Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae.

BACKGROUND: The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. OBJECTIVES: To characterize the genetic pathways leading to high-level azithromycin resistance. METHODS: A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. RESULTS: Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. CONCLUSIONS: This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

Evaluation of the Bio-Rad Dx CT/NG/MG® assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urine.

We evaluated the performance of the Bio-Rad real-time Dx CT/NG/MG® assay for detection of C. trachomatis, N. gonorrhoeae and M. genitalium on a collection of 441 urine samples from sexually transmitted infections, or travellers consultations and from anonymous sperm donors that were previously analysed with the Abbott RealTime CT/NG assay. Samples positive for C. trachomatis or N. gonorrhoeae with the Abbott assay had all previously been confirmed with an in-house real-time PCR assay. Samples positive for M. genitalium with the Bio-Rad assay were subsequently analysed by an in-house real-time PCR. On a total of 441 urines, 104 samples were positive for C. trachomatis, 12 were positive for N. gonorrhoeae and seven were positive for M. genitalium. After retesting of discrepant results, the test results were completely concordant, resulting in a calculated sensitivity and specificity of the Bio-Rad assay of 98.1 % and 100 % for C. trachomatis and of 91.7 % and 100 % for N. gonorrhoeae. Results for M. genitalium with the Bio-Rad assay were also concordant with the results of an in house PCR. We also evaluated the performance of automated nucleic acid extractions of urine samples with the NucliSENS easyMAG (bioMérieux) compared to the manual DNA extraction prescribed by the insert of the kit. The easyMAG extraction gave lower Ct values, relieved inhibition and had a lower hands-on time.

Incidence of HCV and sexually transmitted diseases among hiv positive msm in antwerp, belgium, 2001-2011.

Recurrent Sexually Transmitted Infections (STIs) are an indication of unsafe sexual practices and may be associated with HCV-infection among HIV-positive men who have sex with men. In a retrospective study we analysed the laboratory data of 99 HIV-positive MSM who acquired HCV during the observation period (cases) and 176 HIV-positive MSM who remained HCV negative during the observation period (controls), all followed at the HIV/STI-clinic in Antwerp, Belgium. All laboratory confirmed STI-episodes were recorded since the date of first consultation at our clinic, until the date of HCV-diagnosis of the cases. The HCV incidence varied between 0.24 (2001) and 1.36 (2011) new cases per hundred person-years, with a peak of 2.93 new cases per hundred person-years in 2009. The number of STI-episodes per person-year follow-up was significantly higher for the cases as compared to the controls for syphilis, non-LGV and LGV Chlamydia infections (p < 0.005). When considering the incidence of STIs that occurred 1 year prior to HCV conversion, all laboratory confirmed STIs remained more frequent among cases, but only the difference in syphilis incidence was statistically significant (p < 0.01). Recurrent STIs among HIV positive MSM should be considered as a behavioural and biological risk factor for acquiring HCV and should lead to intensified screening for HCV and counselling of the patient.

Sexually transmitted infections: what's new?

Since the beginning of the third millennium the incidence of Sexually Transmitted Infections (STIs) is rising in Europe and in Belgium, and this after a steady decline in the second half of last century. It concerns new or lesser known diseases such as Hepatitis C and Lymphogranuloma venereum (LGV) and 'old' diseases such as gonorrhoea and syphilis, occurring in specific risk groups. In this article we give an update of the diagnostic means and therapeutic challenges that are of interest for the clinician. Besides these (re)-emerging diseases we touch on Human Papillomavirus (HPV) and Herpes Simplex (HSV). This selection of diseases is based on the daily experience of the clinicians working in the STI clinic of the Institute of Tropical Medicine in Antwerp. Data and clinical guidelines are derived from the Scientific Institute of Public Health in Brussels, the European and American Centers for Disease Control and Prevention, and the Guidelines of the Flemish Agency for Care and Health. New evolutions in diagnostics, prevention and treatment options make it necessary to regularly update the knowledge of this group of diseases, especially when they are complicated by HIV co-infection. As the incidence of neither HIV nor STIs seem to decrease in Belgium and Europe, it remains necessary to stay aware of the state-of-the-art management.

[Incidence of Neisseria gonorrhoeae infections in Belgium: trends 2000-2006]. / Incidence des infections à Neisseria gonorrhoeae en Belgique: tendances 2000-2006.

In Belgium, three registration systems collect epidemiological information on N. gonorrhoeae infections. The descriptive analysis of the data presented in this article allows describing the epidemiology of N. gonorrhoeae infections in Belgium in terms of trends in time, describing the characteristics of the patients, and providing information on resistance to antibiotics. The results on the incidence of N. gonorrhoeae infections show an important increase since the year 2000, and this increase is even more pronounced between 2005 and 2006. The majority of the patients reside in big cities, mainly in the district of Antwerp and in the Brussels-Capital region. Among the N. gonorrhoeae specimens that were sent to the reference laboratory, the proportion of specimens resistant to ciprofloxacine increases each year; this proportion reaches 61.4% in 2006. The increase in the incidence of N. gonorrhoeae infections and in antimicrobial resistance is also observed in other European countries. The increase in incidence may be partly related to the important increase of resistance to ciprofloxacine. It is very important to continue the surveillance of antimicrobial resistance, to adapt treatment in function of the recent evolutions and to inform physicians at a regular basis. The results show that homo- and bisexual men are most at risk for N. gonorrhoeae infections. The prevention campaigns for sexually transmitted infections and screening policy have to be reinforced, particularly among homo- and bisexual men.

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR-restriction fragment length polymorphism.

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR-restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI. It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.

Lack of evidence for the involvement of rectal and oral trichomonads in the aetiology of vaginal trichomoniasis in Ghana.

OBJECTIVE: To investigate the possible involvement of human trichomonads (Pentatrichomonas hominis and Trichomonas tenax) other than Trichomonas vaginalis in the aetiology of vaginal trichomoniasis. METHODS: Vaginal swabs taken from women attending antenatal clinics were tested for Trichomonas vaginalis by traditional assays (wet-mount microscopy and InPouch culture) and nucleic acid amplification (polymerase chain reaction). These swabs were also tested for the presence of P hominis and T tenax by nucleic acid amplification. Oral and rectal swabs from these women were tested for T tenax and P hominis respectively. Data on sociodemographic characteristics, sexual and anogenital hygiene practices likely to seed P hominis and T tenax into the vagina were collected by a questionnaire. RESULTS: 93% (161) of the 173 samples in which T vaginalis was detected by wet preparation or culture was evaluable by PCR. Of this, T vaginalis was detected in 94% (152) by T vaginalis-specific PCR. Neither P hominis nor T tenax was detected in any of the vaginal swab samples. These included nine samples for which T vaginalis had been detected by wet preparation or culture, but were negative by T vaginalis nucleic acid amplification. P hominis and T tenax were not detected in any of the rectal and oral swabs, respectively. CONCLUSION: In this group of women, there was no evidence for the involvement of trichomonads other than T vaginalis in the aetiology of vaginal trichomoniasis.

Detection of Pentatrichomonas hominis DNA in biological specimens by PCR.

AIM: To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens. METHODS: Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs. CONCLUSIONS: A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of P. hominis in vaginal specimens has not been reported before.

Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens.

OBJECTIVES: DNA amplification techniques have become widely used for the diagnosis of sexually transmitted infections. For the detection of Trichomonas vaginalis, PCR techniques are not yet widely used despite the publication of several assays. The sensitivity and specificity of five independent primer sets were determined on self collected vaginal specimens obtained from female commercial sex workers. METHODS: Self collected specimens were obtained from symptomatic and asymptomatic women attending a female sex workers clinic in Abidjan, Côte d'Ivoire. Two vaginal specimens were collected, the first one was processed for culture and the second was processed for PCR analysis. PCR techniques for trichomonads were performed, using the primers as reported by Riley (TVA5/TVA6), Kengne (TVK3/TVK7), Madico (BTUB 9/BTUB 2), Shiao (IP1/IP2), and Mayta (TV1/TV2). An EIA amplicon detection method was designed for each of the primer sets. RESULTS: True positive specimens were defined as culture positive and/or two positive PCR results with EIA amplicon detection in any combination. According to this definition a prevalence of 20% was obtained compared to 7% obtained by culture. The PCR primer set TVK3/TVK7 gave the highest sensitivity (89.2%). Poor sensitivities were obtained with the primer sets TV1/TV2 (60.2%) and TVA5/TVA6 (63.9%). PCR showed a sensitivity improvement of 2.4% up to 12% when EIA was used for amplicon detection. CONCLUSIONS: Overall, the sensitivities of the different PCR assays resulting from this study were lower than those previously described. These findings could be the result of the nature of the specimen population and suggests a strain variability.

High prevalence of sexually transmitted diseases in a rural area in Mozambique.

OBJECTIVE: To assess the extent of the sexually transmitted diseases (STDs) problem in a rural area of Mozambique. METHODS: A cross sectional study among pregnant women and patients presenting with genital complaints. Laboratory confirmation was done for gonorrhoea, chlamydial infection, active syphilis, trichomoniasis and HIV infection. SETTING: A primary health care setting in Vilanculos, Inhambane province, Mozambique. RESULTS: Evidence of one or more of the above STDs was found in 51% of 201 pregnant women, 56% of 85 women and 62% of 77 men with genital complaints. Neisseria gonorrhoea or Chlamydia trachomatis were found in 16% of pregnant women, 23% of female patients and 28% of male patients; genital ulcer disease was present in 6%, 28% and 36%, of respectively pregnant women, female and male patients. The prevalence of active syphilis was about the same in the three groups of study subjects, that is 15%. HIV infection was found in 4% of the male patients; no HIV infection could be detected in the female groups. CONCLUSION: STDs were a major health problem in this rural area in Mozambique. Though HIV infection was still low, the high prevalence of STDs indicates that the potential is there for an explosive spread of the HIV/AIDS epidemic.

Effect of aerobic training on left ventricular diastolic filling.

The effects of aerobic training on left ventricular diastolic filling were determined and related to the effects on maximal oxygen uptake, left ventricular systolic function, and left ventricular mass. Nine untrained healthy males with a mean age of 32 years underwent a ten-week program of bicycle training. Echocardiographic left ventricular mass, maximal oxygen uptake, radionuclide-derived left ventricular ejection fraction, and average left ventricular filling rate were obtained before and after training. Training resulted in significant increases in maximal oxygen uptake (from 30 +/- 6 (mean +/- S.D.) to 40 +/- 7 ml/kg/min, p less than 0.001) and average left ventricular filling rate (from 2.04 +/- 0.36 to 2.22 +/- 0.31 end-diastolic volumes/sec, p less than 0.05), but no changes in heart rate, systolic blood pressure, left ventricular ejection fraction, or left ventricular mass. These data indicate that short-term high-intensity aerobic training enhances average left ventricular diastolic filling independent of any change in left ventricular geometry, systolic function, heart rate, or blood pressure.

Tc-99m RBC blood-pool imaging demonstrates umbilical vein in portosystemic shunt.

Dynamic hepatic scintigraphy in a patient with longstanding ethanolism revealed hepatofugal drainage of the liver area. That this pathway represented the umbilical vein was shown by subsequent blood-pool images. The umbilical vein drained the left portal vein into venous channels in or near the anterior abdominal wall. These channels proceeded through epigastric vessels into the right femoral/iliac venous system. The umbilical vein may serve as a shunt from the portal system to the systemic circulation in cases of portal hypertension. Blood-pool imaging has a potential role in clarifying possible venous channels identified by hepatic scintiangiography, and can suggest the presence of portal hypertension when these vessels are visualized.

Scintigraphic appearance of axillofemoral and femorofemoral bypass grafts.

The scintigraphic appearance of axillofemoral and femorofemoral bypass grafts was reviewed in three cases in which the grafts were patent and intravascular radiotracers defined their course. In a fourth case, the axillofemoral graft was occluded and an aberrant vessel was identified (probably acting as a physiological shunt).