-cadherin, and Vimentin in postmenopausal cervical lesions.

BACKGROUND: To detect the correlation between Lactobacillus vaginalis and the expression of epithelial-mesenchymal transition (EMT)-related factors, E-cadherin, ß-catenin, N-cadherin, and Vimentin, in postmenopausal cervical squamous intraepithelial lesions (SILs) and cervical squamous cell carcinoma (SCC), and to explore the possible mechanism. METHODS: From January 2016 to January 2020, 30 postmenopausal patients with low-grade SIL (LSIL), 18 patients with high-grade SIL (HSIL), and 30 patients with SCC who underwent colposcopy biopsy in the Outpatient Department of the First Affiliated Hospital of Inner Mongolia Medical University were selected as the experimental group, and 30 postmenopausal normal women were selected as the control group. The expression of 16SrRNA of Lactobacillus vaginalis in each group was determined by the 16S third-generation full-length amplification sequencing technique. The mRNA expression levels of E-cadherin, ß-catenin, N-cadherin, and Vimentin were detected by quantitative real-time polymerase chain reaction (qPCR). The correlation between the 16SrRNA expression level of Lactobacillus vaginalis and the mRNA expression level of the EMT-related proteins was compared among all groups. RESULTS: (I) The progression of postmenopausal cervical SILs to cervical SCC was significantly positively correlated with age, number of pregnancies, smoking, pH value, positive rate of HPV16, and negatively correlated with total Lactobacillus 16SrRNA expression (P<0.0001). (II) The level of vaginal microbiota in postmenopausal women showed that Lactobacillus iners was dominant. With the progression of the disease, the expression levels of 16SrRNA in Lactobacillus iners and Lactobacillus total vagina decreased gradually, and the differences were statistically significant (P<0.05). (III) With the disease progresses. The expression of total Lactobacillus 16SrRNA was positively correlated with the mRNA expression of ß-catenin and E-cadherin (r>0; P<0.05), and negatively correlated with the mRNA expression of Vimentin and N-cadherin (r<0; P<0.05). CONCLUSIONS: In postmenopausal women, Lactobacillus vaginalis interacts with HPV and is associated with the occurrence of EMT, promoting the development of cervical lesions.

Altered protein secretion of Chlamydia trachomatis in persistently infected human endocervical epithelial cells.

Chlamydia trachomatis is the most common bacterial infection of the human reproductive tract globally; however, the mechanisms underlying the adaptation of the organism to its natural target cells, human endocervical epithelial cells, are not clearly understood. To secure its intracellular niche, C. trachomatis must modulate the host cellular machinery by secreting virulence factors into the host cytosol to facilitate bacterial growth and survival. Here we used primary human endocervical epithelial cells and HeLa cells infected with C. trachomatis to examine the secretion of bacterial proteins during productive growth and persistent growth induced by ampicillin. Specifically, we observed a decrease in secretable chlamydial protease-like activity factor (CPAF) in the cytosol of host epithelial cells exposed to ampicillin with no evident reduction of CPAF product by C. trachomatis. In contrast, the expression of CopN and Tarp was downregulated, suggesting that C. trachomatis responds to ampicillin exposure by selectively altering the expression of secretable proteins. In addition, we observed a greater accumulation of outer-membrane vesicles from C. trachomatis in persistently infected cells. Taken together, these results suggest that the regulation of both gene expression and the secretion of chlamydial virulence proteins is involved in the adaptation of the bacteria to a persistent infection state in human genital epithelial cells.

Development of a pigtail macaque model of sexually transmitted infection/HIV coinfection using Chlamydia trachomatis, Trichomonas vaginalis, and SHIV(SF162P3).

BACKGROUND: Sexually transmitted infections (STIs) are associated with an increased risk of HIV infection. To model the interaction between STIs and HIV infection, we evaluated the capacity of the pigtail macaque model to sustain triple infection with Trichomonas vaginalis, Chlamydia trachomatis, and SHIV(SF162P3). METHODS: Seven SHIV(SF162P3) -infected pigtail macaques were inoculated with T. vaginalis only (n = 2), C. trachomatis only (n = 1), both T. vaginalis and C. trachomatis (n = 2), or control media (no STI; n = 2). Infections were confirmed by culture and/or nucleic acid testing. Genital mucosa was visualized by colposcopy. RESULTS: Characteristic gynecologic signs were observed for both STIs, but not in control animals. Manifestations were most prominent at days 7-10 post-infection. STIs persisted between 4 and 6 weeks and were cleared with antibiotics. CONCLUSIONS: These pilot studies demonstrate the first successful STI-SHIV triple infection of pigtail macaques, with clinical presentation of genital STI symptoms similar to those observed in humans.

Host chemokine and cytokine response in the endocervix within the first developmental cycle of Chlamydia muridarum.

The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.

Diagnostic implications of 16S ribosomal assay for gonorrhoea.

OBJECTIVES: In the absence of a single nucleic acid amplification test (NAAT) that is both highly specific and sensitive for gonorrhoea, many have put forward the 16S-based assay as a confirmatory test for Neisseria gonorrhoeae. This study was undertaken to evaluate the performance of PCR based on 16S ribosomal gene in comparison with a porA pseudogene-based assay. METHODS: The specificity of both the porA pseudogene-based PCR and 16S ribosomal gene PCR was checked against a panel of strains comprising of non N gonorrhoeae Neisseria sp (NgNS) and other gram-negative and gram-positive bacteria. The sensitivity studies were performed using different dilutions of N gonorrhoeae DNA. PCRs were also done on endocervical and urethral swab samples collected from a total of 100 female and 50 male patients presenting to sexually transmitted disease clinics, Dermatology OPD of AIIMS and Safdarjang Hospital, New Delhi, India, recruited as per inclusion criteria. RESULTS: PCR assay based on 16S ribosomal gene showed cross-reactivity with three of six strains of N sicca. The porA pseudogene-based PCR was highly specific. Analytical sensitivity of 16S-based ribosomal assay was more than that of porA pseudogene-based assay. In clinical samples, for female patients, sensitivity, specificity, positive predictive value and negative predictive value of 16S ribosomal assay was 100% (95% CI 51.7% to 100%), 91.5% (95% CI 83.4% to 96%), 42.9% (95% CI 18.8% to 70.4%) and 100% (95% CI 94.7% to 100%), respectively, while for the male patients it was 100% (95% CI 85% to 100%), 95.5% (95% CI 75.1% to 99.8%), 96.6% (95% CI 80.4% to 99.8%) and 100% (95% CI 80.8% to 100%), respectively. CONCLUSIONS: The data presented in this report supports use of 16S ribosomal assay as a screening assay only. The porA pseudogene target is highly specific for N gonorrhoeae and may be used as a supplemental assay.

Risk factors and algorithms for chlamydial and gonococcal cervical infections in women attending family planning clinics in Thailand.

AIM: To identify risk factors associated with and evaluate algorithms for predicting Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cervical infections in women attending family planning clinics in Thailand. METHODS: Eligible women were recruited from family planning clinics from all regions in Thailand. The women were followed at 3-month intervals for 15-24 months. At each visit, the women were interviewed for interval sexually transmitted infection (STI) history in the past 3 months, recent sexual behavior, and contraceptive use. Pelvic examinations were performed and endocervical specimens were collected to test for CT and NG using polymerase chain reaction. RESULTS: Factors associated with incident CT/NG cervical infections in multivariate analyses included region of country other than the north, age

[The study of expression of apoptosis receptors (CD95+) on the surface of neutrophils from cervical secretion of women with chlamydial infection and the possibility of its correction by magnetic laser radiation].

The present work was designed to study the expression of CD95 antigen (Fas/APO-1) at the surface of neutrophil granulocytes from the cervical secretion. Sixty five female patients with Chlamydia infection available for observation exhibited enhanced CD95+ expression following basic therapy. It was found that combined treatment with the use of magnetic laser radiation normalized the level of CD95+ surface receptors on neutrophils.