Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Background

Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods

This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results

Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion

We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.     

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.     

Predictor of multidrug resistant tuberculosis in southwestern part of Ethiopia: a case control study

Background

Curable disease tuberculosis is becoming incurable or difficult to treat due to drug resistance. Multi drug resistance tuberculosis is a major health problem for less developed countries. Development of drug resistance is mainly as result of man related factors and poor lifestyle. Identifying predictors of drug resistance and working on them is the important way of reducing the expansion in high burden countries. Ethiopia is one of TB, TB/HIV, and multi-drug resistant tuberculosis (MDR-TB) high burden country globally. This study was aimed to assess predictor of MDR-TB in southwest part of Ethiopia.

Methods

Unmatched case control study was conducted in case to control ratio of 1:1.2 in southwest part of Ethiopia. The cases were recruited from confirmed MDR-TB patient enrolled on second line treatment in Shenen Gibe Hospital (MDR-TB treatment center of the prefecture) and the controls were recruited from previously TB patients who cured or patient with smear negative at the end of treatment month during the study period in the same area. The data was collected by structured questionnaire by interview and logistic regression analyses were used to identify predictors of MDR-TB. Odds ratios with 95% CI were computed to determine the predictors.

Result

From the total 132 participants about 45% of them were cases. None disclosed tuberculosis infected to relatives [AOR?=?3.4, 95% CI (1.2–9.8)], insufficient instruction on how to take anti-TB drug [AOR?=?4.7, 95% CI (1.4–14.6)], contact history with MDR-TB [AOR?=?8.5, 95% CI (2.9–25.5)], interruption of first-line anti-TB treatment for at list 1 day [AOR?=?7.9, 95% CI (2.5–24.9)], and having alcohol drinking habits [AOR?=?5.1, 95% CI (1.4–18.7)] were identified predictors for MDR-TB infection in study area.

Conclusion

TB infection disclosure status, insufficient instruction on drug usage, contact history with MDR-TB, interruption of first-line anti-TB drugs, and alcohol drinking habits were identified predictor of MDR-TB case. Therefore, early detection and proper treatment of drug susceptible TB, strengthening directly observed treatment, short-course on daily bases, community involvement, and supporting the patient to intervene identified factors is paramount.

     

Correction: Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Correction to Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial Amina Abdelaal, Hassan Abd El-Ghaffar, Mohammad Hosam Eldeen Zaghloul, Noha El mashad, Ehab Badran, Amal Fathy Annals of Clinical Microbiology and Antimicrobials 2009, 8:4 (30 January 2009)     

基于PARMS技术的结核分枝杆菌利福平耐药基因rpoB分子标记的开发

文中旨在建立结核分枝杆菌利福平耐药基因rpoB的荧光分子标记,为分子药物敏感性试验提供简便、可靠的基因分型检测方法。对比分析利福平耐药菌株rpoB基因531、526、516、511、513等氨基酸位点的基因突变与敏感菌株中等位基因的序列差异,结合PARMS技术 (Penta-primer amplification refractory mutation system),建立rpoB基因的荧光分子标记。利用其对104例结核分枝杆菌临床分离株进行检测,经Sanger测序验证,正确率100%。并采用比例法药敏试验对这104份样本进行了利福平耐药性鉴定,与分子标记结果相符率为94.23%。结果表明,分子标记有较强可靠性,能检出表型药敏无法测出的低浓度耐药样本 (511/533单位点突变)。建立的11组荧光分子标记能覆盖92%–96%的利福平耐药菌株rpoB基因突变类型,为快速检测结核分枝杆菌利福平耐药提供新思路。     

感控干预在控制多重耐药定植菌医院感染的临床研究

目的研究多重耐药定植菌与医院感染的关系以及去定植措施的效果。方法对入住ICU的患者进行多重耐药定植菌筛查,观察阳性患者和阴性患者医院感染发生率;将阳性患者随机分为对照组和试验组,对照组采用常规的预防控制措施,试验组加上安尔碘Ⅲ去鼻腔MRSA、口服金双歧去肠道产ESBLs肠杆菌措施,观察两组患者医院感染发生率以及定植菌与医院感染的关系,对比两组去定植效果。结果ICU多重耐药菌定植率为31. 16% ;多重耐药定植菌筛查阴性患者医院感染率为7. 56%,阳性患者医院感染率为15.47% （P 〈0.01）;对照组医院感染率为20.79%,试验组医院感染率为22. 22% （P 〉0. 05）;对照组定植菌医院感染率为4. 95% ,试验组定植菌医院感染为0（ P 〈0.01）;对照组产ESBLs大肠埃希菌定植自行清除率为26. 31% ,试验组产ESBLs大肠埃希菌去定植率为80. 95% （P 〈 0. 01）。结论ICU患者多重耐药菌定植率高,多重耐药菌定植患者医院感染率高,口服金双歧去肠道产ESBLs细菌干预措施有效,安尔碘Ⅲ去鼻腔MRSA效果需继续研究。     

Rapid diagnosis of tuberculosis by detection of mycobacterial lipoarabinomannan in urine

There is an urgent need for improved tools for laboratory diagnosis of active tuberculosis (TB). Here, we describe two methods, a catch-up ELISA and a dipstick test based on the detection in urine of lipoarabinomannan (LAM). LAM is a major and specific glycolipid component of the outer mycobacterial cell wall. Preliminary experiments showed that LAM is excreted in the urine of mice injected intraperitoneally with a crude cell wall preparation of Mycobacterium tuberculosis. Both methods were highly sensitive, detecting LAM at concentrations of 1 ng/ml and 5 pg/ml, respectively. Of 15 patients with active TB, all showed intermediate to high levels of LAM in their urine (absorbance values from 0.3 to 1.2, mean 0.74). Only one sample showed an absorbance value below the chosen cut off value of 0.4. All but one of the urine samples from 26 healthy nursing workers exhibited OD value below 0.4 cut off. These methods may prove valuable for rapid and simple diagnosis of TB in particular in developing countries lacking biosafety level 3 (BSL3) facilities.     

一株污水源多重耐药大肠杆菌的耐药性检测及全基因组测序分析

【背景】水体环境分布广、流动性强,是耐药菌和耐药基因传播的主要媒介。【目的】了解北方污水厂大肠杆菌携带的耐药基因及可移动遗传元件情况。【方法】从北方污水厂筛选出一株多重耐药大肠杆菌,通过药敏试验进行耐药性检验,采用96孔板法测定菌株的最小抑菌浓度,利用酶标仪探究亚抑菌浓度抗生素对菌株生长的影响,并对菌株进行全基因组测序,对其携带的耐药基因及可移动遗传元件进行预测。【结果】大肠杆菌WEC对四环素、环丙沙星、诺氟沙星和红霉素具有耐药性,亚抑菌浓度的四环素、环丙沙星和诺氟沙星能够延缓或抑制菌株的生长。WEC菌株的基因组中包含一条大小为4 782 114 bp的环状染色体和2个大小分别为60 306 bp (pWEC-1)和92 065 bp (pWEC-2)的环状质粒。菌株共携带129个耐药基因,其中128个位于染色体上,在染色体上预测到原噬菌体、基因岛及插入序列的存在,部分可移动遗传元件携带有耐药基因。质粒pWEC-1中无耐药基因,pWEC-2含有1个耐药基因,在质粒基因组中预测到原噬菌体和插入序列。【结论】污水源大肠杆菌WEC是一株多重耐药菌株,其基因组中携带耐药基因和多种可移动遗传元件...     

Comparative genomic analyses of multi-drug resistant Mycobacterium tuberculosis from Nepal and other geographical locations

Nepal exhibits a tuberculosis (TB) incidence rate that is comparable to neighbouring high TB incidence countries. In addition, it records >500 cases of multi-drug resistant (MDR) TB each year. The objective of this study was to perform whole-genome bioinformatic analysis on MDR-TB isolates from Nepal (n = 19) to identify the specific mutations underlying their phenotypic resistance. In addition, we examined the dominant genotype among the Nepal MDR-TB isolates, the East-Asian Beijing sub-lineage, to determine its relatedness to a panel of 1274 genomes of international strains available from public databases. These analyses provided evidence that the XDR-TB isolates in our collection were not derived from importation of primary XDR-TB to Nepal but were more likely the result of acquisition of second-line drug resistance in Nepal. Resistance to fluoroquinolones was detected among a high proportion of the Nepal isolates. This has implications for the management of TB, including appropriate antimicrobial stewardship and susceptibility testing for fluoroquinolones and other second-line TB drugs, to minimise the development of XDR-TB among Nepal TB cases.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.Download video file.^{(243M, mp4)}     

A case of Kartagener's syndrome: Importance of early diagnosis and treatment

Kartagener''s syndrome is a very rare congenital malformation comprising of a classic triad of sinusitis, situs inversus and bronchiectasis. Primary ciliary dyskinesia is a genetic disorder with manifestations present from early life and this distinguishes it from acquired mucociliary disorders. Approximately one half of patients with primary ciliary dyskinesia have situs inversus and, thus are having Kartagener syndrome. We present a case of 12 year old boy with sinusitis, situs inversus and bronchiectasis. The correct diagnosis of this rare congenital autosomal recessive disorder in early life is important in the overall prognosis of the syndrome, as many of the complications can be prevented if timely management is instituted, as was done in this in this case.     

Detection of mutations associated with resistance to rifampicin and isoniazid in Mycobacterium tuberculosis by polymerase chain reaction-ligase detection reaction

Mycobacterium tuberculosis （MTB） infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB （MDR-TB）, defined as being resistant to at least rifampicin （RMP） and isoniazid （1NH）,     

Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria

In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.     

Ambulatory multi-drug resistant tuberculosis treatment outcomes in a cohort of HIV-infected patients in a slum setting in Mumbai, India

Background

India carries one quarter of the global burden of multi-drug resistant TB (MDR-TB) and has an estimated 2.5 million people living with HIV. Despite this reality, provision of treatment for MDR-TB is extremely limited, particularly for HIV-infected individuals. Médecins Sans Frontières (MSF) has been treating HIV-infected MDR-TB patients in Mumbai since May 2007. This is the first report of treatment outcomes among HIV-infected MDR-TB patients in India.

Methods

HIV-infected patients with suspected MDR-TB were referred to the MSF-clinic by public Antiretroviral Therapy (ART) Centers or by a network of community non-governmental organizations. Patients were initiated on either empiric or individualized second-line TB-treatment as per WHO recommendations. MDR-TB treatment was given on an ambulatory basis and under directly observed therapy using a decentralized network of providers. Patients not already receiving ART were started on treatment within two months of initiating MDR-TB treatment.

Results

Between May 2007 and May 2011, 71 HIV-infected patients were suspected to have MDR-TB, and 58 were initiated on treatment. MDR-TB was confirmed in 45 (78%), of which 18 (40%) were resistant to ofloxacin. Final treatment outcomes were available for 23 patients; 11 (48%) were successfully treated, 4 (17%) died, 6 (26%) defaulted, and 2 (9%) failed treatment. Overall, among 58 patients on treatment, 13 (22%) were successfully treated, 13 (22%) died, 7 (12%) defaulted, two (3%) failed treatment, and 23 (40%) were alive and still on treatment at the end of the observation period. Twenty-six patients (45%) experienced moderate to severe adverse events, requiring modification of the regimen in 12 (20%). Overall, 20 (28%) of the 71 patients with MDR-TB died, including 7 not initiated on treatment.

Conclusions

Despite high fluoroquinolone resistance and extensive prior second-line treatment, encouraging results are being achieved in an ambulatory MDR-T- program in a slum setting in India. Rapid scale-up of both ART and second-line treatment for MDR-TB is needed to ensure survival of co-infected patients and mitigate this growing epidemic.     

Screening for HIV-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the Xpert MTB/RIF assay: a prospective study

Background

The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown.

Methods and Findings

Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102–236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%).

Conclusions

In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal. Please see later in the article for the Editors'' Summary     

1株多重耐药肺炎克雷伯菌耐药基因和整合子、转座子遗传标记研究

目的 了解多重耐药肺炎克雷伯菌的耐药基因存在状况和遗传学背景。方法 聚合酶链反应(RCR)法对多重耐药的肺炎克雷伯菌进行β-内酰胺酶基因、氨基糖苷类修饰酶基因、质粒AmpC酶基因、qacEΔ1-sull耐消毒剂和磺胺基因、整合子遗传标记(整合酶基因)、Tn21/Tn501转座子遗传标记(汞离子还原酶基因)检测。结果 TEM、SHV型β-内酰胺酶基因, DHA型质粒AmpC酶基因,aac(6′)-1型氮基糖苷类修饰酶基因,qacEΔ1-sul1耐消毒剂和磺胺基因,整合子遗传标记(intI1整合酶基因),Tn21/Tn501转座子遗传标记(merA汞离子还原酶基因)检测阳性。结论 多重耐药肺炎克雷伯菌存在多种耐药基因和Ⅰ类整合子、Tn21/Tn501转座子。     

Use of a molecular diagnostic test in AFB smear positive tuberculosis suspects greatly reduces time to detection of multidrug resistant tuberculosis

Background

The WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings.

Methods

The performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program.

Results

Among 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistance directly from the sputum specimen in 159 (89.8%) of 177 specimens and MDR-TB in 109 (95.6%) of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01). The most prevalent INH resistance mutation was S315T (78%) in the katG codon and the most common rifampicin resistance mutation was S531L (68%) in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB). The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days) compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media).

Conclusions

Compared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.