Enumeration of Mycobacterium leprae using real-time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.     

Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlandia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.     

Evaluation of qPCR-based assays for leprosy diagnosis directly in clinical specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5-10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations.     

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood of individuals, eight years after completion of anti-leprosy therapy.

Thirty eight patients with indeterminate leprosy (HI), at least 4 to 6 years after discharge from multibacillary (MB) or paucibacillary (PB) schemes of anti leprosy multidrug therapy (MDT), were submitted to traditional diagnostic procedures for leprosy and to polymerase chain reaction (PCR) analysis of different clinical samples for detection of Mycobacterium leprae DNA. No significant difference was observed for any of the parameters analyzed between PB or MB schemes of treatment and no indications were found for more efficient outcome of HI using the MB scheme. Remarkably, 18 (54.5%) of the individuals were PCR positive in at least one of the samples: positivity of PCR was highest in blood samples and four individuals were PCR positive in blood and some other sample. Upon comparison of PCR results with clinical and histopathological parameters, no correlation was found between PCR-positivity and eventual relapse. This is the first report on detection of M. leprae DNA in PB patients, more than half a decade after completion of MDT, suggesting that live bacilli are present and circulating much longer than expected, although reinfection of the individuals can not be excluded. Overall, we feel that because of the high sensitivity of the assay, extreme care should be taken about association of PCR results, efficacy of treatment and disease status.     

Polymerase chain reaction for the detection of Mycobacterium leprae

A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.     

Advances in the diagnosis and treatment of leprosy

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Over recent years, many important advances have been made in developing molecular diagnostics, in identifying highly effective drugs and designing multidrug regimens for treatment, and in unravelling the genomic structure and functions of the leprosy bacillus. Using the new information about specific sequences of M. leprae, several gene probes and gene amplification systems for confirming diagnosis and monitoring treatment have been developed. Among these, polymerase chain reaction (PCR)-based methods have been useful in confirming the diagnosis in paucibacillary leprosy (where few bacilli are present). RNA-targeting systems for monitoring the progress of treatment, in situ hybridisation techniques for analysing specimens with nonspecific histological features, and molecular methods for direct detection of rifampicin/dapsone resistance are other major technological advances with immense applied value. Several effective regimens for the treatment of leprosy have been developed, which include rifampicin, clofazimine and dapsone as core drugs. Although these regimens are generally satisfactory, limitations in terms of persisting activity and late reactions/relapses in paucibacillary leprosy, and persistence of dead and/or live organisms in multibacillary forms of the disease, have been observed.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.     

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.     

Development of a novel real-time PCR method for detection of HSV types 1 and 2 DNA using HybProbe chemistry

Herpes simplex viruses types 1 and 2 are members of the Alphaherpesviridae subfamily, as they can infect both skin and nerves and develop latent infection within the dorsal root and trigeminal ganglia. Infections with these viruses are common worldwide and cause wide range of clinical syndromes. Although HSV-1/2 infect healthy children and adults, disease is more severe and extensive in the immunocompromised individuals and/or during neuroinfections. The aim of the study was development of real-time PCR assay for detection and differentiation of herpes simplex viruses type 1 and 2. DNA in clinical samples, using specific dual-channel HybProbe chemistry. The nalytical sensitivity of assay was tested using serial dilutions of HSV-1 and HSV-2 DNA in range between 10 degrees and 10(-5). (4.35 x 10(5)-4.00 x 10(2) copies/ml and 4.18 x 10(5)-3.82 x 10(2) copies/ml, respectively). Thirty four cell line isolates and sixteen clinical samples taken from a group of adult patients with neurological signs were tested for the presence of HSV-1/2 DNA in the LightCycler instrument. Described in-house real-time PCR assay detected herpesviral DNA in all cell line isolates (31 of them were HSV-1 positive; 3 were HSV-2 positive) and in 10 clinical samples (positive only for HSV-1). The conclusion is that developed HybProbe-based real-time PCR test is very reliable and valuable tool for detection and differentiation of HSV-1/2 viremia in different kind of samples. The high level of sensitivity and accuracy provided by this assay is favorable for the quantification of herpes simplex virus 1 and 2 DNA in clinical specimens, especially during low-copy infections.     

人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。     

Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR

Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the host's stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.     

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.     

Immunogold labeling method for Mycobacterium leprae-specific phenolic glycolipid in glutaraldehyde-osmium-fixed and Araldite-embedded leprosy lesions

Phenolic glycolipid (PGL)-I, a Mycobacterium leprae-specific antigen currently used for serodiagnosis of preclinical leprosy, has thus far not been localized subcellularly in leprosy bacilli and their host cells. In this study, we developed an immunogold-labeling technique for qualitative identification of PGL-I sites in glutaraldehyde-osmium-fixed and Araldite-embedded M. leprae and host macrophages in human skin biopsies. Such "hard-fixed," plastic-embedded skin and nerve biopsies from patients with varying cell-mediated immunity to leprosy are amply available worldwide. Our method involves etching of plastic sections with H2O2, incubation with swine serum to eliminate nonspecific labeling, and long (22 hr) incubation at room temperature with monoclonal antibodies to PGL-I. Gold labeling was seen predominantly on cell walls of M. leprae, in vacuolar spaces of bacillated phagolysosomes, and occasionally on the cytoplasm and cell membrane of M. leprae. Host macrophage cytoplasm was labeled very infrequently. This technique allows studies on possibly persisting antigenic PGL-I in multibacillary leprosy patients during or after multidrug therapy. The method may also prove useful for subcellular localization of specific bacterial lipids in other mycobacterial diseases, including tuberculosis.     

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.     

Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR

Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.     

Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR

AIMS: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR. METHODS AND RESULTS: A linear range of quantification between 0.1-10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30-4500 to 1.9 x 10(6)-6.0 x 10(6) target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification. CONCLUSIONS: Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.     

Attachment of non-culturable toxigenic Vibrio cholerae O1 and non-O1 and Aeromonas spp. to the aquatic arthropod Gerris spinolae and plants in the River Ganga, Varanasi

Abstract DNA from Mycobacterium leprae , present in non-invasive clinical samples from leprosy patients, such as nasal secretion and hair bulbs, was submitted to amplification by the polymerase chain reaction using a M. leprae -specific repetitive sequence as a target. After optimization of sample processing and of the PCR conditions, we were able to detect DNA from M. leprae in both types of clinical samples, even from paucibacillary leprosy patients. The use of hair bulbs and nasal secretion as clinical samples for screening of household contacts and for the evaluation of a risk population, or for the follow-up of patients under chemotherapy, and monitoring of bacterial load is discussed.     

DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA)

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO, leprosy remains endemic in many countries with approximately 250,000 new cases each year. The entire M. leprae genome has been mapped and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. Variable number tandem repeat (VNTR) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) has been used to study leprosy evolution and transmission in several countries including China, Malawi, the Philippines, and Brazil. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.