SARS-CoV-2 in saliva, oropharyngeal and nasopharyngeal specimens.

INTRODUCTION: The reference test to evaluate patients with suspected respiratory virus infection is a real-time reverse transcription-polymerase chain reaction (RT-PCR) from a nasopharyngeal swab (NPS). However, other specimen collection methods such as an oropharyngeal swab (OPS) or saliva specimen are also used for SARS-CoV-2 testing during the ongoing COVID-19 pandemic. However, it remains unclear if rates of SARS-CoV-2 detection differ between sampling methods. This study will compare the rates of SARS-CoV-2 detection by saliva, OPS, and NPS sampling in a public setting. METHODS: Individuals referred for outpatient SARS-CoV-2 testing will be invited to participate in a prospective clinical study. They will have saliva, OPS and NPS specimens collected that will be analysed separately for SARS-CoV-2 RNA by RT-PCR. The rate of SARS-CoV-2 detection in saliva, OPS and NPS will be compared using a logistic regression mixed-effect model analysis. A sample of 19,110 participants is required at an expected 1.5% test-positive rate in order to detect a 25.6% difference. The total sample size will be adjusted as the test-positive rate changes. CONCLUSIONS: This study will provide evidence for the optimal site of specimen collection to detect SARS-CoV-2. The results may help guide the health authorities. FUNDING: This is an investigator-initiated trial based on an unrestricted grant from the Novo Nordisk Foundation and the Aage og Johanne Louis-Hansens Fond. The foundations have had no say in the decisions on study design or reporting. TRIAL REGISTRATION: ClinicalTrials.gov (ID: NCT04715607).

Isolation of Nontuberculous Mycobacteria from the Environment of Ghanian Communities Where Buruli Ulcer Is Endemic.

UNLABELLED: This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin-supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana. IMPORTANCE: Diseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.

Strengthening the Tuberculosis Specimen Referral Network in Uganda: The Role of Public-Private Partnerships.

BACKGROUND: Diagnosis of multidrug-resistant tuberculosis and prompt initiation of effective treatment rely on access to rapid and reliable drug-susceptibility testing. Efficient specimen transport systems and appropriate training on specimen referral contribute to optimal and timely access to tuberculosis diagnostic services. METHODS: With support and technical assistance from a public-private partnership (PPP) between Becton Dickinson and the US President's Emergency Plan for AIDS Relief, the Uganda National TB Reference Laboratory (NTRL) and National TB and Leprosy Program redesigned the tuberculosis specimen transport network and trained healthcare workers with the goal of improving multidrug-resistant tuberculosis detection. RESULTS: Between 2008 and 2011, the PPP mapped 93% of health facilities and trained 724 healthcare and postal staff members covering 72% of districts. Strengthening the tuberculosis specimen referral system increased referrals from presumptive multidrug-resistant tuberculosis cases by >10-fold, with 94% of specimens reaching the NTRL within the established target transport time. CONCLUSIONS: This study demonstrates the potential of PPP collaborations with ministries of health to positively influence patient care by strengthening laboratory systems through increased access to drug-susceptibility testing in Uganda. Ongoing efforts to integrate specimen transport networks will maximize resources and improve patient management.

Immunofluorescence in dermatology.

Direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) tests on skin biopsy are being done mostly in academic teaching hospitals. These tests provide a useful diagnostic aid to dermatologists. Immunohistology and serology can, in conjunction with histology, provide considerable help in delineation and diagnosis of various skin disorders as well as systemic diseases with skin involvement, e.g. systemic lupus erythematosus. Immunofluorescence (IF) studies have now become an invaluable supplement to clinical and histological examination in a variety of dermatological diseases. These skin diseases now include not only bullous and connective tissue disorders, vasculitides, and conditions such as lichen planus, but also the scaling dermatoses, notably psoriasis. In this review article, we share our experience of providing such a diagnostic facility for more than 30 years in a large tertiary care health center in North India and also help to outline the conditions, which can be diagnosed confidently, and others where IF can help in confirming a diagnosis or the immune component of the disease. The article also deals with handling of skin biopsy specimens and interpretation of biopsy findings on DIF and IIF examination.

FTA card utility for PCR detection of Mycobacterium leprae.

The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA.

Diagnosis of clinical samples spotted on FTA cards using PCR-based methods.

The broad clinical presentation of Leishmaniasis makes the diagnosis of current and past cases of this disease rather difficult. Differential diagnosis is important because diseases caused by other aetiologies and a clinical spectrum similar to that of leishmaniasis (e.g. leprosy, skin cancers and tuberculosis for CL; malaria and schistosomiasis for VL) are often present in endemic areas of endemicity. Presently, a variety of methods have been developed and tested to aid the identification and diagnosis of Leishmania. The advent of the PCR technology has opened new channels for the diagnosis of leishmaniasis in a variety of clinical materials. PCR is a simple, rapid procedure that has been adapted for diagnosis of leishmaniasis. A range of tools is currently available for the diagnosis and identification of leishmaniasis and Leishmania species, respectively. However, none of these diagnostic tools are examined and tested using samples spotted on FTA cards. Three different PCR-based approaches were examined including: kDNA minicircle, Leishmania 18S rRNA gene and PCR-RFLP of Intergenic region of ribosomal protein. PCR primers were designed that sit within the coding sequences of genes (relatively well conserved) but which amplify across the intervening intergenic sequence (relatively variable). These were used in PCR-RFLP on reference isolates of 10 of the most important Leishmania species: L. donovani, L. infantum, L. major & L. tropica. Digestion of PCR products with restriction enzymes produced species-specific restriction patterns allowed discrimination of reference isolates. The kDNA minicircle primers are highly sensitive in diagnosis of both bone marrow and skin smears from FTA cards. Leishmania 18S rRNA gene conserved region is sensitive in identification of bone marrow smear but less sensitive in diagnosing skin smears. The intergenic nested PCR-RFLP using P5 & P6 as well as P1 & P2 newly designed primers showed high level of reproducibility and sensitivity. Though, it was less sensitive than kDNA minicircle primers, but easily discriminated between Leishmania species.

[Summary of questionnaires on slit skin smear test in clinics, hospitals, and university hospitals].

We have performed a questionnaire to survey the present conditions of the slit skin smear test, a method to diagnose leprosy. The answer was obtained from 40 (93.0%) out of 43 clinics, department of dermatology of university and other hospitals and leprosy sanatoriums. Slit skin smear test was carried out in most institutions. However, when inspection frequency was low, a laboratory technician performed Ziehl-Neelsen staining or its variation using a normal slide glass. A few institutions carried out fluorescence staining. Both physician and technician examined the slides in most cases, however, in more than half of the leprosy sanatoriums, technician was the only person examined. Bacterial index was evaluated in most institutions, while only for the presence of bacteria was examined 5 institutions. Slit skin smear test is simple and easy, but accuracy is different with skills, glass slides to be used and methods for staining and inspection. Supply of the glass slide with a marker, the spread of staining methods, technical improvement of an inspection are demanded in future.

Fisioterapia respiratória como método alternativo no diagnóstico da tuberculose num hospital de referência / Chest physical therapy as an alternative method on tuberculosis diagnosis into a reference hospital

Visando avaliar a aplicabilidade da técnica fisioterapêutica de depuraçãobrônquica, a técnica ELTGOL, como método auxiliar para a obtenção de amostras de escarro com finalidade diagnóstica em pacientes com suspeita de tuberculose (TB) pulmonar, foram incluídos 235 pacientes adultosinternados no Instituto de Infectologia Emílio Ribas, identificados pelo pedido médico de pesquisa de BAAR no escarro. Destes, 75 foram excluídos devido a problemas administrativos, alta/óbito precoce, amostras inadequadas, etc. Cinco amostras foram consecutivamente coletadas de cada um dos demais 160 pacientes, a saber: por técnica “espontânea” (ESP) no 1º dia, pelatécnica ELTGOL no 2º dia, novamente por ESP no 3º dia, indução do escarro com solução hipertônica salina (ISHS) no 4º dia e a última amostra por ESP no 5º dia. Todas as amostras também foram semeadas em meio de cultura (LJ). Dos 160 pacientes mantidos, 132 (82,5%) eram hiv-positivos e 28 (17,5%) eram hiv-negativos, 43 (26,9%) confirmaram o diagnóstico de TB (cultura positiva de qualquer uma de suas cinco amostras) e 117 pacientes(73,1%) não confirmaram TB. Existe uma aparente superioridade dos resultados da baciloscopia nas amostras obtidas pela técnica ELTGOL, em especial se comparada a uma amostra única pela ESP ou à ISHS, porém sem diferença estatisticamente significante. Quanto à cultura, o resultado obtido pela coleta espontânea de 3 amostras foi significantemente melhor do que qualquer técnica de coleta única (p<0,05). Esta relação foi mantida nospacientes definidos como sem expectoração espontânea na anamnese e nos já submetidos a tratamento empírico prévio

Evaluation of commercial enzyme-linked immunosorbent assay kits for detection of tuberculosis in Argentinean population.

Pathozyme-Myco G (Myco G), M, A, and TB complex plus (Omega Diagnostics Ltd., Alloa, Scotland) were evaluated for the serological diagnosis of pulmonary tuberculosis (TB) in an Argentinean population. Sera from 58 patients with pulmonary TB, 24 subjects with pulmonary mycobacteriosis or mycoses (pulmonary MM group), and 45 subjects with other underlying disorders (control group) were analyzed. The sensitivities of the tests ranged from 29% (Myco M) to 82% (Myco G) in smear-positive patients (17 subjects) and from 29% (TB complex plus) to 49% (Myco G) in smear-negative patients (41 subjects). The specificities of the assays varied from 93% (Myco M) to 100% (Myco G and TB complex plus) in controls and from 62% (Myco A) to 96% (TB complex plus) in the pulmonary MM group. Overall, for the diagnosis of smear-negative patients, Myco G had the best characteristics, with a sensitivity of 49% and specificities of 100% for controls and 75% for the pulmonary MM group; after its combination with TB complex plus, its sensitivity improved to 59%. Nevertheless, despite its relatively poor capacity to discriminate between pulmonary TB and pulmonary MM, Myco G, alone or in combination with TB complex plus, would be a useful diagnostic tool for patients with suspected pulmonary TB living in areas where the relative prevalence of pulmonary MM was low.

Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization.

Biopsy and skin-scraping specimens from 130 leprosy cases across the disease spectrum (56 TT/BT/I, 73 BB/BL/LL, and 1 neuritic case) and 50 healthy contacts were studied to assess the application of gene amplification. The nucleic acids from these clinical specimens were extracted by an integrated freeze-thawing--optimized lysozyme-/proteinase-k treatment-purification and fractionation procedure. The nucleic acids from cultured organisms were isolated by the stepwise procedure earlier standardized at this laboratory. Gene amplification for a 360-bp fragment of the 18-kDa protein gene was carried out using primer and the procedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain reaction product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in specimens with demonstrable acid-fast bacilli. A positivity of 73% in TT/BT/I specimens and 93% in BB/BL/LL specimens was observed. Four combinations were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B+ (3%) and EB-, B- (12%). By combining the blot hybridization with EB staining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by the absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organisms, grown from these specimens. It is recommended that for optimum sensitivity and specificity both EB staining and blot hybridization should be done.

Inventory of skin smear practices in 6 leprosy control programmes in Nigeria.

A study of slit skin smear (SSS) examination practices in 6 Nigerian Leprosy Control Programmes was undertaken to assess the quality of smearing, staining and reading. Results indicated that the standard of SSS practices fall below the ideal. There is a great need for training as well as supervision and support of laboratory staff if this deplorable situation is to be improved.

Polymerase chain reaction for detection of Mycobacterium leprae in nasal swab specimens.

The polymerase chain reaction based on the selective amplification of a 531-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied to nasal swab specimens from leprosy patients, occupational contacts, and endemic and nonendemic controls. To prevent false-positive amplification, we used dUTP and uracil-DNA-glycosylase in all polymerase chain reactions. False-negative reactions were detected by using a 531-bp modified template as an internal control. Amplification products were found in 55% of untreated patients, in 19% of the occupational contacts, in 12% of endemic controls, and in none of the nonendemic controls. This study strongly suggests that not only leprosy patients but also healthy persons may carry M. leprae. We concluded that polymerase chain reaction is a reliable method to detect M. leprae in nasal specimens. The method holds promise for studying the spread and transmission of M. leprae within a population.