Pragmatic, open-label, single-center, randomized, phase II clinical trial to evaluate the efficacy and safety of methylprednisolone pulses and tacrolimus in patients with severe pneumonia secondary to COVID-19: The TACROVID trial protocol.

INTRODUCTION: Some COVID-19 patients evolve to severe lung injury and systemic hyperinflammatory syndrome triggered by both the coronavirus infection and the subsequent host-immune response. Accordingly, the use of immunomodulatory agents has been suggested but still remains controversial. Our working hypothesis is that methylprednisolone pulses and tacrolimus may be an effective and safety drug combination for treating severe COVID-19 patients. METHODS: and analysis: TACROVID is a randomized, open-label, single-center, phase II trial to evaluate the efficacy and safety of methylprednisolone pulses and tacrolimus plus standard of care (SoC) versus SoC alone, in patients at advanced stage of COVID-19 disease with lung injury and systemic hyperinflammatory response. Patients are randomly assigned (1:1) to one of two arms (42 patients in each group). The primary aim is to assess the time to clinical stability after initiating randomization. Clinical stability is defined as body temperature ≤37.5 °C, and PaO2/FiO2 > 400 and/or SatO2/FiO2 > 300, and respiratory rate ≤24 rpm; for 48 consecutive hours. DISCUSSION: Methylprednisolone and tacrolimus might be beneficial to treat those COVID-19 patients progressing into severe pulmonary failure and systemic hyperinflammatory syndrome. The rationale for its use is the fast effect of methylprednisolone pulses and the ability of tacrolimus to inhibit both the CoV-2 replication and the secondary cytokine storm. Interestingly, both drugs are low-cost and can be manufactured on a large scale; thus, if effective and safe, a large number of patients could be treated in developed and developing countries. TRIAL REGISTRATION NUMBER: NCT04341038 / EudraCT: 2020-001445-39.

Correction to: CSF inflammatory markers and neurocognitive function after addition of maraviroc to monotherapy darunavir/ritonavir in stable HIV patients: the CINAMMON study.

Due to a production error the bottom portion of Figure 1 was omitted. The corrected figure is given below.

CSF inflammatory markers and neurocognitive function after addition of maraviroc to monotherapy darunavir/ritonavir in stable HIV patients: the CINAMMON study.

CINAMMON is a phase IV, open-label, single-arm, pilot study assessing maraviroc (MVC) in the central nervous system (CNS) when added to darunavir/ritonavir monotherapy (DRV/r) in virologically suppressed HIV-infected subjects. CCR5 tropic participants on DRV/r were recruited. Participants remained on DRV/r for 12 week (w) (control phase). MVC 150 mg qd was added w12-w36 (intervention phase). Lumbar puncture (LP) and neurocognitive function (Cogstate) examinations scheduled at baseline, w12 and w36; MRI before w12, again at w36. Primary endpoint was CSF inflammatory marker changes during intervention phase. Secondary endpoints included changes in NC function and MRI parameters. CSF/plasma DRV/r concentrations measured at w12 and w36, MVC at w36. Nineteen patients recruited, 15 completed (17M, 2F). Dropouts: headache (2), knee problem (could not attend, 1), personal reasons (1). Mean age (range) 45.4 years (27.2-65.1), 13/19 white, 10/19 MSM. No changes in selected CSF markers were seen w12-w36. Overall NC function did not improve w12-w36: total age adjusted z score improved by 0.27 (weighted paired t test; p = 0.11); for executive function only, age adjusted z score improved by 0.54 (p = 0.03). MRI brain parameters unchanged. DRV plasma:CSF concentration ratio unchanged between w12 (132) and w36 (112; p = 0.577, Wilcoxon signed-rank). MVC plasma:CSF concentration ratio was 35 at w36. No changes in neuroinflammatory markers seen. In this small study, addition of 24w MVC 150 mg qd to stable DRV/r monotherapy showed possible improvement in executive function with no global NC effect. Learning effect cannot be excluded. This effect should be further evaluated.

Elvitegravir concentrations in seminal plasma in HIV-1-infected men.

OBJECTIVES: The aim of the study was to quantify elvitegravir (EVG) concentrations in the semen of HIV-1-infected men receiving antiretroviral therapy (ART) consisting of an elvitegravir/cobicistat/emtricitabine/tenofovir (EVG/COBI/FTC/TDF) single-tablet regimen. METHODS: A phase IV, cross-sectional study was carried out including HIV-1-infected male adults with suppressed plasma HIV-1 RNA who switched ART to EVG/COBI/FTC/TDF. Total EVG concentrations at the end of the dosing interval (C24 h ) and HIV-1 RNA were measured in paired seminal plasma (SP) and blood plasma (BP) samples 4 weeks after switching to EVG/COBI/FTC/TDF. Validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify EVG concentrations, and HIV-1 RNA was determined by real-time polymerase chain reaction (PCR). RESULTS: Ten men were included. Their median age was 40 years (range 24-47 years), the median time on ART was 50 months (range 10-186 months), the median time with plasma HIV-1 RNA < 40 copies/mL was 37 months (range 7-113 months), and the median CD4 count was 737 cells/µL (range 190-1122 cells/µL). Four weeks after switching to EVG/COBI/FTC/TDF, all subjects had HIV-1 RNA < 40 copies/mL in both BP and SP. Median EVG C24 h was 277 ng/mL (range 64.8-1790 ng/mL) in BP and 169 ng/mL (range 12.8-792 ng/mL) in SP. A significant correlation was observed between BP and SP EVG concentrations (Spearman rho 0.952; P < 0.001). The median SP:BP EVG concentration ratio was 0.39 (range 0.20-0.92). EVG C24 h in SP was at least 23-fold the in vitro protein-unbound 50% effective response (EC50 ) of HIV-1 clinical isolates (0.04-0.55 ng/mL). In all but one individual, EVG C24 h in SP was also higher than the blood plasma protein binding-adjusted 95% inhibitory concentration (IC95 ) of wild-type HIV-1 (45 ng/mL). CONCLUSIONS: Seminal EVG concentrations in HIV-infected men treated with EVG/COBI/FTC/TDF sufficed to contribute to maintaining HIV-1 RNA suppression in this compartment.

Contribution of Population Pharmacokinetics to Dose Optimization of Ganciclovir-Valganciclovir in Solid-Organ Transplant Patients.

Treatment of solid-organ transplant (SOT) patients with ganciclovir (GCV)-valganciclovir (VGCV) according to the manufacturer's recommendations may result in over- or underexposure. Bayesian prediction based on a population pharmacokinetics model may optimize GCV-VGCV dosing, achieving the area under the curve (AUC) therapeutic target. We conducted a two-arm, randomized, open-label, 40% superiority trial in adult SOT patients receiving GCV-VGCV as prophylaxis or treatment of cytomegalovirus infection. Group A was treated according to the manufacturer's recommendations. For group B, the dosing was adjusted based on target exposures using a Bayesian prediction model (NONMEM). Fifty-three patients were recruited (27 in group A and 26 in group B). About 88.6% of patients in group B and 22.2% in group A reached target AUC, achieving the 40% superiority margin (P< 0.001; 95% confidence interval [CI] difference, 47 to 86%). The time to reach target AUC was significantly longer in group A than in group B (55.9 ± 8.2 versus 15.8 ± 2.3 days,P< 0.001). A shorter time to viral clearance was observed in group B than in group A (12.5 versus 17.6 days;P= 0.125). The incidences of relapse (group A, 66.67%, and group B, 9.01%) and late-onset infection (group A, 36.7%, and group B, 7.7%) were higher in group A. Neutropenia and anemia were related to GCV overexposure. GCV-VCGV dose adjustment based on a population pharmacokinetics Bayesian prediction model optimizes GCV-VGCV exposure. (This study has been registered at ClinicalTrials.gov under registration no. NCT01446445.).

Etravirine concentrations in seminal plasma in HIV-infected patients.

OBJECTIVES: To determine etravirine concentrations and the HIV-1 viral load (VL) in blood plasma (BP) and seminal plasma (SP) of HIV-infected patients. METHODS: Ten adult antiretroviral-experienced HIV-1 patients receiving an etravirine-containing regimen for at least 1 month were enrolled. Semen and blood samples were collected ~12 or 24 h after the last etravirine dose, depending on twice-daily or once-daily dosing, respectively. Liquid chromatography tandem mass spectrometry was used to determine etravirine concentrations and HIV-1 VL was determined by real-time PCR (detection limit 40 copies/mL). Results are presented as the median (range) unless otherwise indicated. RESULTS: Ten blood and 20 semen samples were collected. The CD4 count was 502 (252-817) cells/mm(3) and the BP VL was <40 (<40-362) copies/mL. The time on etravirine was 52 (12-124) weeks. The BP etravirine concentration was 452.5 (258-751) ng/mL. The SP etravirine concentration was 62.9 (31.2-166.0) ng/mL and values were above the IC(50) range (0.39-2.4 ng/mL) in all cases. The median etravirine SP:BP ratio was 0.16 (0.07-0.26). The SP VL was <40 copies/mL in all patients, whereas the BP VL was detectable in one patient with poor adherence to treatment. CONCLUSIONS: Etravirine concentrations in male genital secretions are modest, reaching only 16% of the BP concentration. Nevertheless, they are more than 10 times greater than the wild-type IC(50) range (not adjusted for protein binding).

Viral infections of the central nervous system in Spain: a prospective study.

The aim of the study was to determine the incidence of viruses causing aseptic meningitis, meningoencephalitis, and encephalitis in Spain. This was a prospective study, in collaboration with 17 Spanish hospitals, including 581 cases (CSF from all and sera from 280): meningitis (340), meningoencephalitis (91), encephalitis (76), febrile syndrome (7), other neurological disorders (32), and 35 cases without clinical information. CSF were assayed by PCR for enterovirus (EV), herpesvirus (herpes simplex [HSV], varicella-zoster [VZV], cytomegalovirus [CMV], Epstein-Barr [EBV], and human herpes virus-6 [HHV-6]), mumps (MV), Toscana virus (TOSV), adenovirus (HAdV), lymphocytic choriomeningitis virus (LCMV), West Nile virus (WNV), and rabies. Serology was undertaken when methodology was available. Amongst meningitis cases, 57.1% were characterized; EV was the most frequent (76.8%), followed by VZV (10.3%) and HSV (3.1%; HSV-1: 1.6%; HSV-2: 1.0%, HSV non-typed: 0.5%). Cases due to CMV, EBV, HHV-6, MV, TOSV, HAdV, and LCMV were also detected. For meningoencephalitis, 40.7% of cases were diagnosed, HSV-1 (43.2%) and VZV (27.0%) being the most frequent agents, while cases associated with HSV-2, EV, CMV, MV, and LCMV were also detected. For encephalitis, 27.6% of cases were caused by HSV-1 (71.4%), VZV (19.1%), or EV (9.5%). Other positive neurological syndromes included cerebellitis (EV and HAdV), seizures (HSV), demyelinating disease (HSV-1 and HHV-6), myelopathy (VZV), and polyradiculoneuritis (HSV). No rabies or WNV cases were identified. EVs are the most frequent cause of meningitis, as is HSV for meningoencephalitis and encephalitis. A significant number of cases (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) still have no etiological diagnosis.

Successful outcome of ganciclovir-resistant cytomegalovirus infection in organ transplant recipients after conversion to mTOR inhibitors.

Ganciclovir-resistant (GanR) cytomegalovirus (CMV) infection after organ transplantation is emerging as a significant therapeutic challenge. We report two cases of GanR CMV infection successfully managed by switching immunosuppression from calcineurin inhibitors to an mTOR inhibitor-based regimen. This salvage therapy should be considered when other options are not available.

Etravirine concentrations in CSF in HIV-infected patients.

OBJECTIVES: To determine etravirine concentrations in CSF in HIV-infected patients. METHODS: Twelve HIV-1 adult antiretroviral-experienced patients receiving an etravirine-containing regimen for at least 1 month were enrolled. Both CSF and blood samples were taken around 12 h after the last etravirine dose. Liquid chromatography-tandem mass spectrometry was used to determine etravirine concentrations, and HIV-1 viral load was determined by real-time PCR (limit of detection 40 copies/mL). RESULTS: Twelve blood and 12 CSF samples were collected. The median CD4 count was 333 (84-765) cells/mm(3) and the median plasma HIV-1 viral load was <40 (range <40-1777) copies/mL. The median time on etravirine was 34 (range 4-140) weeks. The median etravirine concentration in plasma was 611.5 (range 148-991) ng/mL. The median CSF etravirine concentration was 7.24 (range 3.59-17.9) ng/mL; in all cases, values were above the IC(50) range (0.39-2.4 ng/mL). The median etravirine CSF:plasma ratio was 0.01 (range 0.005-0.03). The CSF viral load was >40 copies/mL in one patient and plasma viral load was still detectable after 4 weeks of therapy. CONCLUSIONS: Etravirine achieves concentrations several times greater than the IC(50) range in CSF. All patients with undetectable plasma viral load were virologically suppressed in CSF while receiving an etravirine-containing regimen. Etravirine may help in controlling HIV-1 in CNS.

Viral response in stable patients switching to fosamprenavir/ritonavir monotherapy (the FONT Study).

OBJECTIVE: The aim of the study was to evaluate the efficacy of fosamprenavir/ritonavir (FPV/r) monotherapy in plasma and reservoirs in virologically suppressed patients. METHODS: A 48-week, prospective, single-arm pilot trial was carried out (trial registration: ISRCTN78584791). Patients receiving triple therapy [FPV/r plus two nucleoside reverse transcriptase inhibitors (NRTIs) for at least the previous month], with viral load (VL) <40 HIV-1 RNA copies/mL and no previous virological failure (VF) on protease inhibitors (PIs), were included in the trial and received FPV/r monotherapy (700/100 mg/12 h). VL and FPV/r levels [by liquid chromatography-tandem mass spectrometry (LC/MS/MS); limit of detection (LOD) 0.5 ng/mL] in cerebrospinal fluid (CSF) were determined at week 24. VF was defined as VL >40 copies/mL in three consecutive samples or >500 copies/mL in two samples. RESULTS: Enrolment was prematurely stopped because of a high percentage of VF. Twenty patients (45% men; median age 43.5 years) were included in the trial. Nine patients (45%) presented therapeutic failure [seven (35%) had VF, and two discontinued therapy]. Resistance testing was available in five patients. One patient presented major PI mutations (54L, 32I and 47V) in addition to one minor mutation (13V), whereas two patients had minor PI mutations (10V+36I and 71T, respectively). The patient with major PI mutations switched from FPV/r to darunavir/r and VL was re-suppressed. In the other six patients with VF, VL was re-suppressed after the reintroduction of NRTIs. VL was <40 copies/mL in all CSF samples (n=10). Median amprenavir plasma levels were 2.5 µg/mL (range 0.7-8.6 µg/mL) at week 24 and 2.5 µg/mL (range 0.4-3.8 µg/mL) at VF. The CSF amprenavir concentration was 28.1 ng/mL (range 6.39-83.6 ng/mL), exceeding the reported 50% inhibitory concentration (IC(50) ) range for CSF in nine of 11 patients. CONCLUSIONS: The high percentage of patients with VF in our study suggests that the use of FPV/r in a simplification monotherapy strategy should be discouraged. Adequate amprenavir levels and undetectable VL in CSF were documented in all samples evaluated.

Sequential treatment of cytomegalovirus infection or disease with a short course of intravenous ganciclovir followed by oral valganciclovir: efficacy, safety, and pharmacokinetics.

Oral (p.o.) or intravenous (IV) ganciclovir (GCV) has been the first-line agent for prevention and treatment of cytomegalovirus (CMV) infection and disease in solid organ transplantation (SOT). The introduction of p.o. valganciclovir, with higher bioavailability than p.o. GCV, has proven to be a suitable approach toward outpatient p.o. therapy for CMV infection/disease. The present single-arm, exploratory pilot trial performed with 21 patients investigates the efficacy and safety of a short therapeutic course (21 days) based on an initial IV treatment with GCV (5 mg/kg twice daily, for 5 days) followed by p.o. valganciclovir (900 mg twice daily, for 16 days) for CMV infection/disease in SOT patients. In all cases, doses were adjusted for renal function. Moreover, the study allowed comparison of exposure to GCV after p.o. valganciclovir with respect to IV GCV in the same patients. Response to treatment was monitored until day 180. Viral load eradication was achieved in 66.7% of patients, on day 21. Although not statistically significant, a trend was seen toward increased persistence of viral load on day 21 for patients with donor positive/recipient negative CMV serostatus or receiving either anti-rejection therapy or polyclonal anti-thymocyte globulin. CMV clinical infection recurred in 14.3% of patients, with higher recurrence rates in patients with risk factors for persistence of viremia. Exposures to GCV after using IV GCV or p.o. valganciclovir showed comparable values (P=0.054). This short course, combining initial IV GCV and subsequent p.o. valganciclovir, may provide effective exposure and therapeutic response in the treatment of CMV infection in SOT patients with adequate drug exposure and with the additional potential benefit of shortening the length of hospital stay, which may result in cost reduction and improved patient comfort.

Impact of hepatitis C virus coinfection on immune restoration during successful antiretroviral therapy in chronic human immunodeficiency virus type 1 disease.

The effect of coinfection with hepatitis C virus (HCV) on immune restoration in 39 human immunodeficiency virus (HIV)-infected patients during treatment with combined antiretroviral therapy (cART) was prospectively evaluated. After 48 weeks of treatment, HCV-coinfected patients had lower increases in CD4% (P = .05), total CD4+ (P = .01), and naïve CD4+ (P = .06) T cells than did single-infected subjects. Higher baseline naïve CD4+ T-cell levels were associated with better CD4+ (P = .05) and naïve CD4+ (P < .001) T-cell recovery. After a 4-year follow up, the differences disappeared (median CD4+ increase: 291 and 306 cells for HCV-positive and HCV-negative patients, respectively, P = .9). No significant differences were seen in memory CD4+ T cells (P = .30), and CD8+ cells expressing CD38 (P = .10) and CD28 (P = .73). These results suggest that, independently of other factors, infection with HCV blunts early CD4+ T-cell recovery in HIV-infected patients treated with combined antiretroviral therapy (cART). However, as good control of viral replication is maintained, satisfactory long-term immune restoration can nonetheless be achieved.

Preliminary results of treatment with pegylated interferon alpha 2A for chronic hepatitis C virus in kidney transplant candidates on hemodialysis.

INTRODUCTION: At present, there is little published information on the outcome of treatment with pegylated interferon (Peg-IF alpha 2a) in hepatitis C virus (HCV)-infected hemodialysis patients awaiting renal transplantation. The objective of this study was to assess the efficacy and tolerance of Peg-IF alpha 2a in this population. PATIENTS AND METHODS: Twelve noncirrhotic HCV-infected patients (10 men, 50 +/- 8 years of age, genotype 1b 84%), were prescribed Peg-IF alpha 2a, at 135 microg/wk for 48 weeks. Liver biopsy was performed in 11 of 12 cases. RESULTS: Six patients completed 48 weeks of treatment, with one end of treatment response (ETR), two sustained viral responses (SVRs), and three HCV relapses. Treatment was shorter in the six remaining patients: two cases 24 weeks (one due to medical reasons with relapse, one due to nonresponse), one patient chose to discontinue at 14 weeks (with relapse), one patient died of stroke at 10 weeks, and in two additional patients interferon was withdrawn at 18 weeks because of severe anemia (SVR) and at 26 weeks due to prolonged fever (relapse). Other secondary treatment-related events included anemia (requiring transfusion in two patients and major erythropoietin administration in six), and fever in four patients. CONCLUSIONS: Peg-IF had limited efficacy in this group, with ETR in 83%, SVR in only 25%, and recurrence in 50%. Tolerance was moderate, with 4/12 (33%) discontinuing treatment due to adverse events, personal decision, or death. Large randomized controlled studies are needed to determine the role of Peg-IF treatment in this population.

Prevalence and patterns of polyomavirus urinary excretion in immunocompetent adults and children.

BK and JC polyomavirus infections are acquired commonly during childhood, mainly asymptomatically. These viruses are thought to remain latent in renal tissue after the primary infection and to reactivate under certain conditions. This reactivation leads to urinary excretion of virus particles, which can be detected by a range of methods. However, while this reactivation has been studied in depth in immunocompromised patients, little information is available about healthy individuals. The present study used PCR-based methods to examine urine samples from healthy individuals (51 adults and 15 children), and found that 62.7% of adults and 13.2% of children excreted polyomaviruses in the urine, mostly JC virus (41.2%). JC virus excretion was continuous, while BK virus excretion was mostly occasional.

[Breast-feeding in southern Catalonia. Epidemiological analysis of sociocultural and health factors influencing choice and duration]. / Lactancia materna en el sur de Cataluña. Estudio de los factores socioculturales ysanitarios que influyen en su elección y mantenimiento.

OBJECTIVE: To study the incidence and prevalence of breast-feeding and to determine the factors that influence the mother's decision to breast-feed or to use adapted milk. MATERIAL AND METHODS: Two hundred families were included in a survey in the hospital's maternity department. Those who breast-fed were followed up by means of a telephone call on days 15, 30, 90, and 180. RESULTS: On leaving hospital 78% of the neonates were receiving breast milk only. After 15 days, 89.7% of the neonates continued to receive breast milk and at 6 months this figure was 39%. Breast-feeding was discontinued after a mean of 2.5 months. The mean age of mothers who breast-fed was 30.2 years and that of mothers using adapted milk was 27.9 years (p,0.05). Mothers decided on the type of feeding before pregnancy (52.5%). This decision was unchanged by prenatal information except in the case of information provided by the family, especially if both parents were breast-fed (p,0.05). Doctors provided little information. The mother's level of education did not influence the decision to breast-feed although the higher the mother's education, the greater the tendency to breast-feed (74.7% with primary education vs 81.5% with higher education). Being in paid employment did not influence the decision to breast-feed (76% of mothers worked vs 79% of mothers who did not). The main reasons for discontinuance were hypogalactia, "feeling hungry", and work. In general, giving up breast-feeding was the mother's decision. CONCLUSIONS: The information pregnant women receive on breast-feeding should be based on unified criteria. The implementation of joint protocols between primary and hospital care as well as breast-feeding support groups help mothers to begin and continue breast-feeding.

Lactancia materna en el sur de Cataluña. Estudio de los factores socioculturales y sanitarios que influyen en su elección y mantenimiento / Breast-feeding in southern catalonia. Epidemiological analysis of sociocultural and health factors influencing choice and duration

Objetivo: Investigar la incidencia y prevalencia de la lactancia materna y conocer los factores que influyen en la decisión materna. Material y método: En la maternidad del hospital se pasó una encuesta a 200 familias. A los que recibían lactancia materna se les hizo un seguimiento mediante encuesta telefónica a los 15, 30, 90 y 180 días. Resultados: El 78% recibían lactancia materna exclusiva al alta. A los 15días seguía el 89,7% y a los 6 meses el 39%. La media de abandono fue 2,5 meses. La edad media de la madre que lacta es 30,2 años y la de artificial es del 27,9 años (p,0,05). Las madres toman la decisión del tipo de alimentación antes del embarazo (52,5%). La información recibida preparto no cambia su decisión, excepto la procedente de su familia, sobre todo si ambos padres fueron amamantados por lactancia materna (p,0,05). El grado de información recibida de los médicos es bajo. El nivel de estudios no influye, aunque la tendencia es cuanto más alto sea éste, más lactancia materna (74,7% primarios frente a 81,5% superiores). El trabajar o no, no influye (76% de las que trabajan frente a 79% de las que no). Los motivos principales de abandono fueron hipogalactia, "se queda con hambre", y trabajo (14%). El abandono en general lo decide la madre. Conclusiones: La información acerca de la lactancia materna que han de recibir las gestantes ha de basarse en criterios unificados. La puesta en marcha de protocolos conjuntos primaria-hospital y la creación de "Grupos de ayuda a la lactancia" facilita a las madres la decisión y el mantenimiento de ésta (AU)

[Evaluation of four rapid methods for the investigation of the toxigenic capacity of Clostridium difficile strains isolated in a selective medium]. / Evaluación de cuatro métodos rápidos para la investigación de la capacidad toxigénica de las cepas de Clostridium difficile aisladas en un medio selectivo.

BACKGROUND: Use of selective Clostridium difficile culture as a diagnostic method for C. difficile associated disease requires to prove the toxigenic ability of the isolates. Toxin B detection by cell culture assay after growing the microorganism in enriched broth is the standard method, but it delays the final diagnosis for 3-5 days. This study compares retrospectively four rapid techniques for detecting these toxigenic C. difficile strains. METHODS: 106 clinical isolates of C. difficile (72 toxigenic and 34 non-toxigenic), these and 16 clinical strains of other species of Clostridium were investigated. The four methods were performed directly from colonies growing on solid agar. They were: a) cytotoxin detection in cell culture; b) two PCR amplifications of toxin A and toxin B, respectively, and c) toxin A detection by an immunoenzymatic method (VIDAS CDA2). All these procedures were completed within a normal working day. RESULTS: Only the 72 toxigenic C. difficile strains gave positive results by cell culture and PCR techniques (sensitivity and specificity: 100%). A total of 14 out of 49 toxigenic C. difficile strains showed negative results by the VIDAS assay in the first run, but all them were positive in repeated tests. CONCLUSIONS: Although all methods performed well, the cytotoxicity assay done directly on colonies growing in CCFA is a simple and rapid technique, and appears to be well-suited for use in laboratories with access to cell cultures.

Recovery and analysis of human immunodeficiency virus type 1 (HIV) RNA sequences from plasma samples with low HIV RNA levels.

Amplification of human immunodeficiency virus type 1 (HIV) reverse transcriptase (RT) and protease (PT) sequences from plasma is difficult when HIV RNA levels are low, and it usually cannot be accomplished in samples with <1,000 HIV RNA copies/ml. Because the RNA extraction step is critical for the success of subsequent amplifications and sequence analyses, two RNA extraction methods were compared to study plasma samples with low HIV RNA levels. Forty-four plasma samples containing <500 HIV RNA copies/ml in a branched-DNA (bDNA) assay (Quantiplex HIV RNA assay version 2.0 [Chiron Corp., Emeryville, Calif.]) were studied. RNA was extracted by using two commercial kits (QIAamp Viral RNA kit [Qiagen, Hilden, Germany] and NucliSens kit [Organon Teknika, Boxtel, The Netherlands]). Fragments (1,144 bp) encompassing HIV PT and RT sequences were amplified by nested PCRs. Amplified products were sequenced by using a commercial kit (Applied Biosystems). HIV RNA was recovered from a total of 21 plasma samples, including 20 samples after extraction by the NucliSens method, and 8 samples after extraction by the QIAamp method (P < 0.05). Mean HIV RNA levels in these samples, measured by an ultrasensitive bDNA assay (Quantiplex HIV RNA assay version 3.0; Chiron Corp., Emeryville, Calif.), were 848 copies/ml (median, 666; range, 154 to 2,606 copies/ml). Analysis of RT and PT sequences in five samples demonstrated an average of 3.8 and 2.4 resistance mutations in these regions, respectively. The NucliSens RNA extraction kit is a valuable method for obtaining HIV RNA for genotypic studies from plasma fractions of individuals with low HIV RNA levels.