Neoehrlichiosis in Symptomatic Immunocompetent Child, South Africa.

We describe a case of neoehrlichiosis in an immunocompetent child with acute febrile illness in South Africa. Neoehrlichiosis was diagnosed by PCR on 16S rDNA from bone marrow aspirate. Phylogenetic analysis indicated an organism closely related to Candidatus Neoehrlichia. Clinicians should be aware of possible ehrlichiosis even in immunocompetent patients.

Biofilm formation in invasive Staphylococcus aureus isolates is associated with the clonal lineage.

The contribution of the genetic background of Staphylococcus aureus to biofilm formation is poorly understood. We investigated the association between the genetic background and the biofilm forming ability of clinical invasive S. aureus isolates. Secondary objectives included investigating any correlation with biofilm formation and methicillin resistance or the source of bacteraemia. The study was conducted at a 1300-bed tertiary hospital in Cape Town, South Africa. S. aureus isolates obtained from blood cultures between January 2010 and January 2012 were included. Genotypic characterization was performed by PFGE, spa typing, SCCmec typing and MLST. Thirty genotypically unique strains were assessed for phenotypic biofilm formation with the microtitre plate assay. All isolates were tested in triplicate and an average optical density, measured at a wavelength of 490 nm, was determined. The biofilm forming ability of isolates with A490 ≤ 0.17 were considered non-adherent, A490 > 0.17 'weak positive' and A490 > 0.34 'strong positive'. Fifty seven percent of isolates formed biofilms. Weak biofilm formation occurred in 40% (n = 12) and strong biofilm formation in 17% (n = 5) of isolates. All 5 isolates capable of strong biofilm formation belong to one spa clonal complex (spa-CC 064). Strains from spa-CC 064 were capable of higher biofilm formation than other spa clonal complexes (p = 0.00002). These 5 strains belonged to MLST CC5 and CC8. Biofilm formation correlates with the spa clonal lineage in our population of invasive S. aureus strains. Biofilm formation did not correlate with methicillin resistance and was not related to the source of bacteraemia.

Commercial nucleic acid amplification tests in tuberculous meningitis--a meta-analysis.

Although nucleic acid amplification tests (NAATs) promise a rapid, definitive diagnosis of tuberculous meningitis, the performance of first-generation NAATs was suboptimal and variable. We conducted a meta-analysis of studies published between 2003 and 2013, using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool to evaluate methodological quality. The diagnostic accuracy of newer commercial NAATs was assessed. Pooled estimates of diagnostic accuracy for commercial NAATs measured against a cerebrospinal fluid Mycobacterium tuberculosis culture-positive gold standard were sensitivity 0.64, specificity 0.98, and diagnostic odds ratio 64.0. Heterogeneity was limited; P value = 0.147 and I(2) = 33.85%. The Xpert MTB/RIF® test was evaluated in 1 retrospective study and 4 prospective studies, with pooled sensitivity 0.70 and specificity 0.97. The QUADAS-2 tool revealed low risk of bias, as well as low concerns regarding applicability. Heterogeneity was pronounced among studies of in-house tests. Commercial NAATs proved to be highly specific with greatly reduced heterogeneity compared to in-house tests. Sub-optimal sensitivity remains a limitation.

Emergence and treatment of multidrug resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in South Africa.

Drug resistant tuberculosis (TB) has reached alarming proportions in South Africa, draining valuable resources that are needed to fight drug susceptible TB. It is currently estimated that 9.6% of all TB cases have multi-drug resistant (MDR)-TB, thereby ranking South Africa as one of the highest MDR-TB burden countries in the world. Molecular epidemiological studies have demonstrated the complexity of the epidemic and have clearly shown that the epidemic is driven by transmission as a consequence of low cases detection and diagnostic delay. The latter has in turn fueled the amplification of drug resistance, ultimately leading to the emergence of extensively drug resistant (XDR)-TB. Despite the introduction of new drugs to combat this scourge, culture conversion rates for XDR-TB remain below 20%. Failure to achieve cure may be explained from DNA sequencing results which have demonstrated mutations in 7 genes encoding resistance to at least 8 anti-TB drugs. This review shows how molecular epidemiology has provided novel insights into the MDR-TB epidemic in South Africa and thereby has highlighted the challenges that need to be addressed regarding the diagnosis and treatment of MDR-TB. An important step towards for curbing this epidemic will be collaboration between clinicians, laboratories and researchers to establish scientific knowledge and medical expertise to more efficiently guide public health policy.

Detecting drug-resistant tuberculosis: the importance of rapid testing.

Despite numerous intervention strategies, including the direct observed short-course treatment strategy and improved diagnostic methods, the incidence of multidrug-resistant and extensively drug-resistant tuberculosis (TB) continues to rise globally. Many treatment policies are based on the model that acquisition of drug resistance in already infected individuals drives the drug-resistant TB epidemic, hence the focus on drug-resistance testing of retreatment cases. However, molecular epidemiology and mathematical modeling suggest that the majority of multidrug-resistant TB cases are due to ongoing transmission of multidrug-resistant strains. This is most likely the result of diagnostic delay, thereby emphasizing the need for rapid diagnostics and comprehensive contact tracing, as well as active case finding. Current diagnosis of TB in low-income, high-burden regions relies on smear microscopy and clinical signs and symptoms. However, this smear-centered approach has many pitfalls, including low sensitivity in HIV patients and children, the inability of smear to reveal drug-resistance patterns, and the need for sampling on consecutive days. In order to address these limitations, efforts have been made to expand access to Mycobacterium tuberculosis culture and drug susceptibility testing. However, the slow growth rate of the causative agent, M. tuberculosis, contributes to significant diagnostic delay. Molecular-based diagnostic methods, targeting mutations that are known to confirm drug resistance, are capable of significantly reducing diagnostic delay. Two such methods, the line-probe assay and the real-time PCR-based Xpert® MTB/RIF assay, have been described. The latter test shows particular promise for smear-negative and extrapulmonary specimens. This may prove especially useful in settings where co-infection rates with HIV are high. However, since most research focuses on the performance of both of these assays, further investigations need to be done regarding the impact of the routine implementation of these assays on TB control programs and the cost effectiveness thereof.

Xpert MTB/RIF for rapid diagnosis of tuberculous lymphadenitis from fine-needle-aspiration biopsy specimens.

This study demonstrates the excellent diagnostic accuracy of the Xpert MTB/RIF test in patients with tuberculous lymphadenitis. The test sensitivity and specificity were 96.7% (95% confidence interval [CI], 86.6 to 100%) and 88.9% (95% CI, 69.6 to 100%), respectively, and it correctly identified 6/6 (100%) of the cytology smear-negative/culture-positive cases and 1 of 2 (50%) rifampin-resistant cases.

Tuberculosis assays: past, present and future.

Recent developments in the field of TB diagnostics, including the introduction of the Xpert MTB/RIF assay in field testing, raise the hope for faster and more accurate identification of active TB patients. However, there are still many issues that need to be addressed as no point-of-care tests are yet available. Furthermore, no tests are available which are universally applicable to all patients. Improvements in the microbiological and molecular-based approaches are promising and the diagnostic pipeline is encouraging. Host markers associated with active disease may hold promise, especially in situations where sputum diagnostics are problematic, including in children, HIV-infected individuals and in the case of extrapulmonary TB.

Combining fine-needle aspiration biopsy (FNAB) and high-resolution melt analysis to reduce diagnostic delay in Mycobacterial lymphadenitis.

Tuberculous lymphadenitis is the most common cause of extra-pulmonary tuberculosis (TB) in developing countries. Lymphadenitis caused by non-tuberculous mycobacteria (NTM) requires consideration, particularly in immunocompromised patients and children in developed countries. Fine-Needle Aspiration Biopsy (FNAB) offers a valuable specimen collection technique, but culture confirmation, mycobacterial speciation and drug resistance testing (if indicated) is often unavailable in TB endemic areas and result in unacceptable diagnostic delay. We evaluated the diagnostic value of high-resolution DNA melting (HRM) analysis in the diagnosis of mycobacterial lymphadenopathy using FNAB and an inexpensive transport medium. Specimens were collected from patients referred to the FNAB Clinic at Tygerberg Hospital (June 2007-May 2008) with clinical mycobacterial lymphadenitis. Cytology, culture, and HRM were performed on all specimens. The reference standard for disease was defined as positive cytology (morphological evidence plus mycobacterial visualization) and/or a positive culture. Specimens were collected from 104 patients and mycobacterial disease was confirmed in 54 (51.9%); 52 Mycobacterium tuberculosis, 1 Mycobacterium Bovis BCG and 1 NTM. Cytology was positive in 83.3% (45/54) and culture in 72.2% (39/54) of patients. HRM identified 57.4% (31/54) of cases. By using the defined reference standard, we recorded 94.0% specificity and 51.9% sensitivity (positive predictive value 90.3%) with HRM analysis.HRM analysis allowed rapid and species specific diagnosis of mycobacterial lymph adenitis in the majority of patients, permitting early institution of appropriate therapy. Optimization of this technique requires further study.