Diagnosing osteoarticular tuberculosis and detecting rifampicin resistance: A comparative analysis of Truenat MTB Plus vs GeneXpert Ultra.

SETTING: Diagnosing osteoarticular tuberculosis (OATB) and detecting drug resistance is a challenge in an endemic country like India. OBJECTIVE: Truenat MTB Plus assay (TruPlus), a chip-based portable machine, was compared with GeneXpert Ultra (GxUltra) for diagnosing drug-resistant OATB. DESIGN: 115 synovial fluid and pus specimens [22 culture-positive confirmed, 58 culture-negative clinically-suspected, 35 non-TB controls] processed between 2017 and 2023 were subjected to TruPlus, GxUltra and multiplex-PCR for diagnosing OATB. They were further screened for rifampicin resistance using TruRif chip. The performance was evaluated against composite reference standard, phenotypic drug susceptibility testing and rpoB gene sequencing. RESULTS: TruPlus, GxUltra and MPCR detected 77.5 %, 71.25 %, and 83.75 %, cases of OATB, respectively. TruPlus detected five additional cases missed by GxUltra. The performance of TruPlus was comparable to GxUltra (p = 0.074) and to MPCR (p = 0.074), while performance of GxUltra was significantly inferior to MPCR (p = 0.004). The overall agreement with reference standard was substantial for TruPlus and MPCR and moderate for GxUltra. Both TruRif and GxUltra reported 4 cases as rifampicin resistant. CONCLUSION: TruPlus along with TruRif offers better sensitivity than GxUltra. Its compact and portable platform allows wider application in peripheral settings, thus making it a pragmatic solution for diagnosing OATB and its drug resistance.

Fine-mapping host genetic variation underlying outcomes to Mycobacterium bovis infection in dairy cows.

BACKGROUND: Susceptibility to Mycobacterium bovis infection in cattle is governed in part by host genetics. However, cattle diagnosed as infected with M. bovis display varying signs of pathology. The variation in host response to infection could represent a continuum since time of exposure or distinct outcomes due to differing pathogen handling. The relationships between host genetics and variation in host response and pathological sequelae following M. bovis infection were explored by genotyping 1966 Holstein-Friesian dairy cows at 538,231 SNPs with three distinct phenotypes. These were: single intradermal cervical comparative tuberculin (SICCT) test positives with visible lesions (VLs), SICCT-positives with undetected visible lesions (NVLs) and matched controls SICCT-negative on multiple occasions. RESULTS: Regional heritability mapping identified three loci associated with the NVL phenotype on chromosomes 17, 22 and 23, distinct to the region on chromosome 13 associated with the VL phenotype. The region on chromosome 23 was at genome-wide significance and candidate genes overlapping the mapped window included members of the bovine leukocyte antigen class IIb region, a complex known for its role in immunity and disease resistance. Chromosome heritability analysis attributed variance to six and thirteen chromosomes for the VL and NVL phenotypes, respectively, and four of these chromosomes were found to explain a proportion of the phenotypic variation for both the VL and NVL phenotype. By grouping the M. bovis outcomes (VLs and NVLs) variance was attributed to nine chromosomes. When contrasting the two M. bovis infection outcomes (VLs vs NVLs) nine chromosomes were found to harbour heritable variation. Regardless of the case phenotype under investigation, chromosome heritability did not exceed 8% indicating that the genetic control of bTB resistance consists of variants of small to moderate effect situated across many chromosomes of the bovine genome. CONCLUSIONS: These findings suggest the host genetics of M. bovis infection outcomes is governed by distinct and overlapping genetic variants. Thus, variation in the pathology of M. bovis infected cattle may be partly genetically determined and indicative of different host responses or pathogen handling. There may be at least three distinct outcomes following M. bovis exposure in dairy cattle: resistance to infection, infection resulting in pathology or no detectable pathology.

[Association of vitamin D receptor gene polymorphisms with susceptibility to bone and joint tuberculosis in Chinese Han population].

OBJECTIVE: To investigate the association between vitamin D receptor (VDR) gene Apa I polymorphism and the susceptibility to bone and joint tuberculosis in Chinese Han population. METHODS: Between May, 2015 and June, 2016, 100 patients with bone and joint tuberculosis and 100 healthy volunteers were recruited concomitantly in Heyuan Hospital of Traditional Chinese Medicine. Vitamin D receptor gene Apa I polymorphisms in these subjects were analyzed using SNaPshot. RESULT: The genotype frequencies of Apa I-AA, Apa I-Aa and Apa I-aa were 51%, 41%, and 8% in the case group and 33%, 55%, and 12% in the control group, respectively, showing significant differences between the two groups (P<0.05). The genotype of Apa I-AA was significantly higher in the case group with an odds ratio (OR) of 2.073 (95% CI: 1.142-3.763). CONCLUSION: The Apa I polymorphisms of the VDR gene are associated with the susceptibility to bone and joint tuberculosis in Chinese Han population, and individuals with a Apa I-AA genotype are at greater risks to develop bone and joint tuberculosis.

Association of tumor necrosis factor-α gene polymorphism with osteoarticular tuberculosis prognosis in a Hebei population.

This study investigated the association of tumor necrosis factor-α (TNF-α)-308, -238, and -863 polymorphisms with osteoarticular tuberculosis (OA-TB) prognosis in a Hebei population. Genomic DNA was extracted from venous blood samples of 120 OA-TB patients and 100 healthy volunteers. TNF-α-308, -238, and -863 were analyzed by PCR-restriction fragment length polymorphism; genotype and allele frequencies were calculated. Serum TNF-α level was significantly higher in OA-TB patients (283.16 ± 51.68 ng/L) than in control (122.54 ± 54.65 ng/L; P < 0.05). Higher frequency of TNF-α-308 GG genotype in healthy volunteers (91.0%) than in OA-TB patients (79.2%) indicated that it was a protective factor against OA-TB (OR = 0.405, 95%CI = 0.147-0.657, P = 0.007). Higher frequencies of TNF-α-308 GA genotype and TNF-α-308 allele (A) in OA-TB patients (20.8 and 10.4%, respectively) than in healthy volunteers (8.0 and 5.0%, respectively) indicated an association with increased risk of OA-TB (OR = 3.112, 95%CI = 1.520-6.343, P = 0.003; OR = 3.109, 95%CI = 1.676-6.538, P = 0.006; respectively). Haplotype association analysis of TNF-α polymorphisms (-308/-238/-863) showed a higher frequency of TNF-α AGA in OA-TB patients (12.1%) than in healthy volunteers (3.5%), indicating that it was a risk factor for OA-TB (OR = 4.201, 95%CI = 1.80-9.91, P = 0.010). TNF-α-308 G/A and TNF-α AGA (-308/-238/-863) were associated with a predisposition to OA-TB, which could aid clinical detection, prevention, and prognosis of OA-TB.

The Early Mediaeval manorial estate of Gars/Thunau, Lower Austria: An enclave of endemic tuberculosis?

In recent decades, an increasing number of studies have aimed to shed light on the origin and spread of tuberculosis in past human populations. Here we present the results of a systematic palaeodemographic and palaeopathological survey of the Early Mediaeval population of Gars/Thunau (Lower Austria), which - at this stage - includes 373 individuals recovered at two archaeological sub-sites: a fortified settlement (including a necropolis) at the top of a hill - probably reserved for social and military elites; and a large riverine settlement at the foot of the hill, a so-called 'suburbium', where burials and an area of 'industrial' character were discovered. We recorded a great number of pathological alterations and a variety of 'classical' features of tuberculosis, such as vertebral destructions (Pott's disease) and joint destructions, and other pathological (unspecific) features probably linked with Mycobacterium tuberculosis infection (e.g. new bone formation at the inner surface of the ribs, endocranial alterations in the form of 'pits', and new bone formation at the cranial base). We hypothesize that the two contemporaneous (∼900-1000 AD) populations of Gars/Thunau differed not only in their social affiliation/condition, but also in the type and frequencies of their population-density-related infectious diseases (in particular tuberculosis). Moreover, we investigated the molecular genetic evidence of the causative organism in a few selected immatures exhibiting pathological changes at the inner wall of the cranium and discuss these findings in regard to the macroscopic features observed. Finally, we analysed carbon and nitrogen stable isotopes of both populations and strontium isotope ratios of the hill-top inhabitants in order to reconstruct certain aspects of diet and mobility to test our hypothesis concerning the specific social and/or military character of the site.

Tuberculosis in Late Neolithic-Early Copper Age human skeletal remains from Hungary.

Alsónyék-Bátaszék in Southern Hungary is one of the largest late Neolithic settlements and cemeteries excavated in Central Europe. In total, 2359 burials from the Late Neolithic - Early Copper Age Lengyel culture were found between 2006 and 2009 [1]. Anthropological investigations previously carried out on individuals from this site revealed an interesting paleopathological case of tuberculosis in the form of Pott's disease dated to the early 5(th) millennium BC. In this study, selected specimens from this osteoarcheological series were subjected to paleomicrobiological analysis to establish the presence of MTBC bacteria. As all individuals showing clear osteological signs of TB infection belonged to a single grave group, 38 individuals from this grave group were analysed. The sample included the case of Pott's disease as well as individuals both with and without osseous TB manifestations. The detection of TB DNA in the individual with Pott's disease provided further evidence for the occurrence of TB in Neolithic populations of Europe. Moreover, our molecular analysis indicated that several other individuals of the same grave group were also infected with TB, opening the possibility for further analyses of this unique Neolithic skeletal series.

Human tuberculosis predates domestication in ancient Syria.

The question of pre-neolithic tuberculosis is still open in paleopathological perspective. One of the major interests is to explore what type of infection could have existed around the early stage of animal domestication. Paleopathological lesions evoking skeletal TB were observed on five human skeletons coming from two PPNB sites in Syria, which belongs to the geographical cradle of agriculture. These sites represent respectively pre-domestication phase (Dja'de el Mughara, Northern Syria, 8800-8300 BCE cal.) and early domestication phase (Tell Aswad, Southern Syria, 8200-7600 BCE cal.). MicroCT scan analyses were performed on two specimens (one per site) and revealed microscopic changes in favor of TB infection. Detection of lipid biomarkers is positive for two specimens (one per site). Initial molecular analysis further indicates the presence of TB in one individual from Dja'de. Interestingly, no morphological evidence of TB was observed on animal remains of wild and newly domesticated species, discovered in these sites. These observations strongly suggest the presence of human tuberculosis before domestication and at its early stages.

Two positive tuberculosis cases in the late Nigrovits family, 18th century, Vác, Hungary.

Two mummies of the Hungarian mummy collection from Vác were the subjects of anthropological, paleopathological, radiological, paleomicrobiological, paleohistological and paleoproteomic studies. Both individuals belonged to the same family. The father, József Nigrovits (No 29), died at the age of 55 on the 11th of November 1793; his son, Antal Nigrovits (No 54), died on the 16th of July 1803, at the age of 22. They lived in the 18th century in Vác, a small town in northern Hungary. The macroscopic examination of the son showed a severely deformed neck and back region; the father has no visible mark of any illnesses. As earlier researches showed that tuberculosis was widespread in the community, the etiology of these deformities was examined. The paleomicrobiological results found that both individuals were infected with tuberculosis. Although they suffered from TB, the CT scan data of the bodies and their 3D reconstructions showed no skeletal evidence of tuberculosis. The deformity of the son turned to be a developmental abnormality of unknown origin, but no Pott's gibbus was present.

Lipid biomarkers provide evolutionary signposts for the oldest known cases of tuberculosis.

Studies on the evolution of tuberculosis, and the influence of this disease on human and animal development and interaction, require the accumulation of indisputable biomarker evidence. Ideally, the determination of full genomes would provide all the necessary information, but for very old specimens DNA preservation may be compromised and only limited DNA amplification may be a possibility. Mycobacterium tuberculosis is characterised by the presence of unusual cell envelope lipids, with specific biomarker potential. Lipid biomarker recognition has been decisive in pinpointing the oldest known cases of human and animal tuberculosis; the former are a woman and child from a pre-pottery settlement at Atlit-Yam, Israel (∼9,000 ka) and the latter is an extinct Bison antiquus from Natural Trap Cave, Wyoming (∼17,000 ka). Including some new data, it is demonstrated how analysis of a combination of mycolic, mycocerosic and mycolipenic acid and phthiocerol biomarkers provide incontrovertible evidence for tuberculosis in these landmark specimens.

Tuberculosis infection in a late-medieval Hungarian population.

The AD 16-17(th) century skeletal series from Bácsalmás-Óalmás (southern Hungary) has already been the subject of previous paleopathological studies concerning TB-related bone lesions. Due to recent development of macroscopic and molecular diagnostic methods in paleopathology and paleomicrobiology, a five-year international research program was recently started in order to re-evaluate the TB-related lesions in the complete series, comprising 481 skeletons. The skeletal material of these individuals was examined using macromorphological methods focusing on both classical/advanced stage skeletal TB alterations and atypical/early-stage TB lesions. Paleomicrobial analysis was used to study the presence of Mycobacterium tuberculosis complex (MTBC) DNA both in morphologically positive and negative cases. Samples were tested for the repetitive element IS6110 and further characterized by spoligotyping. In the whole series, 283 possible cases of TB infections were identified based on morphological alterations. Skeletal samples of eighteen individuals, morphologically positive as well as negative cases, were selected for further biomolecular examinations. Among them, seven individuals were PCR positive for the repetitive IS6110 sequence of the MTBC genome. Compared to the few cases of TB from the Bácsalmás-Óalmás series previously described, a much higher prevalence of MTBC infected skeletons was revealed in this study. The atypical/early stage skeletal lesions occurred significantly more frequently than the so-called classical alterations. Paleomicrobial analysis confirmed a prevalence of MTBC infection nearing 40% among the selected sample. Preliminary results also indicated better preservation of bacterial DNA in the compact layer of long bones and teeth, while spoligotyping suggested infection by different MTBC pathogens.

Tuberculosis origin: The Neolithic scenario.

This paper follows the dramatic changes in scientific research during the last 20 years regarding the relationship between the Mycobacterium tuberculosis complex and its hosts - bovids and/or humans. Once the M. tuberculosis and Mycobacterium bovis genomes were sequenced, it became obvious that the old story of M. bovis evolving into the human pathogen should be reversed, as M. tuberculosis is more ancestral than M. bovis. Nevertheless, the timescale and geographical origin remained an enigma. In the current study human and cattle bone samples were examined for evidence of tuberculosis from the site of Atlit-Yam in the Eastern Mediterranean, dating from 9250 to 8160 (calibrated) years ago. Strict precautions were used to prevent contamination in the DNA analysis, and independent centers used to confirm authenticity of findings. DNA from five M. tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. These, together with pathological changes detected in some of the bones, confirm the presence of the disease in the Levantine populations during the Pre-pottery Neolithic C period, more than 8000 years ago.

7000 year-old tuberculosis cases from Hungary - Osteological and biomolecular evidence.

This study derives from the macroscopic analysis of a Late Neolithic population from Hungary. Remains were recovered from a tell settlement at Hódmezovásárhely-Gorzsa from graves within the settlement as well as pits, ditches, houses and as stray finds. One of the most important discoveries from these remains was evidence of tuberculosis. Pathological analysis of the seventy-one individuals revealed numerous cases of infections and non-specific stress indicators on juveniles and adults, metabolic diseases on juveniles, and evidence of trauma and mechanical changes on adults. Several cases showed potential signs of tuberculosis and further analyses were undertaken, including biomolecular studies. The five individuals were all very young adults and included a striking case of Hypertrophic Pulmonary Osteopathy (HPO) with rib changes, one case with resorptive lesions on the vertebrae, two cases with hypervascularisation on the vertebrae and periosteal remodelling on the ribs, and one case with abnormal blood vessel impressions and a possible lesion on the endocranial surface of the skull. The initial macroscopic diagnosis of these five cases was confirmed by lipid biomarker analyses, and three of them were corroborated by DNA analysis. At present, these 7000-year-old individuals are among the oldest palaeopathological and palaeomicrobiological cases of tuberculosis worldwide.

Morphological and biomolecular evidence for tuberculosis in 8th century AD skeletons from Bélmegyer-Csömöki domb, Hungary.

Macromorphological analysis of skeletons, from 20 selected graves of the 8th century AD Bélmegyer-Csömöki domb, revealed 19 cases of possible skeletal tuberculosis. Biomolecular analyses provided general support for such diagnoses, including the individual without pathology, but the data did not show coherent consistency over the range of biomarkers examined. Amplification of ancient DNA fragments found evidence for the Mycobacterium tuberculosis complex DNA only in five graves. In contrast, varying degrees of lipid biomarker presence were recorded in all except two of the skeletons, though most lipid components appeared to be somewhat degraded. Mycobacterial mycolic acid biomarkers were absent in five cases, but the weak, possibly degraded profiles for the remainder were smaller and inconclusive for either tuberculosis or leprosy. The most positive lipid biomarker evidence for tuberculosis was provided by mycolipenic acid, with 13 clear cases, supported by five distinct possible cases. Combinations of mycocerosic acids were present in all but three graves, but in one case a tuberculosis-leprosy co-infection was indicated. In two specimens with pathology, no lipid biomarker evidence was recorded, but one of these specimens provided M. tuberculosis complex DNA fragments.

Relationship between human LTA4H polymorphisms and extra-pulmonary tuberculosis in an ethnic Han Chinese population in Eastern China.

Two single nucleotide polymorphisms in Leukotriene A4 hydrolase (LTA4H) gene were reported to be associated with protection from pulmonary tuberculosis in Vietnamese population. But these associations were not found in the Russians. To investigate the association of LTA4H polymorphisms with tuberculosis in a Han Chinese population in Eastern China, we genotyped 5 SNPs of LTA4H gene in 743 of pulmonary tuberculosis patients, 372 of extra-pulmonary tuberculosis patients and 888 of healthy controls individuals. The CC and TT homozygotes of rs1978331 and rs2540474 were identified to have higher rates (P < 0.01) and be risk factors in the patients with extra-pulmonary tuberculosis (OR = 1.412; 95% CI = 1.104-1.804 and(OR = 1.380; 95% CI = 1.080-1.764). However, no significant association was found between any of the SNPs and pulmonary tuberculosis. In the extra-pulmonary tuberculosis subgroups. LTA4H gene were significantly associated with tuberculous meningitis, lymph node tuberculosis, bone tuberculosis and other extra-pulmonary tuberculosis except for pleural tuberculosis. The present findings suggest that polymorphisms in the LTA4H gene may affect susceptibility to extra-pulmonary tuberculosis and change the risk of developing the disease in the Han nationality in the East China.

Multifocal Tuberculous osteomyelitis: possible inherited interferon gamma axis defect.

Multifocal Tuberculous (TB) osteomyelitis is an extremely rare entity in immunocompetent individuals. The authors report a 19-mo-old girl with multifocal TB osteomyelitis which resolved completely following institution of four drug antituberculous treatment for 1 y and detailed immunological evaluation was found to be normal but as the response of interferon gamma (IFN γ) in terms of production of IL12p40 was severely impaired, she is suggested to have a possible defect in the interferon gamma axis even though the exact defect in the pathway could not be delineated. This case report reiterates the need for detailed immunological evaluation in children presenting with multifocal TB osteomyelitis.

Human leukocyte antigen typing in tuberculous rheumatism: Poncet's disease.

SETTING: Tuberculous rheumatism (Poncet's disease) is a reactive polyarthropathy associated with extra-pulmonary and pulmonary tuberculosis (TB) without evidence of mycobacterial infection of the involved joints. As all patients with TB do not present with this peculiar clinical feature, a genetic susceptibility is suspected. OBJECTIVE: To determine the major histocompatibility complex (MHC) class I and II alleles in Mexican mestizo patients with Poncet's disease. DESIGN: In this case-control study of 16 Mexican mestizo patients diagnosed with Poncet's disease and 99 ethnically matched healthy controls, high resolution human leukocyte antigen (HLA) typing was performed for HLA-A, B, DR and DQ by polymerase chain reaction. HLA-DRB1 and HLA-DQB1 subtypes were performed by sequence-specific oligonucleotide probe hybridization. RESULTS: A significantly increased frequency of HLA-B27 (corrected P = 0.01) and DQB1*0301 (corrected P = 0.0009) alleles and decreased frequency of HLA-DQB1*0302 (corrected P = 0.00001) were identified in patients compared to healthy controls. CONCLUSION: These data suggest that genes located within the MHC may play a role in the susceptibility to Poncet's disease in patients diagnosed with TB.

Differential regulation of chemokine secretion in tuberculous and staphylococcal osteomyelitis.

Bone infection or osteomyelitis is characterized by uncontrolled inflammation and destructive bone loss although little is known about immunopathogenesis of infection. We investigated control of chemokine secretion from osteoblasts infected with either Mycobacterium tuberculosis, which normally elicits a granulomatous host response, or Staphylococcus aureus, which drives a host response dominated by neutrophil influx. We show that M. tuberculosis infection of cultured and primary osteoblasts induces extensive secretion of the chemokines interleukin (IL)-8, inducible protein (IP) 10, RANTES, and monocyte chemoattractant protein (MCP) 1 within 72 h (1630 +/- 280 pg/ml per 4 x 10(5) cells, 74,130 +/- 8480 pg/ml per 4 x 10(5) cells, 18,330 +/- 3040 pg/ml per 4 x 10(5) cells, and 138,670 +/- 13,340 pg/ml per 4 x 10(5) cells, respectively, for MG-63 osteoblasts). S. aureus infection also results in secretion of these chemokines but secretion is delayed and of lesser magnitude (210 +/- 10 pg/ml per 4 x 10(5) cells, 11,570 +/- 1240 pg/ml per 4 x 10(5) cells, 930 +/- 34 pg/ml per 4 x 10(5) cells, and 13,770 +/- 720 pg/ml per 4 x 10(5) cells for IL-8, IP-10, RANTES, and MCP-1, respectively). The minimal up-regulation of secretion of the neutrophil attractant IL-8 in staphylococcal infection is both striking and unexpected. In both infections, chemokine secretion was dependent on the presence of live organisms. Differences in kinetics and magnitude of chemokine secretion are associated with distinct patterns of mRNA expression, as assessed by ribonuclease protection assay (RPA) and reverse-transcription polymerase chain reaction (RT-PCR). In addition, nuclear localization of the transcription factor activator protein (AP) 1 in M. tuberculosis-infected osteoblasts also is distinct as compared with S. aureus-infected cells. In summary, this study shows that osteoblasts have an important pathogen-specific role in control of chemokine gene expression and secretion during the human immune response to osteomyelitis.

Use of polymerase chain reaction in diagnosis of occult tuberculosis of the fibula.

An unusual case of tuberculosis of the lower end of the fibula in a young patient is reported. The patient presented with symptoms of pain and swelling over the outer aspect of the right ankle with full range of painless ankle movements. The plain radiographs of the ankle were normal but MRI scan showed increased signals within the lower end of the fibula on T2-weighted images. The histology of the lesion showed only a few Langhans giant cells and culture failed to grow any organism. Polymerase chain reaction analysis of the biopsy specimen, however, showed growth of Mycobacterium tuberculosis. The patient responded to antitubercular treatment with complete resolution of symptoms. Polymerase chain reaction analysis should be considered in atypical presentations with bone pain to rule out an occult infectious pathology.