Detection of human papillomavirus DNA in urine specimens from human immunodeficiency virus-positive women

Human immunodeficiency virus (HIV)-positive women may represent one of the fastest-growing populations at risk for acquiring cervical cancer and thus require frequent screening. The purpose of the present studies was to validate a PCR-based urine assay by comparing detection and genotyping of human papillomavirus (HPV) DNA in urine samples and matching cervical swab specimens of HIV-positive women. Despite a difference in amplifiability, the prevalence of any HPV genotype (58% for the cervical swab specimens and 48% for the urine specimens) was not significantly different in this population. The levels of concordance were 70, 71, and 78% for detection of any HPV type, any high-risk HPV type, or any low-risk HPV type in the two specimen types, respectively. While instances of discordant detection were greater for the cervical swab specimens than for the urine specimens, this was not statistically significant. The distributions of HPV genotypes were similar in the cervix and the urine for the majority of types examined. Importantly, detection of HPV DNA in urine was associated with an abnormal Papanicolaou smear to the same extent that detection of HPV DNA in a cervical swab specimen was. These data provide preliminary support for the proposal to use urine testing as a primary or secondary screening tool for cervical cancer in HIV-positive women or as an epidemiological tool. Additional studies with larger sample sizes must be conducted in order to further verify these findings.     

Mycoplasma penetrans is capable of activating V gamma 9/V delta 2 T cells while other human pathogenic mycoplasmas fail to do so

While most mycoplasma species appear to have evolutionarily lost the ability to synthesize isoprenoid precursors, Mycoplasma penetrans has retained the nonmevalonate pathway that proceeds via the immunogenic intermediate (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). Consequently, this pathogen is capable of stimulating human V gamma 9/V delta 2 T cells.     

High prevalence of antibodies to Mycoplasma penetrans in human immunodeficiency virus-seronegative and -seropositive populations in Brazzaville, Congo.

In industrialized countries, the prevalence of antibodies to Mycoplasma penetrans is higher among human immunodeficiency virus (HIV)-seropositive homosexuals than other HIV-seropositive and HIV-seronegative groups. In an African heterosexual population, we found a higher prevalence of M. penetrans antibodies in HIV-seronegative blood donors (15.5%) than in France (0.9%) or the United States (0.3%) and a prevalence of 13.4% in HIV-seropositive individuals. HIV-seropositive individuals with less than 5% CD4 cells had a higher prevalence of M. penetrans antibodies than individuals with 5% or more CD4 cells (25.0 versus 8.5%).     

Lipid-associated membrane proteins of Mycoplasma fermentans and M. penetrans activate human immunodeficiency virus long-terminal repeats through Toll-like receptors

Mycoplasmas are known to enhance human immunodeficiency virus (HIV) replication, and mycoplasma-derived lipid extracts have been reported to activate nuclear factor-kappaB (NF-kappaB) through Toll-like receptors (TLRs). In this study, we examined the involvement of TLRs in the activation of HIV long-terminal repeats (LTR) by mycoplasma and their active components responsible for the TLR activation. Lipid-associated membrane proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with acquired immune-deficiency syndrome (AIDS), were found to activate HIV LTRs in a human monocytic cell line, THP-1. NF-kappaB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. These results indicate that the activation of HIV LTRs by M. fermentans and M. penetrans LAMPs is dependent on NF-kappaB, and that the activation of HIV LTR by M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In contrast, the LTR activation by M. penetrans LAMPs is carried out through TLR1 and TLR2, but not TLR6. Subsequently, the active component of M. penetrans and M. fermentans LAMPs was purified by reverse-phase high-performance liquid chromatography (HPLC). Interestingly, the purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF-kappaB through TLR1 and TLR2. On the other hand, the activation of NF-kappaB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1.     

Gangrenous herpetic whitlow in a human immunodeficiency virus-positive patient

An unusual case of a gangrenous herpetic whitlow is reported. The patient, a 37-year-old man with a ten-year history of intravenous drug abuse, was antibody positive for human immunodeficiency virus. Progressive, extremely painful paronychia of the left third and fourth fingers gradually developed, which persisted despite a variety of treatment protocols, including antibiotics and radiotherapy, ultimately necessitating amputation of the distal portions of the digits. Characteristic herpes-type intranuclear inclusions within epidermal cells were identified in histologic sections of the specimen. Immunohistochemistry using rabbit antihuman herpes virus antibody confirmed the diagnosis. This apparently represents the first documented case of herpetic gangrene in an immunocompromised patient.     

Pneumocystis carinii infection in human immunodeficiency virus-positive patients.

Pneumocystis carinii is a ubiquitous, atypical unicellular fungus. P. carinii pneumonia (PCP) is responsible for considerable morbidity and mortality in acquired immune deficiency syndrome (AIDS) patients, and is the leading complication in advanced human immunodeficiency virus (HIV) infection. Many different host (mammal)-specific species of Pneumocystis exist, but the life-cycle is not understood fully. Human strains are designated as P. carinii f. sp. (special form) hominis (at least 59 different types). P. carinii is spread via the airborne route. Disease is most frequently caused by fresh exposure to a source of P. carinii, rather than by reactivation of latent infection. Asymptomatic carriage among healthy persons may occur. PCP occurs in HIV-infected patients when the CD4+ count falls below a certain threshold; organisms multiply and gradually fill the alveoli. Symptoms, which include a mildly productive cough, progressive dyspnoea and fever, may persist for months prior to diagnosis. Without treatment, progressive respiratory insufficiency invariably ends in death. Pulmonary specimens may be obtained by procedures of varying sensitivity and risk. Diagnosis is usually confirmed by detection of stained organisms; however, staining procedures vary in sensitivity and ease of use. Robust polymerase chain reaction (PCR) protocols with good predictive results may be useful in the future. Therapy falls into two categories: for acute primary infections and for prophylaxis. A confirmed diagnosis ensures that patients do not receive potentially toxic medication (adverse drug reactions can occur). Prophylaxis can dramatically reduce the frequency of PCP in HIV patients, and its more widespread use should lead to a decline in the incidence of PCP in the future.     

Hemolytic and hemoxidative activities in Mycoplasma penetrans

Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H(2)O(2)-mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.     

Genotypic study of Pneumocystis jirovecii in human immunodeficiency virus-positive patients in Thailand

Pneumocystis jirovecii is one of the common opportunistic infections in human immunodeficiency virus (HIV)-infected patients in Thailand. Information regarding genotypic and epidemiological of this organism in Thai patients is not available. We analyzed the genotypes of 28 P. jirovecii-positive specimens from bronchoalveolar lavage and sputum samples from HIV-infected Thai patients based on nucleotide variations of the internal transcribed spacer regions 1 and 2 of the rRNA gene. Thirteen genotypes were the same as previously reported outside Thailand. Ten genotypes, which included Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb, and Qq, were new. Ir and Rp were unique and dominant types observed in HIV-infected Thai patients. Thirteen specimens (46.4%) were infected with a single type of P. jirovecii, and fifteen (53.6%) were mixed infections. These differences may be used as genotypic markers for studying the epidemiology and transmission of P. jirovecii in the Thai population.     

In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals.

Mycoplasma penetrans is a mycoplasma species newly isolated from the urine of human immunodeficiency virus (HIV)-infected individuals and presents the only case in which an association has been found between antibodies against a mycoplasma and HIV infection. To further explore the effects of M. penetrans on the immune system, we studied the influence of this mycoplasma on peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-infected individuals. M. penetrans induced, in addition to blastogenesis of PBMCs, a significant proliferative response associated with the expression of some activation markers such as CD69, HLA-DR, and CD25. This M. penetrans-dependent lymphocyte activation was observed not only in healthy donors but also in HIV-infected persons at different stages of the disease. In addition, our study revealed that both CD4+ and CD8+ T lymphocytes were responsive to M. penetrans. Interestingly, the mitogenic activity of M. penetrans was associated with mycoplasma cells but not with the supernatants of mycoplasma culture. The potent stimulating activity of M. penetrans on T lymphocytes from HIV-infected individuals is of particular interest in view of the supposed contribution of immune activation to HIV replication and disease progression.     

Infertility among human immunodeficiency virus-positive women: incidence and treatment dilemmas

The increasing demand for fertility advice among human immunodeficiencyvirus (HIV)-positive women under our care led us to review theincidence of infertility and the ethical problems associatedwith its management. All HIV-positive women who attended theHIV outpatients clinic from October 1990 to the end of January1996 were studied. The main outcome measures were: the numberof women undergoing infertility investigations before and afterHIV diagnosis, their demographic and social details, and theoutcome of these investigations. Most of the 183 women studiedwere in their reproductive years (mean age 32.7 ± 6.7years). Nine women had undergone infertility investigations,and/or treatment before HIV diagnosis, three of whom were diagnosedwith HIV during routine testing prior to IVF treatment Six declinedfurther infertility treatment after discovering their HIV status.Eight women have undergone infertility investigations afterHIV diagnosis but none have achieved pregnancy to date. Managementdecisions may have been hampered by ethical uncertainties inseveral cases. In conclusion therefore, as requests for infertilitytreatment from HIV-infected women occur and may become morecommon as the prevalence of HIV infection in women continuesto rise, the ethical issues associated with the management ofthis problem demand urgent attention so that clear guidelinesare available to aid treatment decisions.     

Markers of human papillomavirus infection and their correlation with cervical dysplasia in human immunodeficiency virus-positive women

Human papillomavirus (HPV) genotypes and HPV DNA load were analysed in cervical smears from 76 human immunodeficiency virus (HIV)-positive and 54 HIV-negative women. The prevalence of genotypes was similar for all women, with the exception of HPV62, which was over-represented in HIV-positive samples. HIV-positive women showed a higher prevalence of multiple genotypes that correlated neither with CD4(+) T-cell counts nor with cervical dysplasia. No significant differences were observed in terms of total or single-type HPV DNA load. The HPV DNA load in both HIV-positive and HIV-negative women was significantly higher in squamous intra-epithelial lesions than in negative Pap smears.     

Relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women

Human papillomavirus (HPV) is an etiologic agent for both oropharyngeal and cervical cancers, yet little is known about the interrelationship between oral and cervical HPV infections. Therefore, we compared the prevalences and type distributions of oral and cervical HPV infections and evaluated infection concordance in a cross-sectional study within the Women's Interagency HIV Study cohort. Oral rinse and cervical-vaginal lavage samples were concurrently collected from a convenience sample of 172 human immunodeficiency virus (HIV)-positive and 86 HIV-negative women. HPV genomic DNA was detected by PGMY09/11 L1 consensus primer PCR and type specified by reverse line blot hybridization for 37 HPV types and beta-globin. Only 26 of the 35 HPV types found to infect the cervix were also found within the oral cavity, and the type distribution for oral HPV infections appeared distinct from that for cervical infections (P<0.001). Oral HPV infections were less common than cervical infections for both HIV-positive (25.2% versus 76.9%, P<0.001) and HIV-negative (9.0% versus 44.9%, P<0.001) women. Oral HPV infections were more common among women with a cervical HPV infection than those without a cervical HPV infection (25.5% versus 7.9%, P=0.002). The majority of women (207; 93.7%) did not have simultaneous oral and cervical infections by the same HPV type; however, the number of women who did (14; 6.3%) was significantly greater than would be expected by chance (P=0.0002). Therefore, the oral and cervical reservoirs for HPV infection are likely not entirely independent of one another.     

Survival of feline mycoplasmas in urine.

The effects of length of incubation and urine osmolality on the survival of feline mycoplasmas and ureaplasmas and representative gram-positive and gram-negative bacteria in synthetic urine which approximated the osmolality of normal cat urine were investigated. Both Escherichia coli and Staphylococcus aureus withstood the effects of increasing osmotic pressure. In the most concentrated urine, significant decreases (P less than 0.001) in CFU were observed for E. coli at exposure times of 30 min and longer. S. aureus was not affected by longer exposure or increased osmotic strength. Both Mycoplasma felis and Mycoplasma gateae were affected adversely by longer exposure times and high osmotic strength (P less than 0.001). A Ureaplasma sp. was not adversely affected except at very high (greater than or equal to 2,980 mosM) osmotic strengths or after prolonged incubation (120 min) at relatively high (1,976 mosM) osmotic strengths (P less than 0.001). The failure of both M. felis and M. gateae to survive under osmotic conditions present in normal feline urine suggests that it is unlikely that these mycoplasmas are involved in urinary disorders in cats.     

Characterization of a Unique ADP-Ribosyltransferase of Mycoplasma penetrans

Mycoplasma penetrans is a urogenital tract pathogen implicated in the deterioration of the immune system in human immunodeficiency virus-infected AIDS patients. Here, we describe a 78-kDa protein from M. penetrans, designated MYPE9110, that exhibits sequence similarity to known ADP-ribosyltransferases (ADPRTs) such as Bordetella pertussis pertussis toxin and Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin. MYPE9110 possesses key amino acid residues found in all ADPRTs that are essential for ADPRT activity. Several mammalian cell proteins are ADP-ribosylated by MYPE9110, and the full-length recombinant protein exhibits a strong auto-ADP-ribosylating activity. In the absence of target proteins, MYPE9110 demonstrates a NAD-glycohydrolase activity by hydrolyzing NAD. Furthermore, this toxin elicits cytopathology in HeLa cells by inducing cytoplasmic vacuolization in the presence of ammonium chloride. The deletion of the C-terminal region of MYPE9110 significantly diminishes its binding to host cells while still exhibiting an ADPRT activity, suggesting that MYPE9110 is a member of the family of A-B ADPRT toxins.The Mollicutes contain several human-pathogenic mycoplasmas including Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma penetrans. M. penetrans GTU-54 was first isolated from the urine of a human immunodeficiency virus (HIV)-positive homosexual male (20). Subsequent studies reported a higher frequency of antibodies against M. penetrans (40%) in sera of HIV-infected AIDS patients than in sera of HIV-infected non-AIDS and HIV-negative control groups (20% and 0.3%, respectively) (36). It was previously hypothesized that M. penetrans could exist as either an opportunist or a cofactor in AIDS progression for several reasons, including the ability of M. penetrans to activate human T lymphocytes (31). Although considered predominately a urogenital tract pathogen, M. penetrans strains have also been isolated from blood (strain HF-1) and respiratory tract cultures (strain HF-2) of a non-HIV-infected patient with primary antiphospholipid syndrome and bacteremia (37).Among the genome-sequenced pathogenic mycoplasmas, M. penetrans strain HF-2 is the largest, at 1.4 Mbp, with a low G+C content of 25.7% and 1,038 predicted coding sequences (32). The genome consists of many paralogs, including the p35 gene family, which may account for its larger genome size than other pathogenic mycoplasmas. The p35 gene family encodes surface lipoproteins, including the immunodominant P35 protein, which is the basis of the serological diagnosis of M. penetrans infection (32). In silico analysis of the M. penetrans genome indicates the presence of a two-component response regulator (MYPE3960) and a putative sensory transduction histidine kinase (MYPE2360). No such regulatory elements have been found for other sequenced mollicute genomes, suggesting that M. penetrans is unique in that it may be able to sense and synthesize proteins in response to its survival niche (32).Following adherence to host cells, M. penetrans induces cytoskeleton rearrangement, as evidenced by the aggregation of tubulin and α-actinin (13). This event leads to the internalization of M. penetrans, where it resides in the host cell cytosol, membrane-bound vacuoles, or perinuclear region for extended periods of time (2, 10, 13). This intracellular environment offers many benefits to the bacterium, where it can avoid host immune responses and acquire essential nutrients (26). The mechanism by which M. penetrans triggers cytoskeletal rearrangements is still poorly understood. Manipulation of the cytoskeletal network by bacterial toxins, including ADP-ribosylating toxins, was previously reported for both gram-negative and gram-positive bacteria (26). Until recently, the only genome-encoded potential virulence factors of M. penetrans included endonucleases, hemolysins, and proteases (3, 18, 32), and their roles in pathogenesis remain unclear.Recently, an ADP-ribosylating and vacuolating toxin was described for M. pneumoniae (17). This toxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, was first identified by its interaction with the human lung protein surfactant protein A and by its limited but critically relevant sequence similarity to the Bordetella pertussis pertussis toxin S1 subunit ADP-ribosyltransferase (ADPRT) domain (17, 19). ADPRTs are found in a wide range of bacterial pathogens and catalyze the transfer of a single ADP-ribosyl group from β-NAD onto specific amino acid residues of host cell proteins with the release of nicotinamide (24). Mono-ADP-ribosylation by these ADPRTs leads to the modification of various host cell target proteins and their activities, including the inhibition of host protein synthesis by diphtheria toxin (DT) (25), alterations in signal transduction pathways by pertussis toxin (21), and interference of actin polymerization by Clostridium iota and C2 toxins (1, 33). Many members of these ADPRTs, including pertussis toxin and DT, belong to the A-B family, which possesses an active ADPRT subunit, known as the A subunit, and a B subunit responsible for the binding and translocation of the active subunit across host cell membranes (11).Our discovery of CARDS toxin in M. pneumoniae prompted us to look at other mycoplasma genomes for the existence of a related protein. A hypothetical protein in the M. penetrans genome, annotated MYPE9110, shared sequence similarity with CARDS toxin and a conserved domain with the pertussis toxin S1 subunit. In this study we show that MYPE9110 is related to the family of classical A-B toxins, as it possesses an N-terminal ADPRT domain and a C-terminal cell-binding domain. We also show that MYPE9110 elicits cytopathology in host cells, indicating the virulence potential of this newly characterized M. penetrans protein.