Multiple Cross Displacement Amplification Combined With Real-Time Polymerase Chain Reaction Platform: A Rapid, Sensitive Method to Detect Mycobacterium tuberculosis

In this study, we evaluated the diagnostic accuracy of multiple cross displacement amplification (MCDA) combined with real-time PCR platform in pulmonary tuberculosis (PTB) patients. Total 228 PTB patients and 141 non-TB cases were enrolled. Based on the analysis of the first available sample of all participants, MCDA assay showed a higher overall sensitivity (64.0%), with a difference of more than 10% compared with Xpert MTB/RIF (Xpert) assay (51.8%, P < 0.05) and combined liquid and solid culture (47.8%, P < 0.001) for PTB diagnosis. In particular, MCDA assay detected 31 probable TB patients, which notably increased the percentage of confirmed TB from 57.9% (132/228) to 71.5% (163/228). The specificities of microscopy, culture, Xpert and MCDA assay were 100% (141/141), 100% (141/141), 100% (141/141), and 98.6% (139/141), respectively. Among the patients with multiple samples, per patient sensitivity of MCDA assay was 60.5% (52/86) when only the first available sputum sample was taken into account, and the sensitivity increased to 75.6% (65/86) when all samples tested by MCDA assay were included into the analysis. Therefore, MCDA assay established in this study is rapid, accurate and affordable, which has the potential in assisting the accurate and rapid diagnosis of PTB and speed up initiation of TB treatment in settings equipped with real-time PCR platform.

Cytology Reporting System for Lung Cancer from the Japan Lung Cancer Society and the Japanese Society of Clinical Cytology: An Extensive Study Containing More Benign Lesions

<b><i>Introduction:</i></b> The Japan Lung Cancer Society (JLCS) and the Japanese Society of Clinical Cytology (JSCC) have proposed a new four-tiered cytology reporting system for lung carcinoma (JLCS-JSCC system). Prior to the proposal, the Papanicolaou Society of Cytopathology (PSC) had proposed a revised reporting system (PSC system), which comprises the “neoplastic, benign neoplasm, and low-grade carcinoma” category (N-B-LG category), in addition to the 4 categories of the JLCS-JSCC system. This study aimed to evaluate the interobserver agreement of the JLCS-JSCC system with an additional dataset with more benign lesions in comparison with the PSC system. <b><i>Methods:</i></b> We analyzed 167 cytological samples, which included 17 benign lesions, obtained from the respiratory system. Seven observers classified these cases into each category by reviewing one Papanicolaou-stained slide per case according to the JLCS-JSCC system and PSC system. <b><i>Results:</i></b> The interobserver agreement was moderate in the JLCS-JSCC (<i>k</i> = 0.499) and PSC (<i>k</i> = 0.485) systems. Of the 167 samples, 17 samples were benign lesions: 7 pulmonary hamartomas, 5 sclerosing pneumocytomas, 2 squamous papillomas, one solitary fibrous tumor, one meningioma, and one lymphocytic proliferation. There were diverse sample types as follows: 11 touch smears, 3 brushing smears, 2 aspirations, and one sputum sample. Fourteen samples (82.3%) were categorized into “negative” or “atypical” by more than half of the observers in the JLCS-JSCC system. Conversely, 3 samples were categorized as “suspicious” or “malignant” by more than half of the observers in the JLCS-JSCC system. On the other hand, 11 samples (64.7%) were categorized into the N-B-LG category by more than half of the observers in the PSC system. <b><i>Conclusions:</i></b> The concordance rate in the JLCS-JSCC system was slightly higher than that in the PSC system; however, the interobserver agreement was moderate in both the JLCS-JSCC and PSC systems. These results indicate that both the JLCS-JSCC and PSC systems are clinically useful. Therefore, both systems are expected to have clinical applications. It may be important to integrate the 2 systems and construct a universal system that can be used more widely in clinical practice.

Tuberculosis infection among youths in overcrowded university hostels in Kenya: a cross-sectional study

Background Tuberculosis (TB) remains a top global health problem and its transmission rate among contacts is higher when they are cohabiting with a person who is sputum smear-positive. Our study aimed to describe the prevalence of TB among student contacts in the university and determine factors associated with TB transmission. Methods We performed a cross-sectional study with an active contact case finding approach among students receiving treatment at Kilifi County Hospital from January 2016 to December 2017. The study was conducted in a public university in Kilifi County, a rural area within the resource-limited context of Kenya. The study population included students attending the university and identified as sharing accommodation or off-campus hostels, or a close social contact to an index case. The index case was defined as a fellow university student diagnosed with TB at the Kilifi County Hospital during the study period. Contacts were traced and tested for TB using GeneXpert. Results Among the 57 eligible index students identified, 51 (89%) agreed to participate. A total of 156 student contacts were recruited, screened and provided a sputum sample. The prevalence of TB (GeneXpert test positive/clinical diagnosis) among all contacts was 8.3% (95% CI 4.5–14%). Among the 8.3% testing positive 3.2% (95% CI 1.0–7.3%) were positive for GeneXpert only. Sharing a bed with an index case was the only factor significantly associated with TB infection. No other demographic or clinical factor was associated with TB infection. Conclusion Our study identified a high level of TB transmission among university students who had contact with the index cases. The study justifies further research to explore the genetic sequence and magnitude of TB transmission among students in overcrowded university in resource limited contexts.

Efficacy of GeneXpert in Diagnosing Smear Negative Pulmonary Tuberculosis and Comparison to Culture

Objectives: Aim of present study is to observe the efficiency of GeneXpert MTB/RIF Assay in comparison to MTB culture on Lowenstein Jensen media in diagnosis of smear negative pulmonary tuberculosis cases. Methods: This descriptive study was carried out in The University of Lahore in collaboration with King Edward Medical University/Mayo Hospital Lahore 11th September, 2020 to 10th April 2021. Smear negative for acid fast bacilli patients enrolled for anti TB treatment were the target population. After taking informed consent, patients were asked to submit first morning sputum sample for culture on Lowenstein Jensen Medium and GeneXpert. Results: A total of 345 smear negative TB patients were diagnosed clinically and/or on the basis of radiological findings with mean age of 38.28±17.93,consisting of 47.5% male and 52.5% females recruited in this study. History of TB contact was present among 41.4% patients whereas history of smoking and diabetes remained to be 27.2% and 17.4% respectively. Culture showed significantly higher rate (35.1%) (p-value <0.05) of detection of MTB as compared to GeneXpert (21.5%). A sensitivity of 56.01% was calculated for GeneXpert whereas for culture on LJ medium it was 60.63%. Conclusion: Sensitivity of GeneXpert MTB/RIF Assay is a bit low in diagnosing the SNPT patients as compared to the culture but still registers itself as a handsome tool in terms of promptness and definite detection of MTB complex. Further provision of rifampicin susceptibility is bonus in same time.

Pneumocystis Jirovecii Infection in Autologous Hematopoietic Stem Cell Transplant Recipients

Background Infections remain a common cause of morbidity and mortality following hematopoietic stem cell transplant (HSCT). Pneumocystis jiroveci pneumonia (PJP) is an opportunistic infection that can occur in HSCT patients, although the association is best demonstrated in allogeneic HSCT, occurring less commonly in autologous HSCT. However, reports on PJP incidence, timing of infection, and outcomes among autologous HSCT cohorts are limited. Furthermore, while current guidelines recommend 3-6 months of prophylaxis against PJP following autologous HSCT, the optimal duration and even necessity of prophylaxis is not well established. Patients and Methods We performed a retrospective analysis of all consecutive patients who had autologous HSCT at the University of Michigan Blood and Marrow Transplantation program over a 20-year period from 1/1/2000 through 12/31/2019. The cohort consisted of a total of 2082 patients, 1221 male (58.6%), with median age 56 (range 10 months - 77 years, 91.2% ≥ 18 years). Records were searched for use of PJP prophylaxis over 6-month and 2-year follow-up periods post-HCT to determine rates of prophylaxis and choice of agent. Cases of polymerase-chain reaction (PCR)-confirmed PJP occurring within two years of HSCT were identified. The timing, clinical and laboratory features at diagnosis, use of concurrent immunosuppression, treatment, and outcomes were determined. Results Of the 2082 patients undergoing autologous HCT, 704 patients (33.8%) received PJP prophylaxis in the first 6 months following transplant. Prophylaxis rates varied over time, ranging from 14.6% to 80.0% when calculated by year of transplant (Figure 1). Trimethoprim-sulfamethoxazole (TMP-SMX) was the most used prophylaxis agent (70.3%), followed by inhaled pentamidine (31.8%), with intravenous pentamidine (8.1%), dapsone (7.1%), and atovaquone (2.6%) being used less frequently. There were 9 cases of PJP identified in our cohort, with an incidence of 0.43%. There were 6 males, with median age of 50 (range 34 - 69). Cases occurred a median of 126 days following HSCT (range 65 - 496), with 4 cases occurring after 6 months. None of the patients were on PJP prophylaxis at the time of diagnosis, and only 2 patients had received prophylaxis at any point after transplant. In 8 of 9 cases, patients were receiving concurrent pharmacologic immunosuppression in the form of steroids or maintenance brentuximab (Table 1). All patients presented with symptoms compatible with PJP, most often with fevers, dyspnea, and cough. Diagnosis was made by PCR from bronchoalveolar lavage specimen in 8 cases, and from sputum sample in 1 case. All patients were lymphopenic at the time of diagnosis, with median absolute lymphocyte count of 400 cells/µL (range 200 - 1100). Patients were most often treated with TMP-SMX. Three patients required transfer to the intensive care unit and 2 were intubated. Ultimately, 2 patients died from PJP infection; the remaining 7 recovered (Table 2). Conclusion Our analysis reveals that among a large cohort, incidence of PJP following autologous HSCT is low. This was the case even with relatively modest rates of PJP prophylaxis in the first 6 months following transplant. Most cases of PJP occurred in patients receiving additional immunosuppression and often occurred late following transplant. Figure 1 Figure 1. Disclosures Pianko: Karyopharm: Honoraria.

Effectiveness of expiratory technique and induced sputum in obtaining good quality sputum from patients acutely hospitalized with suspected lower respiratory tract infection: a statistical analysis plan for a randomized controlled trial

Background Targeted antimicrobial treatment is essential to avoid unnecessary use of broad-spectrum antibiotics and antimicrobial resistance. Targeted treatment relies on a precise microbiological diagnosis — in pneumonia, this poses a challenge as the usefulness of Gram stains and cultures is highly dependent on the quality of the sputum sample. This study aims to examine adverse effects and quality of sputum samples obtained by expiratory techniques (forced expiratory technique and sputum induction) compared with tracheal suction. The hypothesis is that expiratory techniques are non-inferior to tracheal suction in obtaining samples from the lower respiratory tract. This statistical analysis plan (SAP) describes the study design, method, and data analysis of the trial to increase transparency, avoid reporting bias or data-driven analysis and increase the study’s reproducibility. Method The design is a pragmatic, non-inferiority, parallel-arm randomized controlled trial including 280 patients admitted with suspected lower respiratory infection to two emergency departments. Patients are randomized to a usual care group, where sputum samples are collected by tracheal suction or to an intervention group where sputum samples are collected by forced expiratory technique and sputum induction. The statistical analysis will follow an intention-to-treat protocol. This SAP is developed and submitted before the end of recruitment, database closure, and statistical analyses. Discussion The results of this study will provide valuable knowledge to clinical practice by comparing adverse effects and sputum sample quality associated with different sample methods. Trial registration Clinicaltrials.gov, NCT04595526. Submitted on October 19, 2020

Pyemotes Species Isolated from Sputum Sample of COVID-19 patient- A Case Report

COVID-19, which emerged as a new and unknown disease in December of 2019, has transformed into a full-blown pandemic. It has affected all spheres of life, however, has claimed more lives of immune-compromised. Many opportunistic infections are being reported. We present a case of pyemotes species in sputum sample of a COVID-19 patient

A Rare Case of Primary Pulmonary Amoebiasis without Gastrointestinal Involvement: A Case Report

Introduction: The main pathogen of amoebiasis is Entamoeba histolytica which is very common in tropical and developing countries, where sanitation, hygiene, and low socio-economic status are major problems. The most common site of infection is the intestinal mucosa. For extraintestinal amoebiasis, the most common sites are the liver, followed by the lungs and brain.Case: A 15-year-old male was presented to the ER with chief complaint of breathlessness for the last 10 days, accompanied by productive coughing with dark brown sputum and pain on lower right chest. The patient had fever while the disease progressed. Gastrointestinal symptoms were denied. Physical examination showed lowered breathing sound on the right lung, dullness on lower right chest, and usage of accessory breathing muscles. USG found heteroechoic lesion of 8x7cm in size suspicious of lung abscess, later on confirmed as Entamoeba histolytica found in sputum sample, while abdominal USG showed no abnormality. The patient was admitted for antibiotic therapy and consultation to cardiothoracic surgeon.Discussion: One of the rarest routes of amoebiasis is primary deposition of cysts to the lungs through aspiration. Pulmonary amoebiasis often causes abscess formation, produces brown-colored sputum called “anchovy sauce”. It is notable that we found one of the rarest cases of amoebiasis infection, where there is a pulmonary infection without any intestinal involvement.Conclusion: Whilst infection of primary pulmonary amoebiasis is very rare, it is still an important etiology to put on the differential diagnosis of pulmonary abscess. Therefore, sputum examination or biopsy is required even when there are no gastrointestinal disturbances.

Gemella massiliensis sp. nov., a new bacterium isolated from the human sputum

Thanks to its ability to isolate previously uncultured bacterial species, culturomics has dynamized the study of the human microbiota. A new bacterial species, Gemella massiliensis Marseille-P3249T, was isolated from a sputum sample of a healthy French man. Strain Marseille-P3249T is a facultative anaerobe, catalase-negative, Gram positive, coccus, and unable to sporulate. The major fatty acids were C16:0 (34%), C18:1n9 (28%), C18:0 (15%) and C18:2n6 (13%). Its 16S rRNA sequence exhibits a 98.3% sequence similarity with Gemella bergeri strain 617-93T, its phylogenetically closest species with standing in nomenclature. Its digital DNA–DNA hybridization (dDDH) and OrthoANI values with G. bergeri of only 59.7 ± 5.6% and 94.8%, respectively. These values are lower than the thresholds for species delineation (> 70% and > 95%, respectively). This strain grows optimally at 37 °C and its genome is 1.80 Mbp long with a 30.5 mol% G + C content. Based on these results, we propose the creation of the new species Gemella massilienis sp. nov., strain Marseille-P3249T (= CSUR P3249 = DSMZ 103940).

MOSQUITO-BORNE PARASITES IN PATIENTS NEWLY INFECTED WITH HIV IN RELATIONSHIP WITH CD4 COUNT AND TNF ΑLPHA

Background: Mosquito-borne parasites include the pathogenic protozoa and helminths that are transmitted by the insect vector which may co-infect with other organisms to elicit an immune response. Purpose: To determine the frequency of mosquito-borne parasites in patients newly infected with HIV in relationship with CD4 count and TNFα. Method: Thirty-one (31; aged 15-32 years; male-12; female-19) newly diagnosed HIV positive patients and fifty (50) age-matched HIV negative volunteers were recruited as a control subject for this study. All subjects were negative to anti-HCV/HBsAg ELISA, Plasmodium, Acid-Fast Bacilli (AFB) tests and the control subjects were also negative to HIVP24 Ag-Ab ELISA, Plasmodium spp. and Wuchereria bancrofti microscopy. Venous blood including Night blood samples and sputum samples were obtained from the participants for CD4 count by cyflowmetry, TNFα, HIVP24Ag-Ab, anti-HCV, HBsAg by ELISA and microscopic identification by Giemsa staining while Sputum sample was used for Ziehl Neelsen staining to demonstrate Acid Fast Bacilli (AFB). Result: A lower frequency of 25.8% (Rajan, 2008) Plasmodium spp. and 6.5% (James et al., 2015) W. bancrofti was obtained in newly infected HIV patients compared with 32% (Zeitlmann et al., 2001) Plasmodium spp. and 8% (WHO, 2019) W. bancrofti obtained in the non-HIV infected control subjects. Showed a significant decrease in CD4 count and increase in plasma TNFα in both HIV mono-infection and coinfection with Plasmodium spp. and W. bancrofti compared with the results obtained in the non-HIV infected control subjects (p<0.05) and the results obtained in the newly infected HIV patients without Plasmodium spp. and W. coinfection (p<0.05). Conclusion: There was a significant increase in plasma TNFα and a decrease in CD4 count in both HIV mono-infection and coinfection with Plasmodium spp. and W. bancrofti while a lower frequency of Plasmodium spp. and W. bancrofti was obtained in newly infected HIV patients compared with the results obtained in the non-HIV infected control subjects.