Non-inferiority testing for qualitative microbiological methods: Assessing and improving the approach in USP 1223.

The United States Pharmacopoeia (USP) presents two approaches for showing non-inferiority of an alternate qualitative microbiological method versus a compendial method. One approach compares the positive rates for the alternate and compendial methods at one spike level, while the other one compares multiple most probable number (MPN) estimates from a multi-spike design using a t-test. In this paper, we discuss these approaches under certain assumptions and propose a third approach that can be used for both single and multiple dilutions, which we call the generalized MPN (gMPN) approach. Simulations, using Poisson distributed numbers of microorganisms in test samples, confirm that the USP approach based on rates is not suitable, that the USP approach based on MPNs is appropriate for non-inferiority, but the gMPN approach outperforms the MPN-based approach and is therefore recommended.

A comparison of spiking experiments to estimate the detection proportion of qualitative microbiological methods.

The detection proportion of a qualitative microbiological test method is the probability to detect a single micro-organism. A general expression for the moment estimator of the detection proportion is provided. It depends on the distribution of the spikes used in a validation study through its moment-generating function. Several forms of spiking experiments are compared on their estimation performance using simulations and assuming a generalized Poisson distribution (GPD) for the spikes. The optimal design, which minimizes the mean squared error of our proposed moment estimator, depends on the dispersion parameter of the GPD. The design that uses just one spiked solution instead of multiple solutions is optimal for Poisson and overdispersed Poisson and it is robust against distributions for the spikes.

Validation of qualitative microbiological test methods.

This paper considers a statistical model for the detection mechanism of qualitative microbiological test methods with a parameter for the detection proportion (the probability to detect a single organism) and a parameter for the false positive rate. It is demonstrated that the detection proportion and the bacterial density cannot be estimated separately, not even in a multiple dilution experiment. Only the product can be estimated, changing the interpretation of the most probable number estimator. The asymptotic power of the likelihood ratio statistic for comparing an alternative method with the compendial method, is optimal for a single dilution experiment. The bacterial density should either be close to two CFUs per test unit or equal to zero, depending on differences in the model parameters between the two test methods. The proposed strategy for method validation is to use these two dilutions and test for differences in the two model parameters, addressing the validation parameters specificity and accuracy. Robustness of these two parameters might still be required, but all other validation parameters can be omitted. A confidence interval-based approach for the ratio of the detection proportions for the two methods is recommended, since it is most informative and close to the power of the likelihood ratio test.

A comparison of test statistics for the recovery of rapid growth-based enumeration tests.

This paper considers five test statistics for comparing the recovery of a rapid growth-based enumeration test with respect to the compendial microbiological method using a specific nonserial dilution experiment. The finite sample distributions of these test statistics are unknown, because they are functions of correlated count data. A simulation study is conducted to investigate the type I and type II error rates. For a balanced experimental design, the likelihood ratio test and the main effects analysis of variance (ANOVA) test for microbiological methods demonstrated nominal values for the type I error rate and provided the highest power compared with a test on weighted averages and two other ANOVA tests. The likelihood ratio test is preferred because it can also be used for unbalanced designs. It is demonstrated that an increase in power can only be achieved by an increase in the spiked number of organisms used in the experiment. The power is surprisingly not affected by the number of dilutions or the number of test samples. A real case study is provided to illustrate the theory.

Is it possible to reduce the incidence of weekend oocyte retrievals in GnRH antagonist protocols?

A retrospective analysis of a large, randomized clinical trial (Engage) assessed whether adjusting the start day of ovarian stimulation and/or day of human chorionic gonadotrophin (HCG) trigger could minimize oocyte retrieval during weekends without adverse effects on clinical outcome. Patients received recombinant FSH/gonadotrophin-releasing hormone (GnRH) antagonist regimens, with stimulation starting on day 2 or 3 of menses. HCG was administered when at least three follicles of ⩾ 17 mm were present on ultrasound scan or 1 day later. The frequency distribution of the day of reaching the HCG criterion relative to stimulation initiation was analysed to determine the optimal stimulation start day (cycle day 2 or 3) depending on the weekday at which menses started, to minimize weekend retrieval. The number of oocytes retrieved and pregnancy rates were not affected by start day and/or delay in HCG administration in regularly ovulating women aged 18-36 years with bodyweight 60-90 kg, body mass index 18-32 kg/m(2) and menstrual cycle length 24-35 days. In recombinant FSH/GnRH antagonist regimens, it appears possible to minimize weekend oocyte retrieval by selecting the cycle day to initiate stimulation, day 2 when menses starts Friday-Tuesday, otherwise day 3 and if necessary in combination with a 1-day HCG delay. A retrospective analysis of the data collected from a large, randomized clinical trial (Engage) was conducted to assess whether adjusting the start day of ovarian stimulation and/or the day of human chorionic gonadotrophin (HCG) trigger could minimize the possibility of retrieving the mature egg cells during weekends, without an adverse effect on the clinical outcome. Patients were administered recombinant FSH/gonadotrophin-releasing hormone (GnRH) antagonist on either day 2 or 3 of menses in order to start ovarian stimulation. Ultrasound monitoring was subsequently performed to determine when at least three follicles of ⩾17 mm or more in diameter were present, and final maturation was induced by HCG administration either on that day or 1 day later, if necessary. The frequency distribution of day of reaching HCG criterion relative to ovarian stimulation initiation on cycle day 2 or 3 was analysed to determine the optimal ovarian stimulation day (day 2 or 3) depending on the weekday at which the menses started to minimize the possibility of weekend egg retrieval. Number of eggs retrieved and pregnancy rates were not found to be affected by day of stimulation and/or a 1-day delay in HCG administration. In a recombinant FSH/GnRH antagonist protocol, weekend egg retrieval can be minimized by selecting the appropriate cycle day to initiate ovarian stimulation (cycle day 2 when menses starts Friday to Tuesday, otherwise cycle day 3), and if necessary in combination with a 1-day HCG delay.

Comparative incidence of ovarian hyperstimulation syndrome following ovarian stimulation with corifollitropin alfa or recombinant FSH.

Corifollitropin alfa is a novel recombinant gonadotrophin with sustained follicle-stimulating activity. A single injection can replace seven daily injections of recombinant follicle-stimulating hormone (rFSH) during the first week of ovarian stimulation. All cases of ovarian hyperstimulation syndrome (OHSS) with corifollitropin alfa intervention in a gonadotrophin-releasing hormone antagonist protocol have been assessed in three large trials: Engage, Ensure and Trust. Overall, 1705 patients received corifollitropin alfa and 5.6% experienced mild, moderate or severe OHSS. In the randomized controlled trials, Engage and Ensure, the pooled incidence of OHSS with corifollitropin alfa was 6.9% (71/1023 patients) compared with 6.0% (53/880 patients) in the rFSH group. Adjusted for trial, the odds ratio for OHSS was 1.18 (95% CI 0.81-1.71) indicating that the risk of OHSS for corifollitropin alfa was similar to that for rFSH. The incidence of mild, moderate and severe OHSS was 3.0%, 2.2% and 1.8%, respectively, with corifollitropin alfa, with 1.9% requiring hospitalization, and 3.5%, 1.3% and 1.3%, respectively, in the rFSH arms, with 0.9% requiring hospitalization. Despite a higher ovarian response with corifollitropin alfa compared with rFSH for the first 7days of ovarian stimulation, the incidence of OHSS was similar. Corifollitropin alfa is a new agent used in ovarian stimulation treatment for IVF fertilization. One injection of corifollitropin alfa can replace seven injections of recombinant FSH (rFSH). In three studies of corifollitropin alfa treatment, we assessed all cases of ovarian hyperstimulation syndrome (OHSS), a potentially serious complication of ovarian stimulation treatment. Overall, 5.6% of the patients (95/1701) experienced OHSS. Two of the trials compared corifollitropin alfa versus rFSH. Because OHSS is relatively rare, we pooled the results of these trials to give a more reliable estimate of the incidence of OHSS. In the pooled analysis, 6.9% (71/1023) of patients receiving corifollitropin alfa had signs or symptoms of OHSS, compared with 6.0% in the rFSH group (53/880). The risk of OHSS with corifollitropin alfa treatment was similar to the risk of OHSS in patients who received rFSH: the incidence of mild, moderate and severe OHSS was 3.0%, 2.2% and 1.8%, respectively, in patients in the corifollitropin alfa treatment groups, with 1.9% requiring hospitalisation, and 3.5%, 1.3% and 1.3%, respectively, in patients in the rFSH treatment groups, with 0.9% requiring hospitalization. Although the ovaries respond more to corifollitropin alfa than to rFSH for the first 7days of ovarian stimulation, neither treatment regimen was significantly more likely to cause OHSS.