Risk factors for canine magnesium ammonium phosphate urolithiasis associated with bacterial infection

Background@#With limited information available, the association among urinary tract infections, urease-producing bacteria and the presence of magnesium ammonium phosphate (MAP) urolithiasis in canines in Thailand requires more study. @*Objectives@#This study aimed to investigate the association between demographic characteristics of canines and the presence of MAP urolithiasis in canines, and to evaluate antimicrobial susceptibility patterns of bacteria isolated from canine uroliths. @*Methods@#A total of 56 canines admitted for treatment with surgical removal of uroliths were recruited. Demographic characteristics and clinical chemistry data were recorded.Bacteria isolated from the removed uroliths were identified. Chemical compositions of the uroliths were analyzed by Fourier transform infrared spectrometer. Potential risk factors were determined with univariable and multivariable logistic regression analyses. @*Results@#Of 56 canine urolithiasis, bacteria were isolated from uroliths of 38 canines (27 MAP and 11 non-MAP) but not from uroliths of 18 canines (5 MAP and 13 non-MAP). The most common bacteria found in nidus of MAP uroliths was Staphylococcus pseudintermedius (approximately 51%). An antimicrobial resistance was frequently found in Staphylococci isolates (42.86%). Multivariate logistic regression analysis showed that the predictors of MAP urolith in canine urolithiasis were being female (p = 0.044; adjusted odds ratio [OR], 10.22; 95% confidence interval [CI], 1.06– 98.24) and the positive urolith culture (p = 0.012; adjusted OR, 8.60; 95% CI, 1.60–46.30). @*Conclusions@#Our results indicate that S. pseudintermedius (a urease-producing bacterium) is the major causative bacteria of MAP uroliths. A positive urolith culture and being female are risk factors of MAP urolithiasis in canines.

Thalassemia screening using OF/DCIP tests and turbidimetric assay

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Guideline for setecting left-over blood specimens for use as QC samples for thalassemia screening using OF/DCIP

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Implementation of quality control system for improvement of thalassemia screening at Maung Saung hospital, Roiet province

The prevention and control program of thalassemia and hemoglobinopathies has been carried out at Maung Saung Hospital, Roiet province according to the policy of the Ministry of Public Health. Discrepancy between the result of thalassemia screening and confirmatory test is commonly encountered. This study aimed to implement the quality control system at the hospital for improvement of thalassemia and hemoglobinopathies screening using a combined osmotic fragility test (KKU-OF) and dichlorophenolindophenol test (KKU-DCIP-Clear). Study plan was designed and made into 3 phases. In Phase I, a retrospective data in the year 2006 was collected and analyzed to identify to the screening problems. There were 5.4 % false positive and 5.4 % false negative for DCIP test but the effectiveness of OF test could not be determined as α - thalassemia 1 has not been examined. Phase II was an implementation of quality control system for thalassemia screening. Staffs performing screening tests were re-trained at the Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Khon Kaen University. Standard operating procedures (SOP) for KKU-OF and KKU-DCIP-Clear were then prepared. Phase III was set to evaluate the effectiveness of screening after implementation of the system. Prospective screening was carried out at Maung Saung hospital on 130 subjects and the remaining blood specimens were sent to the CMDL for further confirmatory tests. Among 130 subjects examined,α - thalassemia 1, β - thalassemia and Hb E were identified in 3.9 %, 0.8% and 40.0 %, respectively. The sensitivity, specificity, positive predictive and negative predictive values were found to be 100 %, 95.5 %, 95.0 % and 100 %, respectively. This result indicates that thalassemia and hemoglobinopathies screening at Maung Saung Hospital has been improved which should consequently lead to a more effective prevention and control program.

α/β Globin mRNA of β-thalassemia genes with premature termination

Mutation of β-globin gene causing translation-premature termination results in significant decrease of the mRNA abundance. This phenomenon arises from the degradation of mutant mRNA by nonsense codon-mediated mRNA decay (NMD). In this study, α/β-globin mRNA ratio was determined by semi-quantitative RT-PCR in β-thalassemia carriers with the β17, β41/42, β71/72 and β27 mutations and in a patient with compound heterozygous β17, β27 mutations. Values were compared with normal individual. The α/β-globin mRNA ratio of normal individual was found to be 0.98 whereas those of heterozygous for β17, β41/42, β71/72 and compound heterozygous β17/β27 were 0.89 1.66 1.60 and 3.09, respectively. Direct DNA sequencing of the cDNA demonstrated mutant mRNA only in the carrier with β17 mutation but not other mutations. This result was in concordance with the α/β-globin mRNA ratio observed. This data indicates that the α/β-globin mRNA ratio is dependent on the type and the location of premature termination mutation and related to some other factors involving NMD mechanism in cells.

Problem evaluation and efficiency improvement for thalassemia and hemoglobinopathies screening at community hospitals

To improve the efficiency of thalassemia screening, problems related to the screening procedures had been assessed in 3 community hospitals i.e. Thatako Hospital; Nakornsawan province, Lansaka Hospital; Nakhon Si thammarat province, and Borabue Hospital; Mahasarakham province. The investigation was done in 3 phases. Phase I aimed to identify the problems of routine thalassemia screening. From each hospital, blood samples screened for routine services were collected and sent to the Thalassemia Research Project, the Center for Research and Development in Medical Diagnostic Laboratory (CMDL) at the Khon Kaen University for confirmation. False positive and false negative rates were determined to assess the accuracy of the results. It was found that the range of false positive and false negative rates of the osmotic fragility test (OF test) for screening of α-thalassemia 1 and β-thalassemia was 7.9-29.6% and 0-100%, respectively. The DCIP precipitation test (DCIP-test) used for Hb E screening resulted in a range for false positive results of 1.4-4.0% and for false negative results of 3.6-50%. A combination of the OF/DCIP test revealed 10.5 to 29.6% false positive- and 3.5 to 42.9%, false negative results. In phase II the causes for the measuring errors were identified. These varied from hospital to hospital. Most of the causes were related to mistakes made by the laboratory personnels. A workshop on thalassemia screening was convened to improve the skills of the laboratory staff to perform screening tests correctly. Modified laboratory procedures were implemented in each hospital. Reassessment of the efficiency of thalassemia screening was conducted again by collecting blood samples screened for thalassemia from each hospital and sent to the CMDL for confirmation. The sensitivity and specificity of the combined tests increased considerably from 57.1-96.6% to 97.1-100% and 70.4-88.9% to 81.5-89.9% indicating that the efficiency of thalassemia screening had been improved. The results indicated that the proficiency testing system should be implemented at community hospitals to standardize and improve the quality of thalassemia and hemoglobinopathies screening within Thailand.

Screening for thalassemia and hemoglobin E in pregnant Laos women at the Mother and Child Health Hospital, Vientiane, Lao People’s democratic republic

In order to provide relevant data for implementation of a prevention and control program of thalassemia in Laos population, we have studied the screening for thalassemia and hemoglobin E in pregnant Laos women. Study was conducted on 307 pregnant women attending at the Mother and Child Health hospital, Vientiane, Lao peopleûs Democratic Republic. Initial screening was performed using a combined KKU osmotic fragility (KKU-OF) and KKU dichlorophenolindophenol (KKU-DCIP) tests. Subjects were divided according to the results of OF and DCIP screening tests into 4 groups including 154 (-/-), 58 (+/-), 22 (-/+) and 73 (+/+) individuals. All blood samples were further analyzed on the Hb-HPLC analyzer and by DNA analysis by PCR to identify thalassemia genes. Among 307 subjects examined, 39 cases (12.7 %) had α-thalassemia 1, 11 cases (3.6 %) were β-thalassemia and 93 cases (30.2 %) had Hb E. The effectiveness of screening for the three severe forms of thalassemia namely; α-thalassemia 1, β-thalassemia and Hb E was calculated. The sensitivity, specificity, positive and negative predictive values were 99.2 %, 85.5 %, 83.0 % and 99.4 %, respectively. This result indicates that thalassemia screening is possible and is an effective tool for prevention and control of thalassemia and hemoglobinopathies in Lao P.D.R. With this screening approach, some false positive results might be expected but false negative for the three important forms of thalassemia should be very rare.

The association of Gγ XmnI polymorphism with hemoglobin F levels in homozygous hemoglobin E

The Gγ -Xmn I polymorphism (-158 C → T) in 131 subjects with homozygous Hb E were investigated. The Xmn I (+/+), (+/-) and (-/-) patterns were identified in 77 (58.8 %), 45 (34.3 %) and 9 (6.9 %) cases, respectively. This result indicates that majority of βE globin genes are linked to the Xmn I + allele. Among 61 cases of Hb E homozygote (Hb \> 9 g/dl) who had (bE/bE, aa/aa) genotype, 37 (59.7 %), 20 (32.3 %) and 5 (8.0 %) cases had the Xmn I (+ / +), (+ / -) and (- / -) patterns, respectively. The median levels of Hb E and other Rbc parameters in these groups were not difference. However, the median levels of Hb F as determined by Hb-HPLC analysis, the MCHF (ng) and the Hb F concentration (mg/dl) were significantly higher in the Xmn I (+ / +) group (3.6 %, 731.0 ng and 374.0 mg/dl) as compared to those with the Xmn I (+ / -) (2.4 %, 456.9 ng and 264.4 mg/dl) and the Xmn I (- / -) (2.5 %, 518.0 ng and 245.0 mg/dl) at p-value \< 0.01 using Kruskal Wallis One-way Analysis of Variance and Mann-Whitney U-Test. No difference was found between the Xmn I (+ / -) and the Xmn I (- / -) groups. Although the Gγ -Xmn I (+ / +) polymorphism is associated with high Hb F production in Hb E homozygote, it is apparent that other factors requiring further investigation might also be involved.

Effective routine screening for a - thalassemia 1 carroer with the SEA deletion

In order to provide rapid method for identifying a - thalassemia 1 in massive screening, we have tested a simple screening strategy using simple blood test and real-time PCR.\  Study was done at our ongoing thalassemia screening at the Thalassemia Service Unit, Faculty of Associated Medical Sciences, Khon Kaen University. Initial screening was done for all samples using a modified one tube osmotic fragility test (OF test) and RBC indices obtained using standard blood cell counter. Those who had positive OF test results or MCV less than 80 fl were subjected to further PCR analysis for detection of a - thalassemia 1 (SEA deletion) by two PCR methods. In the first method, a - thalassemia 1 determinant was identified using a conventional GAP-PCR routinely run in our laboratory. In the second method, identification of a - thalassemia 1 (SEA) was carried out using a real-time PCR with SyBr green I and melt curve analysis. The SEA determinant generated specific melt curve with Tm of 86 + 1 ๐ C. Among 98 subjects who were positive at the initial screening, a - thalassemia 1 (SEA deletion) was detected in 22 (22.5 %) of them by both PCR methods. These included 14 a \– thalassemia 1 carriers, 3 patients with Hb H disease and 5 subjects with double heterozygote for a-thalassemia 1 and Hb E. No a - thalassemia 1 was detected in the remaining 76 cases. This data demonstrates that identification of a - thalassemia 1 in routine practice with large number of samples can be effectively done using a combination of OF test or MCV and a real-time PCR which should prove useful in a prevention and control program of thalassemia in area with high prevalence.

Proficiency testing in laboratory diagnosis of common thalassemia and hemoglobinopathies in Thailand

To improve the efficiency of laboratory diagnosis of common thalassemia and hemoglobinopathies in Thailand,the proficiency testing program was set up at the Centre for Research and Development of Medical DiagnosticLaboratories (CMDL), Khon Kaen University. The KKU-Hb controls were sent to laboratory members togetherwith essential hematological parameters. Each time, two control samples designated as husband and wife were sent.Upon receiving, all laboratory members analyzed control samples in their routine practices and interpreted the resultusing hematological data received and result of Hb analyses in their laboratories. The result of laboratory investigationsand the interpretations as well as the risks of having fetuses with 3 severe thalassemia diseases including homozygousα- thalassemia 1, homozygous β-thalassemia and β-thalassemia / Hb E disease were applied into the form providedand sent back to CMDL. Three cycles were investigated with 21, 23 and 66 participant laboratories, respectively.All control samples were received within appropriate times and conditions. It was found that more than 90 % ofparticipant laboratories could report acceptable levels of Hb A2 and Hb F and give accurate interpretation. Memberswere analyzed and grouped into 4 different quality groups;Excellent, Good, Fair and Need improvement.The proportions of members in the Excellent, Good, Fair and Need improvement groups were respectively found tobe (81.0, 9.5, 4.75 \& 4.75 %) in the first cycle and (69.6, 0, 21.7 \& 8.7 %) in the second cycle and (56.0, 18.2,24.3 \& 1.5 %) in the third cycle. It was found that the values of Hb A2 and Hb F were reported quite accuratelyfrom each laboratory member. However, when samples with complicated data were supplied, the increased inmis-interpretation and evaluation of relative risks were observed. This result indicates the requirement of furtherimprovement in the laboratory interpretation and knowledge related to laboratory diagnosis of thalassemiaand hemoglobinopathies of the participants. The evaluation system developed should prove useful in both developmentof external quality control program in laboratory diagnosis and further facilitate the prevention and control programof thalassemia and hemoglobinopathies in Thailand.