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.

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.

Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands.

A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Delta315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8,332 patients in The Netherlands (1993-2002) were screened for the Delta315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Delta315. IS6110 restriction fragment length polymorphism analysis showed that Delta315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Delta315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.

Pneumonia caused by Mycobacterium kansasii in a series of patients without recognised immune defect.

The clinical and epidemiological characteristics of 17 patients diagnosed with Mycobacterium kansasii pneumonia within a limited geographical region over a period of 10 years are described. An in-depth evaluation of the innate and adaptive immune systems was performed for five available patients. A comparison was made of the genetic fingerprint patterns of the isolates obtained by restriction fragment length polymorphism (RFLP) analysis, with the major polymorphic tandem repeat (MPTR) as a probe. Predisposing factors consisted of smoking, airway abnormalities, substance abuse, diabetes or poor general condition, but in two patients no risk factor was identified. In the five patients tested, no abnormalities or deficiencies were detected in the innate or adaptive type-1 immunity. All M. kansasii isolates had identical MPTR RFLP patterns, although no epidemiological connection could be established, and these were identical to those of clinical isolates from Australian patients. These data do not support the theory that defects in the innate or adaptive type-1 immunity have a role in the pathogenesis of invasive M. kansasii infections. The identical fingerprint patterns of the isolates suggested the existence of a virulent strain of M. kansasii.

Investigation of cross contamination in a Mycobacterium tuberculosis laboratory using IS6110 DNA fingerprinting.

SETTING: A laboratory for routine culturing of Mycobacterium tuberculosis. OBJECTIVE: Investigation of an episode of laboratory cross contamination using IS6110 restriction fragment length polymorphism (RFLP) typing. Improvement of laboratory protocols to prevent contaminations in the future. To stress the importance of 'good laboratory practice', and interaction with clinicians about laboratory results. DESIGN: Fingerprinting of mycobacterial isolates from 1) cultures suspected of being contaminated and 2) strains suspected of being the source of the cross-contamination. RESULTS: RFLP typing results indicated that clinical samples were contaminated by strains which had been processed in species identification procedures one day earlier in the same safety cabinet. This cross contamination also resulted in exceptional RFLP typing results--mixed banding patterns. Three patients were treated on the basis of false-positive laboratory results. Because the laboratory results were confusing for the clinicians, the treatment of one true tuberculosis patient was severely delayed. CONCLUSION: 'Good laboratory practice' is very important to prevent cross contamination. RFLP typing proved to be a useful tool to trace the source of contamination. Interaction with clinicians receiving doubtful results is of the utmost importance.

Molecular epidemiology of tuberculosis in Cuba outside of Havana, July 1994-June 1995: utility of spoligotyping versus IS6110 restriction fragment length polymorphism.

SETTING: Molecular typing has become an important tool for examining the extent of active transmission of tuberculosis. OBJECTIVES: To examine transmission of tuberculosis in Cuba using IS6110 restriction fragment length polymorphism (RFLP) typing and to evaluate the utility of spoligotyping. DESIGN: One hundred and sixty Mycobacterium tuberculosis strains isolated over a one year period in Cuba were subjected to RFLP and spoligotyping. RESULTS: Forty-eight percent of the isolates were found in 19 clusters of strains with identical RFLP patterns. In general, cluster sizes were limited, except for two large institutional outbreaks. Age was strongly inversely correlated to clustering. Most streptomycin-resistant isolates were found in clusters. Fifteen spoligotype clusters comprised 78% of the isolates. Significantly different IS6110 RFLP types subdivided 11 spoligotype clusters, whereas none of the IS6110 clusters were subdivided by spoligotyping. CONCLUSIONS: Considering the short study period, 48% clustering is high, indicating that recent transmission plays an important role in Cuba. Although resistance is still a minor problem, transmission of streptomycin-resistant strains occurs. The high polymorphism observed with IS6110 RFLP indicates that this marker is useful for future molecular epidemiological studies in Cuba. Spoligotyping appeared less suitable for population-based studies.

Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar reference strains, human and animal isolates.

SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.

Measles in Tanzania: antibody response in children after vaccination and antibody state of mothers and newborns.

The antibody response to attenuated live measles vaccines was studied in two groups of 29 (A) and 53 (B) African children. In group A 22 sera and in group B 34 sera showed no pre-immunization haemagglutination inhibition (HI) titres. Vaccination resulted in seroconversion in 64 and 85% of groups A and B, respectively. The difference in antibody response between the two groups could be traced to children in the age group seven to eight months, where seroconversion was absent in 67% of group A children and in 0% of group B children. Antibody levels were studied in 234 mothers and their newborns. In 131 serum pairs the antibody levels of mother and child were similar. 78 (33%) of mothers showed a higher titre and 25 (11%) a lower titre than their babies. All newborns except one and all mothers except one possessed antibody titres above 10. Children in the age groups seven to eight months and nine to ten months showed antibody in 12 and 7%, respectively. Over 90% of these children had not been infected in the first ten months of life.

Restriction fragment length polymorphism typing of mycobacteria.

In principle, restriction fragment length polymorphism (RFLP) typing can be applied to strains of all mycobacterial species for which suitable probes have been identified. International consensus has been achieved regarding the methodology of IS6110 RFLP typing of Mycobacterium tuberculosis complex isolates (1) and IS1245 RFLP typing of Mycobacterium avium strains (2). This chapter describes the technical details of these standardized methods regarding the isolation of DNA, restriction enzymes, electrophoresis conditions, internal- and external-size markers, Southern blotting, and several probes used for hybridization. Furthermore, RFLP typing of isolates of some other mycobacterial species is described.

Study of the vaginal mucous membrane following tampon utilisation; aspect on colposcopy, scanning electron microscopy and transmission electron microscopy.

The effect of periodic tampons was studied in 17 young women during the menstrual and inter-menstrual cycle. Biopsies (n = 19) were analysed on scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effect of medium absorbant tampons was compared with that of super-absorbant tampons. On colposcopy, dryness was noted in 89% cases, peeling in 47% cases. Microulcerations were observed only once (5%). Dryness and peeling was most often encountered with the super-absorbant tampons and during the inter-menstrual phase. TEM revealed cellular destruction affecting all layers of the epithelium: lysis of superficial and intermediate cells, destruction of desmosomes accompanied by the creation of inter-cellular spaces, lipidic vacuoles in the lysed cells. SEM revealed epithelial peeling with cleavage and severe cellular desquamation. Cellular anomalies were common at the level of the cytoplasmic membrane, which presented either defects, a porous aspect, or appeared swollen by air-bubbles.

Comparison of the effects of different menstrual tampons on the vaginal epithelium: a randomised clinical trial.

The effects on the vaginal epithelium of three different menstrual tampons in normal conditions of use were studied in 41 women during the menstrual period. The vaginal epithelium was studied in a total of 123 cycles at a structural and ultra-structural level with colposcopy, vaginal smear and biopsy on TEM and SEM. One of the three tampons studied showed a lower level of abnormalities on colposcopy (36.6% vs. 56.1% vs. 68.3%), with an inverse correlation between the severity of the dryness and the quantity of blood absorbed by the tampon (4.2 g when the colposcopy was normal vs. 1.8 g in case of severe dryness). Cytology is not a good test for assessing the effects of tampons because of the high rate of acellular and uninterpretable samplings. The biopsy effects were defined according to their depth in the epithelium. The same tampon showed the lowest level in biopsy abnormalities. No correlation was found between severity of the colposcopy and biopsy results. Colposcopy can demonstrate the degree of severity of dryness or any other effect, but biopsy only confirms the effect and does not correlate the degree of severity. Materials and designs of tampons can play a role in reducing the drying effects to the vaginal epithelium.

[Vaginal ulceration induced by abuse of tampons]. / Ulcération vaginale provoquée par l'emploi abusif de tampons périodiques.

A large vaginal ulcerated area which came about because super-absorbant tampons had been worn for three years is reported. Cure was obtained when the area was excised and sutured. There was no recurrence after the patient stopped using the tampons. There are 36 cases reported in the literature. Ulceration occurs in young women (25 years), nulliparous or primiparous (79%), who use tampons abnormally during the periods and between the periods (75%). The ulceration is always characteristic in appearance: it is a punched-out area, round or oval in shape, and is situated in the upper third of the vaginal barrel near where the tampon presses on to the vagina. Spontaneous cure occurs in 75% of cases when the tampon is no longer used. Physio-pathological hypotheses as to the causation are given. A suggestion is made as to the relationship between this syndrome and the staphylococcal toxic shock syndrome. The discovery of vaginal ulceration is not usually due to use of menstrual tampons. One has to think of adenosis of the vagina, herpetic ulceration, a syphilitic chancre or cancer, the diagnosis depending a little on the age of the patient. In our case that we report, continuous usage of super-absorbant tampons was accompanied by a large vaginal ulcer. Because of this case we have reviewed the literature about the cases that have been reported and have collected 36 cases.

[Exogenous reinfection with Mycobacterium tuberculosis caused by nosocomial transmission by a patient with AIDS]. / Exogene reïnfectie met Mycobacterium tuberculosis door nosocomiale transmissie bij een patiënt met AIDS.

A 55-year-old AIDS patient relapsed with tuberculosis as a result of exogenous reinfection, 1.5 years after a prior diagnosis of tuberculosis, for which he had been treated. He was reinfected after exposure to another AIDS patient, a 25-year-old man with tuberculosis, when they were hospitalized together during 5 days. The diagnosis of tuberculosis in the latter patient was delayed because the clinical picture was obscured by another infection. Reinfection and nosocomial transmission were demonstrated by analysis of the restriction-fragment-length polymorphism patterns on serial isolates of Mycobacterium tuberculosis. Increased alertness to tuberculosis, especially among HIV-infected persons, and implementation of effective infection control precautions are important in the prevention of nosocomial transmission.

[Transmission of sensitive and resistant strains of Mycobacterium tuberculosis in The Netherlands, 1993-1995, studied by means of DNA fingerprinting]. / Transmissie van gevoelige en resistente Mycobacterium tuberculosis-stammen in Nederland, 1993-1995, onderzocht met DNA-'fingerprinting'.

OBJECTIVE: Determination of the contribution of recent transmission of both sensitive and drug-resistant Mycobacterium tuberculosis strains to the number of tuberculosis cases in the Netherlands. DESIGN: Descriptive study. SETTING: National Institute for Public Health and Environment, Bilthoven, the Netherlands. METHODS: Since 1993 all isolates of M. tuberculosis in the Netherlands are sent to the national reference laboratory for surveillance purposes. The strains with IS6110 DNA fingerprint, isolated from January 1993 to July 1995, were analysed for clustering of DNA patterns (clustering of identical DNA patterns was assumed to represent recent transmission of tuberculosis). A transmission index was calculated from the ratio of the number recently infected tuberculosis patients and the number of source patients. RESULTS: Among 2,217 M. tuberculosis isolates, 1,313 unique DNA fingerprints were observed, while 264 DNA patterns occurred more than once. 904 (41%) DNA fingerprints were part of a cluster of identical fingerprints. The mean cluster size was 3.42. The 232 resistant strains showed significantly less clustering (33% versus 42%, p < 0.02) and a smaller transmission index (0.27 versus 0.42, p < 0.02) compared with sensitive strains. CONCLUSION: Recent transmission contributes to the magnitude of the tuberculosis problem in the Netherlands. The epidemiological situation would, however, lead to gradual elimination of the disease were it not for introduction of tuberculosis from other countries. Transmission of resistant strains is relatively limited. Micro-epidemics caused by resistant M. tuberculosis strains were not observed.

[Intravenously-administered immunoglobulins as first-choice agent in juvenile dermatomyositis]. / Intraveneus toegediend immunoglobuline als middel van eerste keuze bij juveniele dermatomyositis.

Dermatomyositis is an acquired disease characterised by symmetric predominantly proximal muscle weakness of the arms and legs, and misery. It may be associated with myalgia and there is often a characteristic rash. The mainstay of therapy is corticosteroids. Recently efficacy of intravenous immunoglobulin (IVIg) in chronic refractory dermatomyositis was reported. Because corticosteroids can cause serious side effects, we treated a seven-year-old girl suffering from dermatomyositis with IVIg as initial therapy. After two courses of IVIg infusions at a dose of 0.4 g/kg/day for five consecutive days, the patient made a rapid and complete recovery. This case shows that IVIg may be effective as initial therapy in patients with dermatomyositis. Whether IVIg is really a better treatment than corticosteroids should be investigated in a randomised study.

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.