Distinct Effects of the Cervicovaginal Microbiota and Herpes Simplex Type 2 Infection on Female Genital Tract Immunology.

Background: Genital inflammation is a key determinant of human immunodeficiency virus (HIV) transmission, and may increase HIV-susceptible target cells and alter epithelial integrity. Several genital conditions that increase HIV risk are more prevalent in African, Caribbean, and other black (ACB) women, including bacterial vaginosis and herpes simplex virus type-2 (HSV-2) infection. Therefore, we assessed the impact of the genital microbiota on mucosal immunology in ACB women and microbiome-HSV-2 interactions. Methods: Cervicovaginal secretions and endocervical cells were collected by cytobrush and Instead Softcup, respectively. T cells and dendritic cells were assessed by flow cytometry, cytokines by multiplex enzyme-linked immunosorbent assay (ELISA), and the microbiota by 16S ribosomal ribonucleic acid gene sequencing. Results: The cervicovaginal microbiota of 51 participants were composed of community state types (CSTs) showing diversity (20/51; 39%) or predominated by Lactobacillus iners (22/51; 42%), L. crispatus (7/51; 14%), or L. gasseri (2/51; 4%). High-diversity CSTs and specific bacterial phyla (Gardnerella vaginalis and Prevotella bivia) were strongly associated with cervicovaginal inflammatory cytokines, but not with altered endocervical immune cells. However, cervical CD4+ T-cell number was associated with HSV-2 infection and a distinct cytokine profile. Conclusions: This suggests that the genital microbiota and HSV-2 infection may influence HIV susceptibility through independent biological mechanisms.

[IS A NONBACTERIAL PROSTATITIS NONBACTERIAL?].

The study involved 287 patients with chronic recurrent urethroprostatitis. In 83 (28.9%) of them the bacterial microflora in prostatic secretions was detected. The remaining 204 patients with nonbacterial prostatitis underwent in-depth laboratory testing. Cytological and immunological methods, electron microscopy, PCR, McCoy cell culture and developed by the authors the pre-culture pathogen accumulation technique were used to investigate urethral and prostatic smears. As a result C. trachomatis was detected in 84.8% of patients, T. vaginalis--75.5%, and viruses of the Herpesviridae family--in 68.6%. Co-infection was diagnosed in 82.4% of patients, no infection was found in 6.9% of patients. The highest diagnostic sensitivity and specificity were observed in the pre-culture pathogen accumulation technique. According to electron microscopy, T. vaginalis was presented in several morphological shapes: flagellated, spherical and amoeboid. Thus, in 93.1% of patients who had previously been diagnosed with nonbacterial prostatitis by standard bacterial culture, the in-depth microbiological examination identified atypical infections (chlamydia, herpes virus, trichomonas), revealing that the rate of real nonbacterial (non-infectious) prostatitis did not exceed 6.9%.

CD18 deficiency evolving to megakaryocytic (M7) acute myeloid leukemia: case report.

Leukocyte adhesion deficiency type 1 (LAD 1 - CD18 deficiency) is a rare disease characterized by disturbance of phagocyte function associated with less severe cellular and humoral dysfunction. The main features are bacterial and fungal infections predominantly in the skin and mucosal surfaces, impaired wound healing and delayed umbilical cord separation. The infections are indolent, necrotic and recurrent. In contrast to the striking difficulties in defense against bacterial and fungal microorganisms, LAD 1 patients do not exhibit susceptibility to viral infections and neoplasias. The severity of clinical manifestations is directly related to the degree of CD18 deficiency. Here, a 20 year-old female presenting a partial CD18 deficiency that developed a megakaryocytic (M7) acute myeloid leukemia is described for the first time. The clinical features of the patient included relapsing oral thrush due to Candida, cutaneous infections and upper and lower respiratory tract infections, followed by a locally severe necrotic genital herpetic lesion. The patient's clinical features improved for a period of approximately two years, followed by severe bacterial infections. At that time, the investigation showed a megakaryocytic acute myeloid leukemia, treated with MEC without clinical improvement. The highly aggressive evolution of the leukemia in this patient suggests that adhesion molecules could be involved in the protection against the spread of neoplastic cells.

Vulvar pseudoepitheliomatous hyperplasia associated with herpes simplex virus type II mimicking cancer in an immunocompromised patient.

We report an exaggerated dermatological inflammatory condition in an immunocompromised patient. The patient was a 51-year-old woman who had HIV infection and a history of cervical cancer. Three years after highly active antiretroviral therapy with an improved immune status, and 2 years after remission of cervical cancer, she developed verrucous perineal masses. Provisional diagnosis was recurrent cervical cancer or primary vulvar cancer. Pathological features revealed pseudoepitheliomatous hyperplasia associated with herpes viral infection. After minimal response to systemic oral antiviral drugs and topical imiquimod, she had clinical resolution with the addition of systemic oral corticosteroid.

Recalcitrance of bacterial vaginosis among herpes-simplex-virus-type-2-seropositive women.

AIM: The multifactorial etiology of bacterial vaginosis (BV) impedes development of effective treatment and prevention strategies. Herein, we evaluated the effects of herpes simplex virus type 2 (HSV-2), a suspected BV risk factor, on vaginal flora composition. MATERIALS AND METHODS: Correlations between HSV-2 infection and BV were prospectively explored among 12 HSV-2-seropositive women with asymptomatic BV who were asked to collect daily vaginal swab specimens for Gram stain analysis of vaginal flora and determination of HSV-2 shedding frequencies during the 1month before and after metronidazole therapy. RESULTS: Unlike prior longitudinal studies that reported rapid fluctuations in vaginal flora composition and frequent episodes of spontaneously resolving BV, we found that 99.4% (310/312) of vaginal smears collected before initiation of metronidazole were consistent with a diagnosis of BV. Effectiveness of metronidazole therapy was also much lower than previously reported in studies not restricting enrollment to HSV-2-seropositive women; we observed a BV recurrence rate of 89% in the first month after completion of therapy while the median time to this recurrence occurred only 14days after treatment. CONCLUSIONS: Our study demonstrates BV recalcitrance among HSV-2-infected women and provides additional evidence for a linkage between this chronic viral infection and abnormal vaginal flora. Additional work will be needed to define mechanisms responsible for this correlation and to determine if vaginal flora health of HSV-2-infected women is improved by medications that suppress HSV-2 shedding.

Expression of the herpes simplex virus type 2 latency-associated transcript enhances spontaneous reactivation of genital herpes in latently infected guinea pigs.

The latency-associated transcript (LAT) is the only herpes simplex virus (HSV) gene product detectable in latently infected humans and animals. In this report, we show that a 624-bp deletion in the promoter of the HSV-2 LAT had no discernable effect on viral growth in tissue culture or in acute genital infection of guinea pigs, but impaired LAT accumulation and led to a marked decrease in spontaneous genital recurrences when compared with the behavior of wild-type and rescuant strains. Differences in the ability of the mutant to replicate, or in how readily it established or maintained latency did not account for this finding. Thus, HSV LAT expression facilitates the spontaneous reactivation of latent virus.

The characteristic site-specific reactivation phenotypes of HSV-1 and HSV-2 depend upon the latency-associated transcript region.

After replication at sites of initial inoculation, herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) establish lifelong latent infections of the sensory and autonomic neurons of the ganglia serving those sites. Periodically, the virus reactivates from these neurons, and travels centripetally along the neuronal axon to cause recurrent epithelial infection. The major clinically observed difference between infections with herpes simplex virus type 1 and type 2 is the anatomic site specificity of recurrence. HSV-1 reactivates most efficiently and frequently from trigeminal ganglia, causing recurrent ocular and oral-facial lesions, while HSV-2 reactivates primarily from sacral ganglia causing recurrent genital lesions. An intertypic recombinant virus was constructed and evaluated in animal models of recurrent ocular and genital herpes. Substitution of a 2.8-kbp region from the HSV-1 latency-associated transcript (LAT) for native HSV-2 sequences caused HSV-2 to reactivate with an HSV-1 phenotype in both animal models. The HSV-2 phenotype was restored by replacing the mutated sequences with wild-type HSV-2 LAT-region sequences. These sequences or their products must act specifically in the cellular environments of trigeminal and sacral neurons to promote the reactivation patterns characteristic of each virus.

[Comprehensive examination of the female urogenital tract infection by polymerase chain reaction using multiplex test systems].

The results of the polymerase chain reaction studies performed in 2006-2008 were used to make a retrospective analysis of the detection of urogenital herpesvirus infections in reproductive-aged women constituting the urban population of the central region of Russia. The study used both monotarget and mutiplex test systems to detect herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus types 6 and 7. A total of about 7.500 referrals; the detection rate for HSV was about 1%; that for CMV was from 0.3 in 2006 to 1% in 2008; that for EBV was from 0.1% in 2006 to 0.3% in 2008. More than a half of HSV-, CMV-, or EBY-positive samples also contained DNA of other causative agents and some samples did two pathogens or more. Multiplex test systems for herpesviruses considerably enhance the efficiency of diagnostic studies and reduce the material and time costs of diagnosis.

Microbial Diversity of Genital Ulcers of HSV-2 Seropositive Women.

We measured the microbial community structure of genital ulcers in women. Swabs from clinically detected ulcers were tested for HSV-2 and Treponema pallidum by polymerase chain reaction (PCR). HSV-2 and T. pallidum were detected by serum antibody testing. Microbial community structure was characterized by high-throughput 16 s rRNA gene amplicon sequencing. Multiple group testing and Elastic net and Lasso regressions identified taxa associated with differences in factors of interest. Among 49 ulcer specimens from 49 HSV-2 seropositive women, by PCR HSV-2 was recovered from 28 (57%) specimens and T. pallidum from none; one woman showed serologic evidence of syphilis. Overall, 63% of women were HIV-positive and 49% had an uncircumcised male sex partner. By both multiple group testing and regression, Porphyromonas (FDR p-value = 0.02), Prevotella (FDR p-value = 0.03), Anaerococcus (FDR p-value = 0.07), and Dialister (FDR p-value = 0.09) were detected at higher relative abundance in HSV-2 PCR-positive than negative ulcers. The presence of HSV-2 in a lesion was associated with presumed bacterial agents of Bacterial vaginosis. Differences in bacterial communities may contribute to HSV-2 ulcer pathogenesis, severity, or prolonged healing. If these results are confirmed, future studies may consider the influence of BV treatment on women's GUD and HSV-2 incidence and recurrence.

Association of HPV infection and clearance with cervicovaginal immunology and the vaginal microbiota.

Cervical human papillomavirus (HPV) infection may increase HIV risk. Since other genital infections enhance HIV susceptibility by inducing inflammation, we assessed the impact of HPV infection and clearance on genital immunology and the cervico-vaginal microbiome. Genital samples were collected from 65 women for HPV testing, immune studies and microbiota assessment; repeat HPV testing was performed after 6 months. All participants were HIV-uninfected and free of bacterial STIs. Cytobrush-derived T cell and dendritic cell subsets were assessed by multiparameter flow cytometry. Undiluted cervico-vaginal secretions were used to determine cytokine levels by multiplex ELISA, and to assess bacterial community composition and structure by 16S rRNA gene sequence analysis. Neither HPV infection nor clearance were associated with broad differences in cervical T cell subsets or cytokines, although HPV clearance was associated with increased Langerhans cells and HPV infection with elevated IP-10 and MIG. Individuals with HPV more frequently had a high diversity cervico-vaginal microbiome (community state type IV) and were less likely to have an L. gasseri predominant microbiome. In summary, HPV infection and/or subsequent clearance was not associated with inflammation or altered cervical T cell subsets, but associations with increased Langerhans cells and the composition of the vaginal microbiome warrant further exploration.

Pseudoepitheliomatous hyperplasia causing a painful plaque in a HIV-infected female.

Dermatological conditions are more common and can present atypically, in human immunodeficiency virus-infected individuals. This case report describes a 22-year-old human immunodeficiency virus-positive Caucasian female who presented with a vulval lesion eight weeks after starting antiretroviral treatment. Clinical examination revealed a 2 cm well-demarcated plaque on the outer aspect of the left labium minus. The lesion was tender, no contact bleeding or ulceration present. She was presumptively treated for chancroid and herpes simplex with 500 mg ceftriaxone IM stat, 1 g azithromycin PO stat, and valacyclovir 500 mg BD for five days. The lesion persisted despite treatment, and during follow-up, a punch biopsy was carried out. She was diagnosed with pseudoepitheliomatous hyperplasia of the epidermis. In addition to highlighting this condition that has been previously reported in human immunodeficiency virus/herpes simplex virus co-infection, this case demonstrates that unusual skin presentations must be considered in human immunodeficiency virus-infected individuals and illustrates the importance of biopsy for any non-healing lesions.

Chlamydial Pre-Infection Protects from Subsequent Herpes Simplex Virus-2 Challenge in a Murine Vaginal Super-Infection Model.

Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singly-infected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans.

Poor correlation between genital lesions and detection of herpes simplex virus in women in labor.

OBJECTIVE: To estimate the accuracy of clinical diagnosis of genital herpes for herpes simplex virus (HSV) detection among women in labor. METHODS: Viral detection by culture and HSV DNA polymerase chain reaction (PCR) among women who underwent cesarean delivery for genital herpes was compared with women without HSV symptoms in labor who had genital swabs collected for HSV culture and to a subset of these women who had genital specimens available for PCR analysis, regardless of culture results. RESULTS: From 1989 to 1999, 126 of 19,568 (0.6%) women underwent cesarean delivery for HSV. Twenty-six percent of 110 of these women had HSV detected by culture from at least 1 genital specimen and 46% of 70 of these women had HSV detected by PCR. During the same period, 61 of 12,623 (0.5%) asymptomatic women had HSV detected by culture. Between 1995 and 1996, 57 of 2,109 (2.7%) asymptomatic women had HSV detected by PCR. Thus, the presence of genital lesions had a sensitivity for HSV detection of 37% by culture and 41% by PCR. The amount of HSV present in asymptomatic women with HSV detected in genital secretions by PCR was often as high as those with genital lesions, although the median amount of HSV DNA detected was greater in women with lesions. CONCLUSION: Clinical diagnosis of genital herpes at the time of labor correlates relatively poorly with HSV detection from genital sites or lesions by culture or PCR and fails to identify asymptomatic women who have HSV in their genital secretions at the time of labor.

Genital herpes simplex infections: effect of asymptomatic shedding and latency on management of infections in pregnant women and neonates.

Asymptomatic shedding of herpes simplex virus (HSV) at delivery occurred in 2.4% of women with a past history of recurrent genital infections; using current methods, this could not be predicted in advance. In addition, only 22% of the mothers of infected infants had an elicitable history of recurrent genital infections. Passively derived neutralizing antibody titers of 1:106 to HSV-1 and 1:67 to HSV-2 were found in 22 exposed infants who remained asymptomatic as compared with 1:8 and 1:8, respectively, in ill infants, suggesting that transfer of antibody from the mother may be an important host defense. Among treated infants, a more rapid rise in antibody titer was seen in infants receiving adenine arabinoside than in those receiving acyclovir; two of the latter infants developed a severe infection in a second organ following cessation of the drug. Exposure of infants to HSV appears inevitable at this time. The extreme variability in outcome is probably related to host factors that are poorly understood at present.

Lymphocyte responses to herpes simplex virus isolates from patients with primary and recurrent herpes simplex genitalis.

The ability of herpes simplex (HSV) isolates from patients with herpes simplex genitalis (HSG) to induce lymphocyte transformation in cells from unrelated, healthy, seropositive donors was examined in a standard lymphocyte transformation assay. All HSV isolates were obtained, before the initiation of therapy, from patients who were enrolled in a placebo-controlled trial of acyclovir in the treatment of HSG. Isolates from patients with primary infections were used, as well as those from patients with frequent (eight or more episodes per year) or infrequent (two or fewer per year) recurrent disease. Blastogenic responses to isolates from patients with infrequent HSG recurrences were significantly less than those to isolates from patients either primary infections or frequent recurrences. No differences between the latter two groups of isolates were seen. These observations demonstrate that differences among naturally occurring HSV isolates exist as determined by this in vitro assay of host-virus interactions. Differences among strains may be important in the pathogenesis of HSV infections, particularly with respect to latency.

Effect of acyclovir on genital infection with herpes simplex virus types 1 and 2 in the guinea pig.

The pathogenesis of genital infection with three different strains of herpes simplex virus type 1 (HSV-1) and three strains of herpes simplex virus type 2 (HSV-2) was compared in the guinea pig. Strain differences in severity of clinical disease and mortality were noted. HSV-1 strains generally produced milder disease than HSV-2. Both HSV-1 and HSV-2 infections resulted in acute and chronic changes in the cervix. Virus recovery during latent infection was more frequently obtained from the spinal cord in HSV-1-infected animals and from lumbosacral ganglia in HSV-2-infected animals. Systemic treatment with acyclovir, after the onset of clinical disease, had minimal, if any, effect on genital infection with HSV-1 (NYU-78), but similar treatment of HSV-2 (WT-186) infection resulted in decreased lesion scores, paralysis, and mortality during acute infection. A reduction in virus isolations from lumbosacral ganglia was noted during both acute and latent infection with HSV-2 (WT-186) in the acyclovir-treated groups.

Spectrum of sensitivity of acyclovir of herpes simplex virus clinical isolates.

HSV-1 and HSV-2 clinical isolates were tested for their in vitro sensitivity to acyclovir. The median ID50 for 32 genital HSV-2 isolates was 0.215 micrograms/ml and this was not significantly higher than a median value of 0.125 micrograms/ml for 28 oral HSV-1 isolates (p = 0.08). A wider range of ID50s was observed with the HSV-2 isolates compared with the HSV-1 isolates. The concentration of drug required to inhibit virus replication by 90 and 99 percent was approximately 10- and 100-fold greater than that producing 50 percent inhibition.

A point mutation in the thymidine kinase gene is responsible for acyclovir-resistance in herpes simplex virus type 2 sequential isolates.

A number of HSV-2 isolates, sequentially recovered from ulcerative ano-genital lesions of an AIDS patient during a prolonged treatment with acyclovir (ACV), have been studied at the molecular level. All of them were highly resistant to ACV (ACV-r) and shown to be virtually deficient in thymidine kinase (TK) activity. The ACV-r phenotype was demonstrated to be due to the production of truncated TK polypeptide. Structural alteration of this gene, as shown in one isolate, was caused by a chain-terminating mutation that originated from a cytidine deletion at position 520 of the TK open reading frame. This mutation generated a TGA stop codon 27 nucleotides downstream. An additional isolate was also recovered following ACV discontinuation and after a cycle of treatment with foscarnet. This isolate had lost the ACV-r trait and was characterized by a wild type TK sequence and by the production of a functional enzyme. Data presented confirm that a prolonged treatment with acyclovir can easily select ACV-r HSV-2 isolates carrying a TK- phenotype caused by a frameshift mutation. Although recovered from lesions tributary of different myelomers, these isolates may belong to the same strain that has undergone multiple cycles of reactivation and has possibly mutated during its axonal route to the skin.

Recurrences after first episodes of genital herpes in patients treated with topical acyclovir cream.

The effect of topical acyclovir treatment of first episode genital herpes on the time to first recurrence in a group of 42 patients receiving either acyclovir or placebo was investigated. Topical acyclovir treatment had no effect on time to first recurrence in patients with either first episode HSV-1 or HSV-2 infections. There was no significant difference in the time to first recurrence in patients with either true primary or initial genital infections. However, the time to first recurrence in patients with first episode HSV-2 was significantly shorter than in patients with first episode HSV-1. Acyclovir treatment appeared to have no effect on the development of neutralising antibody in patients with either virus type.

Efficacy of 5-methoxymethyl-2'-deoxyuridine in combination with arabinosyladenine for the treatment of primary herpes simplex genital infection of mice and guinea pigs.

The relative efficacy of 5-methoxymethyl-2'-deoxyuridine (MMdUrd), arabinosyladenine (ara-A) and the combination of MMdUrd and ara-A in the treatment of experimental genital herpes (GH) was investigated using mouse and guinea pig models. The infection was initiated by intravaginal inoculation using either HSV-2, strain X-265 or HSV-2, strain MS. Treatment was initiated 3 h post virus inoculation. The parameters used to evaluate efficacy were: percent mortality; mean day of death; virus yield from the vaginal secretions; and mean lesion score. The simultaneous application of 5% MMdUrd and 5% ara-A was an effective treatment for controlling primary GH in both animal models. Combination chemotherapy was also effective in preventing recurrence of infection as well as the emergence of drug resistant virus. At 20% concentration, ara-A was effective in providing protection against GH. However, lesions due to recurrent GH appeared after cessation of treatment and the virus isolated from vaginal secretions of ara-A treated animals required higher concentration of drug for inhibition of virus replication in cell culture. 20% MMdUrd was only partially effective in controlling GH. The production of infectious virus particles (virus yield) in cell culture after exposure to either ara-A of MMdUrd alone or in combination was determined. When MMdUrd and ara-A were used together, a substantially lower amount of each drug was needed to inhibit virus production completely and removal of drugs did not result in an increase in virus yield.