Quality Management Systems (QMSs) of Human-Based Tissue and Cell Product Manufacturing Facilities.

In the broadest sense, a quality management system (QMS) runs by continuous interaction of elements based on processes, procedures, policies, guidelines, and resources that are compiled to guide an organization under the scope of its operational mission, vision, and objectives. QMSs in the biopharmaceutical sector defines a written and applied set of rules which aids in improvement of the quality of biopharmaceutical process engineering, while primarily assuring human tissue- and cell-based starting material and end product safety and minimizing the risk of human medicinal product recall, in the most cost-effective ways possible. This chapter aims to outline the crucial position of a QMS under the scope of good practices (GxPs) in the biopharmaceutical industry, as regards human tissue- and cell-based products.

Cultivo mediante hisopado superficial versus cultivo de la biopsia de tejidos profundos en la infección del pie diabético / Culture through superficial swab versus deep tissue biopsy in the diabetic foot infection

RESUMEN Introducción: para lograr el adecuado y precoz diagnóstico de la infección en pie diabético, es necesario la obtención de una muestra bacteriológica de calidad para la identificación del germen causal. Objetivo: identificar posibles relaciones entre los resultados obtenidos, en el cultivo realizado mediante hisopado superficial versus el obtenido mediante biopsia de los tejidos profundos en la infección del pie diabético. Materiales y métodos: se realizó un estudio explicativo observacional, longitudinal, prospectivo en el Servicio Provincial de Angiología y Cirugía Vascular del Hospital Provincial Clínico Quirúrgico Universitario "Comandante Faustino Pérez", durante un periodo de 3 años desde enero del 2016 hasta diciembre del 2018. Una selección muestral no probabilística determinó una muestra constituida por 138 extremidades en 132 pacientes con diagnóstico clínico de pie diabético infectado, que requirieron cirugía para desbridamiento de la lesión. Aceptaron ser incluidos en la investigación y para el aislamiento del germen causal fueron empleados ambos métodos de cultivo: hisopado superficial y biopsia de los tejidos profundos. Resultados: el promedio de microorganismos aislados se incrementó en relación con la severidad de la infección del pie diabético, con mayor incremento en el aislamiento hecho por el hisopado superficial. El hisopado superficial posee pobre correlación con los gérmenes aislados mediante el cultivo de la biopsia de los tejidos profundos. Conclusiones: las muestras deben ser obtenidas preferentemente por curetaje. En el diagnóstico de la infección del pie diabético es de gran utilidad, por su rapidez y concordancia con los resultados del cultivo, efectuar siempre una tinción de Gram a partir del mismo sitio (AU).

ABSTRACT Introduction: to arrive to an adequate and precocious diagnosis of the diabetic foot infection, it is necessary to obtain a qualitative bacteriological sample to identify the causing germ. Objective: to identify possible relationships between the results obtained both, in the culture made through superficial swab and the culture obtained from deep tissues biopsy in the diabetic foot infection. Materials and methods: a prospective, longitudinal, observational, explicative study was carried out in the Provincial Service of Angiology and Vascular Surgery of Provincial University Clinical Surgical Hospital "Comandante Faustino Pérez", in a period of three years, from January 2016 to December 2018. A non-probabilistic sampling choose a sample of 138 lower limbs in 132 patients with clinical diagnosis of infected diabetic foot, who required surgery for lesion debridement. They gave their consent to be included in the research; for the isolation of the casual germ were used both culture methods, superficial swab and deep tissues biopsy. Results: the average of isolated microorganism increased in relation to the severity of the diabetic food infection, with higher increase in the isolation obtained by superficial swab. The superficial swab shows poor correlation with the germ isolates by the culture the deep tissue biopsy. Conclusions: the samples should be gathered preferably by curettage. In the diagnosis of the diabetic foot infection, it is very useful, due to its speed and concordance with the culture results, to make always a Gram staining beginning from the same place (AU).

Standardized bacteriophage purification for personalized phage therapy.

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward-by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 1011 plaque-forming units (PFUs) per milliliter for Pseudomonas, Klebsiella, and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 109 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 106-fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.

A Way Forward for Culturing Plasmodium vivax.

Trager and Jensen established a method for culturing Plasmodium falciparum, a breakthrough for malaria research worldwide. Since then, multiple attempts to establish Plasmodium vivax in continuous culture have failed. Unlike P. falciparum, which can invade all aged erythrocytes, P. vivax is restricted to reticulocytes. Thus, a constant supply of reticulocytes is considered critical for continuous P. vivax growth in vitro. A critical question remains why P. vivax selectively invades reticulocytes? What do reticulocytes offer to P. vivax that is not present in mature erythrocytes? One possibility is protection from oxidative stress by glucose-6-phosphate dehydrogenase (G6PD). Here, we also suggest supplements to the media and procedures that may reduce oxidative stress and, as a result, establish a system for the continuous culture of P. vivax.

Comprehensive evaluation of complex polymicrobial specimens using next generation sequencing and standard microbiological culture.

Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S identified more organisms per sample and allowed identification of fastidious organisms, while culture was better at capturing organisms when bacterial load was low, and allowed incidental recovery of non-bacterial pathogens. Molecular and culture-based methods together detect more organisms than either method alone.

Routine In Vitro Culture of Plasmodium falciparum: Experimental Consequences?

The advent of Plasmodium falciparum (Pf) in vitro culturing opened the door for malaria research, yielding dramatic advancements in our understanding of the parasite. However, fundamental foundations taken for granted in our research endeavors can unknowingly be an Achilles heel, resulting in potential misdirection. In relation to malaria research, this could be our nonquestioning acceptance of routine in vitro culture of Pf. There is nothing routine or straightforward regarding the dynamic and intimate relationship between the parasite and the in vitro environment. Here, we discuss recent studies demonstrating the impact that slight variations in in vitro Pf culture parameters can have on scientific conclusions. We reason that culture conditions should be re-established as a primary consideration in in vitro malaria experimentation.

A new enrichment diagnostic platform for semen culture.

Urogenital bacterial infections have been described in literature as a potential cause of infertility. For the consequences that a failure in diagnosis could have on the evolution of male urogenital infectious disease, an accurate microbiological procedure to investigate the bacterial species composition of seminal fluid plays a crucial role to better understand the eventual correlation with infertility. In order to improve the quality of semen culture investigations, we have developed a new enrichment diagnostic platform. Semen samples of 540 infertile men were simultaneously analyzed using the standard microbiological semen culture method and an alternative new experimental technique (Brain Heart Infusion broth, BHI, enrichment). Our results established the possibility to apply BHI enrichment to detect bacteria from semen samples with higher sensitivity (100%) and negative predictive value (100%) than the standard technique.

International Society for Cell Therapy Facility Sanitization Survey Laboratory Practices Committee Report.

BACKGROUND AIMS: Quality cell manufacturing processes require a clean laboratory environment. METHODS: This report was aimed at describing current cleaning and sanitization practices reported by facilities that manufacture many types of cellular therapy products for clinical use. It is our hope that this report may provide the groundwork for guidance recommendations directed at developing consensus standards for cleaning and sanitization practices across the globe. Facility sanitization is a central issue to regulatory and accreditation bodies. Facilities are required to develop plans to assess sanitization practices and test cleaning effectiveness. RESULTS: This document provides information on how this is performed in different facilities and may allow newer, smaller or less developed facilities to build, enhance or revise their current quality program by using experience and expertise in facility sanitization reported herein. CONCLUSIONS: This report summarizes the results of the latest survey and compares results with those previously reported. New and relevant trends in the field provide important information and will provide important information for establishing guidelines.

Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

Micropropagation of Cordyline terminalis.

This protocol describes an efficient and rapid method for large-scale multiplication of Cordyline terminalis in a cost-effective manner. Actively growing shoot tips were selected as explants. Murashige and Skoog (MS) basal medium was supplemented with different plant growth regulators at various developmental stages of C. terminalis. The highest percentage of regeneration (95 ± 2.8) and average number of shoot buds (60.2 ± 4.4) per explant were obtained in medium containing 80 mg /L adenine sulfate (AdSO(4)), 2 mg/L 6-benzyladenine (BA), and 0.1 mg/L indole-3-acetic acid (IAA). Thousands of micropropagated plants were produced within 4-5 months using this protocol.

Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood.

We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

Amendments to sterility test requirements for biological products. Final rule.

The Food and Drug Administration (FDA) is amending the sterility test requirements for biological products. This rule provides manufacturers of biological products greater flexibility, as appropriate, and encourages use of the most appropriate and state-of-the-art test methods for assuring the safety of biological products. FDA is taking this action as part of its ongoing efforts to comprehensively review and, as necessary, revise its regulations related to biological products.

Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens? A clinical investigation using PCR and culture techniques.

BACKGROUND: Non-union continues to be one of the orthopedist's greatest challenges. Despite effective culture methods, the detection of low-grade infection in patients with non-union following tibial fracture still presents a challenge. We investigated whether "aseptic" tibial non-union can be the result of an unrecognized infection. METHODS: A total of 23 patients with non-union following tibial shaft fractures without clinical signs of infection were investigated. Intraoperative biopsy samples obtained from the non-union site were examined by means of routine culture methods and by polymerase chain reaction (PCR) for the detection of 16 S ribosomal RNA (rRNA). Control subjects included 12 patients with tibial shaft fractures. RESULTS: 23 patients (8 women and 15 men; mean age: 47.4 years) were included into this study. Preoperative C-reactive protein levels (mean: 20.8 mg/l) and WBC counts (mean: 8,359/µl) in the study group were not significantly higher than in the control group. None of the samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were found by conventional culturing methods in only 1 case of an open fracture from the control group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue specimens from 2 patients (8.7%) with non-union. The analysis of these variable species-specific sequences enabled the identification of specific microorganisms (1x Methylobacterium species, 1x Staphylococcus species). Both PCR-positive patients were culture-negative. CONCLUSIONS: The combination of microbiological culture and broad-range PCR seems to substantially add to the number of microbiological diagnoses obtained and may improve the clinician's ability to tailor therapy to the individual patient's needs.

Impacts of culture-independent diagnostic practices on public health surveillance for bacterial enteric pathogens.

For decades, culture has been the mainstay of diagnostic testing for bacterial enteric pathogens. This paradigm is changing as clinical laboratories adopt culture-independent methods, such as antigen-based tests and nucleic acid-based assays. Public health surveillance for enteric infections addresses 4 interrelated but distinct objectives: case investigation for localized disease control; assessment of disease burden and trends to prioritize and assess impact of population-based control measures; outbreak detection; and microbiologic characterization to improve understanding of pathogens, their virulence mechanisms, and epidemiology. We summarize the challenges and opportunities that culture-independent tests present and suggest strategies, such as validation studies and development of culture-independent tests compatible with subtyping, that could be adopted to ensure that surveillance remains robust. Many of these approaches will require time and resources to implement, but they will be necessary to maintain a strong surveillance system. Public health practitioners must clearly explain the value of surveillance, especially how outbreak detection benefits the public, and collaborate with all stakeholders to develop solutions.

Defining the true sensitivity of culture for the diagnosis of melioidosis using Bayesian latent class models.

BACKGROUND: Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests. METHODS/PRINCIPAL FINDINGS: Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard. CONCLUSIONS/SIGNIFICANCE: Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections.

Thin cell layers: power-tool for organogenesis of floricultural crops.

Thin cell layers or TCLs have been a fundamental corner-stone of tissue culture of all plant species, including floricultural and ornamental plants (FOPs). In this review, the current status of the use of TCL technology to specific FOPs will be outlined while certain successes and difficulties will be focused. The ability to control developmental and morphogenetic processes in vitro through the use of TCLs, especially where conventional methods have not always produced ideal results, is the key in taking lab-based theory to business-based applications. TCL technology, which focuses more on the size rather than the origin of the explant, is a gateway for FOP regeneration and transformation studies. Some general principles and guidelines for quality verification will be provided.

Evaluación de la calidad del diagnóstico micológico: cultivos / Quality assessment of the mycological diagnosis: cultures

Se evaluó la capacidad de los laboratorios participantes del Sub Programa Micología para identificar diferentes cepas de hongos al nivel de género o especie. Tomados los resultados en conjunto, el 66% de los laboratorios identificó en forma correcta las cepas, mientras que otro 25% lo hizo en forma parcial. En el caso de los dermatofitos hubo un mayor porcentaje de respuestas correctas para el reconocimiento de Trichophyton mentagrophytes (76%) respecto de las especies de Microsporum evaluadas: M. canis (66%) y M. gypseum (65%). Los resultados de la identificación de las especies de levaduras mostraron dos grupos diferentes: uno conformado por Candida albicans y Cryptococcus neoformans, con elevados porcentajes de respuestas correctas (87 y 95%, respectivamente) y otro por C. tropicalis y C. glabrata, que ocasionó dificultades para su correcta identificación (sólo 28 y 32% de respuestas correctas). Las especies de Aspergillus evaluadas (A. fumigatus y A. niger) fueron reconocidas en forma correcta por el 63 y 72% de los participantes. Los resultados obtenidos, si bien son considerados como aceptables, evidencian la necesidad de mejorar la capacidad evaluada, teniendo en cuenta su influencia decisiva sobre la calidad del diagnóstico micológico.

The ability to identify different fungal strains at genera or specie level for participant laboratories of the Mycology Sub-Program, was evaluated. Taking into account all results as a whole, 66% of laboratories identified the strains correctly, while another 25% recognized the strains partially. Regarding dermatophytes, a higher percentage of correct responses for Trichophyton mentagrophytes recognition was observed with respect to (76%) those of the evaluated Microsporum species: M. canis (66%) and M. gypseum (65%). The results produced by the identification of yeasts species showed 2 different groups: one constituted by Candida albicans and Cryptococcus neoformans, with higher percentages of correct responses (87 and 95%, respectively) and another one constituted by C. tropicalis and C. glabrata, which presented difficulties for their correct identification (28 and 32% correct responses). The evaluated species of Aspergillus (A. fumigatus and A. niger) were recognized by 63% and 72% of the participants. The results obtained, considered as acceptable, show the need to improve the evaluated capacity, taking into account their decisive influence on mycologic diagnosis quality.

Identification of Candida dubliniensis in a diagnostic microbiology laboratory.

Candida dubliniensis is an emerging yeast pathogen isolated mainly from immunocompromised patients. As molecular tests are currently unsuitable for use in routine diagnostic laboratories, we compared a variety of phenotypic techniques for differentiating C. albicans and C. dubliniensis. The tests included: colony colour on CHROMagar Candida medium; growth at 37 degrees C and 45 degrees C; ability to produce germ tubes and chlamydospores; and the Auxacolor system. The organisms included 105 isolates previously identified as C. albicans, 10 reference strains of C. albicans, 2 reference strains of C. dubliniensis and 102 fresh clinical isolates identified as C. albicans. None of the tests alone was satisfactory but a combination of 3 tests may be suitable for presumptive identification of C. dubliniensis.