Background Mycoplasma genitalium is implicated in genitourinary disorders in both men and women as a sexually transmitted infection (STI). This study aimed to ascertain the prevalence of M. genitalium and identify associated risk factors among women. Aim To investigate the prevalence of M. genitalium and identify various risk-factors associated with M. genitalium infection in women attending the clinic in Western Cameroon. Methods A cross-sectional study was conducted in hospitals from five districts of Western Cameroon on sexually active and non-menstruating women attending for antenatal, prenuptial and contraception consultations,between January 2020 and July 2020. Endocervical swabs (n = 680) were collected, and M. genitalium was detected using real-time PCR targeting the MgPa and pdhD genes. Results A total of 680 women, characterised by a mean age of 27.4 ± 7.5 years, were included in this study. The overall prevalence of the M. genitalium infection was 5.2%. Bivariate analysis revealed that having more than one sexual partner was independently associated with three times higher odds of prevalent M. genitalium infection (OR 2.9, 95% CI: 1.03-8.56). Limitation Cross-sectional design limits exploring temporal relationships with other STIs. Freezing specimens for a year until PCR testing may have compromised detection rates of M. genitalium. Conclusion This study contributes valuable data to the limited understanding of M. genitalium epidemiology. The findings may aid in the formulation of national clinical standards for testing and screening strategies, emphasising the importance of addressing associated risk factors in the targeted population.

Abstract Background Leprosy is a chronic infectious disease, still remains a significant health problem in several parts of the world. A rapid and accurate diagnosis of the disease is essential for better management of Leprosy cases. In this study we compared three molecular methods i.e. PCR, Real TIME PCR and Loop-mediated isothermal amplification (LAMP) assay for detection of mycobacterium leprae. Methods A total of 114 samples were taken from 92 clinically leprosy suspected patients and contacts. DNA was extracted from the samples and molecular assays i.e. PCR, Real TIME PCR and LAMP was performed in parallel. Diagnostic accuracy of each test and Cohen's kappa coefficient was calculated using medcal online software. Results Among the 114 samples tested, the M leprae was detected in 35.09%(40/114), 34.2% (39/114) and 32.4%(37/114) by LAMP, conventional PCR, and real-time PCR, respectively. The overall agreement between conventional PCR and LAMP for detection of M leprae was 97.3% with a Cohen's kappa coefficient of 0.94, indicating perfect agreement. Similarly, the agreement between real-time PCR and LAMP for detection of M leprae was 97.3% with a Cohen's kappa coefficient of 0.94. However, the overall diagnostic accuracy of PCR, Real Time PCR and LAMP for detection of M leprae was 83.2%, 81.2% and 81.2%, respectively. Conclusion All molecular assays i.e. PCR, Real TIME PCR and LAMP had shown a similar diagnostic accuracy for detection of mycobacterium leprae. Disclosures All Authors: No reported disclosures

The World Health Organization (WHO) recommends Xpert® MTB/RIF (Xpert) and its advanced version, Xpert® MTB/RIF Ultra (Xpert Ultra), as first-line diagnostic tests for detecting pulmonary tuberculosis (PTB) and rifampicin resistance in children suspected of having the disease. Respiratory specimens (gastric lavage/bronchoalveolar lavage/sputum/endotracheal aspirate) obtained from 116 children with presumptive PTB were simultaneously processed using liquid medium culture, Xpert assay, and Xpert Ultra assay. Among the specimens from 116 children, six were excluded due to culture contamination (n = 5) or error in Xpert Ultra results (n = 1). Among the remaining 110 specimens, 20 were positive by liquid culture. The former and latter, of the two comparator tests gave a sensitivity of 90% and 95%, respectively. The respective specificity was 93.3% and 88.9%. Xpert Ultra showed a statistically significant slightly higher sensitivity than Xpert. Xpert Ultra showed slightly higher sensitivity than Xpert, with a minimal loss in specificity, partly due to the inclusion of trace results, which help detect paucibacillary cases.

Tuberculous pyomyositis is an uncommon form of extrapulmonary tuberculosis (EPTB). We report a rare case of EPTB in a 34-year-old male post-renal transplant recipient. He presented with pain in the left hip region and fever 9 years after the renal transplantation. A tender swelling was noted over the left hip region, visualized as a loculated hyperintense lesion on MRI. Ultrasound-guided aspiration revealed a purulent discharge which showed acid-fast bacilli on Ziehl-Neelsen staining. GeneXpert MTB confirmed the presence of Mycobacterium tuberculosis, sensitive to Rifampicin. The patient was diagnosed with tuberculous pyomyositis. Antitubercular treatment (ATT) was started and the abscess was drained.

Extracellular vesicles (EVs) have recently emerged as a source of microbe-specific biomarkers for disease diagnosis. In the present study, we evaluated the utility of pleural fluid-derived extracellular vesicles (pEVs) as a source of Mycobacterium tuberculosis (M. tb.) antigens for pleural TB (pTB) diagnosis. EVs were isolated from pleural fluid (PF) samples and were characterized by scanning electron microscopy, and immunoblotting by targeting CD63 and LAMP2 markers. Antigen-detection ELISAs were developed for 2 M. tb.-specific antigens, MPT51 and MPT64 in pEVs (pEV-ELISA) and direct PF samples (PF-ELISA), and were evaluated on n = 86 samples in a blinded manner. Cut-off values were calculated by ROC-curve analysis to achieve 90 % (95% CI:73.47-97.89) and 86.67 % (95%CI:69.28-96.24) specificity for MPT51 and MPT64 pEV-ELISA respectively. The sensitivity of pEV-ELISA was 71.43 % (95%CI; 29.04-96.33) for MPT51 antigen and 57.14 % (95%CI; 18.41-90.1) for MPT64 antigen in the 'Definite' pTB group, while in the 'Definite and Probable' pTB group, the sensitivity was 62.86 % (95%CI:44.92-78.53) for MPT51 and 65.71 % (95%CI:47.79-80.87) for MPT64. The performance of PF-ELISA was sub-optimal, with 28.57 % (95%CI:3.67-70.96) and 14.29 % (95%CI:0.36-57.87) sensitivity for MPT51 and MPT64 in 'Definite' pTB group respectively. We conclude that M. tb.-antigens are concentrated in the EV-fraction of PF samples and EVs can be utilized for antigen-detection assays for pTB diagnosis.

INTRODUCTION:Fetal hemoglobin (HbF) is known to cause method-specific interference in glycated hemoglobin A1c (HbA1c) measurement. Data on HbF interference, however, is currently either lacking for some platforms (eg, the Abbott Core Laboratory Alinity c and Beckman Coulter AU5800) or available only for HbF levels lower than the maximum claimed by the manufacturer (eg, Bio-Rad D-100). METHODS:We examined the effect of HbF interference on 7 HbA1c platforms using a series of spiked adult whole blood samples with increasing HbF levels (0% to 35%) and either low (approximately 5%) or high (approximately 9%) HbA1c levels, using isotope dilution mass spectrometry as a reference measurement procedure. RESULTS:For the high-performance liquid chromatography (HPLC)-based platforms (Bio-Rad VARIANT II TURBO 2.0 and D-100), relative deviation from expected values was not clinically significant, even at an HbF level of 35%. In contrast, immunoassay (AU5800; Roche Diagnostics cobas c311 and cobas b101; and Siemens Healthineers DCA Vantage) and enzymatic (Alinity c) methods showed clinically significant deviation at HbF levels above 10%. DISCUSSION:Our data suggest that the HPLC methods tested can be used for the majority of patients with elevated HbF. For patients with HbF levels above 10%, immunoassay and enzymatic methods appear to be unsuitable, and alternative HbA1c methods are still advised.

Objectives Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis are sexually transmitted pathogens that are highly prevalent in developing countries and are strongly associated with pregnancy complications. In Chad, screening for these sexually transmitted infections (STIs) in pregnant women is based solely on patient-reported symptoms, even though these infections are frequently asymptomatic. This study aims to determine the prevalence of M. genitalium, C. trachomatis, N. gonorrhoeae and T. vaginalis infections, as well as their associated risk factors.Methods In this cross-sectional study, we recruited pregnant women attending antenatal clinics at seven hospitals in N’Djamena. Endocervical swabs were collected, and DNA was extracted. Infections were diagnosed using PCR. Risk factors were identified using a structured questionnaire, and associations were assessed using logistic regression.Results A total of 525 pregnant women were enrolled, of whom 78.5% resided in urban areas, with a mean age of 25.16±5.54 years. Overall, 23.99% of the study population were diagnosed with at least one STI. The individual prevalence of M. genitalium, N. gonorrhoeae, C. trachomatis and T. vaginalis infections was 13.33%, 5.14%, 0.95% and 4.57%, respectively. Coinfections were low, with M. genitalium-T. vaginalis at 0.95%, M. genitalium-N. gonorrhoeae at 0.38% and other combinations at 0.19% each. Women residing in rural areas had nearly two times the odds of M. genitalium infection compared with urban residents (OR=1.98), indicating a higher risk. Age<25 years (OR=1.76), employment outside the home (OR=1.70), lack of STI awareness (OR=6.74) and no prior history of M. genitalium infection (OR=1.71) were also associated with significantly increased risk.Conclusions This study demonstrates a high prevalence of STIs among pregnant women in Chad, underscoring the need for systematic screening rather than solely relying on syndromic management.

Rationale: Prevalence of nontuberculous mycobacterial (NTM) disease is increasing globally. In high-TB-burden countries because of similarities with TB, NTM disease is usually not considered in the differential diagnosis and treated as TB. A prospective multicentre study was conducted at six sites in five states of India between 2021-2024 to determine prevalence of NTM disease and geographic variation of NTM species/subspecies among TB-suspects. Methods: The study was performed on GeneXpert MTB/RIF (Cepheid, USA) negative pulmonary and extrapulmonary specimens applying inclusion and exclusion criteria. All participating centres obtained ethical clearance. Written informed consent was obtained from all study patients. The confirmed NTM isolates (negative on SD-MPT64TB Ag immunochromatography kit, Standard Diagnostics, South Korea) were subjected to GenoType®-mycobacterium common mycobacteria/additional species and GenoType®-NTM-DR line probe assay (LPA) kits (Hain Lifescience, Germany) for NTM species/subspecies identification and were later validated by whole genome sequencing (WGS, Illumina-MiSeq sequencing using Nextra XT-DNA Library Prep protocol, USA). Demographic and other clinical details were carefully recorded. American Thoracic Society 2007 and 2020 guidelines were followed for NTM diagnosis. Results: NTM were isolated from 226 patients [NTM-pulmonary-disease (NTM-PD)=187; extrapulmonary-NTM (EPNTM)=39] among 71,143 TB-suspects with a prevalence of 0.32%. Of these 138(61%) were male. The median time to diagnose NTM was 119.7 days (IQR=57.1-132.1). The mean age of patients was 46.1 years (range=12-73). The mean body mass index (BMI) was 23.1 kg/m2(SD=3.1) and 39.8% (90/226) patients had BMI <18.5 kg/m2. Overall isolated NTM species included: M.avium complex (MAC) (n=58; M.intracellulare=38, M.avium=20), M.abscessus (n=56; M.abscessus subsp. abscessus=29, M.abscessus subsp. massiliense=25, M.abscessus subsp. bolletii=2), M.fortuitum (n=46), M. kansasii (n=45), M. simiae (n=11), M. gordonae (n=7) and one each of M.lentiflavum, M.senegalense and M.mucogenicum. LPA showed 97.6% (122/125) concordance with WGS results in NTM species/subspecies identification. One clarithromycin-resistant M.abscessus subsp. abscessus isolate [T28 sequevar of erm (41) gene] was identified by LPA and WGS. In NTM-PD, species isolated were MAC (27.8%;52/187), M.kansasii (24.1%;45/187), M.abscessus (21.4%; 40/187), M.fortuitum (16%;30/187), M.simiae (5.9%;11/187), M.gordonae (3.7%;7/187), M.mucogenicum (0.5%;1/187) and M.lentiflavum (0.5%;1/187). In EPNTM, M.abscessus (41%;16/39), M.fortuitum (41%;16/39), MAC (15.4%;6/39) and M.senegalense (2.6%;1/39) were commonest species. Following regional variations of NTM species were noted: MAC was predominantly isolated from north and south Indian states, M.kansasii, and M.abscessus from western and central states respectively. Conclusion: This is the first comprehensive Indian study describing prevalence and geographical variation of NTM species/subspecies among TB-suspects.LPA can be safely recommended for NTM diagnosis in high-TB-burden countries.

Chest wall tuberculosis is a very rare clinical entity that accounts for 1 -5% of musculoskeletal tuberculosis (TB). Here we present a case of chest wall TB. A 16 -year -old boy presented with complaints of progressive increasing swelling over the right hemithorax following a history of trauma. Radiologically it was a localized liquid collection with no connection to the thoracic cavity. Pus sample was sent to the mycobacteriology laboratory. The Gene Xpert result came as positive, and sensitive to rifampicin while the acid -fast bacilli (AFB) smear test was negative. Diagnosing a case of chest wall TB is always challenging for clinicians.

There are reports that the neglected zoonotic tropical disease brucellosis is reemerging today. Serological tests are being widely used in the diagnosis of brucellosis. In the present study, we performed a standard agglutination test (SAT) on 1348 suspected cases of brucellosis during the period of four years from April 2018 to March 2022. We noticed an increase in seropositivity from 2.6% in the year 2018-19 to 7.4% in the year 2021-22. We also noticed a spike in seropositivity in the years 2019-20 (12.5%). Our study shows the recent trend in seropositivity of the disease in Chandigarh and, hence, can be a meaningful addition to the existing serological diagnostic data related to brucellosis.

PURPOSE:The identification of anaerobes, Mycobacterium and Nocardia species, and moulds by MALDI-TOF-MS remains a challenge. This study aimed to evaluate the performance of MALDI-TOF in the identification of these organisms. METHODS:A total of 382 strains, comprising 128 (33.5 %) anaerobes, 126(33.0 %) mycobacterial, 113(29.6 %), mycelial fungi, and 15(3.9 %) Nocardia species were evaluated by VITEK MS Version 3.0. The results were compared with the identification of the isolates by DNA sequence analysis. The DNA sequences used for analysis were the 16S rRNA for anaerobic bacteria, hsp65 gene for mycobacteria, whereas both 16S rRNA and hsp65 gene for Nocardia species, and internal transcribed spacer (ITS) and 28S rRNA gene's D1/D2 regions of fungi. RESULTS:The VITEK-MS accurately identified 78.3 % (299/382) of the strains at the species, and 9.4 % (36/382) at the genus level. Misidentifications were observed in 3.9 % (15/382) isolates. Of isolates tested, 8.4 % (32/382) were not identified by the system, and 7.06 % (27/382) were not included in the IVD database. CONCLUSION:An upgraded VITEK MS V3.0 database provides reasonably accurate and rapid identification of clinically relevant anaerobes, mycobacteria, Nocardia species, and moulds to the species level.

Despite the advent of Xpert MTB/RIF, pleural tuberculosis (TB) diagnosis in pleural fluid is still difficult. Hence, we assessed the diagnostic efficacy of its advanced version, Xpert MTB/RIF Ultra, for pleural TB diagnosis using pleural fluid as a sample. Tuberculosis pleuritis (TBP) suspects ( n = 261) were enrolled in the study, of which 29 were excluded. The remaining patients ( n = 232) were categorized into definite TBP ( n = 31), probable TBP ( n = 28), and non-TB controls ( n = 173) based on the composite reference standard consisting of smear, culture, histopathology, and Xpert MTB/RIF as well as follow-up/clinical response to anti-TB therapy. Among the TBP suspects, 59 were diagnosed as TBP patients. The sensitivity of Xpert MTB/RIF Ultra (52.5%) using pleural fluid for TBP diagnosis was higher than sensitivity obtained with smear (22.4%), culture (17.6%), and Xpert MTB/RIF (25%) alone, carried out using either pleural fluid or pleural biopsy or both the samples. In cases of probable TBP, where none of the laboratory tests were positive, Xpert MTB/RIF Ultra use led to an increased diagnostic percentage of definite TBP from 52.5% to 69.4%. Overall, Xpert MTB/RIF Ultra showed promising results for a definitive diagnosis of TBP in pleural fluid samples.

INTRODUCTION:With the development of Xpert MTB/RIF (Xpert) and its latest version Xpert MTB/RIF Ultra (Ultra), diagnosis of tuberculosis has improved dramatically. Only few have compared the Ultra to Xpert, especially in varied geographic locations. The ability of Ultra to detect tuberculosis in pulmonary samples was thus compared in the current study to the Xpert for the quick identification of MTBC and resistance to rifampicin (RR) in sputum samples. METHODOLOGY:Adults and adolescents (>10 years) with suspicion of pulmonary tuberculosis were enrolled for the study. All the samples were subjected to smear microscopy, liquid culture and molecular methods Xpert MTB/RIF as well as Xpert Ultra in parallel. RESULTS:A total of 143 sputum samples were cultured in liquid media, with 52 of them proved to be culture positive. Using culture as the "reference standard," the Xpert Ultra detected MTBC in 45 samples, with sensitivity and specificity of 86.5% (74.2%-94.4%) and 89.9% (81.0%-95.5%), respectively. While the sensitivity and specificity, of Xpert was found to be 76.9% (63.2%-87.5%) and 93.7% (85.8%-97.9%) respectively. Xpert Ultra also detected 78.8% smear negative culture positive samples in comparison to Xpert MTB/RIF which detected 63.6% smear negative samples. CONCLUSION:Xpert Ultra can offer a valid TB diagnosis in a fraction of the time it takes to perform a culture and has higher sensitivity than Xpert MTB/RIF. This is especially true for paucibacillary samples with negative sputum smear.

An elderly patient who presented with 1 week of fever and respiratory symptoms was tested for dengue infection with an Abbott Bioline™ Dengue Duo immunochromatographic assay. Unexpectedly, the control line of the dengue non-structural protein 1 (NS1) component was absent, necessitating result invalidation. It remained absent when the test was repeated on the SD Biosensor Standard™ Q Dengue Duo, but present on the Wells Bio careUSTM Dengue Combo and Asan Easy Test® Dengue DUO assays, suggesting potential interference. Dilution, polyethylene glycol (PEG) precipitation and centrifugation with a 100 kDa filter were performed to reduce/remove the potential interferent. Sera from other patients that showed a control line, and a test line that was either positive or negative for NS1, were used as controls. Upon dilution with negative control serum, a faint control line emerged. PEG precipitation resulted in disappearance of control and test lines in the positive control. Filtration led to emergence of the control line for the patient's serum but caused the test line for the positive control serum to disappear. Overall, investigations suggested the presence of a high molecular weight (>100 kDa) substance which interferes with chicken IgY-anti-chicken IgY binding at the control line of affected assays. Our results highlight two important points: firstly, some commonly used laboratory procedures (e.g. PEG or filtration) may inadvertently remove the target biomarker (e.g. multimeric NS1) and should be interpreted with appropriate controls. Secondly, alternative kits that use a different antigen-antibody combination for the control line can be considered when similar patients are encountered in future.

Background: Neisseria gonorrhoeae is one of the most important causative organisms in causing sexually transmitted infections. The clinical presentation of gonorrhoea mimics the symptoms of other sexually transmitted infections, and a proper diagnosis of the same is therefore crucial in patient management. The current study intended to compare different in-house molecular methods: that is, conventional PCR, real-time PCR, and LAMP assay for detection of N. gonorrhoeae. Methods: A total of 163 samples were collected from 145 patients who presented with urethral and vaginal discharge. Collected samples were processed for culture on GC agar base, and three different molecular diagnostic tests (conventional PCR, real-time PCR, and LAMP assay) were performed simultaneously on all the samples. Results: Culture of N. gonorrhoeae was positive in 17 out of 21 (80.9%) swab samples. With culture as the gold standard method, conventional and real-time PCR had a sensitivity of 94.1%, whereas the sensitivity of the LAMP assay was found to be 88.2%. All three methods had a specificity of 100%. In addition to swab samples, evaluation of urine samples by different molecular methods yielded a good concordance with a kappa value of 0.85 by conventional PCR and real-time PCR showing a perfect level of agreement, while the LAMP assay was found to have a substantial level of agreement. Conclusion: LAMP assay had a comparable diagnostic accuracy to other molecular methods for the detection of N. gonorrhoeae and can be used as a point-of-care test in resource-limited settings.

The nucleic acid amplification tests (NAATs) such as Xpert MTB/RIF have transformed the TB diagnostic field by significantly increasing the case detection. However, newer improved diagnostic assays are still needed to meet the WHO targets to end TB. Present study is based on a novel approach of utilizing the in-vivo expressed specific mycobacterial transcriptomic biomarkers for the diagnosis of pulmonary tuberculosis (PTB). Total 61 subjects were recruited including smear positive (smear+; n = 15), smear negative (smear-; n = 30) PTB patients and disease controls (n = 16). Transcripts of three mycobacterial genes Rv0986, Rv0971c and Rv3121 were analyzed using real time PCR (qRT-PCR) in sputum samples. qRT-PCR with Rv0986, Rv0971c and Rv3121 identified smear + PTB patients with 100 %, 78.6 % and 86.7 % sensitivity respectively. In smear-PTB patients, both Rv0986 and Rv0971c based qRT-PCR resulted in 63 %, sensitivity whereas Rv3121 identified these patients with similar to 40 % sensitivity only. The sensitivity of the assay for smear-patients increased to 85 % when combinatorial analysis of qRT-PCR data for all the three genes was used. Thus, in-vivo expressed mycobacterial transcripts have promising potential as biomarkers for PTB diagnosis.