Ph. Eur. testing for histamine and depressor substances using guinea-pigs and cats: the end of an era. Strategy for removal of animal tests for histamine and depressor substances and their vestiges from the Ph. Eur.

For more than 50 years, in vivo assays have been used for testing pharmaceutical product safety due to their assumed ability to broadly detect potential unidentified contaminants. As part of these in vivo tests, the animal tests for depressor substances and histamine have been described in the European Pharmacopoeia since its first edition in 1977. Both tests measure the effect of histamine and histamine-like substances, using guinea-pigs and cats respectively. In 2024, the Histamine (2.6.10) general chapter is referenced in the Production section of four monographs and 10 monographs have variations of a sentence on designing the manufacturing process to eliminate or minimise substances lowering blood pressure in this same section, without referencing the chapter. The Depressor substances (2.6.11) chapter is referenced only in the Histamine (2.6.10) chapter as a next step if the histamine test is invalid. A historical search was performed and it has shown that the tests for histamine and for depressor substances were introduced by different groups of experts in an inconsistent way at different times, and for different reasons, leading to non-harmonised approaches across monographs. The control of histamine and other depressor substances has been the subject of numerous debates where their use was questioned. During these discussions, reports on positive cases or batches failing the test for histamine or depressor substances were anecdotal. In addition, in vivo tests can be considered non-specific, very variable, time-consuming, costly and ethically doubtful. More importantly, the majority of in vivo methods originate from a time when good manufacturing practice was not widely used and formal method validation requirements were not yet established. In view of the above, the removal of all references to animal tests for histamine or depressor substances from all texts still referring to them is proposed. Since the sentences in the Production section referring to the control of "substances lowering blood pressure", "vasoactive substances" or "hypotensive substances" appeared as remainders of the animal test without further guarantee of safety, it will also be proposed to remove all these sentences from the concerned monographs. Ultimately, the suppression of general chapters 2.6.10 and 2.6.11 from the Ph. Eur. is envisaged. Independently from the above, it is also envisaged to elaborate a new general chapter Histamine in active substances (2.5.47) to include physicochemical or immunochemical methods enabling the detection of histamine. This new text would aim at supporting manufacturers in their histamine control strategy following the inclusion of precaution statements in the general monograph on Products of fermentation (1468); these statements had been added in Ph. Eur. Supplements 9.6 and 10.4, following adverse events related to a GMP issue with gentamicin sulfate. This strategy has been endorsed by the European Pharmacopoeia Commission at its 177th Session in November 2023. The concerned monographs would be a subject of public consultation in Pharmeuropa 36.2 (April 2024).

Role of calcifying nanoparticle in the development of hyperplasia and vascular calcification in an animal model.

OBJECTIVE: Calcifying nanoparticles (NPs) have been detected recently in calcified human arterial specimens and are involved in the process of calcification. This study was designed to test the hypothesis that human-derived NPs could worsen the response to arterial endothelial injury and induce vascular calcification. METHODS: The right carotid artery of 24 New Zealand rabbits was injured with an angioplasty balloon. Animals were perfused intravenously with saline (100 mL) during the experiment and divided into three groups: group-A, control; group-B, exposed to NPs (2 mL) obtained from calcified aortic valves; and group-C, exposed to NPs (2 mL) and treated postoperatively with atorvastatin (2.5 mg/kg/24 h). At 30 days, both carotid arteries were removed and examined histologically. Blood measurements were monitored during the study. RESULTS: The intimal hyperplasia area was significantly larger in the injured right carotid artery compared with the left unoperated carotid artery in all groups. There was no significant variation in medial area between groups. Morphometrically, the intima/media ratio (IMR) was significantly higher in damaged carotids compared with controls. A significant increase of IMR was found in group-B (1.81 ± 0.41) compared with group-A (0.38 ± 0.59; p = .004) or group-C (0.89 ± 0.79; p = .035). Differences between groups C and A were not significant (p = .064). Calcifications were observed in six animals, all of which had been exposed to NPs (4 in group-B, 2 in group-C, p = .027). Plasma levels of cholesterol and triglycerides remained stable. CONCLUSIONS: This research confirms the ability of systemic inoculation of human-derived NPs to accelerate hyperplasia and stimulate calcification in localized areas of arteries previously submitted to endothelial damage, while it was harmless in healthy arteries. Atorvastatin was demonstrated to slow down this process.

Comparison of the physicochemical properties of a biosimilar filgrastim with those of reference filgrastim.

Recombinant human granulocyte-colony stimulating factor (filgrastim) is a therapeutic protein used primarily to reduce incidence and duration of severe neutropenia and its associated, and serious, complications. We have developed a biosimilar filgrastim (Hospira filgrastim; Nivestim) designed to be comparable to Amgen filgrastim (Neupogen). An extensive characterization study assessed the physiochemical similarity of Hospira filgrastim to Amgen filgrastim. Both drugs were supplied in 1 ml glass, single-use, prefilled syringes (five batches of each product at 480 microg/0.5 ml and one batch of each product at 300 microg/0.5 ml). Samples were evaluated using state-of-the-art analytical methods (validated in accordance with International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use or Pharmeuropa guidelines) to determine physicochemical properties, molecular characteristics, purity and biological activity. Samples were compared after long-term storage at 2-8 degrees C and storage at 40 degrees C (stress conditions) to evaluate their degradation impurity profiles. Hospira filgrastim and Amgen filgrastim were shown to have similar physicochemical properties, molecular characteristics, purity and biological activity. No significant differences in product-related impurities were recorded between Hospira filgrastim and Amgen filgrastim following storage for 12 weeks under stress conditions. These data show that the physicochemical profile of Hospira filgrastim is similar to that of Amgen filgrastim.

Evaluation of an immunocapture-agglutination test (Brucellacapt) for serodiagnosis of human brucellosis.

We evaluated the validity and the usefulness of a new test for the diagnosis of human brucellosis based on an immunocapture-agglutination technique. A total of 315 sera from 82 patients with a diagnosis of brucellosis, 157 sera from patients in whom brucellosis was suspected but not confirmed, and 412 sera from people living in rural areas with endemic brucellosis were studied. The seroagglutination test (SAT), Coombs anti-Brucella test, and Brucellacapt test were evaluated. All the initial sera from the 82 patients proved to be positive in Brucellacapt and Coombs tests, while only 75 (91.4%) were positive in the SAT. If a >/=1/160 diagnostic threshold titer was defined for the Brucellacapt test, Coombs test, and SAT, the sensitivities were 95.1, 91.5, and 65.8%, respectively. Taking the same diagnostic threshold titer for the 157 sera from the unconfirmed but suspected patients, the specificities of the Brucellacapt, Coombs, and SAT were 81.5, 96.2, and 100%, respectively; for the 412 control sera, the specificities were 99.0, 99.8, and 100%. The diagnostic efficiency (area below the receiver operating characteristic curve) of Brucellacapt was 0.987852 (95% confidence interval [CI], 0.95109 to 0.99286), very similar to the diagnostic efficiency of the Coombs test (0.97611; 95% CI, 0.94781 to 0.99146) and higher than that of SAT (0.91013; 95% CI, 0.86649 to 0.94317). The results of the Brucellacapt test were compared with those of the Coombs test (correlation coefficient, 0.956; P = 0.000) and SAT (correlation coefficient, 0.866; P = 0.000). The study shows very good correlation between the Brucellacapt and Coombs tests, with a high concordance between titers obtained in the two tests. Nevertheless, lower correlation and concordance were found between the Brucellacapt and Coombs tests when the results for titers of >/=1/160 were compared (0.692; P = 0.000). In acute brucellosis, the Brucellacapt and Coombs tests render positive titers of >/=1/160. When the titers are lower, they increase significantly in the following 30 days, despite the evolution of SAT titers. In contrast, Brucellacapt and Coombs titers are always high (>/=1/640) in brucellosis with long evolution, whether SAT titers are higher or lower than 1/160.

[Laboratory diagnosis and serologic course in patients with tularemia]. / Diagnóstico de laboratorio y evolución serológica de pacientes con tularemia.

BACKGROUND: Tularemia was practically unknown in Spain until the end of 1997, when an epidemic outbreak was declared. This paper presents the data on microbiological diagnosis of 55 patients who suffered from tularemia. PATIENTS AND METHODS: Thirty-two samples from 19 patients and 151 serum samples from 55 patients were obtained for culture. Serologic diagnosis was performed by tube sero-agglutination and microagglutination. Three types of tests were performed on all sera: Wright sero-agglutination (WSA), Coombs test against Brucella spp. and sero-agglutination against Yersinia enterocolitica O:3, Yersinia enterocolitica O:3, and Proteus OX 19. RESULTS: F. tularensis was found in two samples (6.25%) of the 32 received. Titers > or = 1/160 were obtained in 78.2% and 74.5% of the initial sera by tube sero-agglutination and microagglutination, respectively. Correlation between the two tests was 0.80 (p < 0.001). Prozone phenomenon was observed in 59.9% of the sera, while crossed reactivity to Brucella spp. and Proteus spp. was found in 9.3% and 22.8%, respectively. No crossed reactivity was observed with Yersinia spp. CONCLUSIONS: Culture of F. tularensis has low sensitivity. The correlation obtained between tube sero-agglutination and microagglutination is good. Both techniques are useful in routine diagnosis of tularemia, although microagglutination has some advantages over tube agglutination.

Evaluation of six serological tests in diagnosis and postoperative control of pulmonary hydatid disease patients.

Latex agglutination (LA), passive hemagglutination (PHA), immunoelectrophoresis (IEP) and specific IgE, IgM, IgG enzyme-linked immunosorbent assay (ELISA) tests for diagnosis and postoperative follow-up of 79 patients with surgically confirmed pulmonary hydatidosis were evaluated. Specific IgG ELISA was the most sensitive test (83.5%) and the least sensitive tests were specific IgE ELISA (44.3%) and IEP (50.6%). The specificity obtained for all the serologic test was above 97% in all cases. The greatest number of false positives in all tests (except IEP) occurred in patients with Taenia saginata and Taenia solium cysticerci infestations and in patients with lymphoma and leukemia. Specific IgG ELISA demonstrated the highest negative predictive value (93.8%). No statistically significant differences (p > 0.050) were found in the sensitivity of the tests when patients with only one cyst and patients with various cysts were compared. Considering only the patients without relapse, the percentage of seropositive patients increased in all tests at 1 and 3 months after surgery. After that time the percentage of seropositive patients decreased. At 48 months after surgery all patients without relapse became negative in IEP, specific IgE ELISA, and specific IgM ELISA. The antibody titers in all seropositive patients increased during the 3 months after surgery. From these 3 months onward, antibody levels decreased in all serologic tests studied in the group of patients without relapse. The patients who had relapses during the first year after surgery presented persistently elevated antibody titers in all postoperative sera. The antibody titers of the patients who relapsed between the third and fourth years after surgery decreased progressively the third month after surgery, and increased in the serum obtained at the moment of relapse diagnosis. Our results show that persistence of elevated antibody titers in patients with pulmonary hydatidosis in the year after surgery or titer increase after a progressive decrease are indicative of relapse or reinfection.

Clinical evaluation of a commercial ligase-based gene amplification method for detection of Mycobacterium tuberculosis.

The purpose of this study was to evaluate the clinical usefulness of a commercial ligase-based gene amplification method (LCx Mycobacterium tuberculosis test; Abbott Laboratories, USA) for detection of Mycobacterium tuberculosis. The tuberculosis infection rate among clinical samples was 10.6%. The sensitivity, specificity, and positive and negative predictive values were 23.5%, 100%, 100%, and 91.7%, respectively, with the fluorochrome auramine stain; 32.4%, 100%, 100%, and 92.6%, respectively, with culture; and 76.5%, 95.8%, 68.4% and 97.2%, respectively, with the gene amplification method. When only samples from patients without current or previous treatment were studied, the sensitivity was 36.4% with the auramine stain, 63.6% with culture, and 100% with the gene amplification assay. The mean treatment time for culture-negative and assay-negative samples was greater than that of culture-negative and assay-positive samples. The LCx Mycobacterium tuberculosis test is a sensitive method for detection and identification of Mycobacterium tuberculosis. It produces few false-positive results. However, as it can remain positive after the culture becomes negative, it is not recommended for evaluation of treatment efficiency.

[Genotypes of hepatitis C virus: their relationship with risk factors, the severity of liver disease, and the serologic response]. / Genotipos del virus de la hepatitis C: relación con los factores de riesgo, con la gravedad de la enfermedad hepática y con la respuesta serológica.

BACKGROUND: The objective of our study was to ascertain the prevalence of different HCV genotypes between the hepatitis C patients in the health area of Monforte de Lemos, Spain, as well as the possible influence of risk factors on their distribution and their relation with hepatic disease and with the serologic response. PATIENTS AND METHODS: We have studied 128 patients with hepatitis C. Of these, 41 were intravenous drug users (IVDU), 19 had received transfusions, 7 were hemodialyzed and in 61 the risk factors were unknown. Antibodies against HCV were detected by second-generation enzyme immunoassay (EIA) and confirmed by immunoblot. RNA-HCV presence was studied by reverse transcription-PCR (RT-PCR), and a reverse hybridization test of the amplifications was used for the genotyping. RESULTS: Hepatitis C genotypes 1b (46.1 [8.6%]), 1a (23.4 [7.3%]) and 3a (13.3 [5.9%]) were the most frequently encountered genotype. Genotype 1a (48.8 [15.3%]) was the most prevalent genotypes in IVDU patients, while 1b was the most frequent in patients of unknown risk factors (62.3 [12.1%]). Alanine-aminotransferase (ALT) was elevated in 66.6 (17.7%) of patients with genotype 1a, in 87.5 (8.6%) of patients with genotype 1b (p = 0.0367) and in 94.1 (11.2%) of patients with genotype 3a (p = 0.0347). Subtype 1b was present in 6 of 7 cases of cirrhosis (85.7%) and in 7 of 12 cases of active chronic hepatitis (58.3%). No significant statistical differences were observed between the genotypes and the specific IgM response against core antigen of HCV, neither we observed differences in the serologic response against C1, C2, NS3 and NS4 peptides. CONCLUSIONS: Hepatitis C genotypes 1a and 3a were the most prevalent genotypes between IVDU patients while genotype 1b was the most frequent between non-IVDU patients. Genotype 1b was associated to severe liver disease. Percentage of positivity or the reactivity against HCV peptides was independent of the genotype encountered in the patient.

[Variants of hepatitis C virus in different risk groups. Comparative study of a method for genotyping and another for serotyping]. / Variantes del virus de la hepatitis C en diferentes grupos de riesgo. Estudio comparativo de un método de genotipificación y otro de serotipificación.

AIMS: The aim of this study was to know the prevalence of the different variants of HCV in the Health Care area of Monforte de Lemos (Lugo, Spain) and its distribution according to risk factors and to compare the results obtained with one genotyping and one serotyping technique. PATIENTS AND METHODS: Eighty-four patients with hepatitis C were studied, 25 of whom were IVDA, 14 had received blood transfusions, 4 hemodialysis and the risk factor was unknown in 41. The antibodies against HCV were studied by second generation EIA and confirmed by an immunoblot technique. Serotyping was carried out by an ELISA test. Genotyping was undertaken with a reverse hybridation test of the amplification obtained by polymerase chain reaction prior to reverse transcription (RT-PCR). RESULTS AND CONCLUSIONS: The genotypes most frequently observed were 1b (47.6%), 1a (20.2%) and 3 (14.3%). In the IVDA patients the genotypes 1a (40%) and 3 (24%) predominated. The 1b genotype was the most prevalent in the patients of unknown risk (68.3%) and patients with a history of blood transfusion (50%). The prevalence of the different serotypes was similar to that of the corresponding genotypes, with nearly 100% agreement. The number of untypable cases was greater in the serotyping technique (20.2%) than in the genotyping (2.4%). A greater number of mixed infections was detected with serotyping (7 cases, 8.3%) than with genotyping (1 case, 1.2%). Lesser sensitivity of the serotyping test was observed in the patients lacking anti-NS4 antibodies.

Relation of hepatitis C virus genotypes to risk factors and hepatic disease in Spanish patients.

OBJECTIVE: To ascertain the prevalence of hepatitis C virus (HCV) genotypes in Spain and their distribution by risk factors. METHODS: The study covered 216 patients with hepatitis C. Of these, 63 were intravenous drug users (IVDU), 44 had received transfusions, and 30 were hemodialyzed, and in 79 the risk factors were unknown. Antibodies against HCV were detected by second-generation enzyme immunoassay (EIA) and confirmed by immunoblot. HCV RNA presence was investigated by reverse transcription---polymerase chain reaction (RT-PCR), and a reverse hybridization test of the amplifications was used for the genotyping. RESULTS: The most frequently encountered genotypes were 1b (48.1%), 1a (21.3%) and 3a (11.1%). HCV genotypes 1a (42.8%) and 3a (20.6%) were the most prevalent genotypes in IVDU patients, while 1b was the most frequent in patients with unknown risk factors (62.0%), transfused patients (68.1%) and hemodialyzed patients (50.0%). Mixed infections were detected in nine cases (4.1%); three appeared in IVDU patients (4.7% of the total IVDUs), two in transfused patients (4.5%) and four (50%) in patients with unknown risk factors. No statistically significant differences were found in average ages of the IVDU patients with different genotypes. Non-IVDU patients having genotype 3a presented the lowest average age of all. No significant statistical differences were observed in alanine aminotransferase levels among patient groups with different genotypes (p>0.05 in all cases). Subtype 1b was present in six of the seven cases of cirrhosis (85.7%) and in nine of the 18 cases of active chronic hepatitis (50.0%).

Influence of syphilis in hepatitis B transmission in a cohort of female prostitutes.

BACKGROUND AND OBJECTIVES. Prostitutes are a greater risk for hepatitis B virus (HBV) infection than the general population. We studied the influence of age and time as prostitute on HBV infection. We also examined the relationship between syphilis and HBV infection in a cohort of female prostitutes. STUDY DESIGN. The presence of hepatitis B surface antigen (HBsAg), antibodies to hepatitis B core antigen (anti-HBc), antibodies to hepatitis D virus (anti-HD) and treponemal antibodies (FTA-ABS) were determined in 368 prostitutes, of whom 147 were submitted to medical and serological follow-up every six months to evaluate the influence of syphilis in the transmission of hepatitis B. RESULTS AND CONCLUSION. The prevalence of HBsAg was 4.6%, of anti-HBc 31.2%, anti-HD 0.5% and FTA-ABS 35.0%. There was a statistical association between the presence of treponemal antibodies and anti-HBc (P = 0.022). The cohort study performed shows that the accumulated incidence of HBV infection in the FTA-ABS positive prostitutes (24.6%) was significantly higher than that of the FTA-ABS negative group (9.7%) (RR = 2.544; P = 0.034). Our results indicate that syphilis could facilitate the heterosexual transmission of HBV infection.

[A e year study of the electrophoretic RNA patterns of rotavirus in Valladolid]. / Estudio de 3 años de los patrones de electroforesis de ARN de rotavirus en Valladolid.

BACKGROUND: To study the electrophoretic RNA patterns of human rotavirus (RV) during three years in Valladolid (Spain). MATERIAL AND METHODS: From october 1986 to october 1989 we have studied the electrophoretic RNA patterns of RV in 104 fecal samples. Stools were first screened by a latex agglutination test (LA) and reactive samples were confirmed by a commercial enzymeimmunoassay (EIA) and electron microscopy (EM) (negative stain). Electropherotyping was made according to conventional methods. RESULTS: 96.2% of the samples studied yield a defined electropherotype. We have found 8 different types of patterns of genomic RV-RNA circulating in these three years. Two of them belonged to short pattern (C and E) and six to long pattern (A, B, D, F, G and H). CONCLUSIONS: 98.0% of the genomic RNA founded were of long pattern, from which pattern A was the most prevalent (72%) during the study, without a seasonal distribution. Pattern B was found in 20% of cases, while the rest were found only occasionally.

Infection by hepatitis B and C virus in non-intravenous drug using female prostitutes in Spain.

We have studied the prevalence of hepatitis B (HBV) and hepatitis C virus (HCV) serologic markers in female blood donors and in female prostitutes and the relationship of antibodies to hepatitis B core antigen (anti-HBc) and of antibodies to HCV (anti-HCV) with the presence of treponemal antibodies (FTA-ABS) in non-intravenous drug using female prostitutes. Hepatitis B surface antigen (HBsAg) was found in 1.0% of the female blood donors, anti-HBc in 15.6% and anti-HCV in 0.7%. In the prostitutes, the prevalence of HBsAg was 6.1%, anti-HBc was positive in 29.0% and anti-HCV in 8.8%. No significant statistical association between the prevalence of anti-HBc or anti-HCV and the age of prostitutes (p = 0.9111 and p = 0.8254 respectively) or the length of time as prostitutes (p = 0.3583 and p = 0.5770) was found. FTA-ABS positive prostitutes had a significantly higher prevalence of anti-HCV than FTA-ABS negative prostitutes (p < 0.001). No statistical association was found between anti-HBc antibodies and positive FTA-ABS prostitutes (p = 0.336).

Prevalence of anti-hepatitis C virus antibodies in positive FTA-ABS non-drug abusing female prostitutes in Spain.

The prevalence of antibodies against Hepatitis C Virus (anti-HCV antibodies) and its association with the presence of treponemal antibodies (FTA-ABS) was studied in 227 non-drug abusing female prostitutes. The overall anti-HCV antibody prevalence was 8.8%, and a significantly higher prevalence of anti-HCV antibodies was found in prostitutes testing positive for FTA-ABS (19.4%) than in prostitutes testing negative for FTA-ABS (3.9%) (PR = 5.023, P less than 0.001). No statistical association was found between patient age or the duration of prostitution and the presence of anti-HCV antibodies.

Inhibition of the degranulation and myeloperoxidase activity of human polymorphonuclear neutrophils by Brucella melitensis.

The degranulation and myeloperoxidase-H2O2-halide activities of human polymorphonuclear leukocytes from healthy donors were tested after co-incubation with either Brucella melitensis 16M, Staphylococcus aureus or Staphylococcus aureus in presence of lipopolysaccharide, protein fraction, native hapten and soluble fractions released at 65 degrees C from smooth strain of Brucella melitensis 16M. The degranulation and myeloperoxidase activities of polymorphonuclear leukocytes were significantly higher when co-incubated with Staphylococcus aureus than with Brucella melitensis. The presence of lipopolysaccharide, protein fraction, and native hapten did not cause significant modification of either degranulation or myeloperoxidase activities of polymorphonuclear leukocytes against Staphylococcus aureus. Soluble fraction released at 65 degrees C produced a significant reduction in the myeloperoxidase activity but did not alter the degranulation of polymorphonuclear leukocytes triggered by Staphylococcus aureus.

[Anderson's disease. Clinical and morphologic study of 7 cases]. / La maladie d'Anderson. Etude clinique et morphologique de 7 cas.

Anderson's disease is a rare autosomic recessive condition involving the transport of fat through the intestinal mucosa, which could be due to a defect in the intestinal form (B48) of apolipoprotein B. Isolated cases and one important series only have been reported. We wish here to complete the description of the disease. Seven children (age 6 months to 13 years at time of diagnosis) were followed for one month to 15 years. They presented with a malabsorption syndrome, malnutrition, fatty diarrhea (steatorrhea 4-18 g/24 h), failure to thrive (height -1 to -5.5 SD for age) and sometimes disappearance of deep tendon reflexes. Biologically they had signs of malabsorption, hypocalciuria, osteoporosis, low serum iron, decreased levels of vitamins A and E, and hypo-alpha- (50-127 mg/100 ml) and beta- (73-175 mg/100 ml) lipoproteinemia due to decreased levels of plasma cholesterol (40-70 mg/100 ml), and phospholipids (34-67 mg/100 ml); apolipoproteins A1 (26-69 mg/100 ml and B (21-44 mg/100 ml) were also low. After a fatty meal, triglycerides and apolipoproteins did not increase and chylomicrons did not appear. Jejunal biopsies showed the characteristic aspect of enterocytes loaded with lipid droplets. On electron microscopy, these fat droplets were seen in the cytoplasm but neither in the endoplasmic reticulum and the Golgi complex nor in the intercellular spaces. They did not appear to be enclosed in membranes and differed from chylomicrons by their size and density. The disease could thus be due to an abnormal apolipoprotein B48, which would prevent its binding to triglycerides and thus the formation of chylomicrons.