Sex is associated with the persistence of non-optimal vaginal microbiota following treatment for bacterial vaginosis: a prospective cohort study.

OBJECTIVE: Determine the associations between factors and sexual practices and the composition of the vaginal microbiome (VM) of women treated for bacterial vaginosis (BV). DESIGN: Prospective cohort study. SETTING: The Melbourne Sexual Health Centre, Melbourne, Australia. POPULATION: Seventy-five reproductive-age women diagnosed with clinical BV, treated with first-line antibiotics and followed for up to 6 months. METHODS: Women self-collected vaginal swabs and completed questionnaires at enrolment, the day following antibiotics and monthly for up to 6months until BV recurrence or no BV recurrence (n = 430 specimens). Bacterial composition was determined using 16S rRNA gene amplicon sequencing. The effects of ongoing factors on VM composition (utilising 291 monthly specimens) were assessed using generalised estimating equations population-averaged models, which accounted for repeated measures within individuals. MAIN OUTCOME MEASURES: The relative abundance of vaginal bacterial taxa. RESULTS: Women who reported ongoing sex with a regular sexual partner (RSP) had a VM comprised of increased relative abundance of non-optimal BV-associated bacteria (Adjusted co-efficient [Adjusted co-eff] = 11.91, 95% CI 3.39to20.43, P = 0.006) and a decreased relative abundance of optimal, Lactobacillus species (Adjusted co-eff = -12.76, 95% CI -23.03 to -2.49, P = 0.015). A history of BV was also associated with a decreased relative abundance of Lactobacillus spp. (Adjusted co-eff = -12.35, 95% CI -22.68, P = 0.019). The relative abundance of Gardnerella, Atopobium and Sneathia spp. increased following sex with an RSP. CONCLUSIONS: Sex with an untreated RSP after BV treatment was associated with a VM comprised of non-optimal BV-associated bacteria. BV treatment approaches may need to include partner treatment if they are to achieve a sustained optimal VM associated with improved health outcomes. TWEETABLE ABSTRACT: Sex drives a return to a 'non-optimal' vaginal microbiota after antibiotics for bacterial vaginosis.

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.

Disruption of the MRP-L23 gene encoding the mitochondrial ribosomal protein L23 is lethal for Kluyveromyces lactis but not for Saccharomyces cerevisiae.

The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated K1L23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a "petite-positive" strain carrying a rho o-lethality suppressor atp mutation rendered cells unable to grow on a nonfermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis.

Viability after myocardial infarction: can it be assessed within five minutes by low-dose dynamic iodine-123-iodophenylpentadecanoic acid imaging with a multicrystal gamma camera?

Although positron emission tomography (PET) assesses myocardial viability (V) accurately, a rapid, inexpensive substitute is needed. Therefore, the authors developed a low-dose (1 mCi) Iodine-123-Iodophenylpentadecanoic Acid (IPPA) myocardial viability scan requiring analysis of only the first three minutes of data acquired at rest with a standard multicrystal gamma camera. Twenty-one patients > 2 weeks after myocardial infarction (MI) (24 MIs, 10 anterior, 14 inferoposterior, 21 akinetic or dyskinetic) had cardiac catheterization and resting IPPA imaging. V was determined by either transmural myocardial biopsy during coronary bypass surgery (12 patients, 14 MIs) or reinjection tomographic thallium scan (9 patients, 10 MIs), and 50% of MIs were viable. The IPPA variables analyzed were: time to initial left ventricular (LV) uptake in the region of interest (ROI), the ratio of three-minute uptake in the ROI to three-minute LV uptake, three-minute clearing (counts/pixel) in the ROI (decrease in IPPA after initial uptake), and three-minute accumulation (increase in IPPA after initial uptake) in the ROI. Rules for detecting V were generated and applied to 10 healthy volunteers to determine normalcy. While three-minute uptake in nonviable MIs was only 67% of volunteers (P < 0.0001) and 75% of viable MIs, uptake alone identified only 50% of viable MIs and 75% of nonviable MIs. IPPA clearing, however, was > or = 13.5 counts/pixel in 10/12 (83%) of viable MIs, and IPPA accumulation > or = 6.75 counts/pixel identified one more viable MI, for a sensitivity for V of 11/12 (92%), with a specificity of 11/12 (92%), and a 100% normalcy rate. The authors conclude low-dose IPPA (five-minute acquisition with analysis of the first three minutes of data) has potential for providing rapid, inexpensive V data after MI. Since newer multicrystal cameras are mobile, IPPA scans can be done in emergency rooms or coronary care units generating information that might be useful in decisions regarding thrombolysis, angioplasty, or bypass surgery.

Effects of drainage and temperature on carbon balance of tussock tundra micrososms.

We examined the importance of temperature (7°C or 15°C) and soil moisture regime (saturated or field capacity) on the carbon (C) balance of arctic tussock tundra microcosms (intact blocks of soil and vegetation) in growth chambers over an 81-day simulated growing season. We measured gaseous CO2 exchanges, methane (CH4) emissions, and dissolved C losses on intact blocks of tussock (Eriophorum vaginatum) and intertussock (moss-dominated). We hypothesized that under increased temperature and/or enhanced drainage, C losses from ecosystem respiration (CO2 respired by plants and heterotrophs) would exceed gains from gross photosynthesis causing tussock tundra to become a net source of C to the atmosphere. The field capacity moisture regime caused a decrease in net CO2 storage (NEP) in tussock tundra micrososms. This resulted from a stimulation of ecosystem respiration (probably mostly microbial) with enhanced drainage, rather than a decrease in gross photosynthesis. Elevated temperature alone had no effect on NEP because CO2 losses from increased ecosystem respiration at elevated temperature were compensated by increased CO2 uptake (gross photosynthesis). Although CO2 losses from ecosystem respiration were primarily limited by drainage, CH4 emissions, in contrast, were dependent on temperature. Furthermore, substantial dissolved C losses, especially organic C, and important microhabitat differences must be considered in estimating C balance for the tussock tundra system. As much as ∼ 20% of total C fixed in photosynthesis was lost as dissolved organic C. Tussocks stored ∼ 2x more C and emitted 5x more methane than intertussocks. In spite of the limitations of this microcosm experiment, this study has further elucidated the critical role of soil moisture regime and dissolved C losses in regulating net C balance of arctic tussock tundra.

Detection of early systolic dysfunction in ischemia by sequential radionuclide imaging of ejection rates.

The effectiveness of sequential imaging of early regional left ventricular contraction in the detection of ischemic abnormalities was assessed in 47 patients (15 with previous infarction) with angiographically proven coronary artery disease, and 11 normal volunteers, undergoing first pass radionuclide angiography with a multielement gamma camera at rest and at peak exercise. Global left ventricular hemodynamic parameters, and functional images of regional ejection fraction and ejection rate were compared to 6 pairs of sequential rate images showing the decrease and the increase of regional left ventricular volume during a time-interval of 80-280 ms (at rest) and 50-175 ms (at stress) from end-diastole. Diagnostic accuracy of sequential images (67-91%) was higher than that of ejection rate image (71-72%), and of global hemodynamic parameters (33-60%), in the detection of coronary patients. Regional sensitivity of stress sequential increase and decrease image achieved 77% and 100%, respectively. During early systole sensitivity of stress sequential increase and decrease images approached 100% even at rest, subsequently decreasing because of normalizing contraction. Thus, the analysis of early systolic dysfunction provided by dynamic sequential functional images of ejection rates proved to be an effective diagnostic tool in the detection of myocardial ischemic dysfunction.

Myocardial viability assessment with dynamic low-dose iodine-123-iodophenylpentadecanoic acid metabolic imaging: comparison with myocardial biopsy and reinjection SPECT thallium after myocardial infarction.

Aggressive cardiac revascularization requires recognition of stunned and hibernating myocardium, and cost considerations may well govern the technique used. Dynamic low-dose (1 mCi) [123I]iodophenylpentadecanoic acid (IPPA) metabolic imaging is a potential alternative to PET using either 18FDG or 15O-water. Resting IPPA images were obtained from patients with severe ischemic cardiomyopathy, and transmural myocardial biopsies were obtained during coronary bypass surgery to confirm viability. Thirty-nine of 43 (91%) biopsies confirmed the results of the IPPA images with a sensitivity for viability of 33/36 (92%) and a specificity of 6/7 (86%). Postoperatively, wall motion improved in 80% of IPPA-viable, dysfunctional segments. Furthermore, when compared to reinjection thallium (SPECT-TI) scans after myocardial infarction, IPPA-SPECT-TI concordance occurred in 27/35 (77%) (K = 0.536, p = 0.0003). Similar to PET, IPPA demonstrated more viability than SPECT-TI, 26/35 (74%) versus 18/35 (51%) (p = 0.047). Metabolic IPPA cardiac viability imaging is a safe, inexpensive technique that may be a useful alternative to PET.

Intravenous dipyridamole thallium-201 SPECT imaging methodology, applications, and interpretations.

Dipyridamole TI-201 imaging is an ideal alternative to exercise TI-201 scintigraphy in patients who are unwilling or unable to perform maximum exercise stress. The use of intravenous dipyridamole, alone or in combination with exercise, has not been approved for clinical practice by the Food and Drug Administration. Once approval is granted, the test will become a widely used and important component of the cardiac work-up. The indications, methodology, side effects, and utility of dipyridamole cardiac imaging in the clinical setting are discussed and a variety of examples presented.

Functional cardiac imaging in coronary disease: increased sensitivity of first-pass radionuclide angiography utilizing sequential regional left ventricular early diastolic filling rate images.

In 127 patients, 113 with greater than or equal to 50% coronary artery stenosis (CAD), 14 with normal coronaries, cardiac catheterization and first-pass radionuclide angiography (RNA) utilizing left ventricular (LV) regional ejection fraction, first half systolic LV regional mean transit time and ejection rate images were performed. Additionally, the incremental value of a new technique, sequential regional LV filling rate images focusing on the first third of diastole, was established. Diastolic imaging improved RNA sensitivity from 88% (100/113) to 96% (109/113). Single vessel disease sensitivity increased from 77% (23/30) to 90% (27/30), whereas multivessel disease RNA positivity changed from 93% (77/83) to 99% (82/83). LAD system (LAD/D) sensitivity improved by 24% to 94% (79/84); RCA system (RCA/PDA) sensitivity increased 17% to 84% (59/70); circumflex system (CFX/OM) sensitivity was 83% (67/81), an improvement of 5%. Specificity was well maintained despite the increased sensitivity, as 86% (12/14) of patients with normal coronaries were normal by RNA. Furthermore, in the 113 CAD patients, 81% (84/104) of the vessels with insignificant or no stenosis were normal by RNA. We conclude sequential regional LV diastolic filling images substantially increase RNA sensitivity for CAD, while specificity is satisfactorily maintained.

The different radionuclide angiography (RNA) responses to upright bicycle ergometer exercise in a population with low probability coronary disease.

One hundred and one subjects (52 men, 49 women) with low probability coronary disease and normal resting left ventricular ejection fraction performed upright bicycle exercise to target without angina, ischemic ECG change, or segmental wall motion abnormality. Fifteen millicuries of Tc-99m pertechnetate were injected at rest and during peak exercise, and images were processed with first pass technique (RNA). The responses were not uniform. Group 1 was characterized by the resting end diastolic volume index (EDVI) usually greater than 80 ml/M2, a peripheral resistance drop (decreases R) of 0.20-0.50, an increase in cardiac index (increases CI) of 2.8-5.5 1/minute/M2, accompanied by decreasing EDVI and ESVI with exercise, stroke volume index (SVI) remaining essentially unchanged, and left ventricular ejection fraction (EF) usually increasing. Group 2 was characterized by the resting EDVI less than 80 ml/M2, and with the same decreases R and increases CI; EDVI, ESVI, SVI all increased and EF tended to decrease. Group 3 was characterized by decreases R greater than 0.50, increases CI greater than 5.5 1/minute M2, the exercise EDVI usually increasing, ESVI usually decreasing, SVI usually increasing, and EF usually increasing. There were no significant differences (alpha = 0.01) in exercise-related changes between men and women within each group.