Use of stool swabs in molecular transport media increases access to Xpert Ultra testing for TB in children.

SETTING: Tertiary level hospital in Lusaka, Zambia.OBJECTIVE: To measure concordance between Xpert® MTB/RIF Ultra (Ultra) results of stool with and without transport media, and compare Ultra results from the two stool processing methods to Ultra and culture results using gastric aspirates (GA).DESIGN: This was a cross-sectional study collecting stool and GA from children 0-5 years presenting with signs and symptoms of TB. Stool was processed for Ultra testing by two methods: the Simple-One-Step (SOS) on an aliquot of stool and PrimeStore® MTM Molecular Transport Medium (PS-MTM) using a stool swab.RESULTS: A total of 114 children (median age: 17 months, IQR 7-30) provided both a stool and a GA sample. Stool Ultra results processed using the PS-MTM method showed high concordance with stool Ultra results processed by the SOS method, with only 1/114 discordant results. Concordance with GA Ultra was high as well, as 9/13 Mycobacterium tuberculosis (MTB) cases detected were identified by all three methods.CONCLUSION: Ultra results from stool swabs collected using PS-MTM were equivalent to results from stool using the SOS method and GA. Given that PS-MTM inactivates MTB and stabilises DNA without cold chain, using it for stool has the potential to increase access to a TB diagnosis for children in underserved areas.

Introduction of the Simple One-Step stool Xpert Ultra method to detect TB in children and adults.

SETTING: In 2020, the National TB Programme (NTP) of Vietnam conducted an implementation pilot of the Simple One-Step (SOS) stool processing method using Xpert® MTB/RIF Ultra (Ultra) among children and people living with HIV (PLHIV) with signs and symptoms of TB.DESIGN and OBJECTIVES: Using data from this pilot and collecting information on healthcare workers´ (HCWs) perceptions, we assessed the feasibility, acceptability and potential impact of routine stool testing for TB.RESULTS: HCWs perceived collection of stools from children as least stressful of all sample types, stool processing as acceptable and the SOS stool method as easy to perform. After a 3-month induction period, the proportion of initial non-determinate Ultra stool tests was less than 5%. Combined Ultra testing of a respiratory sample and stool resulted in an increase in the proportion of bacteriologically confirmed TB among PLHIV and children by respectively 4.1% (95% CI 1.6-6.6) and 3.9% (95% CI 1.6-6.2). Among children, Mycobacterium tuberculosis was more often detected in stool (26.1%) than in respiratory samples (23.4%) (P = 0.06), including one child with rifampicin resistance.CONCLUSION: Stool testing can be feasibly implemented both in adult PLHIV and in children in routine settings, providing a non-invasive alternative sample type for the diagnosis of TB for patients who cannot produce sputum.

SARS-CoV-2 seroprevalence and associated risk factors in periurban Zambia: a population-based study.

BACKGROUND: We nested a seroprevalence survey within the TREATS (Tuberculosis Reduction through Expanded Antiretroviral Treatment and Screening) project. We aimed to measure the seroprevalence of SARS-CoV-2 infection and investigate associated risk factors in one community (population ∼27,000) with high prevalence of TB/HIV in Zambia. METHODS: The study design was cross-sectional. A random sample of 3592 individuals aged ≥15 years enrolled in the TREATS TB-prevalence survey were selected for antibody testing. Randomly selected blocks of residence were visited between October 2020 and March 2021. Antibodies against SARS-CoV-2 were detected using Abbott- ARCHITECT SARS-CoV-2 IgG assay. RESULTS: A total of 3035/3526 (86.1%) individuals had a blood sample taken. Antibody testing results were available for 2917/3035 (96.1%) participants. Overall, 401/2977 (13.5%) individuals tested positive for IgG antibodies. Seroprevalence was similar by sex (12.7% men vs 14.0% women) and was lowest in the youngest age group 15-19 years (9.7%) and similar in ages 20 years and older (∼15%). We found no evidence of an association between seroprevalence and HIV-status or TB. There was strong evidence (p <0.001) of variation by time of enrollment, with prevalence varying from 2.8% (95% CI 0.8-4.9) among those recruited in December 2020 to 33.7% (95% CI 27.7-39.7) among those recruited in mid-February 2021. CONCLUSION: Seroprevalence was 13.5% but there was substantial variation over time, with a sharp increase to approximately 35% toward the end of the second epidemic wave.

Candida keratitis and endopthalmitis after corneal transplantation; two case reports, a novel regimen and literature overview of therapeutic options.

PURPOSE: To evaluate treatment options for candida keratitis and endopthalmitis after corneal transplantation. METHODS: Case reports and literature review. RESULTS: Two patients with keratitis due to Candida glabrata/parapsilosis after corneal transplantation were successfully treated with a combination of topical voriconazole, intracameral voriconazole and amphotericin B, and systemic treatment with flucytosine. CONCLUSIONS: Natamycine and voriconazole topically are preferred therapeutic options for the treatment of fungal keratitis. Systemic flucytosine is a useful alternative additive, particularly for countries where natamycine is not registered as a pharmaceutical agent.

Renal transplant patient survives a donor-derived abdominal invasive mucormycosis (Lichtheimia ramos a).

Mucormycosis is a life-threatening invasive fungal infection, most commonly described in severely immunocompromised patients. It is characterized by rapid invasive growth of the fungus and often with fatal outcome. We report a case of a renal transplant recipient diagnosed with a donor-derived invasive mucormycosis. In this patient, we used a step-wise approach of withdrawal of immunosuppressants, antifungal induction therapy, extensive surgical debridement of all (potentially) infected tissue, abdominal irrigation of liposomal amphotericin B and interferon gamma. Due to rapid diagnosis and intensive therapy the patient survived.

Attenuation correction for confocal laser scanning microscopy and its application in chromatography.

The applicability of confocal laser scanning microscopy is limited, e.g. by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on beam modelling and on a differential form of the modified Beer-Lambert law. It turns out that the intensity decay can be modelled as a double convolution of the microscopic image with the intensities of the excitation semibeam and the emission beam. Under weak assumptions made for the intensities of the fluorescent radiation and the detected signal, formulas for the attenuation correction and the attenuation simulation are derived. The method traces back to that one published by Roerdink which is modified concerning a more realistic beam modelling, avoiding the so-called weak attenuation expansion and considering fluorescence excitation throughout the light cone of the excitation beam. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation. LAY DESCRIPTION: The applicability of confocal laser scanning microscopy is limited by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on modeling the excitation as well as the emission beam and on a modified Beer-Lambert law for beam attenuation. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation.

High prevalence of azole resistance in Aspergillus fumigatus isolates from high-risk patients.

OBJECTIVES: Aspergillus fumigatus is the leading cause of invasive aspergillosis. Adequate treatment is complicated by an increase in azole resistance. Here, the incidence of voriconazole, posaconazole and itraconazole resistance in clinical isolates from high-risk patients from either the haematology ward or the ICU of the University Medical Center Utrecht in the period 2011-13 is analysed. Putative clonality of resistant strains was tested through cyp51A and microsatellite typing. METHODS: Primary A. fumigatus isolates from 105 patients were collected by an unbiased routine diagnostic-driven approach and phenotypically tested for azole susceptibility. Of the 105 isolates, 5 were from patients with a proven invasive A. fumigatus infection, 48 were from patients with a probable invasive A. fumigatus infection and 52 were from patients with non-invasive infections. Real-time PCR and cyp51A gene and strain typing were performed. RESULTS: Twenty-one out of 105 (20.0%) isolates were resistant to at least one of the three clinical azoles and 17/105 (16.2%) isolates were resistant (MIC >2 mg/L) to voriconazole, the empirical drug of choice for treatment of aspergillosis. There was a striking difference in the prevalence of triazole resistance, with 15.9% resistant isolates (25.0% in proven/probable patients) in the haematology population and 4.5% (10% in proven/probable) in the ICU. While the majority of isolates with elevated MICs of voriconazole were cyp51A related (17/23), both microsatellite and cyp51A sequence typing argue against clonal spread of resistant strains. CONCLUSIONS: This study reveals a high incidence of voriconazole resistance (16.2%) in A. fumigatus in high-risk patients. Our data stress the need for laboratory detection of azole resistance prior to treatment.

Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.

SETTING: The molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular tests. OBJECTIVE: We quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics. DESIGN: Consecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP. RESULTS: Among 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found. CONCLUSIONS: Strains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.

Dedicated vertical wind tunnel for the study of sedimentation of non-spherical particles.

A dedicated 4-m-high vertical wind tunnel has been designed and constructed at the University of Geneva in collaboration with the Groupe de compétence en mécanique des fluides et procédés énergétiques. With its diverging test section, the tunnel is designed to study the aero-dynamical behavior of non-spherical particles with terminal velocities between 5 and 27 ms(-1). A particle tracking velocimetry (PTV) code is developed to calculate drag coefficient of particles in standard conditions based on the real projected area of the particles. Results of our wind tunnel and PTV code are validated by comparing drag coefficient of smooth spherical particles and cylindrical particles to existing literature. Experiments are repeatable with average relative standard deviation of 1.7%. Our preliminary experiments on the effect of particle to fluid density ratio on drag coefficient of cylindrical particles show that the drag coefficient of freely suspended particles in air is lower than those measured in water or in horizontal wind tunnels. It is found that increasing aspect ratio of cylindrical particles reduces their secondary motions and they tend to be suspended with their maximum area normal to the airflow. The use of the vertical wind tunnel in combination with the PTV code provides a reliable and precise instrument for measuring drag coefficient of freely moving particles of various shapes. Our ultimate goal is the study of sedimentation and aggregation of volcanic particles (density between 500 and 2700 kgm(-3)) but the wind tunnel can be used in a wide range of applications.

Effects of perfusion and cyclic compression on in vitro tissue engineered meniscus implants.

PURPOSE: The purpose of this study was to investigate the influence of continuous perfusion and mechanical stimulation on bone marrow stromal cells seeded on a collagen meniscus implant. METHODS: Bone marrow aspirates from 6 donors were amplified in vitro. 10(6) human BMSC were distributed on a collagen meniscus implant. Scaffolds were cultured under static conditions (control) or placed into a bioreactor system where continuous perfusion (10 ml/min) or perfusion and mechanical stimulation (8 h of 10% cyclic compression at 0.5 Hz) were administered daily. After 24 h, 7 and 14 days, cell proliferation, synthesis of procollagen I and III peptide (PIP, PIIIP), histology, and the equilibrium modulus of the constructs were analyzed. RESULTS: Proliferation demonstrated a significant increase over time in all groups (p < 0.001). PIP synthesis was found to increase from 0.1 ± 0.0 U/ml/g protein after 24 h to 2.0 ± 0.5 (perfusion), 3.8 ± 0.3 (mechanical stimulation), and 1.8 ± 0.2 U/ml/g protein (static control, lower than perfusion and mechanical stimulation, p < 0.05). These differences were also evident after 2 weeks (2.7 ± 0.3, 4.0 ± 0.6, and 1.8 ± 0.2 U/ml/g protein, p < 0.01); PIIIP synthesis was found to increase from 0.1 ± 0.0 U/ml/g protein after 24 h to 2.9 ± 0.7 (perfusion), 3.1 ± 0.9 (mechanical stimulation), and 1.6 ± 0.3 U/ml/g protein (controls) after 1 week and remained significantly elevated under the influence of perfusion and mechanical stimulation (p < 0.01) after 2 weeks. Mechanical stimulation increased the equilibrium modulus more than static culture and perfusion after 2 weeks (24.7 ± 7.6; 12.3 ± 3.7; 15.4 ± 2.6 kPa; p < 0.02). CONCLUSION: Biomechanical stimulation and perfusion have impact on collagen scaffolds seeded with BMSCs. Cell proliferation can be enhanced using continuous perfusion and differentiation is fostered by mechanical stimulation.

Multiple strains of Mycobacterium avium subspecies hominissuis infections associated with aborted fetuses and wasting in pigs.

A herd of pigs being reared for breeding and fattening, in which there had been incidences of abortion and wasting, reduced growth rates and an increase in mortality for the past year, were tested for Mycobacterium infection by pathological examinations, skin test, serology and Mycobacterium culture. In one placenta, and also in the lung tissues of fetuses, Ziehl-Neelsen staining revealed acid-fast bacilli in combination with infiltrations of neutrophils, macrophages and multinucleated giant cells. Acid-fast bacilli were also found in the mesenteric lymph nodes, liver and/or spleen and jejunum of pigs with wasting and in slaughtered animals. The specimen cultures were identified as Mycobacterium avium subspecies hominissuis using IS1245-specific PCR and IS1245 restriction fragment length polymorphism (RFLP). IS1245 RFLP revealed that the herd was infected with multiple M avium subspecies hominissuis strains belonging to at least two different clades. It is suggested that this infection may have played a more important role in the economic losses of the pig farm than had been assumed previously.

Intra-assay reliability and robustness of QuantiFERON(R)-TB Gold In-Tube test in Zambia.

BACKGROUND: Interferon-gamma (IFN-gamma) release assays (IGRAs), such as the QuantiFERON-TB Gold In-Tube test (QFT-GIT), are becoming a preferred method for diagnosis of tuberculosis (TB) infection in many industrialised countries. However, data on the effectiveness of IGRAs in high TB-HIV (human immunodeficiency virus) endemic and resource-limited settings, such as Zambia, are limited. OBJECTIVE: To determine the intra-assay reliability and robustness of QFT-GIT in a field setting in Zambia. DESIGN: During July-October 2007, 109 adult smear-positive TB patients were recruited to determine QFT-GIT reliability and the effect of a 24-h delay in incubation. Two simulated laboratory experiments were also performed using 9-14 volunteers, to explore the effect of power outages during incubation and storage temperature of collection tubes on IFN-gamma responses. RESULTS: QFT-GIT intra-assay concordance was 91.7% (kappa = 0.8). Discordance was observed for nine patients, of whom six were HIV-positive. There was evidence of an association between HIV status and discordant results (OR 1.98, 95%CI 1.06-3.67, P = 0.03). A 24-h delay in incubation changed results for 25 of the 109 (22.9%) patients. Power outages that altered incubation time reduced IFN-gamma responses. CONCLUSION: Although QFT-GIT seems reliable in this setting, we have identified operational factors that affect its robustness. These factors may influence the effectiveness of this test in similar resource-limited settings.

Papiloma Vírus Humano (HPV) / Human Papillomavirus (HPV)

Está estabelecido que o HPV (Human Papillomavirus) é o causador de cerca de 99 dos casos de câncer de colo de útero e de uma fração variável de câncer de vagina, vulva, pênis e ânus. Este dado está relacionado a mais de 100 tipos de HPV existentes com diferentes seqüências de DNA. A incidência de casos de câncer de colo de útero atinge principalmente mulheres de 20 anos a 29 anos de idade, sendo que a morbidade nesta faixa etária pode ser diminuída com o rastreamento de lesões do colo de útero e células infectadas pelo HPV, bem como, com a imunização através da vacina bivalente ou quadrivalente. A vacinação preventiva deve ser dada antes da infecção pelo HPV, devido a seu efeito profilático, para que o sistema imune reconheça e evite a infecção viral antes daentrada do vírus na célula. Conseqüentemente, as lesões de colo de útero classificadas como NIC II, NIC III e câncer de colo de útero serão impedidas de instalar-se. Existem duas vacinas desenvolvidas atualmente, a Gardasil® – Merck (quadrivalente) e a Cervarix® – GlaxoSmithKline (bivalente), sendo que ambas utilizam partículas de L1 viral específicas contra o genótipo do HPV, evitando odesenvolvimento de infecção. Estima-se que a eficácia seja de até 5 anos, os quais ainda estão em curso para comprovação. A vacina deverá ser incorporada ao novo calendário brasileiro de imunização, sendo obrigatório para pessoas com doze anos ou mais, assim que sua eficácia e o custo benefício forem comprovados. Desta forma, pretende-se diminuir a estimativa de câncer de colo de útero,infecções genitais e esterilidade decorrente destas doenças. Entretanto, o preventivo da citologia cervico-vaginal continua sendo necessário para a triagem e diagnóstico de possíveis lesões decorrentes de outros motivos que não o HPV.

Comparison of four culture systems for Mycobacterium tuberculosis in the Zambian National Reference Laboratory.

SETTING: National TB Reference Laboratory, Zambia. OBJECTIVE: To compare four TB culture systems when used in a resource-limited setting. DESIGN: Comparison of four culture systems: automated Mycobacterium Growth Indicator Tube (AMGIT) 960, manual MGIT (MMGIT) and two Löwenstein-Jensen (LJ) culture media-commercial (CLJ) and homemade (HLJ). RESULTS: A total of 1916 sputum specimens were received, of which 261 (13.6%) were positive on microscopy. Mycobacterium tuberculosis complex (MTC) was isolated on at least one of the media in 410 (21.4%) specimens: MMGIT recovered 336 (17.5%) MTC, AMGIT 329 (17.2%), CLJ 192 (10.0%) and HLJ 184 (9.6%). The median time to detection for smear-negative specimens was 14 days for AMGIT, 16 days for MMGIT and 34 days for both LJ. Isolation of non-tuberculous mycobacteria (NTM) was more frequent in both MGIT systems (3.5%) than in CLJ (0.9%) and HLJ (0.8%). Contamination rates were high: 29.6% on AMGIT, 23.8% on MMGIT, 14.9% on CLJ and 12.5% on HLJ. CONCLUSION: Despite high contamination rates, either MGIT system considerably improved both the yield and the time to detection of MTC compared to LJ media. Investments in infrastructure and training are needed if culture is to be scaled up in low-income settings such as this.

[Prevention of infections and thromboses after splenectomy or because of functional loss of the spleen]. / Prävention von Infektionen und Thrombosen nach Splenektomie oder bei Funktionsverlust der Milz.

Overwhelming Post-Splenectomy Infection (OPSI or PSS), most frequently caused by encapsulated Gram-positive pathogens, is a complication after splenectomy. Reasons for splenectomy include trauma, or malignant and non-malignant hematologic diseases. OPSI-inducing bacteria are mainly Streptococcus pneumoniae and less frequently Haemophilus influenzae, Neisseria meningitides and Gram-negative bacilli. There exist very efficient--albeit often neglected--strategies, how to prevent infections in patients after splenectomy. These include vaccination, prophylactic antibiotics (always for 3 years during childhood and adolescence) and prompt antibiotic treatment, if an infection is suspected. Patients need to know the nature and likelihood of PSS and they should seek immediate medical attention if they become ill or febrile. Each patient should carry at all times a letter or card documenting the splenectomy. With these measures and precautions, the PSS-risk can be significantly reduced or at best be completely avoided.

Effectiveness of whole grain consumption in the prevention of colorectal cancer: meta-analysis of cohort studies.

The present work aimed to evaluate the effectiveness of whole grain consumption in preventing colorectal cancer. A systematic review with meta-analysis of 11 cohort studies was carried out. The age group of the population studied (1,719,590 participants) was between 25 and 76 years of age. The review evaluated the relative risks with the Cox proportional hazard model. The period of study varied from 6 to 16 years, where 7,745 persons developed colorectal cancer during the follow-up period. In the multivariate analysis, the highest quintile relative risk was 0.94 (95% confidence interval, 0.85-1.03), whereas that for the lowest quintile was 0.96 (95% confidence interval, 0.88-1.04). The location of tumors was also evaluated, with tumors in the colon demonstrating a relative risk of 0.93 (95% confidence interval, 0.83-1.02) and tumors in the recto a relative risk equal to 0.89 (95% confidence interval, 0.79-1.00). In this multivariate analysis, consumption of whole grains was inversely associated with the risk of developing colorectal cancer.

Complement factor H gene polymorphisms and Chlamydia pneumoniae infection in age-related macular degeneration.

PURPOSE: To investigate the association of the complement factor H gene (CFH)Y402H polymorphism and age-related macular degeneration (AMD) in the Austrian population (Caucasoid descent), and to determine whether there is an association between exposure to Chlamydia pneumoniae-responsible for up to 20% of community-acquired pneumoniae-and the AMD-associated CFHrisk polymorphism. METHODS: Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis in 75 unrelated AMD patients and compared with 75 healthy, age-matched control subjects. C. pneumoniaeserum IgG was tested by ELISA (R&D) in both groups. The association between the CFHY402H genetic polymorphism and the disease was examined by chi (2)-test and logistic regression. RESULTS: CFH Y402H genotypefrequencies differed significantly between AMD patients and healthy controls (1277 TT, 22.7%; 1277 TC, 53.3%; and 1277 CC, 22.7% in the AMD group; 1277 TT, 48.0%; 1277 TC, 38.7%; and 1277 CC, 13.3% in the control group) showing a P-value <0.005 (OR:2.920/3.811).No association was found between a positive C. pneumoniae titre and AMD (P=0.192), nor was any association found between C. pneumoniae and the CFH Y402H polymorphism. CONCLUSIONS: Our data confirm that the CFHY402H polymorphism is a risk factor for AMD in the Austrian population with a higher frequency of the Y402 polymorphism in AMD patients. No association between preceding C. pneumoniaeinfection and diagnosed AMD was found.

Studies on travel-related thrombosis.

Due to increased long-distance travelling, travel-related thrombosis and its prevention are frequently discussed between patients and their doctors. There is now well accepted evidence that thromboembolic events can occur during or after long journeys, but despite a plethora of studies on the subject, still very little is known about the height of the absolute risk, the underlying triggering factors and, especially, about the efficacy of specific prophylactic methods. Therefore the recommendations for the prevention of travel-related thrombosis, developed and published by experts in this field, are necessarily based on the risk assessment of the individual traveller and the methods of prevention supported by the experience in medical patients.