Population Structure of Mixed Mycobacterium tuberculosis Infection Is Strain Genotype and Culture Medium Dependent

Background

Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection.

Aim

This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein–Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes.

Method

A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes.

Results

The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with “other genotype” strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype.

Conclusion

The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.     

Time to Culture Positivity and Sputum Smear Microscopy during Tuberculosis Therapy

Sputum smear microscopy is widely used for tuberculosis diagnosis and treatment monitoring. We evaluated the correlation between smear microscopy and time to liquid culture positivity during early tuberculosis treatment. The study included patients with smear-positive pulmonary tuberculosis hospitalized at a tuberculosis reference centre in Germany between 01/2012 and 05/2013. Patient records were reviewed and clinical, radiological and microbiological data were analysed. Sputum samples were collected before treatment initiation and weekly thereafter. A number of 310 sputum samples from 30 patients were analysed. Time to liquid culture positivity inversely correlated with smear grade (Spearman''s rho −0.439, p<0.001). There was a better correlation within the first two months vs. after two months of therapy (−0.519 vs. −0.416) with a trend to a more rapid increase in time to positivity between baseline and week 2 in patients who culture-converted within the first two months (5.9 days vs. 9.4 days, p = 0.3). In conclusion, the numbers of acid-fast bacilli in sputum smears of patients with pulmonary tuberculosis and time to culture positivity for M. tuberculosis cultures from sputum are correlated before and during tuberculosis treatment. A considerable proportion of patients with culture conversion after two months of therapy continued to have detectable acid-fast bacilli on sputum smears.     

Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of Drug-Resistant Tuberculosis in Harare,Zimbabwe

Introduction

Limited data exist on use of the microscopic-observation drug-susceptibility (MODS) assay among persons suspected of MDR-TB living in high HIV-prevalence settings.

Methods

We retrospectively reviewed available clinical and drug susceptibility data for drug-resistant TB suspects referred for culture and drug-susceptibility testing between April 1, 2011 and March 1, 2012. The diagnostic accuracy of MODS was estimated against a reference standard including Löwenstein-Jensen (LJ) media and manual liquid (BACTEC MGIT) culture. The accuracy of MODS drug-susceptibility testing (DST) was assessed against a reference standard absolute concentration method.

Results

One hundred thirty-eight sputum samples were collected from 99 drug-resistant TB suspects; in addition, six previously cultured MDR isolates were included for assessment of DST accuracy. Among persons with known HIV infection status, 39/59 (66%) were HIV-infected. Eighty-six percent of patients had a history of prior TB treatment, and 80% of individuals were on antituberculous treatment at the time of sample collection. M. tuberculosis was identified by reference standard culture among 34/98 (35%) MDR-TB suspects. Overall MODS sensitivity for M. tuberculosis detection was 85% (95% CI, 69–95%) and specificity was 93% (95% CI, 84–98%); diagnostic accuracy did not significantly differ by HIV infection status. Median time to positivity was significantly shorter for MODS (7 days; IQR 7–15 days) than MGIT (12 days; IQR 6–16 days) or LJ (28 days; IQR 21–35 days; p<0.001). Of 33 specimens with concurrent DST results, sensitivity of the MODS assay for detection of resistance to isoniazid, rifampin, and MDR-TB was 88% (95% CI, 68–97%), 96% (95% CI, 79–100%), and 91% (95% CI, 72–99%), respectively; specificity was 89% (95% CI, 52–100%), 89% (95% CI, 52–100%), and 90% (95% CI, 56–100%), respectively.

Conclusion

In a high HIV-prevalence setting, MODS diagnosed TB and drug-resistant TB with high sensitivity and shorter turnaround time compared with standard culture and DST methods.     

Blood Neutrophil Counts in HIV-Infected Patients with Pulmonary Tuberculosis: Association with Sputum Mycobacterial Load

Background

Increasing evidence suggests that neutrophils play a role in the host response to Mycobacterium tuberculosis. We determined whether neutrophil counts in peripheral blood are associated with tuberculosis (TB) and with mycobacterial load in sputum in HIV-infected patients.

Methodology/Principal Findings

Adults enrolling in an antiretroviral treatment (ART) clinic in a Cape Town township were screened for TB regardless of symptoms. Paired sputum samples were examined using liquid culture, fluorescence microscopy, and the Xpert MTB/RIF assay. Absolute neutrophil counts (ANC) were measured in blood samples. Of 602 HIV-infected patients screened, 523 produced one or more sputum samples and had complete results available for analysis. Among these 523 patients, the median CD4 count was 169×10⁹/L (IQR, 96–232) and median ANC was 2.6×10⁹/L (IQR, 1.9–3.6). Culture-positive pulmonary tuberculosis was diagnosed in 89 patients. Patients with TB had a median ANC of 3.4×10⁹/L (IQR, 2.4–5.1) compared to 2.5×10⁹/L (IQR, 1.8–3.4) among those who were culture negative (p<0.0001). In multivariable analyses, having pulmonary TB was associated with an adjusted risk ratio (aRR) of 2.6 (95%CI, 1.5–4.5) for having an ANC level that exceeded the median value (ANC ≥2.6×10⁹/L; p = 0.0006) and an aRR of 6.8 (95%CI, 2.3–20.4) for having neutrophilia defined by a neutrophil count exceeding the upper limit of the normal range (ANC >7.5×10⁹/L; p = 0.0005). Patients were then classified into four mutually exclusive groups with increasing sputum mycobacterial load as defined by the results of culture, Xpert MTB/RIF and sputum smear microscopy. Multivariable analyses demonstrated that increasing sputum mycobacterial load was positively associated with blood ANC ≥2.6×10⁹/L and with neutrophilia.

Conclusions/Significance

Increased blood neutrophil counts were independently associated with pulmonary TB and sputum mycobacterial burden in this HIV-infected patient group. This observation supports the growing body of literature regarding the potential role for neutrophils in the host response to TB.     

Development of a One-Step Probe Based Molecular Assay for Rapid Immunodiagnosis of Infection with M. tuberculosis Using Dried Blood Spots

Background

Antigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with M. tuberculosis. Compared to Interferon-γ release assays (IGRA), IP-10 is released in high levels enabling novel approaches such as field friendly dried blood spots (DBS) and molecular detection.

Aim

To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS.

Method

We developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay was validated and applied for the diagnosis of M. tuberculosis infection in DBS samples from 43 patients with confirmed TB, 13 patients with latent TB and 96 presumed uninfected controls. In parallel, IP-10 and INF-γ levels were measured in Quantiferon (QFT-TB) plasma supernatants.

Results

IP-10 mRNA upregulation was detectable at 4 hours after stimulation (6 fold upregulation) peaking at 8 hours (108 fold upregulation). IFN-γ expression occurred in concert but levels were lower (peak 6.7 fold upregulation). IP-10 gene expression level was significantly higher in patients with tuberculosis (median 31.2, IQR 10.7–67.0) and persons with latent tuberculosis infection (LTBI) (41.2, IQR 9.8–64.9) compared to healthy controls (1.6, IQR 1.1–2.4; p<0.0001). The IP-10 mRNA and protein based tests had comparable diagnostic accuracy to QFT-TB, sensitivity (85% and 88% vs 85%) and specificity (96% and 96% vs 97%, p = ns.).

Conclusion

We developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce the local technological requirements everywhere and make it possible to offer highly accurate immunodiagnostic tests in low resource settings.     

Clinical Utility of a Novel Molecular Assay in Various Combination Strategies with Existing Methods for Diagnosis of HIV-Related Tuberculosis in Uganda

Background

Low income, high-tuberculosis burden, countries are considering selective deployment of Xpert MTB/RIF assay (Xpert) due to high cost per test. We compared the diagnostic gain of the Xpert add-on strategy with Xpert replacement strategy for pulmonary tuberculosis diagnosis among HIV-infected adults to inform its implementation.

Methods

The first diagnostic sputum sample of 424 HIV-infected adults (67% with CD4 counts ≤200/mm³) suspected for tuberculosis was tested by direct Ziehl-Neelsen (DZN) and direct fluorescent microscopy (DFM); concentrated fluorescent microscopy (CFM); Lowenstein-Jensen (LJ) and Mycobacterial Growth Indicator Tube (MGIT) culture; and Xpert. Overall diagnostic yield and sensitivity were calculated using MGIT as reference comparator. The sensitivity of Xpert in an add-on strategy was calculated as the number of smear negative but Xpert positive participants among MGIT positive participants.

Results

A total of 123 (29.0%) participants were MGIT culture positive for Mycobacterium tuberculosis. The sensitivity (95% confidence interval) was 31.7% (23.6–40.7%) for DZN, 35.0% (26.5–44.0%) for DFM, 43.9% (34.9–53.1%) for CFM, 76.4% (67.9–83.6) for Xpert and 81.3% (73.2–87.7%) for LJ culture. Add-on strategy Xpert showed an incremental sensitivity of 44.7% (35.7–53.9%) when added to DZN, 42.3% (33.4–51.5%) to DFM and 35.0% (26.5–44.0%) to CFM. This translated to an overall sensitivity of 76.4%, 77.3% and 79.0% for add-on strategies based on DZN, DFM and CFM, respectively, compared to 76.4% for Xpert done independently. From replacement to add-on strategy, the number of Xpert cartridges needed was reduced by approximately 10%.

Conclusions

Among HIV-infected TB suspects, doing smear microscopy prior to Xpert assay in add-on fashion only identifies a few additional TB cases.     

Impact of Xpert MTB/RIF Testing on Tuberculosis Management and Outcomes in Hospitalized Patients in Uganda

Rationale

The clinical impact of Xpert MTB/RIF for tuberculosis (TB) diagnosis in high HIV-prevalence settings is unknown.

Objective

To determine the diagnostic accuracy and impact of Xpert MTB/RIF among high-risk TB suspects.

Methods

We prospectively enrolled consecutive, hospitalized, Ugandan TB suspects in two phases: baseline phase in which Xpert MTB/RIF results were not reported to clinicians and an implementation phase in which results were reported. We determined the diagnostic accuracy of Xpert MTB/RIF in reference to culture (solid and liquid) and compared patient outcomes by study phase.

Results

477 patients were included (baseline phase 287, implementation phase 190). Xpert MTB/RIF had high sensitivity (187/237, 79%, 95% CI: 73–84%) and specificity (190/199, 96%, 95% CI: 92–98%) for culture-positive TB overall, but sensitivity was lower (34/81, 42%, 95% CI: 31–54%) among smear-negative TB cases. Xpert MTB/RIF reduced median days-to-TB detection for all TB cases (1 [IQR 0–26] vs. 0 [IQR 0–1], p<0.001), and for smear-negative TB (35 [IQR 22–55] vs. 22 [IQR 0–33], p = 0.001). However, median days-to-TB treatment was similar for all TB cases (1 [IQR 0–5] vs. 0 [IQR 0–2], p = 0.06) and for smear-negative TB (7 [IQR 3–53] vs. 6 [IQR 1–61], p = 0.78). Two-month mortality was also similar between study phases among 252 TB cases (17% vs. 14%, difference +3%, 95% CI: −21% to +27%, p = 0.80), and among 87 smear-negative TB cases (28% vs. 22%, difference +6%, 95% CI: −34 to +46%, p = 0.77).

Conclusions

Xpert MTB/RIF facilitated more accurate and earlier TB diagnosis, leading to a higher proportion of TB suspects with a confirmed TB diagnosis prior to hospital discharge in a high HIV/low MDR TB prevalence setting. However, our study did not detect a decrease in two-month mortality following implementation of Xpert MTB/RIF possibly because of insufficient powering, differences in empiric TB treatment rates, and disease severity between study phases.     

High Tuberculosis Prevalence in a South African Prison: The Need for Routine Tuberculosis Screening

Background

Tuberculosis is a major health concern in prisons, particularly where HIV prevalence is high. Our objective was to determine the undiagnosed pulmonary tuberculosis (“undiagnosed tuberculosis”) prevalence in a representative sample of prisoners in a South African prison. In addition we investigated risk factors for undiagnosed tuberculosis, to explore if screening strategies could be targeted to high risk groups, and, the performance of screening tools for tuberculosis.

Methods and Findings

In this cross-sectional survey, male prisoners were screened for tuberculosis using symptoms, chest radiograph (CXR) and two spot sputum specimens for microscopy and culture. Anonymised HIV antibody testing was performed on urine specimens. The sensitivity, specificity and predictive values of symptoms and investigations were calculated, using Mycobacterium tuberculosis isolated on sputum culture as the gold standard.From September 2009 to October 2010, 1046 male prisoners were offered enrolment to the study. A total of 981 (93.8%) consented (median age was 32 years; interquartile range [IQR] 27–37 years) and were screened for tuberculosis. Among 968 not taking tuberculosis treatment and with sputum culture results, 34 (3.5%; 95% confidence interval [CI] 2.4–4.9%) were culture positive for Mycobacterium tuberculosis. HIV prevalence was 25.3% (242/957; 95% CI 22.6–28.2%). Positive HIV status (adjusted odds ratio [aOR] 2.0; 95% CI 1.0–4.2) and being an ex-smoker (aOR 2.6; 95% CI 1.2–5.9) were independently associated with undiagnosed tuberculosis. Compared to the gold standard of positive sputum culture, cough of any duration had a sensitivity of 35.3% and specificity of 79.6%. CXR was the most sensitive single screening modality (sensitivity 70.6%, specificity 92.2%). Adding CXR to cough of any duration gave a tool with sensitivity of 79.4% and specificity of 73.8%.

Conclusions

Undiagnosed tuberculosis and HIV prevalence was high in this prison, justifying routine screening for tuberculosis at entry into the prison, and intensified case finding among existing prisoners.     

A Meta-Analysis of P2X7 Gene-762T/C Polymorphism and Pulmonary Tuberculosis Susceptibility

AimWe performed a comprehensive meta-analysis to determine the association between P2X7 -762T/C polymorphism and pulmonary tuberculosis susceptibility.MethodologyBased on comprehensive searches of the PubMed, SCI, Elsevier, China National Knowledge Infrastructure (CNKI) and Wanfang Database, we identified eligible studies about the association between P2X7 -762T/C polymorphism and pulmonary tuberculosis risk. Pooled odds ratio (ORs) and 95% confidence intervals (95%CIs) were calculated in random-effects model.ResultsA total of 2207 tuberculosis cases and 2220 controls in 8 case-control studies were included in this meta-analysis. Allele model (C vs. T: p = 0.15; OR = 0.83, 95% CI = 0.65–1.07), homozygous model (CC vs. TT: p = 0.23; OR = 0.73, 95% CI = 0.44 to 1.22), and heterozygous model (CT vs. TT: p = 0.57; OR = 0.92, 95% CI = 0.68 to 1.24) did not show increased risk of developing pulmonary tuberculosis. Similarly, dominant model (CC+CT vs. TT: p = 0.32; OR = 0.84, 95% CI = 0.59 to 1.19) and recessive model (CC vs. CT+TT: p = 0.08; OR = 0.77, 95% CI = 0.57 to 1.04) failed to show increased risk of developing pulmonary tuberculosis. Subgroup analysis by ethnicity did not detect any significant association between P2X7–762T/C polymorphism and pulmonary tuberculosis susceptibility.ConclusionsP2X7 -762T/C gene polymorphism is not associated with pulmonary tuberculosis susceptibility.     

Culture Conversion Rate at 2 Months of Treatment According to Diagnostic Methods among Patients with Culture-Positive Pulmonary Tuberculosis

Introduction

The culture-negative conversion rate of sputum after 2 months of treatment in patients with pulmonary tuberculosis (TB) is used as a reliable surrogate marker for relapse after completion of treatment. We hypothesized that culture conversion of sputum at 2 months of anti-TB treatment and the time to culture conversion are different among pulmonary TB patients who are diagnosed using different methods.

Methods

Culture-confirmed pulmonary TB patients who were diagnosed between 1 January, 2011 and 31 December, 2012 were classified into three groups based on the diagnostic method that prompted treatment initiation: positive acid-fast bacilli (AFB) staining of sputum (smear-positive group), negative AFB staining, but Mycobacterium tuberculosis was cultured from sputum (culture-positive group), and positive AFB staining, positive polymerase chain reaction (PCR) for M. tuberculosis, or culture of M. tuberculosis from a bronchoscopic specimen (bronchoscopy group). Rates of negative mycobacterial culture conversion at 2 months of anti-TB treatment and the time to negative culture conversion of sputum were compared among the three groups.

Results

A total of 203 patients with culture-confirmed pulmonary TB were included in the final analysis. TB patients in the culture-positive group (94.1%) and the bronchoscopy group (97.6%) showed a higher culture conversion rate at 2 months of treatment than those in the smear-positive group (78.7%, P = 0.001). Additionally, the time to culture conversion was longer in the smear-positive group (median, 40 days) than in the culture-positive (median, 19 days; P = 0.009) and bronchoscopy groups (median, 29 days; P = 0.004).

Conclusions

The higher culture conversion rate at 2 months and the shorter time to culture conversion among pulmonary TB patients with a negative AFB smear suggests the feasibility of shortening treatment duration and isolation in these patients.     

IFNγ Response to Mycobacterium tuberculosis,Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia

Objectives

Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development.

Methods

A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.

Results

Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007).

Discussion

CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.     

Association of Genetic Variants in Wnt Signaling Pathway with Tuberculosis in Chinese Han Population

Compelling studies have implicated that the Wnt signaling pathway plays an important role in the development and progression of tuberculosis, however, there is little literature addressing the role of polymorphisms in Wnt pathway on tuberculosis. We took a pathway based candidate gene approach to investigate the possible correlation between genetic variants in Wnt pathway and tuberculosis. Three single nucleotide polymorphisms (SNPs) in Wnt pathway (rs4135385 in CTNNB1 gene, rs7832767 in SFRP1 gene, and rs11079571 in AXIN2 gene) were genotyped in 422 Chinese Han tuberculosis patients and 402 frequency matched (age, gender, and ethnicity) controls using high-resolution melting analysis. The genotype and allelic frequencies of rs4135385 and rs7832767 were significantly different among patients and controls. The dominant model of rs4135385 was significantly associated with an increased risk of tuberculosis (AG/GG versus AA: OR = 1.49, 95% CI = 1.06–2.09, p = 0.019). The recessive model of rs7832767 posed a significant higher risk for tuberculosis (TT versus TC/CC, OR = 2.70, 95% CI = 1.41–5.18, p = 0.002). These SNPs were further evaluated whether they were correlated with the site of tuberculosis and the level of inflammatory markers. Rs7832767 was significantly associated with the level of CRP (p = 0.014), and the patients carrying T allele might present with elevated CRP values (OR = 1.90, 95% CI = 1.21–2.96, p = 0.005). Our study provided the first evidence that rs4135385 and rs7832767 were associated with tuberculosis risk, and genetic variants in Wnt signaling pathway might participate in genetic susceptibility to tuberculosis in Chinese Han population. Further epidemiological and functional studies in larger populations are warranted to verify our results.     

Time to Treatment and Patient Outcomes among TB Suspects Screened by a Single Point-of-Care Xpert MTB/RIF at a Primary Care Clinic in Johannesburg,South Africa

Introduction

In December 2010, the World Health Organization recommended a single Xpert MTB/RIF assay as the initial diagnostic in people suspected of HIV-associated or drug resistant tuberculosis. Few data are available on the impact of this recommendation on patient outcomes. We describe the diagnostic follow-up, clinical characteristics and outcomes of a cohort of tuberculosis suspects screened using a single point-of-care Xpert.

Methods

Consecutive tuberculosis suspects at a primary care clinic in Johannesburg, South Africa were assessed for tuberculosis using point-of-care Xpert. Sputum smear microscopy and liquid culture were performed as reference standards. Xpert-negatives were evaluated clinically, and further assessed at the discretion of clinicians. Participants were followed for six months.

Results

From July-September 2011, 641 tuberculosis suspects were enrolled, of whom 69% were HIV-infected. Eight percent were positive by a single Xpert. Among 116 individuals diagnosed with TB, 66 (57%) were Xpert negative, of which 44 (67%) were empirical or radiological diagnoses and 22 (33%) were Xpert negative/culture-positive. The median time to tuberculosis treatment was 0 days (IQR: 0–0) for Xpert positives, 14 days (IQR: 5–35) for those diagnosed empirically, 14 days (IQR: 7–29) for radiological diagnoses, and 144 days (IQR: 28–180) for culture positives. Xpert negative tuberculosis cases were clinically similar to Xpert positives, including HIV status and CD4 count, and had similar treatment outcomes including mortality and time to antiretroviral treatment initiation.

Conclusions

In a high HIV-burden setting, a single Xpert identified less than half of those started on tuberculosis treatment, highlighting the complexity of TB diagnosis even in the Xpert era. Xpert at point-of-care resulted in same day treatment initiation in Xpert-positives, but had no impact on tuberculosis treatment outcomes or mortality.     

Potential Role for IL-2 ELISpot in Differentiating Recent and Remote Infection in Tuberculosis Contact Tracing

Interferon (IFN)-γ release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-γ ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-γ ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (≥100 hours) to the index case increased the risk of positive results in the IFN-γ ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-γ ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-γ ELISpot/positive IL-2 ELISpot result (median = 24 hours; 20_130; p-value = 0.047). Combination of a M. tuberculosis specific IFN-γ ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy.     

Reduction of Maternal Mortality with Highly Active Antiretroviral Therapy in a Large Cohort of HIV-Infected Pregnant Women in Malawi and Mozambique

Background

HIV infection is a major contributor to maternal mortality in resource-limited settings. The Drug Resource Enhancement Against AIDS and Malnutrition Programme has been promoting HAART use during pregnancy and postpartum for Prevention-of-mother-to-child-HIV transmission (PMTCT) irrespective of maternal CD4 cell counts since 2002.

Methods

Records for all HIV+ pregnancies followed in Mozambique and Malawi from 6/2002 to 6/2010 were reviewed. The cohort was comprised by pregnancies where women were referred for PMTCT and started HAART during prenatal care (n = 8172, group 1) and pregnancies where women were referred on established HAART (n = 1978, group 2).

Results

10,150 pregnancies were followed. Median (IQR) baseline values were age 26 years (IQR:23–30), CD4 count 392 cells/mm³ (IQR:258–563), Viral Load log₁₀ 3.9 (IQR:3.2–4.4), BMI 23.4 (IQR:21.5–25.7), Hemoglobin 10.0 (IQR: 9.0–11.0). 101 maternal deaths (0.99%) occurred during pregnancy to 6 weeks postpartum: 87 (1.1%) in group 1 and 14 (0.7%) in group 2. Mortality was 1.3% in women with <than 350 CD4 cells/mm³ and 0.7% in women with greater than 350 CD4s cells/mm³ [OR = 1.9 (CL 1.3–2.9) p = 0.001]. Mortality was higher in patients with shorter antenatal HAART: 22/991 (2.2%) if less than 30 days and 79/9159 (0.9%) if 31 days or greater [OR = 2.6 (CL 1.6–4.2) p<0.001]. By multivariate analysis, shorter antenatal HAART (p<0.001), baseline values for CD4 cell count (p = 0.012), hemoglobin (p = 0.02), and BMI (p<0.001) were associated with mortality. Four years later, survival was 92% for women with shorter antenatal HAART and 98% for women on established therapy prior to pregnancy, p = 0.001.

Conclusions

Antiretrovirals for PMTCT purposes have significant impact on maternal mortality as do CD4 counts and nutritional status. In resource-limited settings, PMTCT programs should provide universal HAART to all HIV+ pregnant women given its impact in prevention of maternal death.     

Assessment of the N-PCR Assay in Diagnosis of Pleural Tuberculosis: Detection of M.tuberculosis in Pleural Fluid and Sputum Collected in Tandem

Background

The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized.

Methodology/Principal Findings

Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation.

Conclusions/Significance

To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.     

Contact Investigation in Households of Patients with Tuberculosis in Hanoi,Vietnam: A Prospective Cohort Study

Setting

Existing tuberculosis control strategies in Vietnam are based on symptomatic patients attending health services for investigation. This approach has not resulted in substantial reductions in the prevalence of tuberculosis disease, despite the National Tuberculosis Program achieving high treatment completion rates. Alternative approaches are being considered.

Objective

To determine the feasibility and yield of contact investigation in households of patients with smear positive pulmonary tuberculosis among household members of tuberculosis patients in Hanoi, Vietnam.

Methods

Household contacts of patients with smear positive pulmonary tuberculosis were recruited at four urban and rural District Tuberculosis Units in Hanoi. Clinical and radiological screening was conducted at baseline, six months and 12 months. Sputum microscopy and culture was performed in contacts suspected of having tuberculosis. MIRU-VNTR molecular testing was used to compare the strains of patients and their contacts with disease.

Results

Among 545 household contacts of 212 patients, four were diagnosed with tuberculosis at baseline (prevalence 734 cases per 100,000 persons, 95% CI 17–1451) and one was diagnosed with tuberculosis during the subsequent 12 months after initial screening (incidence 180 cases per 100,000 person-years, 95% CI 44–131). Two of these cases were culture positive for M. tuberculosis and both had identical or near-identical MIRU-VNTR strain types.

Conclusion

Household contacts of patients with potentially infectious forms of tuberculosis have a high prevalence of disease. Household contact investigation is feasible in Vietnam. Further research is required to investigate its effectiveness.     

Yield of Two Consecutive Sputum Specimens for the Effective Diagnosis of Pulmonary Tuberculosis

Background

From long instances, it is debatable whether three sputum specimens are required for the diagnosis of pulmonary tuberculosis (TB) or TB can be diagnosed effectively using two consecutive sputum specimens. This study was set out to evaluate the significance of examining multiple sputum specimens in diagnosis of TB.

Methods

We retrospectively reviewed the acid-fast bacillus (AFB) smear and culture results of three consecutive days’ sputum specimens from 413 confirmed TB patients which were detected as part of a larger active case finding study in Dhaka Central Jail, the largest correctional facility in Bangladesh.

Results

AFB was detected from 81% (n = 334) patients, of which 89% (n = 297) were diagnosed from the first and additional 9% (n = 30) were from the second sputum specimen. M. tuberculosis growth was observed for 406 patients and 85% (n = 343) were obtained from the first sputum and additional 10% (n = 42) were from the second one. The third specimen didn’t show significant additional diagnostic value for the detection of AFB by microscopy or growth of the M. tuberculosis.

Conclusions

We concluded from our study results that examining two consecutive sputum specimens is sufficient enough for the effective diagnosis of TB. It can also decrease the laboratory workload and hence improve the quality of work in settings with high TB burden like Bangladesh.     

Evaluation of Giant African Pouched Rats for Detection of Pulmonary Tuberculosis in Patients from a High-Endemic Setting

BackgroundThis study established evidence about the diagnostic performance of trained giant African pouched rats for detecting Mycobacterium tuberculosis in sputum of well-characterised patients with presumptive tuberculosis (TB) in a high-burden setting.MethodsThe TB detection rats were evaluated using sputum samples of patients with presumptive TB enrolled in two prospective cohort studies in Bagamoyo, Tanzania. The patients were characterised by sputum smear microscopy and culture, including subsequent antigen or molecular confirmation of Mycobacterium tuberculosis, and by clinical data at enrolment and for at least 5-months of follow-up to determine the reference standard. Seven trained giant African pouched rats were used for the detection of TB in the sputum samples after shipment to the APOPO project in Morogoro, Tanzania.ResultsOf 469 eligible patients, 109 (23.2%) were culture-positive for Mycobacterium tuberculosis and 128 (27.3%) were non-TB controls with sustained recovery after 5 months without anti-TB treatment. The HIV prevalence was 46%. The area under the receiver operating characteristic curve of the seven rats for the detection of culture-positive pulmonary tuberculosis was 0.72 (95% CI 0.66–0.78). An optimal threshold could be defined at ≥2 indications by rats in either sample with a corresponding sensitivity of 56.9% (95% CI 47.0–66.3), specificity of 80.5% (95% CI 72.5–86.9), positive and negative predictive value of 71.3% (95% CI 60.6–80.5) and 68.7% (95% CI 60.6–76.0), and an accuracy for TB diagnosis of 69.6%. The diagnostic performance was negatively influenced by low burden of bacilli, and independent of the HIV status.ConclusionGiant African pouched rats have potential for detection of tuberculosis in sputum samples. However, the diagnostic performance characteristics of TB detection rats do not currently meet the requirements for high-priority, rapid sputum-based TB diagnostics as defined by the World Health Organization.