Biotinidase Activity Research Articles

Comprehensive analysis of genotypic and phenotypic characteristics of biotinidase deficiency patients in the eastern region of Türkiye.

Biotin is a water-soluble vitamin that plays a key role in carboxylation. The formation of free biotin is impaired in biotinidase deficiency (BD), resulting in impaired biotin-dependent carboxylase functions. Based on the percentage of residual serum enzyme activity, BD is classified as partial and profound. Retrospective data including gender, age, parental consanguinity, family history, biotinidase activity analyses, type of deficiency (partial-profound), physical examination, treatment, and genotypes were evaluated in patients diagnosed with biotinidase deficiency in a single center in the eastern region of Türkiye. Patients whose biotinidase enzyme activity was below 30% with biallelic variants in the BTD gene were diagnosed as BD. A total of 302 patients were included in the study. Parental consanguinity was present in 135 (44.7%) of them. Two hundred eighty-six (94.7%) were diagnosed by neonatal screening, 14 (4.6%) by family screening and two (0.06%) by clinical symptoms. Ninety-two (30.5%) of the patients were followed-up with profound deficiency and 210 (69.5%) with partial deficiency. A total of 306 variants were detected. Twenty different variants (3 novel - 3 rare) and 31 different genotypes were detected. The 3 most frequently detected variants were c.410G>A (p.Arg137His; 47.3%), c.1270G>C (p.Asp424His; 29.7%), and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36; 15.3%). The 3 most frequently identified genotypes were c.410G>A (p.Arg137His) / c.1270G>C (p.Asp424His) compound heterozygous (32.4%), c.410G>A (p.Arg137His) homozygous (24.8%), and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) / c.1270G>C (p.Asp424His) compound heterozygous (12.2%). Patients with c.410G>A (p.Arg137His) homozygous variant, c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) homozygous variant and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) / c.410G>A (p.Arg137His) compound heterozygous variant were statistically significantly associated with profound deficiency. Compound heterozygosity of c.410G>A (p.Arg137His) / c.1270G>C (p.Asp424His) variants were significantly associated with partial deficiency. The association between the BTD genotype and biochemical phenotype is not always consistent. Our study provides valuable data by adding variants with genotype-phenotype correlations to the literature and three novel variants, which can provide significant guidance in clinical follow-up.

A retrospective study on biotinidase deficiency: analysis of the Eastern Anatolia region patient cohort

ABTRACT This study retrospectively reviews individuals diagnosed with biotinidase deficiency in Eastern Anatolia to analyze the genetic variants and their relationship with biotinidase activity levels. The research focuses on determining the percentage impact of different variants on enzyme activity. The study included 357 patients who presented to Erzurum City Hospital with symptoms of biotinidase deficiency between 2018 and 2023 and were referred to the medical genetics department. Biotinidase enzyme levels were determined using spectrophotometric and colorimetric techniques, while Sanger sequencing analyzed the four exons and intron boundaries of the BTD gene. In the analysis of 357 patients (181 boys, 176 girls), the most frequent variant was c.1270G > C | p.Asp424His. Biotinidase enzyme activity was above 30% in 97.3% of patients with a homozygous p.D424His mutation. The mutations that caused the most significant decrease in enzyme activity were c.410G > A p.Arg137His, c.38_delinsTCC p.Cys13phefs*36, and c.1535C > T p.Thr512Met. Hearing loss (4 patients) and optic atrophy (1 patient) were mainly observed in patients with the c.38_delinsTCC mutation (homozygous or heterozygous). Most patients were asymptomatic, and mild symptoms were effectively prevented with biotin treatment. This study provides a detailed analysis of genetic diversity and clinical presentation in biotinidase deficiency cases in Eastern Anatolia, demonstrating the efficacy of biotin treatment. It highlights the significant role of genetic variants in phenotypic diversity and the need for personalized treatment, calling for further genetic research to enhance understanding of variant diversity and its impact on enzyme activity.

Incidence of Inborn Errors of Metabolism and Endocrine Disorders Among 40965 Newborn Infants at Riyadh Second Health Cluster of the Ministry of Health Saudi Arabia.

Inborn errors of metabolism (IEM) and endocrine disorders are common genetic conditions in the Saudi population with the incidence rate often underestimated. Newborn screening (NBS) using various disease panels provides the first line in the early detection and intervention among infants with a high risk of IEM. Here we aim to assess the incidence of screening disorders and provide an overview of the NBS program at the Ministry of Health Tertiary Care King Fahad Medical City. Dried blood spots (DBS) from 40,965 newborn infants collected on the second day after birth were analyzed for 20 disorders. The total number of positive screen ("repeat") samples over 10 years was about 1% (n = 382/40,965). The true positive result rate was 15.3% (n = 46/301) with the recall rates of individual disorders ranging from 0.26% (95% CI, 0.17-0.69) to 2.6% (95% CI, 2.19-3.05). The false positive result rate was 84.7% (n = 255/301) with biotinidase activity found to be the most common cause of the second sample repeat. The overall incidence of the screened diseases was 1:891 (95% CI, 11.61-12.47). CH and CAH are the most prevalent among endocrine disorders with an incidence of 1:4097 (95% CI, 2.19-3.05), and PA and ASA among the IEM with an incidence of 1:10,241 (95% CI, 0.09-0.95). In summary, we provide updated data and our experience on the incidence of various IEM and endocrine disorders among the Saudi population, highlight the role of false positive results of biotinidase activity that can increase the recall rate and lead to overestimation of the incidence data, and recommend multicenter studies to achieve a successful national NBS program.

Biallelic loss-of-function variations in BTD cause profound biotinidase deficiency in an Indian patient.

Biotinidase deficiency (BD) is a rare, autosomal recessive metabolic disorder characterized by neurocutaneous symptoms. This study investigates a case of profound BD in an Indian infant and the underlying genetic basis. A 10-month-old male presenting with seizures, hypotonia, ataxia, visual impairments, and developmental delay underwent biochemical and genetic analysis. Biotinidase activity was measured using an ELISA kit. Sanger sequencing of the biotinidase (BTD) gene was performed to identify genetic variations. In silico analysis was employed to assess the potential impact of the identified variants. The infant biotinidase activity was undetectable and its suggest profound biotinidase deficiency. Novel biallelic loss-of-function variations (c.903G > A and c.946C > T) in the BTD gene were identified, leading to premature stop codons and truncated, non-functional protein fragments. This case expands our knowledge of BD genetic diversity and underscores the critical role of early diagnosis and newborn screening programs in managing this treatable condition.

Long-term personalized high-protein, high-fat diet in pediatric patients with glycogen storage disease type IIIa: Evaluation of myopathy, metabolic control, physical activity, growth, and dietary compliance.

Dietary lipid manipulation has recently been proposed for managing glycogen storage disease (GSD) type IIIa. This study aimed to evaluate the myopathic, cardiac, and metabolic status, physical activity, growth, and dietary compliance of a personalized diet high in protein and fat for 24 months. Of 31 patients with type IIIa GSD, 12 met the inclusion criteria. Of these, 10 patients (mean age 11.2 ± 7.4 years) completed the study. Patients were prescribed a personalized high-protein, high-fat diet, comprising 3.0-3.5 g/kg/day of protein and 3.0-4.5 g/kg/day of fat, constituting 18.5%-28% and 70.5%-75.7% of daily energy, respectively. Dietary compliance was ensured and assessed via the regular administration of questionnaires. Our results revealed consistent and significant decreases of 22%, 54%, and 30% in the creatinine kinase, creatine kinase-myocardial band, and lactate dehydrogenase levels, respectively. Echocardiography revealed improvements in the Z-scores of the left ventricular mass and interventricular septum thickness. A significant increase in body muscle mass was observed, and a higher score was achieved using the Daily Activity Questionnaire. Growth monitoring revealed an arrest in the height-SDS at the 6th and 12th months, followed by subsequent improvement at the end of the second year. A gradual and persistent decline in the periods of hypo- and hyperglycemia has been reported. Biotinidase activity decreased, whereas hepatosteatosis increased and then decreased by the end of the study. Implementing a high-protein, high-fat diet and monitoring key parameters in patients with type IIIa GSD can lead to myopathic and cardiac improvements and increased physical activity.

Diagnosis and Treatment of Newborns Referred to the Metabolism Department From the National Newborn Screening Program in Türkiye: A 5-Year Single-Center Experience

Objective: The aims of this study were to investigate biochemical and genetic tests and treatment plans of newborns referred to our center with inherited metabolic disorders screened in Türkiye National Newborn Screening Program (NNSP). Material and Methods: The medical records of babies referred by the NNSP between January 2019 and November 2023 were scanned retrospectively. Plasma biotinidase activity and the biotinidase gene (BTD) analysis results for suspected biotinidase deficiency (BD), the plasma phenylalanine and phenylalanine hydroxylase gene (PAH) analysis for a suspicion of phenylketonuria (PKU) were documented with treatment information. Results: A total of 143 babies, 78 (54.5%) with suspected BD and 65 (45.5%) with suspected PKU were included. A PAH gene analysis was performed on 23 (35.4%) of those had high plasma phenylalanine levels, among which 86.9% were identified with the biallelic variant. Five patients were started on sapropterin-diet combined therapy, three on diet therapy and one on sapropterin therapy. In the first serum biotinidase activity measurement of babies referred with suspected BD, a heterozygous deficiency was detected in 48.7%, partial deficiency in 39.7% and profound deficiency in 10.3%. A BTD gene analysis was performed on 79.5% of those with suspected BD, and biallelic variants were detected in 50%. Forty-six patients (59.0%) underwent biotin treatment. Conclusion: In our study, approximately one-third of the babies referred from NNSP over the five-year course of the study had biallelic variants of the relevant disease. Our research is one of the few studies on NNSP in our country and presents the diagnosis and treatment process of PKU and BD.

Evaluation of Adult Biotinidase Activity in Patients with Idiopathic Inflammatory Demyelinating Diseases

Evaluation of Adult Biotinidase Activity in Patients with Idiopathic Inflammatory Demyelinating Diseases

Can biotinidase activity be used as a new inflammatory marker in multiple sclerosis?

In this study, the relationship between known inflammatory markers and serum biotidinase activity in multiple sclerosis (MS) patients was evaluated. This study was conducted between June 2022 and December 2022 in a tertiary referral hospital. MS patients with active infection, end-stage disease, malignancy, and trauma or infection in the last month were excluded. Complete blood count was performed by automated hematology analyzer, C-reactive protein (CRP) was determined by turbidimetric method with serum sample, and serum biotidinase activity was studied by enzymatic spectrophotometric method. The relationship between neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), CRP, immature granulocyte (IG) percentage, and serum biotidinase activity were evaluated with Spearman correlation analysis. A total of 126 patients (mean age was 36.5±9.2 years) were included in our study, and 86 of them (68%) were female. Eighty-four patients (66%) had low biotidinase activity. The patients with low and normal biotidinase activity were compared and there was no significant difference between age, NLR, PLR, CRP levels, and incidence of optic neuritis and myelitis (p=0.509, p=0.548, p=0.883, p=0.911, p=0.403, p=0.892 respectively). By Spearman's correlation analysis, there was no significant correlation between biotidinase activity and CRP, IG percentage, NLR and PLR. A significant correlation was found between NLR with PLR and IG [(p<0.001, r=0.756); (p=0.007, r=0.245), respectively]. This study suggests that biotidinase activity is not an inflammatory enzyme. More comprehensive prospective studies are needed on this subject.

Alopecia, Dermatitis and Seizures since Infancy: A Case of Biotinidase Deficiency

Abstract Background: Biotinidase deficiency is a rare autosomal recessive metabolic disorder, having a wide spectrum of neurological, cutaneous, and immune-dysfunction-related clinical manifestations, which may be overlooked as common pediatric ailments. Clinical Description: A 2-year–7-month-old girl child, second born to third-degree consanguineous parents, presented with fast breathing and altered sensorium for a day, with a history of seizures and skin lesions since early infancy, developmental delay, and ataxia. Management and Outcome: Investigations revealed a normal septic screen, with metabolic acidosis which was unresponsive to therapy. After stabilizing the child with respiratory and vasopressor support, investigations for immunodeficiency were sent. Tandem mass spectrometry revealed increased C5OH levels, suggestive of holocarboxylase deficiency. Specific enzyme assay showed biotinidase activity deficiency. The child was initially empirically treated with metabolic cocktail comprising carnitine, biotin, thiamine, C0-enzyme Q10, and riboflavin, followed by specific therapy with biotin. The child showed dramatic clinical improvement and normalization of blood gas. Treatment has been advised lifelong with biotin supplementation. Conclusion: Careful observation by a pediatrician can identify the typical combination of clinical features in a child, suggestive of biotinidase deficiency. Evaluation and management of the condition are simple and rewarding.

Accurate determination of Biotinidase activity in serum by HPLC and its utilization as second tier test for the confirmation of initial positive newborn screening results

Diagnosis of Biotinidase deficiency (BTD) is extremely important to avoid several neurodevelopmental problems in early childhood. Colorimetric and fluorometric methods lack specificity and selectivity due to several interferences resulting in a high number of false positive results. We developed an HPLC method for BTD activity in serum with fluorescent detection. In colorimetric assays, biotinidase attacks the amide linkage of the artificial substrate biotinidyl-4-aminobenzoic acid (B-PABA) and releases p-aminobenzoic acid (PABA), which is converted to a purple dye by diazotization reaction. The newly developed method injects the reaction mixture directly into the HPLC column and quantifies using a six-point calibration curve without coupling and diazotization reaction. The method is linear over the 5–1000 μmol/L range. The detection and quantitation limits were 2.5 μmol/L and 5.0 μmol/L, respectively. When compared with colorimetric assay, the correlation coefficient (R2) was 0.9963. The within-assay and between-assay precision was <10.0% for four levels of quality control samples. No significant variation in BTD activity was detected due to hemolysis, icteric, and lipemic samples. The newly developed method eliminates the potential interference due to the presence of aromatic amines and significantly reduces the false positive results observed with the colorimetric method. It is simple, specific, sensitive, faster in sample preparation, and requires a small sample volume. The newly developed HPLC method was used in our laboratory as a secondary tier test for initial positive BTD samples from newborn screening programs. To our knowledge, no similar HPLC method has been reported to date.

Analysis of Biotinidase Activity in Serum by Digital Imaging Colorimetry Detection.

Biotinidase deficiency (BD) is an autosomal recessive inherited disorder of biotin recycling that leads to neurological and cutaneous consequences if left untreated. The clinical features of BD can be ameliorated or prevented by the administration of pharmacological doses of the vitamin biotin. Since it is a treatable disorder, BD is included in the newborn screening program in Türkiye as in many other countries. Therefore, monitoring of biotinidase enzyme activity (BEA) is of vital importance, especially for patients. The aim of this study was to develop a simple and reliable colorimetric method based on digital imaging for the analysis of BEA in serum samples. To determine the optimum distance and LED light source in the analyzer box that we fabricated in the laboratory, images of the solutions in a 96-well microplate were taken with a mobile phone camera, and each color space was examined. The most reliable relationship was between blank subtracted intensities of green channel and analyte concentrations, which was in the range of 35-400 ng/mL p-aminobenzoic acid (r2 = 0.999). The limit of detection and limit of quantification were 11 and 35 ng/mL, respectively. The proposed method was successfully applied to serum samples of 60 patients with BD and 60 healthy controls. We claim that the method can be easily performed for determination of BEA anywhere without needing expensive instruments.

Complex metabolic disharmony in PMM2-CDG paves the way to new therapeutic approaches.

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients.

West syndrome and multiple sclerosis association

Introduction. West syndrome is a rare and severe infantile epileptic encephalopathy, beginning around the age of six months, characterized by a classic electro-clinical triad. This is a pathology totally different from multiple sclerosis (MS) which is a demyelinating disease of the central nervous system caused, affecting young adults, especially females. The association of these two pathologies has never been described. Observation. We report here an exceptional presentation of MS in a 14-year-old girl with a history of West syndrome. She had normal development until the age of six months, when she began to have flexion spasms. The diagnosis of West syndrome was made with a normal MRI. The infantile spasms disappeared after treatment with vigabatrin and adrenocorticotropic hormone (ACTH). It had generally progressed to Lennox Gastaut encephalopathy, with delayed psychomotor development and epileptic sequelae. At 14, she presented with left hemiparesis within a few days. A cerebral MRI showed multiple nodular hyperintensities of the supra and infratentorial white matter, with the presence of an active lesion, fulfilling the diagnostic criteria for multiple sclerosis. CSF analysis was normal. Anti-AQP4, anti-MOG, anti-NMDA and anti-GABA (AB) antibodies were absent in the blood. Antibodies against HIV and viral hepatitis were. Biotinidase activity and autoimmunity tests were correct. The patient received high doses of methylprednisolone IV (1g/day) for three days with remarkable clinical improvement after 15 days. Discussion. MS is a complex and heterogeneous central nervous system (CNS) demyelinating disease. It is not uncommon for epilepsy to be the first symptom of multiple sclerosis. Seizures, on the other hand, are more common after disease progression. Although the disease is characterized by inflammatory lesions of the white matter, various neuropathological and radiological studies have shown that the disease also affects the grey matter. Several studies have shown that seizures are three to six times more common in MS patients than in the general population. Even though MS can start with epilepsy and a seizure may be the only symptom of a relapse of MS, it is still not known whether the two diseases coexist or whether MS predisposes to seizures. Conclusion. The association of these two totally different pathologies can lead us to say that the mechanism of multiple sclerosis may begin in childhood and that the clinical signs appear in adulthood.

Tetraparesis as an initial manifestation of biotinidase deficiency: a case report.

The authors present a case of 2.5-year-old boy complained of progressive weakness in all four limbs, with difficulties in breathing. Abdominal examination revealed hepatomegaly and splenomegaly. Also, her parents were first-degree cousins. Therefore, tandem mass spectroscopy and urine organic acid analysis were planned to exclude metabolic disorders. Urinary organic acid analysis revealed elevated levels of methylmalonic acid and 3-hydroxyisovaleric acid. Serum biotinidase activity was found to be 3.9nmol/min/ml. Oral biotin at a dose of 1mg/kg daily was initiated. A marked improvement of his neurological deficit was noted over a period of 15 days after treatment and cutaneous manifestations resolved within 3 weeks. Myelopathy due to BTD is a challenging diagnosis. Spinal cord impairment is a rare complication of this disease and is frequently unrecognized. BTD should be included in the differential diagnosis of children presenting with demyelinating spinal cord disease.

A Rare Cause of Paroxysmal Movement Disorder Associated with TBC1D24 Gene Mutation in Two Siblings.

A Rare Cause of Paroxysmal Movement Disorder Associated with TBC1D24 Gene Mutation in Two Siblings.

High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy.

Biotinidase deficiency (BD) is an autosomal recessive inherited disorder in which the enzyme biotinidase is totally or partially defective and the vitamin biotin is not recycled. BD meets the major criteria for a population screening program. Newborn bloodspot screening (NBS) allows early diagnosis of BD, thus preventing the high morbidity and mortality associated with untreated disease. Both profound and partial BD variant can be detected by NBS test, and serum enzyme activity and/or mutational analysis are required for definitive diagnosis. In Italy, BD is included in the screening panel for inborn errors of metabolism (IEMs) that has been declared mandatory in 2016. We analyzed the data of the first 3 years of the NBS for BD in our region (Abruzzo, Italy), with the aim to describe the outcomes of this recently introduced screening program. In over 26,393 newborns screened, we found 2 carriers and 16 cases with genotype associated with partial BD. Since the serum biotinidase assay has been recently introduced in our algorithm, only three of our newborns met the criteria of genetic and biochemical confirmation, with an incidence of 1:8797, which is in the high range of what has been reported in the literature. All affected infants carried the 1330G>C (D444H) variant in compound heterozygosis, with variants known to be associated with profound BD. A variant previously not described and likely pathogenic was found in one newborn. None of the infants had signs or symptoms. The study of the distribution of the enzyme activity in our population allowed us to validate the adopted cutoff with which the program has a positive predictive value of 18% and to analyze some preanalytical factors influencing biotinidase activity: A correlation of the enzyme activity with gestational age and time at specimen collection was found. Lower mean values of enzyme activity were found in infants born in the summer.

Establishing biotinidase reference interval: A foundation stone for newborn screening of biotinidase deficiency in Pakistan.

To determine the reference interval of biotinidase activity in healthy neonates. The cross-sectional study was conducted at the Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from May to November 2019, and comprised blood samples collected from healthy neonates aged 2-6 days. The samples were collected on filter paper and analysed on genetic screening processor based on dissociation-enhanced lanthanide flouroimmunoassay. Data was analysed using SPSS 21. Of the 120 dried blood spot specimens, 81(67.5%) were from male babies and 39(32.5%) from female babies. Reference interval for biotinidase activity, based on 2.5th and 97.5th percentiles, was from 3.0 to 11.0 nmol/ml/min. Screening of newborns for biotinidase deficiency is crucial to prevent irreversible neurological damage.

Evaluation of Adult Biotinidase Activity in Patients with Idiopathic Inflammatory Demyelinating Diseases (P1-1.Virtual)

Evaluation of Adult Biotinidase Activity in Patients with Idiopathic Inflammatory Demyelinating Diseases (P1-1.Virtual)

Molecular Background and Disease Prevalence of Biotinidase Deficiency in a Polish Population-Data Based on the National Newborn Screening Programme.

Biotinidase deficiency (BD) is a rare autosomal recessive metabolic disease. Previously the disease was identified only by clinical signs and symptoms, and since recently, it has been included in newborn screening programs (NBS) worldwide, though not commonly. In Europe, BD prevalence varies highly among different countries, e.g., from 1:7 116 in Turkey to 1:75 842 in Switzerland. This paper aimed to present the molecular spectrum of BD (profound and partial forms) in Polish patients diagnosed within the national NBS of 1,071,463 newborns. The initial suspicion of BD was based on an abnormal biotinidase activity result determined in a dry blood spot (DBS) by colorimetric and by fluorimetric methods while biochemical verification was determined by serum biotinidase activity (as quantitative analysis). The final diagnosis of BD was established by serum enzyme activity and the BTD gene direct sequencing. The obtained results allowed for the estimation of disease prevalence (1:66,966 births, while 1:178,577 for profound and 1:107,146 for partial forms), and gave novel data on the molecular etiology of BD.

Biotinidase activity is affected by both seasonal temperature and filter collection cards

This study set out to examine pre-analytical factors affecting the frequency of positive results in newborn screening for biotinidase deficiency. This investigation was prompted by an increase in the annual screen positive rate for biotinidase deficiency in Ontario from 2.65x10−4 in 2016 to 6.57x10−4 in 2017.Season and trend decomposition was used to separate seasonality from an underlying trend in the time series of biotindase activity measurements for the period 2014–01-12 to 2019–07-27 (n = 798,770). This analysis revealed a marked seasonal effect (winter = median + ⩽ 17 MRU, summer = mean - ⩽20 MRU) and a non-linear negative trend. Seasonal temperature was correlated with biotinidase results (Pearson’s r = 0.79) but not with the observed negative trend (Pearson’s r = 0.0025). Time series analysis of biotinidase results grouped by print lot of filter paper revealed that recently printed filter paper cards inhibit biotinidase and that this inhibition resolved over time. This study demonstrates that biotindase activity is inhibited by both increased seasonal temperature and collection on newly printed filter cards.