A custom amplicon sequencing approach to detect resistance associated mutations and sequence types in Mycoplasma genitalium.

BACKGROUND: Mycoplasma genitalium resistance to antibiotic treatments is increasing, with very limited treatment alternatives on the horizon. Surveillance via sequencing of multiple M. genitalium loci would allow: monitoring of known antibiotic resistance mutations, associations between resistance/treatment failure and specific mutations, and strain typing for epidemiological purposes. In this study we assessed the performance of a custom amplicon sequencing approach, which negates the cost of library preparation for next generation sequencing. METHODS: Fifty-two M. genitalium positive samples (cervical, vaginal, anal and rectal swabs, and urine) were used. Three regions associated with M. genitalium antibiotic resistance (23S rRNA, parC and gyrA genes) were targeted, in conjunction with a locus used for differentiation of sequence types in the mgpB gene, and findings compared to Sanger sequencing. RESULTS: Amplicon sequencing provided adequate sequence read coverage (>30×) for the majority of samples for 23S rRNA gene (96%) and mgpB (97%), parC (78%) and gyrA (75%). Single nucleotide polymorphisms (SNPs) were characterised in samples for 23S rRNA gene (94%), parC (56%) and gyrA (4%). Unlike Sanger sequencing, mixed mutations could be identified by the amplicon sequencing method, and ratios of mutation types determined. All results, with one exception, were concordant to Sanger sequence results. Sequence diversity in the mgpB region was represented by 15 sequence types, 4 being observed in multiple samples. CONCLUSIONS: We have demonstrated the utility of this custom amplicon sequencing approach for generating highly informative datasets with the capacity to identify and determine ratios of mixed sequences. The use of this customisable amplicon sequencing method enables cost effective, scalable amplicon sequencing of multiple target regions of interest in M. genitalium.

The impact of sample storage on molecular-based detection of Mycoplasma genitalium.

AIMS: Mycoplasma genitalium causes a common, sexually transmitted bacterial infection. This study assessed the detection of M. genitalium in stored urine samples to understand the impact of sample storage on M. genitalium detection. METHODS: Aliquots of M. genitalium-positive urine (n = 20 patients) were stored at either room temperature (22°C) or 4°C, without a preservative. At weekly intervals, samples were tested using the commercial test ResistancePlus MG® (SpeeDx® , Australia). We report the analysis at 1 week, an acceptable collection-to-test turnaround time, with further analysis over 5 weeks to illustrate degradation trends. RESULTS: After storing at 4°C, the proportion of specimens that remained positive for M. genitalium was 100% after 1 week and 95% after 4 weeks. Storage at 22°C led to more rapid decline in detection in the first 4 weeks, with 95% detected after 1 week and 85% at 2 weeks onwards. At 5 weeks, samples stored at both temperatures had an 85% M. genitalium detection rate, with increase in crossing points (Cq) of 0·72 (95% confidence interval (CI) 0·01-1·43; P-trend = 0·027) at 4°C, and 1·75 ((95% CI 0·79-2·71), P-trend <0·001) at 22°C. CONCLUSIONS: Urine samples stored without preservative, and unfrozen, retained high M. genitalium detection levels over the short term (up to 5 weeks). To minimize degradation, storing at 4°C is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: There is little known about the stability of clinical samples for M. genitalium detection. This study found that a high proportion (85-100%) of samples are still suitable for M. genitalium detection after storage for up to 5 weeks.

Duration of syphilis symptoms at presentations in men who have sex with men in Australia: are current public health campaigns effective?

The rapid rise in syphilis cases has prompted a number of public health campaigns to assist men who have sex with men (MSM) recognize and present early with symptoms. This study aimed to investigate the temporal trend of the duration of self-report symptoms and titre of rapid plasma reagin (RPR) in MSM with infectious syphilis. Seven hundred and sixty-one syphilis cases in MSM diagnosed at the Melbourne Sexual Health Centre (MSHC) from 2007-2013 were reviewed. Median duration of symptoms and RPR titres in each year were calculated. The median durations of symptoms with primary and secondary syphilis were 9 [interquartile range (IQR) 6-14] days and 14 (IQR 7-30) days, respectively. The overall median titre of RPR in secondary syphilis (median 128, IQR 64-256) was higher than in primary syphilis (median 4, IQR 1-32) and in early latent syphilis (median 32, IQR 4-64). The median duration of symptoms for primary syphilis, secondary syphilis and titre of RPR level did not change over time. Public health campaigns were not associated with a significant shorter time from onset of symptoms to treatment. Alternative strategies such as more frequent testing of MSM should be promoted to control the syphilis epidemic in Australia.

Ratio of anogenital warts between different anatomical sites in homosexual and heterosexual individuals in Australia, 2002-2013: implications for susceptibility of different anatomical sites to genital warts.

There is little known regarding the transmissibility of human papillomavirus (HPV) between different sites in men who have sex with men (MSM) and heterosexual individuals. We conducted a retrospective analysis investigating all new patients attending the Melbourne Sexual Health Centre in Australia between 2002 and 2013. We describe the prevalence and ratio of the first episode of anogenital warts in MSM and heterosexual males and females. The proportion of new MSM clients with anal and penile warts was 4·0% (362/8978) and 1·6% (141/8978), respectively; which gave an anal-to-penile wart ratio of 1:2·6. About 13·7% (1656/12112) of heterosexual males had penile warts and 10·0% (1121/11166) of females had vulval warts, which yielded a penile-to-vulval wart ratio of 1:0·7. Penile-anal transmission has a higher ratio than penile-vulval transmission, suggesting that the anal epithelium may be more susceptible to HPV infection than the vulval epithelium in females; these ratios are important in modelling the control of HPV in MSM.

Factors associated with neurocognitive test performance at baseline: a substudy of the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

OBJECTIVES: We describe neuropsychological test performance (NP) in antiretroviral treatment (ART)-naïve HIV-positive individuals with CD4 cell counts above 500 cells/µL. METHODS: In a neurology substudy of the International Network for Strategic Initiatives in Global HIV Trials (INSIGHT) Strategic Timing of AntiRetroviral Treatment (START) study, eight neurocognitive tests were administered. The primary measure of NP was the quantitative NP z-score (QNPZ-8), the average of the z-scores for the eight tests. Associations of baseline factors with QNPZ-8 scores were assessed by multiple regression. Mild neurocognitive impairment (NCI) was defined as z-scores < -1 in at least two of six cognitive domains. RESULTS: A total of 608 participants had a median age of 34 years; 11% were women and 15% were black; the median time since HIV diagnosis was 0.9 years; the median CD4 cell count was 633 cells/µL; 19.9% had mild NCI. Better NP was independently associated with younger age, being white, higher body mass index (0.10 per 10 kg/m(2) higher), and higher haematocrit percentage (0.19 per 10% higher). Worse NP was associated with longer time since HIV diagnosis (-0.17 per 10 years), diabetes (-0.29) and higher Framingham risk score (-0.15 per 10 points higher). QNPZ-8 scores differed significantly between geographical locations, with the lowest scores in Brazil and Argentina/Chile. CONCLUSIONS: This is the largest study of NP in ART-naïve HIV-positive adults with CD4 counts > 500 cells/µL. Demographic factors and diabetes were most strongly associated with NP. Unmeasured educational/sociocultural factors may explain geographical differences. Poorer NP was independently associated with longer time since HIV diagnosis, suggesting that untreated HIV infection might deleteriously affect NP, but the effect was small.

Routine CD4 cell count monitoring seldom contributes to clinical decision-making on antiretroviral therapy in virologically suppressed HIV-infected patients.

OBJECTIVES: In Australia, CD4 cell count is monitored approximately every 6 months in HIV-infected patients during antiretroviral therapy (ART). The aim of this study was to determine if routine CD4 monitoring contributed to decisions on changes to ART, and to estimate how reduced CD4 monitoring could contribute to cost savings in Australia. METHODS: We conducted a retrospective cohort analysis investigating all HIV-infected patients who attended the Melbourne Sexual Health Centre (MSHC) in Australia from 1 April 2011 to 1 October 2013. We reviewed the electronic medical records of all patients who changed or stopped antiretroviral regimens during this time period to determine whether CD4 cell count could have contributed to this clinical decision. RESULTS: Among 1004 patients with HIV infection on ART, none [95% confidence interval (CI) 0-2.3%] of the 162 clinical decisions to change or stop treatment were influenced by CD4 cell counts. Reducing the current biannual CD4 monitoring strategy to annually could potentially save ∼AU$ 1.5 million (US$ 1.4 million) each year in Australia [i.e. ∼AU$ 74 700 (US$ 67 700) could be saved per 1000 HIV-infected patients during ART]. CONCLUSIONS: Routine CD4 monitoring in HIV-infected patients during ART could be reduced from biannually to annually, as it rarely influences clinical decisions in patients' management. Not only could this avoid patients being unnecessarily anxious about normal fluctuations in their CD4 counts but it would also result in cost savings.

Size of anal squamous cell carcinomas at diagnosis: a retrospective case series.

Anal squamous cell carcinoma is more common in HIV-positive homosexual men than in the general population and prognosis worsens with increasing tumour size. To identify opportunities for earlier diagnosis, we aimed to determine size and visibility of anal squamous cell carcinoma at diagnosis. We conducted a retrospective review of medical records between 1992 and 2010 from one hospital radiotherapy centre, a major centre for HIV care, in Melbourne, Australia. Of 128 cases of anal squamous cell carcinoma, 24 (19%) were in HIV-positive men. At diagnosis, half (52%) of the tumours were externally visible and mean estimated tumour size was 36 mm (29 mm in HIV-positive and 38 mm in HIV-negative patients; p = 0.04) and 114/121 (94%) tumours were 1 cm or larger. The most frequent symptoms were bleeding (43%) and pain (36%) and mean duration of symptoms was 22 weeks. This suggests most anal squamous cell carcinoma were visible or palpable for some time before diagnosis, meaning that screening high-risk groups by anal inspection and palpation is plausible.

Acceptability of digital anal cancer screening examinations in HIV-positive homosexual men.

OBJECTIVES: Anal cancer is more common in HIV-positive homosexual men than in HIV-negative homosexual men and the general population. Earlier diagnosis leads to improved prognosis. We aimed to determine if regular anal inspection and digital examination of asymptomatic homosexual men attending for routine HIV care were acceptable and to record the rate of referral for diagnosis of potentially malignant anal lesions. METHODS: We offered anal examinations to consecutive homosexual men with HIV infection aged ≥ 35 years during their routine HIV clinic visits, aiming to complete three examinations over a 12-month period. Acceptability questionnaires were completed at baseline and after each examination and doctors recorded examination findings and all resulting interventions. Hospital referral outcomes were collected and interventions were costed using the Australian Medical Benefits Schedule. RESULTS: Of 142 men who were offered enrolment in the study, 102 [72%; 95% confidence interval (CI) 64-79%] participated. Following the initial anal examinations, four men were referred to surgeons. Cancer was excluded in three men (3%; 95% CI 1-8%) and one was diagnosed with anal squamous cell carcinoma (SCC). Three men had anoscopy performed at the time and two were referred for colonoscopy. Ninety-eight per cent (95% CI 93-100%) of respondents said that they would probably have the examination next time. The intervention was estimated to cost approximately Australian $16 per examination. CONCLUSIONS: Regular anal digital examinations are an acceptable and inexpensive addition to the routine care of homosexual men with HIV infection.

Topical imiquimod 5% as a treatment for localized genital Kaposi's sarcoma in an HIV-negative man: a case report.

We report a case of Kaposi's sarcoma (KS) in an HIV-negative man who has sex with men (MSM) that was successfully treated by topical application of imiquimod 5% cream (Aldara). Our case highlights a potentially effective and non-invasive method of treatment of Kaposi's sarcoma in the shortest timeframe yet recorded. The location of the lesions on the patient's penis also highlights the need for a non-invasive treatment.

Definitions of antiretroviral treatment failure for measuring quality outcomes.

OBJECTIVES: Our aim was to compare three different definitions of treatment failure and discuss their use as quality outcome measures for a clinical service. METHODS: Data for treatment-naïve patients who attended the Melbourne Sexual Health Centre (MSHC) between 1 January 2000 and 31 December 2008 were analysed. Definition 1 was the strict Food and Drug Administration (FDA) definition of treatment failure as determined using the time to loss of virological response (TLOVR) algorithm. Definition 2 defined treatment failure as occurring in those whose viral load never fell to <400 HIV-1 RNA copies/mL or who developed two consecutive viral loads > or =400 copies/mL on any treatment (switching or stopping treatment with a viral load <400 copies/mL was permitted). Definition 3 was the same as definition 2 except that individuals were also deemed to have failed if they stopped treatment for 6 months or longer. RESULTS: There were 310 antiretroviral-naïve patients who started treatment in the study period. Of these, 156 [50.3%; 95% confidence interval (CI) 42.1-53.3%] experienced treatment failure under definition 1, 10 (3.2%; 95% CI 1.5-5.8%) experienced treatment failure under definition 2, and 16 (4.5%; 95% CI 2.5-7.4%) experienced treatment failure under definition 3 over the 108 months of follow-up. The probability of failing definition 1 was statistically different from the probability of failing definition 2 or 3 (P=0.01). CONCLUSION: There were significant differences in treatment failure for the three definitions. If definition 1 were used, the outcomes would be sufficiently common to enable clinics to be compared but would be less meaningful. If definition 2 or 3 were used, the events would be too rare to enable clinics to be compared, but it would be possible to set a benchmark level of success that clinics could aim to reach.

Long-term utility of measuring adherence by self-report compared with pharmacy record in a routine clinic setting.

OBJECTIVES: To compare long-term adherence to antiretroviral therapy in an HIV service, as measured by self-report and by pharmacy records. To determine the level of adherence by each measure required to suppress viral load in a majority of patients. METHODS: The percentage of prescribed doses taken was calculated from (a) the number of missed doses in the previous 28 days reported by patients in a questionnaire at each clinic visit, and (b) pharmacy dispensing records. These were compared with each other and with HIV viral load data. RESULTS: Mean adherence was 96.2% by pharmacy record over 44 months and 98.6% by self-report over 25 months. The two methods correlated with each other (P<0.001) and the proportion of patients with viral load <400 HIV-1 RNA copies/mL increased with adherence as measured by self-report (P=0.001) and pharmacy record (P=0.004). Fewer than 60% of patients always had viral loads <400 copies/mL if adherence fell below 95% (pharmacy record) or 97% (self-report). Adherence was higher for once-daily than for twice-daily therapy (by pharmacy record: 97.2% vs. 96.0%; P<0.001). Adherence by both measures increased over time. CONCLUSIONS: Self-reported antiretroviral adherence correlates with pharmacy dispensing records and predicts suppression of viral load at levels >or=97%. It is practical to adopt this into routine HIV clinical care.