Jojula M, Rao V. K, Vihnudas J. Diagnosing Sputum/smear-negative Pulmonary Tuberculosis in PLHIV Based on Culture a Study from MGM District Hospital. Biomed Pharmacol J 2011;4(2)

---

Manuscript received on :July 12, 2011
Manuscript accepted on :August 04, 2011
Published online on: 02-12-2015

---

How to Citeclose    |   Publication History close

 Views: (Visited 731 times, 1 visits today)    PDF Downloads: 494

Malathi Jojula¹*, V. Kondal Rao² and Jyotsna Vihnudas³

¹Department of Pharm. Microbiology, Sri Shivani College of Pharmacy, Warangal India. ²Department of Microbiology, Kakatiya Medical College, Warangal India. ³Department of Pharm. Biochemistry, Sri Shivani College of Pharmacy, Warangal India.

Abstract

Diagnosis of sputum/smear-negative pulmonary tuberculosis patients can be both challenging and time consuming with many patients being put on empirical anti-tubercular treatment. The study was conducted on100 suspected sputum smear negative pulmonary tuberculosis causes attending ICTC of MGM District Hospital Warangal. All the suspect’s sputum was screened for AFB staining were shown negative. Their mode of clinical presentation, site of TB, CD4 cell count, dysentery in HIV, TB, studied. Tuberculosis (TB) was found in 15% of HIV infected patients those were showing smear negative for AFB. TB were significantly more common in HIV seropositive patients (p< 0.02) in whom the CD4 cell count was also low (less than 100/cmm).8% of dysentery was found among them. The result of this study showed the increased vulnerability of the HIV infected persons to TB infection as seen in other studies in India and abroad. Disseminated TB with lower CD4 cell count is also shown. Smear negative persons were more prone for tuberculosis infection which may become problem in diagnosis and treatment of tuberculosis.

Keywords

HIV/AIDS;Smear negative; TB; CD4 Counts; diherial

Introduction

India has 20% of the global burden of tuberculosis infections, with 1.9 million new cases each year, as well as a large pool (2.5 million) of HIV-infected individuals (1, 2). Out of 1.9 million registered for treatment of tuberculosis in India 28.7% of them were new smear negative cases (3).  Tuberculosis (TB) is a leading cause of HIV-related deaths worldwide. Approximately 30% of HIV-infected persons are estimated to have latent TB infection through out world (4). The mortality associated with TB is considerably higher in HIV-infected than HIV-negative patients (5). The HIV epidemic which is leading to increase in the frequency of smear-negative pulmonary tuberculosis, compared to the smear positive cases, it becomes a complication in diagnosis of HIV persons. HIV-infected patients are twice as likely to have sputum smear-negative, culture-positive pulmonary TB (PTB). The sputum smear has traditionally been used as the method for making an early diagnosis of PTB. Sputum smear examination for acid- fast bacilli (AFB) may detect up to 15-40% of pulmonary tuberculosis cases in laboratories and large portion may remain negative in spite of clinical profile and radiological lesions being consistent with diagnosis of pulmonary tuberculosis (6). The reduction in sensitivity of this test in HIV-infected patients leads to diagnostic delay. In the TB screening process the Chest X-ray (CXR) is an indispensable tool in PLHIV, for whom sputum is often negative (7). Chest X-ray shortens delays in diagnosis of TB and non-TB chest diseases common which are among people living with HIV. HIV TB Co-infected patients with signs and symptoms may die before sputum or culture is available by using Digital Radiology, Chest images are instantly available and abnormalities consistent with TB can be detected on the spot. When  patients  showing with CD4 cell counts less than 200 cells/uL show less typical abnormalities in their Chest image, then it becomes complicated in identify the positive ness of TB. Sputum culture is a more sensitive method of diagnosing PTB in such cases, but will takes up to 8 weeks for the result to be available (8). Early diagnosis of pulmonary tuberculosis prevents progression of disease, morbidity, spread of disease and permanent damage by fibrosis with normal individual to HIV positive individuals (9). HIV as a syndrome associated with other bacterial infections that were mainly belong to the class of Mycobacterium and Enterobacteriaeceae members, it is obvious that HIV positive patients were more susceptible to mycobacterial and diarrheal infections(10). CD4 and CD 8 counts vary in pure HIV positive patient and HIV patient with other co infections (11). Observations from many parts of the world have shown higher incidence of TB among HIV infected individuals, ranging from 5 to 10 per year of observation (12). The mortality associated with TB is considerably higher in HIV-infected than HIV-negative patients (10). The present study aims to assess the role of culture in the diagnosis of sputum/smear-negative pulmonary tuberculosis.

Materials and Methods

For the assessment of the process we have compiled nearly 100 HIV positive patients ’blood samples, from district hospital, Warangal and subjected to CD4 count and also recognition of the other bacterial infections in HIV patients. This method consists of series of steps mainly Sample collection and handling, CD4 cell counting in blood using Flow cytometry, Identification of other bacterial infections associated with HIV and confirmatory tests to determine the particular organism coupled with HIV infection.

Sample collection and Handling

For CD4 enumeration by flow cytometry, whole blood specimens was collected by venipuncture using evacuated blood collections tubes with K3-EDTA or K2-EDTA as the anticoagulant (13). All blood samples labeled at the time of specimen collection, and transported to the Flow cytometry chamber the temperature maintained is18°c to 22°c. Clotted specimens were not used for the study.

CD4 enumeration by Flow cytometer

For the CD4 cell enumeration BD FACS calibur cytometer were used, each patient sample labeled by BD trucount tube with sample identification number. 20µL of BD TRITEST CD3/CD4/CD45 reagent pipetted out in to the bottom of the tube (14). Using the reverse pipetting technique sample is pipetted out on to the side of the tube just above the retainer. 50µL of well mixed anti coagulated whole blood pipetted out in to the bottom of the tube. Tube and vortex capped and mixed gently, after this tube is incubated for 15 minutes in the dark room temperature 20°c-25°c. Later 450µL of 1X BD FACS lysing solution is added in the tube and mixed gently; the sample is incubated for 15 minutes at dark room temperature 20°c-25°c. Finally the sample is analyzed on the flow cytometer (15).

Identification of ohe other Bacterial Infections and Confirmatory Examinations

Sputum and stool sample were collected. Sputum specimens were collected was about 5-10 ml which was processed on the same day as per guidelines (16).  The clinical sputum specimens submitted for the determination of possible mycobacterial infections were examined first for acid-fast bacilli. Smear made from the collected sputum specimen were stained by the Ziehl- Neelsen and examined by microscopy to detect the cases of mycobacterial infections, this method served as an adjunct to culture for determining the acid- fast characteristics of bacteria (17). For the betterment of acid-fast staining of a smear and to reduce the viscosity of specimen, the specimen to be examined was initially treated with 5% of sodium hypochlorite with an equal volume of specimen (18). The concentration of bacilli by centrifugation was done by the Petroff”s method (19). The homogenized sputum was cultured on, Lowenstein Jensens (LJ) media (20). LJ media slopes were used for inoculation of sample and incubated as, first slope at 37 ºC, second slope wrapped in black paper at 37 ºC, third slope at 25 ºC and fourth slope at 44 ºC. Bacteria obtained after incubation on different slants were subcultured and different parameters viz., Rate of growth (21), colony morphology (22), temperature at which growth occurs (23), production of pigment in light and dark (24), Niacin test (25), Nitrate reduction test (26), Catalase test (27), Growth on MacConkey agar(28), Tellurite reduction test (29), Urease test (30)and Tween80 hydrolysis test (31) were performed on the isolates, which are 3-4 weeks old to identify the type of mycobacterium. In the same way diarrheal have been identified by culturing the stool sample on Nutrient agar media, confirmatory test have   performed by biochemical and motility tests, reveals diarrheal infection along with HIV is caused by E. coli.

Results and Discussion

 Of the 100 seropositive cases, 56 were males and 44 were females.  There CD levels had shown a vared in proportions during there attempt to ICTCs in MGM Warangal, district hospital. Out of the 100 patients shown lower CD4+ counts below 200 rate is high in males, 9 HIV seropositive males were found and 5 cases of females were found in our study. The age range for seropositive patients was from 16 – 46 year.  The highest number of HIV infection was found in the age group of 21 – 30 years 60. The seropositivity rate was highest among those who were unemployed is about 40.Tuberculosis was found in 20 patients in the seropositive group (age ranged from 21 – 36 years). HIV seropoistive patients shown a negative AFB smears. Out of 100 HIV Seropositive patients 15 where positive for the culture on L-J medium. Based on  morphology, biochemical tests, and growth rates of the bacterium it was identified as M.tuberulosis.   Of the 100 HIV positive, 8 patients shown diherial infections, when stool samples were subjected for the culture on MacConkey, Nutrient Agar medium, growth was found to be E.coli.

In Asia where HIV epidemic is at an early stage, surveillance data show that the rates of HIV infection had remained lower in-patients with TB compared to that seen in Africa. Studies form Uganda and Zambia have recorded HIV rates of 50-70% among TB patients (32). In Tanzania, the survey conducted during 1994-l 998 on 10,612 new smear positive TB patients revealed 40% HIV prevalence. HIV sero-prevalence rates among patients with extra-pulmonary TB are even higher extra pulmonary TB has been reported in upto 70% of HIV related TB cases when the CD4 lymphocyte counts falls below 10025. Studies indicates that the HIV sero-positivity in TB patients show a wide variation ranging from 0.4% in a study in Delhi to 28.75% in a study conducted in Pune. Moreover, periodic studies from some centres indicate that the HIV prevalence is rapidly increasingamong TB patients (33).In our experience, about 2/3rd of HIV infected individuals have a CD4 < 200 cells/mm3 when they present with TB. Hence, the earlier use of ART is likely to reduce mortality and morbidity in this group of patients (34).

In this study, TB was found in 55 % of the HIV positive patients and in 25% of the HIV negative patients which was significant statistically (p < 0.001). This is consistent with the findings in another study7, but higher than that of a Kolkata study with 27.7% only. Our study was based on the patients admitted in the hospital for evaluation of fever. This may explain the higher incidence of TB in those seronegative patients.

Conclusion

HIV has a world wide distribution and is already a huge problem of over stretching the fragile health infrastructure in most of the countries, like India, with the increasing tuberculosis cases load with HIV infections there will be greater demand to d diagnose and treat HIV TB cases. The diagnosis of TB in HIV patients is difficult for many reasons. Our study had fouced on the seropositive for HIV and when they were subjected for testing for oppurtunictic infections surpricingly to our knowleged we found smear negative for AFB staining TB were shown culture.  Present study revealed evidence of CD4 levels in HIV-TB infections was low CD4 > 200 cells /mm. Out of 100 positive HIV causes there were 15 smear negative but culture positive cases for tuberculosis bacilli. 15 isolates were identified as Mycobacterium tuberculosis, 8 isolstes of E.coli, were also found in our study.

Acknowledgements

The implementation and success of this project could not have been possible with out the patient guidance of Dr. Kondal Rao Department of Microbiology, KMC warangal. Professor S. Ramreddy Department of Microbiology Kakatiya University, warangal. We are indebted to V.Jyostna, Head of Biochemistry Department, Shivani Pharmacy College, Warangal for the full fledged co operation. We express our profound gratitude to Dr.V. Rajukumar Principal of Shivani Pharmacy College, for providing excellent lab facility. It’s our privilege to keep on record our deep sense of gratitude and warm regards to all IEC members for clearing ethical issues and approving for working on this topic. Last but lest I would like to thanks for all the lab technicians for their encouragement and support.

References

1. World Health Organization. WHO report 2008.Global tuberculosis control: surveillance,planning,financing.http://www.who.int/tb/  publications/global_report/2008/pdf/fullreport.pdf. Accessed 3 March 2009.
2. Sharma SK, Alladi Mohan, Tamilarasu Kadhiravan. HIV-TB co-infection: epidemiology, diagnosis, and management. Indian J Med Res 2005; 121:550–567.
3. K. Siddiqi, M. Lambert, J. Walley The Lancet Infectious Diseases, Volume 3, Issue 5, Pages288-296;2009
4. UNAIDSAIDS epidemic update, December 2009. UNAIDS Geneva 2009.
5. WHO, Improving the diagnosis and treatment of smear-negative pulmonary and extra-pulmonary tuberculosis among adults and adolescents, WHO/HTM/HIV/2007.01
6. Bock NN, Mallory JP, Mobley N, DeVoe B, Taylor BB: Outbreak of tuberculosis associated with a floating card game in the rural south: lessons for tuberculosis contact investigations. Clin Infect Dis 1998, 27:1221-1226.
7. Dr Haileyesus Getahun MD a, Mark Harrington MA b, Rick O’Brien MD c, Paul Nunn FRCP a Diagnosis of smear-negative pulmonary tuberculosis in people with HIV infection or AIDS in resource-constrained settings: informing urgent policy changes The Lancet, Volume 369, Issue 9578, Pages 2042 – 2049, 16 June 2007
8. Sarah Howie, Robert ramage, and Tim Hewson.2000. Innate immune system  damage in human immuno deficiency virus type I Infection. Am.J.respir, Care Med, Vol 162, No 4, 141-145.
9. Jens J.Pindborg, Hons Drsci, HonLLD, et al.1989.Classification of oral lesions  Associated with HIV infection. Elsevir inc, Vol 67, issue 3,292-295.
10. S.S. Uppal, S. C. Tewari, Shashiverma,P.S. Dhot.2004.Comparision of CD4 and  CD8 lymphocyte counts in HIV negative Pulmonary TB Patients with those in  normal blood donors and the effect of antituberucular treatment: Hospital based  Flow cytometric study, Wileg-lissinc, Vol 618, issue1, 20-26.
11. Lynns zijenah, Gerard Kadzirange, Simm Madzime et al. 2006. Affordable flow Cytometry for enumeration of absolute CD4+T lymphocytes to identify sub type C HIV infected adults requiring anti retro viral therapy (ART) and monitoring response to ART in resource-Limited setting,Journal of translational medicine,            4:33.
12. Diag bouga S, Chazallon C, Kazatchkine, et al. 2003. Successful implementation of low cost method for enumerating CD4+T lymphocyte in resource –limited setting: The ANSR 12-26 study, AIDS, 17:2201-2208.
13. Lyamuga EF, Kagoma C, Mbena EC, UrassaW.K, et al.1996. Evaluation of the FAS Count, TRAX CD4 and Drynabead methods for  cd4 lymphocyte  determination. J. Immunol methods, 195: 103-112.
14. Patricia TK, Georgia PK.1985. Public health mycobacteriology: a guide for the  level III laboratory, Atlanta: US Department of health and Human Services,   Public Health Service, CDC: 71-146
15. Herbert MS and Robert CG.1985. In Mycobacterium, Manual of Clinical Microbiology, Ed. Edwin H. Lennette, American Society for Microbiology,  Michigan, pp 216-247.
16. Krasnow, I., &L. G. Wanye.1969. Comparison of method for tuberculosis bacteriology. Appl. Microbiol. 18:915-917.
17. Monica Cheesbrough.1991. Mycobactria in medical laboratory manual for tropical countries, ELBS edition, Microbiology (II).
18. Krasnow, I., and G.C. Kidd.1965. The effect of a buffer wash of sputum  Sediments digested with Zephiran trisodium phosphoate on the recovery of Acid- fast bacilli.Am. J. Clin.Pathol.
19. Minnikin DE 1982 in Ratledge C, Stanford JL (eds) The biology of the  mycobacteria, vol. 1. Academic Press, New York, p.95
20. David HL, Jahan M-T et al. 1978 International Journal of Systematic Bacteriology 28: 46.
21. Kilburn, J.O., K, D. Stottmeier, & G.P. Kubica. 1968. Aspartic acid as a precursor for niacin synthesis by tubercle bacilli grown on 7H10 agar medium, Am. Rev.  Resir. Dis. 129: 264- 268.
22. Jenkins PA 1985 Tubercle 66: 193
23. Kubica, G. P., & G. L. Pool.  1960. Studies on the catalase activity of acid- fast           bacilli. I. An attempt to subgroup these: organisms on the basis of their catalase  activities at different temperatures and pH. Am. Rev. Respir, Dis. 81: 737- 740.
24. Jones, W.D.,   & G. p. Kubica. 1964. The use of MacConkeys agar for  Differential typing of Mycobacterium fortutitum. Am.  J. Med. Technol. 30: 187-195
25. Kilburn, J. O., V.A. Silcox, and G.P. Kubica. 1969. Differential identification of  mycobacteria. V. The tellurite reduction test .Am. Rev. Respir. Dis. 99:94- 100.
26. Collins CH, Grange JMet al.1985b journal of Hygiene 94: 135.
27. Vestal A 1975 Procedure for isolation  and identification of mycobacteria. US  Dept of Health, Education and Welfare, Publication no, CDC79-8230.
28. Kilburn, J,O., K. F. O Donnell, V. A. Silcox, and H. L. David.1973 Preparation of a stable mycobactreial  Tween hydrolysis test substrate. Appl. Microbial. 26:826.
29. Anuradha S, Soloman S, Rajasekaran S: HIV seropositivity in patients with  respiratory disease. Ind J Tub 40: 13-15
30. George Bicego, J. Ties Boerma and Carine Ronsmans.   2002.  The effect of AIDS  on maternal mortality in Malawi and Zimbabwe. AIDS, Vol 16, No 7,1078- 1081.
31. S.K. Sharma, Alladi Mohan* & Tamilarasu Kadhiravan. 2005. HIV-TB co-infection: Epidemiology, diagnosis & management. Indian J Med Res 121, 550-567.
32. John L Ho. 1996. Co-Infection with HIV and Mycobacterium tuberculosis:  immunologic interactions, disease progression, and survival. Mem. Inst. Oswaldo Cruz, Vol.91, No.3.
33. Liberato IR, de Albuquerque Mde F, Campelo AR, de Melo HR.2004. Characteristics of pulmonary tuberculosis in HIV seropositive and seronegative     patients in a Northeastern region of Brazil. E pub;37 (1):46-50. 19.