Therapeutic drug monitoring and TB treatment outcomes in patients with diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) increases the risk of TB disease and poor treatment outcomes such as delayed sputum culture conversion due to inadequate drug exposure. Therapeutic drug monitoring (TDM) has improved these outcomes in some settings.METHODS: To compare treatment outcomes in programs with routine TDM vs. programs that did not use TDM, we conducted a retrospective study among people with DM and TB at health departments in four US states.RESULTS: A total of 170 patients were enrolled (73 patients in the non-TDM group and 97 patients in the TDM group). Days to sputum culture conversion and total treatment duration were significantly shorter in the TDM group vs. the non-TDM group. In adjusted analyses, patients who underwent TDM were significantly more likely to achieve sputum culture conversion at 2 months (P = 0.007).CONCLUSION: TDM hastened microbiological cure from TB among people with DM and a high risk for poor treatment outcomes in the programmatic setting.

Clofazimine for Treatment of Cryptosporidiosis in Human Immunodeficiency Virus Infected Adults: An Experimental Medicine, Randomized, Double-blind, Placebo-controlled Phase 2a Trial.

BACKGROUND: We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis. METHODS: We performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis. RESULTS: Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls. CONCLUSIONS: Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy. CLINICAL TRIALS REGISTRATION: NCT03341767.

Discordance in Xpert® MTB/RIF assay results among low bacterial load clinical specimens in Bangladesh.

BACKGROUND: The Xpert® MTB/RIF assay detects Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. RIF-resistant (RIF-R) MTB cases detected using Xpert on sputum specimens at three private-sector TB screening centres in Dhaka, Bangladesh, were subjected to consecutive confirmatory Xpert testing, the results of which were MTB-positive/RIF-susceptible, MTB-positive/RIF-indeterminate or MTB-negative. OBJECTIVE: To assess the possible causes of discordant MTB and RIF-R results. METHODS: Discordant confirmatory Xpert test results were subjected to further investigations using the GenoType® MTBDRplus assay, culture and rpoB gene sequencing. RESULTS: The confirmatory Xpert test was performed on a remnant or a second specimen collected from individuals with an initial RIF-R result (n = 69); 22 (32%) results were discordant, 20 of which had an 'MTB detected-very low' result. Further investigations were mostly concordant with the confirmatory Xpert test. Average variability in paired cycle threshold (Ct) values were higher in 'MTB detected-very low' results vs. specimens with low, medium or high detected MTB results (P < 0.05); discordant results were mostly observed in specimens with 'MTB detected-very low' (20/22). CONCLUSIONS: Repeating the Xpert test and comparing with other available tests should be considered in case of 'MTB detected-very low, RIF resistance detected' results on Xpert.

Comparison of TaqMan(®) Array Card and MYCOTB(TM) with conventional phenotypic susceptibility testing in MDR-TB.

BACKGROUND: Although phenotypic drug susceptibility testing (DST) is endorsed as the standard for second-line drug testing of Mycobacterium tuberculosis, it is slow and laborious. METHODS: We evaluated the accuracy of two faster, easier methodologies that provide results for multiple drugs: a genotypic TaqMan(®) Array Card (TAC) and the Sensititre(®) MYCOTB(TM) plate. Both methods were tested at three central laboratories in Bangladesh, Tanzania, and Thailand with 212 multidrug-resistant tuberculosis (MDR-TB) isolates and compared with the laboratories' phenotypic method in use. RESULTS: The overall accuracy for ethambutol, streptomycin, amikacin, kanamycin, ofloxacin, and moxifloxacin vs. the phenotypic standard was 87% for TAC (range 70-99) and 88% for the MYCOTB plate (range 76-98). To adjudicate discordances, we re-defined the standard as the consensus of the three methods, against which the TAC and MYCOTB plate yielded 94-95% accuracy, while the phenotypic result yielded 93%. Some isolates with genotypic mutations and high minimum inhibitory concentration (MIC) were phenotypically susceptible, and some isolates without mutations and low MIC were phenotypically resistant, questioning the phenotypic standard. CONCLUSIONS: In our view, the TAC, the MYCOTB plate, and the conventional phenotypic method have similar performance for second-line drugs; however, the former methods offer speed, throughput, and quantitative DST information.

Undertreated HIV and drug-resistant tuberculosis at a referral hospital in Irkutsk, Siberia.

SETTING: A referral hospital for tuberculosis (TB) in Irkutsk, the Russian Federation. OBJECTIVE: To describe disease characteristics, treatment and hospital outcomes of TB-HIV (human immunodeficiency virus). DESIGN: Observational cohort of HIV-infected patients admitted for anti-tuberculosis treatment over 6 months. RESULTS: A total of 98 patients were enrolled with a median CD4 count of 147 cells/mm(3) and viral load of 205 943 copies/ml. Among patients with drug susceptibility testing (DST) results, 29 (64%) were multidrug-resistant (MDR), including 12 without previous anti-tuberculosis treatment. Nineteen patients were on antiretroviral therapy (ART) at admission, and 10 (13% ART-naïve) were started during hospitalization. Barriers to timely ART initiation included death, in-patient treatment interruption, and patient refusal. Of 96 evaluable patients, 21 (22%) died, 14 (15%) interrupted treatment, and 10 (10%) showed no microbiological or radiographic improvement. Patients with a cavitary chest X-ray (aOR 7.4, 95%CI 2.3-23.7, P = 0.001) or central nervous system disease (aOR 6.5, 95%CI 1.2-36.1, P = 0.03) were more likely to have one of these poor outcomes. CONCLUSION: High rates of MDR-TB, treatment interruption and death were found in an HIV-infected population hospitalized in Irkutsk. There are opportunities for integration of HIV and TB services to overcome barriers to timely ART initiation, increase the use of anti-tuberculosis regimens informed by second-line DST, and strengthen out-patient diagnosis and treatment networks.

Administrative interventions associated with increased initiation on antiretroviral therapy in Irkutsk, Siberia.

A bundle of initiatives to integrate human immunodeficiency virus (HIV) and tuberculosis (TB) services was assessed for the impact on antiretroviral therapy (ART) initiation at a TB referral hospital in Irkutsk, Russian Federation, from February 2014 to December 2015. The ART initiation rates in 166 ART-naïve patients undergoing anti-tuberculosis treatment (34.1% with multidrug or extensively drug-resistant TB) increased significantly from 14 (17%) pre-intervention to 44 (54%) post-intervention (P < 0.001). A survey of TB hospital staff identified administrative prioritisation as the most important initiative for increasing ART initiation.

Un ensemble d'initiatives visant à intégrer les services relatifs au virus de l'immunodéficience humaine (VIH) et à la tuberculose (TB) a été évalué en termes d'impact sur la mise en route du traitement antirétroviral (TAR) dans un hôpital de référence de la TB à Irkoutsk, Fédération de Russie, entre février 2014 et décembre 2015. Les taux de mise en route du TAR chez 166 patients n'en ayant jamais reçu et traités pour TB (34,1% avec une TB multi-résistante ou ultra-résistante) ont significativement augmenté de seulement 14 (17%) avant l'intervention à 44 (54%) après l'intervention (P < 0,001). Une enquête auprès du personnel de la TB a identifié la priorisation administrative comme l'initiative la plus importante dans l'augmentation de l'initiation du TAR.

Se evaluó un conjunto de iniciativas encaminadas a integrar los servicios de atención de la infección por el virus de la inmunodeficiencia humana (VIH) y la tuberculosis (TB), con el objeto de determinar la repercusión de la integración sobre el comienzo del tratamiento antirretrovírico (TAR) en el hospital de referencia de la TB de Irkutsk, en la Federación de Rusia, de febrero del 2014 a diciembre del 2015. La tasa de iniciación del TAR en 166 pacientes que nunca lo habían recibido y que estaban en curso de tratamiento antituberculoso (34,1% con TB multirresistente o extremadamente multirresistente) aumentó de manera significativa de solo 14 pacientes antes de la intervención (17%) a 44 pacientes después de la misma (54%; P < 0,001). Al interrogar al personal encargado de la TB en este hospital de referencia, se puso en evidencia que la priorización administrativa del TAR constituía la iniciativa de más había influido en el incremento de su utilización.

Outbreak of pyrazinamide-monoresistant tuberculosis identified using genotype cluster and social media analysis.

SETTING: Monoresistance to pyrazinamide (PZA) has infrequently been associated with Mycobacterium tuberculosis. OBJECTIVE: To report an outbreak of PZA-monoresistant M. tuberculosis in Virginia involving two genotype clusters from December 2004 to August 2010. RESULTS: Thirty cases were identified involving a predominantly young, US-born population with histories of substance use and incarceration and a large proportion of children aged <15 years (n= 6, 20%); of these, 23 cases (77%) were culture-confirmed as M. tuberculosis complex. DNA fingerprinting and molecular analysis of the PZA resistance gene, pncA, demonstrated a clonal strain that was not M. bovis. Genotypic data provided the initial link between seemingly unrelated cases, and helped reveal a historic genotype cluster of cases from 2004. Further genotype cluster and contact investigation procedures, including the novel use of the social networking website Facebook.com, revealed additional links between the 2004 and 2009 genotype clusters and described an ongoing, extensive outbreak necessitating an enhanced screening and treatment protocol for contacts. CONCLUSIONS: This outbreak demonstrates how tuberculosis can spread through a young, vulnerable population. The use of genotypic data and the novel incorporation of social media investigations were critical to understanding the settings and context of infectivity.

New concepts in diagnostics for infectious diarrhea.

Conventional approaches to the diagnosis of infectious diarrhea must include several modalities to detect an array of potential viruses, bacteria, and parasites. We will provide a general overview of the wide range of diagnostic modalities available for enteropathogens, briefly discuss some of the limitations of conventional methods, and then focus on new molecular methods, including real-time PCR and next-generation sequencing. In particular, we will discuss quantitation of pathogen load with these techniques. We will then describe examples whereby novel diagnostics may help illuminate the etiology of infectious diarrhea, where they may not, and how they may benefit studies of immunity to enteric infections.

Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica.

Leptin is an adipocytokine that links nutrition to immunity. Previous observation that a genetic polymorphism in the leptin receptor affected susceptibility to Entamoeba histolytica infection led to the hypothesis that leptin signaling has a protective role during intestinal amebic infection. In this study we show that mice lacking the functional leptin receptor developed devastating mucosal destruction after E. histolytica infection. Bone marrow chimera experiments demonstrated that leptin receptor expressed on hematopoietic cells was not sufficient to confer resistance. Similarly, peripheral knockout of the leptin receptor rendered animals susceptible, indicating that central expression of the leptin receptor was not sufficient to confer protection. The site of leptin action was localized to the gut via an intestinal epithelium-specific deletion of the leptin receptor, which rendered mice susceptible to infection and mucosal destruction by the parasite. Mutation of tyrosine 985 or 1138 in the intracellular domain of the leptin receptor, which mediates signaling through the SH2-containing tyrosine phosphatase/extracellular signal-regulated kinase (SHP2/ERK) and signal transducer and activator of transcription 3 (STAT3) pathways, respectively, demonstrated that both were important for mucosal protection. We conclude that leptin-mediated resistance to amebiasis is via its actions on intestinal epithelium rather than hematopoietic cells or the brain, and requires leptin receptor signaling through both the STAT3 and SHP2/ERK pathways.

Persistence of Entamoeba histolytica infection in CBA mice owes to intestinal IL-4 production and inhibition of protective IFN-gamma.

The mechanisms whereby certain mouse strains develop persistent intestinal infection with Entamoeba histolytica remain unclear. In this work, we characterized the kinetic pattern of cytokine responses during the course of natural infection in CBA mice and showed that intracecal amebic infection led to a rapid and sustained upregulation of Th2 (IL-4, IL-5, IL-13) and Th17 cytokine responses while Th1 cytokines, IL-12p35 and interferon (IFN)-gamma, were suppressed. Depletion of IL-4 cleared infection by 14 days post-challenge, and this clearance correlated with and was mediated by IFN-gamma. The protective role for IFN-gamma was not strain-specific, as 129 background IFN-gammaR knockout mice exhibited a higher infection rate than their wild-type littermates. These studies indicate that IL-4 plays a critical pathogenic role in the persistence of E. histolytica infection through suppression of protective IFN-gamma and provide a possible explanation for why certain humans spontaneously clear amebiasis while others progress to invasive disease.

Gender and genetic control of resistance to intestinal amebiasis in inbred mice.

Resistance to the establishment of intestinal Entamoeba histolytica infection is dependent on the inbred mouse strain. In this work we used the inbred strains B6 (resistant), CBA (susceptible), B6CBAF(1) and a backcross of B6CBAF(1) to CBA to further examine the genetic basis of resistance. Mouse genotype was assessed with single nucleotide polymorphism and microsatellite markers and infection assessed by culture 9 days after intracecal E. histolytica challenge. The backcross population showed a male predisposition to culture positivity (P<0.002). F1 genotype at two loci on chromosomes 1 and 2 exhibited suggestive linkage with resistance to infection (P=0.0007 and 0.0200). Additional suggestive quantitative trait locus were observed on chromosomes 1, 9 and 13 for cecal parasite antigen load and histologic evidence of inflammation. Infection in C3H x B6 recombinant inbred mice supported the mapping data. Candidate B6 genes on chromosomes 1 and 2 were examined by microarray analysis of epithelial tissues from B6 vs CBA mice. This work shows a male predisposition to intestinal amebiasis and suggests that relatively few B6 loci can confer resistance in inbred mice. Future identification of regional candidate genes has implications for understanding the human variability to amebic infection.

Entamoeba histolytica: genetic diversity of African strains based on the polymorphism of the serine-rich protein gene.

The polymorphism of the serine-rich Entamoeba histolytica protein (SREHP) among isolates obtained from different geographic regions was analyzed by a nested PCR followed by restriction analysis. Thirteen different profiles were generated from 23 E. histolytica isolates from Cameroon, Zimbabwe and South Africa while 20 others were generated from 38 E. histolytica PCR positive stool samples from South Africa. One of the profiles was common to isolates from Cameroon, Zimbabwe and South Africa and constituted the most prevalent (26.1%) of all the profiles. However, profiles unique to each country were also observed amongst the samples. A non-significant difference was observed between isolates from diarrheic and non-diarrheic samples. Of interest, of the five HIV positive stool samples three had the same profile indicating the possibility that some E. histolytica strains might be more common/pathogenic in immuno-compromised individuals. The results obtained showed that African isolates of E. histolytica may possess extremely complex genetic structures independent of geographic location. This study indicates that certain profiles might be responsible for the presentation of intestinal amoebic symptoms. However, more extended studies need to be performed in order to confirm these observations.

Bronchoalveolar neutrophils, interferon gamma-inducible protein 10 and interleukin-7 in AIDS-associated tuberculosis.

During advanced AIDS tuberculosis (TB) often presents atypically with smear-negative and non-cavitary disease, yet immune features associated with this change are poorly characterized. We examined the local immune response in a cohort of Tanzanian AIDS-associated TB patients who underwent bronchoalveolar lavage. TB infection was confirmed in bronchoalveolar lavage (BAL) fluid by culture, probe and polymerase chain reaction (PCR). Among TB patients CD4 count correlated positively with the extent of cavitary disease as well as BAL TB load (qPCR C(T)). TB patients had significantly higher granulocyte-macrophage colony-stimulating factor (GM-CSF) than non-TB patients, and those with non-cavitary TB had significantly higher BAL interferon gamma-inducible protein (IP-10) and interleukin (IL)-7 than those with cavities. BAL neutrophils were as prevalent as monocytes/macrophages or epithelial cells, and immunohistochemistry revealed that neutrophils, monocytes/macrophages, and epithelial cells were major sources of the IP-10 and IL-7. These data suggest a dysregulated cytokine profile may contribute to the TB of advanced AIDS.

Cryptosporidium species: preliminary descriptions of the prevalence and genotype distribution among school children and hospital patients in the Venda region, Limpopo Province, South Africa.

In the present study, the prevalence and species distribution of Cryptosporidium among school children and hospital patients in the Venda region of South Africa was determined. Real time PCR (qPCR) was used for initial screening to detect positive samples while a nested PCR followed by restriction fragment length polymorphism was used to determine the species genotype. From a total of 244 stool samples tested, 44 (18%) had Cryptosporidium with no significant difference (chi(2)=0.04; P=0.841) between samples collected from patients attending hospitals 36/197 (18%) and the samples from primary schools 8/47 (17%). The age groups most affected were those from 2 to 5 years old (28.6%) and 50 to 59 years old (50.0%). Cryptosporidium was detected in 4 (12.5%) of the 31 HIV positive individuals. Fifty-seven percent of the Cryptosporidium positive samples were diarrheic and 26 (59.1%) had elevated lactoferrin content. C. hominis (82%) was more common than C. parvum (18%). This study has demonstrated the high prevalence of Cryptosporidium infections in the Venda region and its implications in causing diarrhea and inflammation.

Caspase 3-dependent killing of host cells by the parasite Entamoeba histolytica.

The parasite Entamoeba histolytica is named for its ability to lyse host tissues. To determine the factors responsible, we have initiated an examination of the contribution of parasite virulence factors and host caspases to cellular destruction by the parasite. Amoebic colitis in C3H/HeJ mice was associated with extensive host apoptosis at sites of E. histolytica invasion. In vitro studies of E. histolytica-Jurkat T-cell interactions demonstrated that apoptosis required contact via the amoebic Gal/GalNAc lectin, but was unaffected by 75% inhibition of the amoebic cysteine proteinases. Parasite-induced DNA fragmentation was unaffected in caspase 8-deficient Jurkat cells treated with the caspase 9 inhibitor Ac-LEHD-fmk. In contrast, caspase 3-like activity was observed within minutes of E. histolytica contact and the caspase 3 inhibitor Ac-DEVD-CHO blocked Jurkat T cell death, as measured by both DNA fragmentation and 51Cr release. These data demonstrate rapid parasite-induced activation of caspase 3-like caspases, independent of the upstream caspases 8 and 9, which is required for host cell death.