Laboratory Cross-contamination Research Articles

Mycobacterium tuberculosis pseudo-outbreak due to laboratory cross-contamination: A molecular epidemiology outbreak investigation.

Mycobacterial culture is routinely performed to diagnose tuberculosis (TB) in Canada. Globally, meta-analyses suggest that up to 2% of positive cultures are falsely positive for Mycobacterium tuberculosis due to laboratory cross-contamination. Five patients from distinct clinical institutions in Montréal were diagnosed with culture-positive TB as their clinical samples were processed in a centralized mycobacteria laboratory. Cross-contamination was suspected due to culture positivity in an organ donor with low TB pre-test probability. We describe a TB pseudo-outbreak due to laboratory cross-contamination and assess the role of conventional typing (i.e., mycobacterial interspersed repetitive unit variable number of tandem repeats [MIRU-VNTR]) and whole-genome sequencing (WGS) in supporting the investigation. Patients' epidemiological risk factors and clinical presentations were reviewed. The trajectories of pre- and per-analytic samples were retraced to identify potential cross-contamination events. Tuberculosis isolates were characterized by MIRU-VNTR and WGS using Oxford Nanopore Technology (ONT). The bioinformatic pipeline tbpore (v0.7.1) cluster was used for phylogenetic analyses. Two patients had previous exposure to endemic settings and clinical symptoms compatible with TB. Culture media inoculation overlapped in time for four patients, including one with suspected pulmonary cavitary disease and an organ donor whose organs had been transplanted in three different receivers. The MIRU-VNTR and WGS typing confirmed isolates from those four patients to be identical. Clinical, laboratory and molecular typing data, including results from ONT sequencing, were considered sufficiently robust to confirm laboratory cross-contamination and TB therapy was discontinued including in all organ transplant recipients.

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants.

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false-negative viral PCR test results. Such tests are also susceptible to false-positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses, and nonviral conditions (n = 318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to diagnose COVID-19. We find that optimal classifiers include an interferon-stimulated gene that is strongly induced in COVID-19 compared with nonviral conditions, such as IFI6, and a second immune-response gene that is more strongly induced in other viral infections, such as GBP5. The IFI6+GBP5 classifier achieves an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n = 553). We further provide proof-of-concept demonstration that the classifier can be implemented in a clinically relevant RT-qPCR assay. Finally, we show that its performance is robust across common SARS-CoV-2 variants and is unaffected by cross-contamination, demonstrating its utility for improved accuracy of COVID-19 diagnostics. IMPORTANCE In this work, we study upper respiratory tract gene expression to develop and validate a 2-gene host-based COVID-19 diagnostic classifier and then demonstrate its implementation in a clinically practical qPCR assay. We find that the host classifier has utility for mitigating false-negative results, for example due to SARS-CoV-2 variants harboring mutations at primer target sites, and for mitigating false-positive viral PCR results due to laboratory cross-contamination. Both types of error carry serious consequences of either unrecognized viral transmission or unnecessary isolation and contact tracing. This work is directly relevant to the ongoing COVID-19 pandemic given the continued emergence of viral variants and the continued challenges of false-positive PCR assays. It also suggests the feasibility of pan-respiratory virus host-based diagnostics that would have value in congregate settings, such as hospitals and nursing homes, where unrecognized respiratory viral transmission is of particular concern.

Cross-Contamination versus Outbreak: Pre-XDR Mycobacterial Strains Confirmed by Whole-Genome Sequencing.

Whole-genome sequencing (WGS) is promising for the quality control of laboratory facilities for Mycobacterium tuberculosis (MTB) strains. We describe the clinical and laboratory characteristics of false positive versus true positive MTB cultures based on WGS, which were experienced in a real clinical setting. Strain harvest and DNA extraction from seven isolates from pre-extensive drug-resistant (pre-XDR) TB patients transferred to the Korea University Ansan Hospital were performed, and epidemiologic links and clinical information, including the phenotypic drug susceptibility test (pDST), were investigated. WGS was performed using Ion GeneStudio with an ION530tm chip (average sequencing depth, ~100-fold). In the phylogenetic tree, identical and different strains were distributed separately. Five of the seven isolates were identical; the remaining two isolates differed from the others. The images of the referred pre-XDR-TB patients with false positive MTB that were analyzed were of regions close to old TB scars. Further, the results of WGS gene mutation analysis for ethambutol, streptomycin, and fluoroquinolone resistance in all six patients were not concordant with the pDST results. WGS and clinical information were useful in differentiating laboratory cross-contamination from true positive TB, thereby avoiding the unnecessary treatment of false positive patients and delay in treating true positive TB patients, with reliable genotypic drug resistance results.

Modelling and magnitude estimation of cross-contamination in the kitchen for quantitative microbiological risk assessment (QMRA).

In the kitchen of the consumer, two main transmission routes are relevant for quantitative microbiological risk assessment (QMRA): the cross‐contamination route, where a pathogen on a food product may evade heating by transmission via hands, kitchen utensils and other surfaces, e.g. to non‐contaminated products to be consumed raw; and the heating route, where pathogens remain on the food product and are for the most part inactivated through heating. This project was undertaken to model and estimate the magnitude of cross‐contamination in the domestic environment. Scientific information from the relevant literature was collected and analyzed, to define the cross‐contamination routes, to describe the variability sources and to extract and harmonise the transfer fractions to be included as model parameters. The model was used to estimate the relative impact of the cross‐contamination routes for different scenarios. In addition, the effectiveness of several interventions in reducing the risk of food‐borne diseases due to cross‐contamination was investigated. The outputs of the model showed that the cutting board route presents a higher impact compared to other routes and replacement of the kitchen utensils is more effective than other interventions investigated; the transfer to other surfaces and objects, which can house bacteria in the environment, is also described. Laboratory cross‐contamination trials have been performed to estimate bacterial transfer via cutting, from the external surface of the meat to the cutting surfaces and to the knife. The results, obtained from the laboratory trials, show magnitudes of and differences in the bacterial transfer fraction to the knife and the cutting surface in relation to which side of the meat is contaminated. Despite the complexity of factors which influence bacterial transfer, the combination of laboratory work with mathematical modelling enhanced scientific understanding and appreciation of the uncertainty of the estimates. QMRA methodology results in magnitude estimation of cross‐contamination in the kitchen and evaluation of intervention strategies.

Microbiological Assessment of Selected Laboratories at a Local University in Malaysia

Microbiological contaminations in the laboratories create not only diagnostic issues but also pose a major health risk to lab users. This study was conducted to determine the airborne microbial contamination in seven selected laboratories (KA, KP, PB, NA, BP, CR and MB) at a local public university in Malaysia and to assess the level of contamination on the contact surfaces of the studied laboratories. Two types of sampling methods were used in this study; passive air sampling and contact surface swab sampling. The total microbial counts were determined using Tryptone Soya agar (bacterial count) and Potato Dextrose agar (fungal count). Results showed that NA laboratory had the highest level of total microbial contamination (20.33 ± 3.35 CFU/cm3 ). Most laboratories had significantly higher (p < 0.05) air fungal contamination level compared to bacterial contaminations except for PB and NA laboratories. Significant differences were observed for airborne bacterial contamination readings between sampling time (during working hours vs after hours) for all laboratories except for BP and CR. Overall, bacterial contamination was the highest for incubator door handles’ samples from MB laboratory with an average reading of 93.00 ± 1.43 CFU/cm2 whereas the highest fungal contamination level was obtained from door knobs and work benches, both from CR laboratory with an average reading of 73.33 ± 6.67 CFU/cm2 and 73.33 ± 0.58 CFU/cm2 respectively. Findings from this study could assist in monitoring the efficacy of the existing laboratory management systems namely on the good laboratory practices including aseptic techniques, care for laboratory hygiene and cross-contamination control practices by the laboratory users to ensure that the microbiological contaminations in the laboratories are minimized.

Laboratory Cross-Contamination of Mycobacterium tuberculosis: A Systematic Review and Meta-analysis.

Microbiological cultures are the mainstay of the diagnosis of tuberculosis (TB). False-positive TB results lead to significant unnecessary therapeutic and economic burden and are frequently caused by laboratory cross-contamination. The aim of this meta-analysis was to quantify the prevalence of laboratory cross-contamination. Through a systematic review of five electronic databases, we identified studies reporting rates of laboratory cross-contamination, confirmed by molecular techniques in TB cultures. We evaluated the quality of the identified studies using the National Institute of Health (NIH) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and conducted a meta-analysis using standard methodology recommended by the Cochrane Collaboration. Based on 31 eligible studies evaluating 29,839TB cultures, we found that 2% (95% confidence intervals [CI] 1-2%) of all positive TB cultures represent false-positive results secondary to laboratory cross-contamination. More importantly, we evaluated the rate of laboratory cross-contamination in cases where a single-positive TB culture was available in addition to at least one negative TB culture, and we found a rate of 15% (95% CI 6-33%). Moreover, 9.2% (91/990) of all patients with a preliminary diagnosis of TB had false-positive results and received unnecessary and potentially harmful treatments. Our results highlight a remarkably high prevalence of false-positive TB results as a result of laboratory cross-contamination, especially in single-positive TB cultures, leading to the administration of unnecessary, harmful treatments. The need for the adoption of strict technical standards for mycobacterial cultures cannot be overstated.

A case of Mycobacterium tuberculosis laboratory cross-contamination

SettingA laboratory cross-contamination event was suspected because Mycobacterium tuberculosis was unexpectedly detected at a high incidence in the cultures of several clinical specimens at the National Hospital Organization, Tokyo National Hospital, Japan. ObjectiveTo describe a case of Mycobacterium tuberculosis laboratory cross-contamination. DesignWe reviewed the medical records of 20 patients whose clinical specimens were suspected to have been contaminated by Mycobacterium tuberculosis. Variable number of tandem repeat analysis with 15 loci, the Japan Anti-Tuberculosis Association-12, and three additional hyper-variable loci, was performed to identify the cross-contamination event. ResultsThe clinical, laboratory, and variable number of tandem repeat data revealed that the cross-contamination had possibly originated from one strongly positive specimen, resulting in false-positive results in 11 other specimens, including a case treated with anti-tuberculosis drugs. ConclusionClinical and laboratory data must be re-evaluated when cross-contamination is suspected and variable number of tandem repeat analysis should be used to confirm cross-contamination. Furthermore, original isolates should be stored appropriately, without sub-culturing and genotyping should be performed at the earliest possible for better utilization of variable number of tandem repeat for the identification of cross-contamination.

Whole genome sequencing used in an industrial context reveals a Salmonella laboratory cross-contamination

In 2013, during a routine laboratory analysis performed on food samples, one finished product from a European factory was tested positive for Salmonella Hadar. At the same period, one environmental isolate in the same laboratory was serotyped Salmonella Hadar. Prior to this event, the laboratory performed a proficiency testing involving a sample spiked with NCTC 9877 Salmonella Hadar. The concomitance of Salmonella Hadar detection led to the suspicion of a laboratory cross-contamination between the Salmonella Hadar isolate used in the laboratory proficiency testing and the Salmonella Hadar isolate found on the finished product by the same laboratory. Since the classical phenotypic serotyping method is able to attribute a serotype to Salmonella isolates with a common antigenic formula, but cannot differentiate strains of the same serotype within the subspecies, whole genome sequencing was used to test the laboratory cross-contamination hypothesis. Additionally, 12 Salmonella Hadar from public databases, available until the time of the event, were included in the whole genome sequencing analysis to better understand the genomic diversity of this serotype in Europe. The outcome of the analysis showed a maximum of ten single nucleotide polymorphisms (SNPs) between the isolates coming from the laboratory and the finished product, and thus confirmed the laboratory cross-contamination. These results combined with all additional investigations done at the factory, allowed to release finished product batches produced and thus circumvented unnecessary food waste and economic losses for the factory.

Detection of tuberculosis laboratory cross-contamination using whole-genome sequencing

ObjectiveDetection of tuberculosis laboratory cross-contamination using whole-genome sequencing. MethodsA total of 22 M. tuberculosis strains with high genotypic homology from one hospital were collected during the drug resistance surveillance. Genome sequencing and epidemiological investigation were conducted to determine the occurrence of cross-contamination. ResultsThe pair wise comparison between the genomes in each cluster indicated that 15 (71.4%) of 21 strains with available genomic data had no SNP differences with at least one other strain within the same cluster. The analysis of the specimen collection time found that, among the 16 strains collected on the same day, 14 (87.5%) of them had no SNP differences with one another strain; meanwhile, among the strains within the same cluster whose SNP distance was 0, 93.3% (14/15) of them had the same collection time, suggesting that these findings were most likely caused by cross contamination. ConclusionA high proportion of M. tuberculosis strains with genotypic homology from the single institute that shared the same process time period was most likely caused by the cross contamination. Whole genome sequencing analysis can help to determine the occurrence of cross contamination.

Identifying Mixed Mycobacterium tuberculosis Infection and Laboratory Cross-Contamination during Mycobacterial Sequencing Programs.

The detection of laboratory cross-contamination and mixed tuberculosis infections is an important goal of clinical mycobacteriology laboratories. The objective of this study was to develop a method to detect mixtures of different Mycobacterium tuberculosis lineages in laboratories performing mycobacterial next-generation sequencing (NGS). The setting was the Public Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive mycobacterial growth indicator tubes. We analyzed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, which were obtained during development and production use of a diagnostic process using NGS. The counts of the most common (major) variant and all other variants (nonmajor variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development. For each sample, we determined the nonmajor variant proportions at 55 sets of lineage-defining positions. The nonmajor variant proportion in the two most mixed lineage-defining sets (F2 metric) was compared with that of the 47 least-mixed lineage-defining sets (F47 metric). The following three patterns were observed: (i) not mixed by either metric; (ii) high F47 metric, suggesting mixtures of multiple lineages; and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevations relative to F47. Pattern ii was observed in batches, with similar patterns in the M. tuberculosis H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in the patterns were 97%, 2.8%, and 0.001%, respectively. The F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis genomes.

A case of laboratory cross-contamination of Mycobacterium tuberculosis identified using comparative genomics.

Two false-positive tuberculosis (TB) cases in Yamagata Prefecture, Japan, 2016. To report the effectiveness of comparative genomics of Mycobacterium tuberculosis for identification of cross-contamination cases. Case report of laboratory cross-contamination. Beginning with detection of an identical genotype in two M. tuberculosis strains using variable number of tandem repeat typing, we suspected M. tuberculosis cross-contamination of specimens collected in a mycobacteriology laboratory based on epidemiological investigations. This suspicion was confirmed using comparative genomics of the two M. tuberculosis strains and a strain from an epidemiologically unrelated specimen from the same batch as the two strains in the mycobacteriology laboratory. All strains had an identical genomic sequence with no single nucleotide variants. Comparative genomics, which offers the highest discrimination power, is a potent tool for identifying laboratory cross-contamination using epidemiological investigations.

A comparison of liquid and solid culture for determining relapse and durable cure in phase III TB trials for new regimens

BackgroundTuberculosis kills more people than any other infectious disease, and new regimens are essential. The primary endpoint for confirmatory phase III trials for new regimens is a composite outcome that includes bacteriological treatment failure and relapse. Culture methodology is critical to the primary trial outcome. Patients in clinical trials can have positive cultures after treatment ends that may not necessarily indicate relapse, which was ascribed previously to laboratory cross-contamination or breakdown of old lesions. Löwenstein-Jensen (LJ) medium was the previous standard in clinical trials, but almost all current and future trials will use the Mycobacteria Growth Indicator Tube (MGIT) system due to its simplicity and consistency of use, which will affect phase III trial results.LJ was used for the definition of the primary endpoint in the REMoxTB trial, but every culture was also inoculated in parallel into the MGIT system. The data from this trial, therefore, provide a unique opportunity to investigate and compare the incidence of false ‘isolated positives’ in liquid and solid media and their potential impact on the primary efficacy results.MethodsAll post-treatment positive cultures were reviewed in the REMoxTB clinical trial. Logistic regression models were used to model the incidence of isolated positive cultures on MGIT and LJ.ResultsA total of 12,209 sputum samples were available from 1652 patients; cultures were more often positive on MGIT than LJ. In 1322 patients with a favourable trial outcome, 126 (9.5%) had cultures that were positive in MGIT compared to 34 (2.6%) patients with positive cultures on LJ. Among patients with a favourable outcome, the incidence of isolated positives on MGIT differed by study laboratory (p < 0.0001) with 21.9% of these coming from one laboratory investigating only 4.9% of patients. No other baseline factors predicted isolated positives on MGIT after adjusting for laboratory. There was evidence of clustering of isolated positive cultures in some patients even after adjusting for laboratory, p < 0.0001. The incidence of isolated positives on MGIT did not differ by treatment arm (p = 0.845, unadjusted). Compared to negative MGIT cultures, positive MGIT cultures were more likely to be associated with higher grade TB symptoms reported within 7 days either side of sputum collection in patients with an unfavourable primary outcome (p < 0.0001) but not in patients with a favourable outcome (p = 0.481).ConclusionsLaboratory cross-contamination was a likely cause of isolated positive MGIT cultures which were clustered in some laboratories. Certain patients had repeated positive MGIT cultures that did not meet the definition of a relapse. This pattern was too common to be explained by cross-contamination only, suggesting that host factors were also responsible. We conclude that MGIT can replace LJ in phase III TB trials, but there are implications for the definition of the primary outcome and patient management in trials in such settings. Most importantly, the methodologies differ in the incidence of isolated positives and in their capacity for capturing non-tuberculosis mycobacteria. It emphasises the importance of effective medical monitoring after treatment ends and consideration of clinical signs and symptoms for determining treatment failure and relapse.

Use of M2e ELISAs for longitudinal surveillance of commercial poultry in Indonesia vaccinated against highly pathogenic avian influenza

In countries where highly pathogenic avian influenza virus (HPAIV) H5N1 is endemic and controlled by vaccination, post-vaccination serological monitoring is essential to differentiate vaccinated poultry from those that are infected. The objectives of this study were to validate two experimental ELISAs that detect antibodies raised against the M2e protein of avian influenza virus that can be used for DIVA purposes. Results from the sM2e and tM2e ELISAs were compared with other conventional tests for the detection of H5N1influenza virus (virus isolation and RT-PCR) using samples collected from 16 commercial flocks in Indonesia. These comprised vaccinated layers aged between 18 and 68 weeks old that were sampled at ten-weekly intervals. A small number of sera were positive in sM2e and tM2e ELISA, 14 (0.6%) and 17 (0.7%) respectively, with low OD420 (0.1–0.3), but only 4 sera were positive in both tests. At the flock level, the incidence of M2e positive sera was low (4%), well below previously established minimum of 40% for an HPAIV H5N1-infected flock. Conventional M and H5 gene RT-PCRs indicated that none of 16 flocks were infected at any time during the study. No virus was isolated from any of the 480 pooled swab samples, except from one, for which the combined data analysis suggest to be the result of a laboratory cross-contamination. Clinical disease, mortalities or reduction in production performance, indicative of field H5N1 challenge, were not observed either in any of the flocks. Birds from two surveyed flocks, challenged in the laboratory with an Indonesian HPAIV H5N1 developed M2e antibodies in 50% and 55% of surviving birds with OD420 in the range of 0.35–1.47 in tM2e ELISA, confirming the validity of the criteria established for use of M2e ELISA for DIVA purposes. Overall these results showed that the tM2e ELISA could be a useful monitoring tool to ascertain freedom from H5N1 infections in vaccinated commercial poultry.

Clonal Complexity in Mycobacterium tuberculosis Can Hamper Diagnostic Procedures

Clonal complexity is increasingly accepted in Mycobacterium tuberculosis infection, including mixed infections by ≥2 strains, which usually occur in settings with a high burden of tuberculosis and/or a high risk of overexposure to infected patients. Mixed infections can hamper diagnostic procedures; obtaining an accurate antibiogram is difficult when the susceptibility patterns of the strains differ. Here, we show how mixed infections can also prove challenging for other diagnostic procedures, even outside settings where mixed infections are traditionally expected. We show how an unnoticed mixed infection in an HIV-positive patient diagnosed in Madrid, Spain, with differences in the representativeness of the coinfecting strains in different sputum samples, markedly complicated the resolution of a laboratory cross-contamination false positivity alert.

Molecular epidemiology of tuberculosis in Victoria, Australia, reveals low level of transmission.

Victoria, Australia. To measure the level of Mycobacterium tuberculosis transmission in Victoria. Retrospective analysis of mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing profiles from all first M. tuberculosis complex (MTC) isolates obtained from patients residing in Victoria from 2003 to 2010 was performed. State TB reference laboratory records were matched with Department of Health notification records to obtain further laboratory, demographic, contact investigation, clinical and treatment data. These data were used to assign patients to one of four categories: 1) no epidemiological link, 2) possible link, 3) likely transmission event and 4) laboratory contamination. A total of 2377 MTC isolates were genotyped using 15-locus MIRU-VNTR. Of the 2298 M. tuberculosis isolates, 1029 (44.8%) had unique genotypic profiles and were considered epidemiologically unrelated, while 1269 (55.2%) isolates shared a profile with one or more other strains, defined as a genotypic cluster. Systematic investigation of all 268 genotypic clusters, including 24-locus MIRU-VNTR on selected isolates, led to a further 862 patients being classified as unrelated, bringing the total number of patients with no epidemiological links to 1891 (82.3%). Of the remaining patients, 294 (12.8%) were classified as having possible epidemiological links, 96 (4.2%) were classified as having known epidemiological links representing likely transmission events and 17 (0.7%) as the result of laboratory cross-contamination. There is considerable genotypic diversity among Victorian MTC isolates, and the level of transmission is low.

Laboratory cross-contamination of Mycobacterium tuberculosis in Northwest of Iran

IntroductionGold standard method for Mycobacterium tuberculosis identification is microbial culture, but this method can cause cross-contaminations. In the present study we aimed to investigate possible cross-contaminations in two main tuberculosis laboratories in Northwest of Iran. MethodsOne hundred and fifty-six isolates from two central tuberculosis laboratories were investigated by IS6110-RFLP and VNTR-ETR methods. Results53 isolates were clustered in 18 clusters. 15 isolates were smear negative and single culture positive among which four isolates were suspected to be contaminated and in two isolates (1.28%) contaminations were confirmed. ConclusionWith this confirmation clustered isolates reduced from 53 isolates to 49 isolates and recent transmission rate of tuberculosis reduced to 21.2%. This study showed genotyping smear negative and single culture positive isolates can prevent unnecessary treatments and for this purpose VNTR-ETR is a suitable method.

Rotary-based platform with disposable fluidic modules for automated isolation of nucleic acids.

We describe the development and evaluation of a rotary-based platform with multiple disposable fluidic modules for simultaneous automatic nucleic acid (NA) isolation from up to 24 biological samples. The procedure is performed inside insulated individual disposable modules, which minimizes both the risk of infection of personnel and laboratory cross-contamination. Each module is a segment of a circular cylinder containing a leak-proof inlet port for sample input, reservoirs with lyophilized chemicals and solvents, fluidic channels, stoppers, valves, a waste reservoir and an outlet port equipped with the standard micro test tube for NA collection. The entire platform, apart from the rotor that accommodates 24 modules, consists of functional elements that provide spinning of the rotor, reagent mixing, pressure delivery, and heating of reaction mixtures. The transfer of the reaction mixtures inside the modules is performed either with rotation of the rotor or with excessive air pressure applied to the module's reservoirs. The entire process takes less than 40 min, starting from the sample loading to the recovery of the purified NA, and it allows NA isolation both from bacterial cells and viral particles. The feasibility and reproducibility of the developed platform was demonstrated by the NA isolation from suspensions of Bacillus thuringiensis and Mycobacterium tuberculosis cells within a concentration range of 10(8) to 10(2) cells/ml. Isolation of NAs from blood plasma samples with varying concentration of hepatitis B and C viruses from 10(7) to 10(2) particles/ml were also successful. The purity and integrity of the extracted NAs were both reliable for performing quantitative PCR.

Molecular typing of Mycobacterium bovis isolates: a review.

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

Comparison of Ligation-Mediated PCR Methods in Differentiation ofMycobacterium tuberculosisStrains

Fast and inexpensive identification of epidemiological links between limited number of Mycobacterium tuberculosis strains is required to initially evaluate hospital outbreaks, laboratory crosscontaminations, and family or small community transmissions. The ligation-mediated PCR methods (LM-PCR) appear sufficiently discriminative and reproducible to be considered as a good candidate for such initial, epidemiological analysis. Here, we compared the discriminative power of the recently developed in our laboratory fast ligation amplification polymorphism (FLAP) method with fast ligation-mediated PCR (FLiP). Verification of the results was based on analyzing a set of reference strains and RFLP-IS6110 typing. The HGDI value was very similar for both LM-PCR methods and RFLP-IS6110 typing. However, only 52% of strains were correspondingly grouped by both FLiP and FLAP methods. Differentiation by FLAP method demonstrated a limited similarity to IS6110-RFLP (37,7%). As much as 78,7% of strains were grouped identically when differentiated by FLiP and IS6110-RFLP methods. The analysis differentiated 31, 35, and 36 groups when using FLAP, FLiP, and RFLP-IS6110 methods, respectively.

Recognizing Laboratory Cross-Contamination

Mycobacterium tuberculosis (MTB) isolation from clinical specimens is the standard for TB diagnosis. Positive MTB cultures are rarely questioned, but false-positive culture rates range from 2% to 4%. In December 2011, two smear-negative, culture-positive TB cases were reported to the Oklahoma State Department of Health (OSDH) in people without TB signs or symptoms. OSDH TB control officers interviewed physicians and laboratory personnel, reviewed patient charts, traced epidemiologic links, and performed microbiologic studies to determine if these were true TB cases. Both specimens were found to have been processed on the same day, at the same laboratory, under the same hood, and by the same technician sequentially after a strongly smear-positive TB specimen. No epidemiologic links were identified among the three patients. Spoligotyping and 24-locus mycobacterial interspersed repetitive unit typing of the three specimens were identical. Only liquid media grew MTB in the two questionable specimens. A laboratory splash incident was suspected, whereby all three liquid media sample lids were open during inoculation rather than being opened one at a time, causing cross-contamination. Also, the two specimens were incubated for 2-3 weeks longer than standard protocol before MTB growth was observed. Patient 1 was not treated for TB because her physician doubted the culture result. Patient 2, an organ transplant recipient, began four-drug anti-TB therapy, and an investigation was initiated for transplant-associated TB. Adherence to strict laboratory techniques and recognizing the possibility of false-positive MTB cultures, especially when inconsistent with clinical data, are essential in preventing erroneous TB diagnoses.