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 ~  Abstract
 ~  Materials and Me...
 ~  Culture of sputum
 ~  Blood
 ~  Results
 ~  Discussion
 ~  References

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ORIGINAL ARTICLE
Year : 2004  |  Volume : 22  |  Issue : 1  |  Page : 28-33
 

Prevalence of bacterial and fungal agents causing lower respiratory tract infections in patients with human immunodeficiency virus infection


Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, India

Correspondence Address:
Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, India

 ~ Abstract 

PURPOSE: This study was designed to document the prevalence of HIV associated respiratory infections in Hyderabad. Such studies have not been worked out before in this region. METHODS: The study included specimens from 130 patients with complaints suggestive of lower respiratory tract infection. Among them 100 were HIV reactive and 30 were HIV nonreactive. Both the expectorated as well as induced sputum samples were collected and processed to examine for the bacterial and fungal pathogens including Pneumocystis carinii. RESULTS: Sputum samples from 63% of HIV reactive and 33.3% of HIV nonreactive patients were culture positive. In all, there were 70 pathogens isolated from the HIV reactive subjects, 44.3% were bacteria, 42.9% were Mycobacteria and 12.8 % were fungi. CONCLUSIONS: Lower respiratory tract infection is a common problem among HIV reactive patients and majority are bacterial infections. Polymicrobial isolation was observed only among the HIV reactive patients. PCP was not documented in our series.

How to cite this article:
Shailaja V V, Pai L A, Mathur D R, Lakshmi V. Prevalence of bacterial and fungal agents causing lower respiratory tract infections in patients with human immunodeficiency virus infection. Indian J Med Microbiol 2004;22:28-33


How to cite this URL:
Shailaja V V, Pai L A, Mathur D R, Lakshmi V. Prevalence of bacterial and fungal agents causing lower respiratory tract infections in patients with human immunodeficiency virus infection. Indian J Med Microbiol [serial online] 2004 [cited 2019 Aug 22];22:28-33. Available from: http://www.ijmm.org/text.asp?2004/22/1/28/8058


The acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is the most important public health problem of the 20th century.[1] Though HIV made a delayed entry into India, its spread has been very rapid and at present is in an advanced stage of the epidemic in some states of the country.[2] The infection is alarming due to the unique pathogenesis of the virus that decreases the CD4 cells, signaling the emergence of opportunistic infections in the host.[3] Among the various opportunistic infections, respiratory infections account for upto 70% of AIDS defining illness.[4] Their relative importance differs in different parts of the world.[5]
The present study was undertaken to note the prevalence of various bacterial and fungal pathogens of lower respiratory tract in HIV reactive patients and an attempt was made to specifically examine for P.carinii in induced sputum specimens. No viral cultures were performed due to lack of facilities.

 ~ Materials and Methods Top

Patients clinically diagnosed to have lower respiratory tract infections attending the Andhra Pradesh Chest Hospital, Hyderabad were included in the study. All these patients were initially screened for anti HIV antibodies before being enrolled in the study.
One hundred HIV reactive [UBI HIV 1/2 EIA (United Biomedical, Inc., Beijing) and HIV TRIDOT (J.Mitra & Co. Ltd., New Delhi) confirmed] and 30 nonreactive patients with lower respiratory tract infection formed the study groups.
Specimens
An early morning expectorated sputum as well as induced sputum were collected separately in sterile containers from all patients included in the study. Induction of sputum was done using a Nebulizer (model - Medel Aero Family) and 3% hypertonic saline for 15 minutes. In addition to sputum 5 mL of blood was collected from all patients included in the study.
Processing of specimens
Microscopic examination of sputum
Induced sputum was examined for the presence of trophozoites and cysts of P.carinii while the expectorated sputum was examined for bacterial and fungal pathogens. The quality of the expectorated sputum was assessed both by macroscopic and microscopic examination. Any sample that was thin, watery and with no purulent matter was considered unsuitable for further processing. Bartlett's scoring method was used for microscopic evaluation of the expectorated sputum.[6]
A sputum was considered unsuitable if it had a final score of 0 or less. All unsuitable specimens were discarded and a repeat specimen was collected. Staining for the several organisms was carried out as mentioned in [Table - 1].

 ~ Culture of sputum Top

The sputum specimens were inoculated into blood agar with 10% sheep blood, Chocolate agar with 10% sheep blood, McConkey's agar and Brain Heart Infusion (BHI) agar.
Any significant bacterial growth was further processed as per the standard procedure to identify the pathogens.[7] The species identification of Enterobacteriaceae was performed using the Entero rapid identification kit (Mikro LA test kit - Entero Rapid 24).[8]
A portion of the specimen was concentrated by the modified Petroff's method and inoculated in the Lowenstein Jensen's medium[9] and in the Bactec-12B vials for culturing Mycobacteria. The latter was processed in the BACTEC 460 TB system (Becton Dickinson, USA).[10]
The sputum was also inoculated onto Sabouraud dextrose agar (SDA) with antibiotics, SDA without antibiotics in duplicate (incubated at 37°C and 25°C) and BHI agar (incubated at 37°C). Any significant growth of a fungal species was further identified as per standard protocol.[11]
A portion of the induced sputum specimen was washed with saline and centrifuged (1500 rpm / 10 minutes) and the sediment was smeared and stained for P.carinii [Table - 1]. The remaining portion of induced sputum was used for indirect immunofluorescence staining with monoclonal antibodies specific for P. carinii (Monoflou KIT P.carinii, Sanofi Pasteur, France).

 ~ Blood Top

Serum from all the 130 patients included in the study was evaluated for the levels of lactate dehydrogenase enzyme (LDH). (LDH levels are increased to > 450 U/L in 90% of patients with P.carinii pneumonia).[4],[12]

 ~ Results Top

[Table - 2] through 4 show the number and the nature of the isolates from both the categories of patients included in the study. The results were statistically evaluated. The isolation of mycobacteria and other bacteria among HIV reactive patients was found to be significantly higher (P=0.01) than in HIV nonreactive patients. No significant difference was found between the two groups of patients in the prevalence of fungal and polymicrobial infections.
Seventy pathogens were isolated from 63 patients of the HIV reactive group. 44.31% of the isolates were bacteria, other than Mycobacteria [Table - 2].
There were 30 mycobacterial isolates from the HIV reactive group. 29/30 (96.67%) were identified as M.tuberculosis by the BACTEC 460 TB, while one patient had a florid infection with an atypical Mycobacterium, which was presumptively identified as M.avium complex. This patient was an Indian student from Canada.
 M.catarrhalis   was repeatedly isolated from the sputum of three patients with HIV. In all these patients there were plenty of gram negative diplococci in the Gram smear along with polymorphs. Repeat sampling was performed to prove and confirm the organism as the etiologic agent of the respiratory infection.
Among the 27 fungal isolates from HIV reactive patients, 9 were pathogenic (12.83%), 6/9 were Candida albicans, 2/9 were Cryptococcus neoformans and one was Aspergillus niger. The rest of the 18 isolates were non albicans Candida spp. that were considered as colonizers of the oropharynx. Unlike the cases with C.albicans, which showed plenty of pseudohyphae, there were no pseudohyphae demonstrated by the Gram stain and the candidal growth was insignificant and mixed with normal flora organisms of the oropharynx in all these 18 patients. They were hence considered as colonizers of the oropharynx.
Serum LDH, in all the patients from both groups, was below the optimum cutoff value for P.carinii pneumonia (450 U/L).[4],[12]

 ~ Discussion Top

The importance of respiratory infections in HIV patients is well documented.[1] The true incidence of these infections is difficult to assess and varies with the population surveyed.[5] In the present study, of the 100 HIV reactive patients, the etiologic agent could be identified in 63 patients. The clinical and radiological correlation among the culture positive cases in both the study groups is shown in [Table - 5] and [Table - 6] respectively.
Among the opportunistic infections associated with HIV, diseases like pneumonia of bacterial origin occur at a rate many times higher in the HIV infected patients than in the general population.[13] In the present study, the bacterial isolates from the HIV group were much higher and of varied etiology than those of the HIV nonreactive group [Table - 2], 3). Also the polymicrobial etiology in eight of the HIV reactive patients is a significant finding, indicating the severity of the infection in this group.
As per the figures from National AIDS control organization (NACO), bacterial infections constituted 7% of opportunistic infections and the common organisms encountered in pulmonary infections were S.pneumoniae, H.influenza and  S.aureus  .[2] In the present study, out of all the pathogens isolated, bacterial isolates constituted 44.28% among which K.pneumoniae was isolated in 32.26% of cases and S.pneumoniae in 25.81% and S.aureus in 12.90%. Of all the infections reported, risk or susceptibility to infections with encapsulated organisms such as S.pneumoniae and C.neoformans appears to be the most.[13] Tchamran in his study on the lung diseases due to common bacteria in HIV infected individuals in African adults, noted 81% of infections due to S.pneumoniae and reported it to be the most offending pathogen in HIV reactive patients.[14]
 P.aeruginosa   infection in patients with HIV is often community acquired and is associated with substantial mortality. Dropulic in his study on the clinical manifestations of P. aeruginosa infection among patients with AIDS found that of the 73 episodes of P.aeruginosa infections, 13 were that of pneumonia.[15] In the present study, 3 (9.68%) of the isolates were P.aeruginosa and one of the patients expired due to severe pneumonia.
M.catarrhalis is generally considered a commensal in the upper respiratory tract of adults, and its isolation from sputum is often reported as “normal flora of the oropharynx”. This appears to be a misconception, as Sehgal and Shaimy in 1994,[16] reported this organism as the second most common isolate from patients suffering with lower respiratory tract infections. In the present study, M.catarrhalis was repeatedly isolated in 3(9.68%) cases and was considered a pathogen. In all these cases the sputum was heavily loaded with the organisms. This organism needs to be looked for in HIV reactive patients when LRTI is suspected.
Khan and others reviewed the emergence of Nocardia species as human pathogens, especially in those who were immunocompromised.[7] Case reports of pulmonary nocardiosis in patients with HIV infection were also documented by Lee and others from Singapore.[18] Uttam Chandani in his study on Nocardiosis in HIV infections reported 30 cases, of which 21 were that of pulmonary Nocardiosis.[19] Chest radiographs showed either consolidation, alveolar or the reticulonodular pattern of infiltration. In the present study, one of the patients had clinically, radiologically and microbiologically proven Nocardiosis. The isolate was identified as  N.asteroides  .
Tuberculosis (TB) ranks as the most common infection seen in the developing countries. About 55 - 89% of AIDS cases in India, were found to be suffering from extensive pulmonary TB.[2],[20],[21] In the present study, M. tuberculosis was isolated in 29 HIV positive cases (42.89%). However atypical mycobacteria, commonly reported from AIDS cases in the Western countries, have not been seen in India.[2] In the HIV nonreactive group, 3/30 patients had infection with M.tuberculosis. These figures clearly show an increased rate of TB in HIV reactive (28%) than among the HIV nonreactive patients (10%).
Although less common than bacterial infections, serious fungal infections occur in the immunocompromised patient both as new infection and as reactivation of latent disease. Fungal pulmonary infections often precede the appearance of other opportunistic infections, but frequently co-exist with other pathogens.[1] Though pulmonary candidiasis is documented to be a very rare disease, occurring in late stages of AIDS, oral and oesophageal candidiasis is reported as the second most common (58%) opportunistic infection among HIV patients, from India. In the present study, 25 fungal isolates were recovered from the sputum. C.albicans has been identified in 6/25 fungal isolates. To rule out the possibility of oropharyngeal colonization, a common feature among the HIV reactives, those cases with plenty of pseudohyphae on smear examination were considered as significant pathogens. No Candida species were isolated from the sputum of HIV nonreactive patients.
C. neoformans is distributed through out the world and is the most common fungus causing life threatening illness in patients with HIV. Though the meningeal involvement is the commonest presentation, pulmonary cryptococcosis has been documented in various studies. In one study from Rwanda, pulmonary cryptococcosis was associated with HIV 1 infection in 37 patients, 29 of whom had primary pulmonary cryptococcosis. It was concluded that the disease is not a rare complication of HIV infection.[22] In the present study, two of the fungal isolates were C.neoformans and the clinico radiographic findings were correlated. One of the patients succumbed to the infection. C. neoformans was also isolated from one case in a known diabetic who was HIV nonreactive.
Meyohas, in a 7 year study on AIDS patients, reported isolated cases of aspergillosis from patients with a predisposing neutropenia due to HIV or steroid therapy.[23] Shivananda in his study in 1992 on 825 patients with pulmonary infections found 15.39% of isolates to be Aspergillus species. Of these, A.fumigatus were 11.15%, A.niger were 3.2% and  A.flavus   was 0.96%.[24] In her study of repeated sputum samples, Geetalakshmi from Chennai, documented pulmonary aspergillosis in 36 samples. The isolates from this study were A.fumigatus, A.niger and Candida species.[25] Punkajalakshmi et al from Chennai, in their review, emphasized the emergence of candidiasis, cryptococcosis, aspergillosis, penicillosis and other pheohyphomycosis in patients infected with HIV.[26] In the present study, one of the HIV patients on steroid therapy, had a dual infection with A.niger and S.aureus.
Multiple defects in the normal host immune system caused by infection with HIV, predispose to an increased frequency of a variety of opportunistic infections. The infections are different in different geographical regions. In the developing countries of Asia and Africa, TB and other tropical parasitic diseases are highly prevalent than P.carinii pneumonia (PcP), which is the commonest infection in the Western world.[2] Very few Indian studies have reported PcP in HIV. The reported incidence of PcP is about 4% of opportunistic infections in HIV patients and three cases have been reported from Delhi.[27] Unlike in the West, the prevalence of PcP is low or negligible in India. This probably could be explained by the extensive use of cotrimoxazole in the prophylaxis of PcP in HIV. In the present study, a thorough attempt was made to demonstrate the presence of trophozoites / cysts of P.carinii in the induced sputum. However, none of the sputum samples were positive for P.carinii.
This study demonstrates that lower respiratory tract infection is a common problem among the HIV reactive patients and majority are bacterial and mycobacterial infections. Polymicrobial isolation is seen only among the HIV reactive patients. 

 ~ References Top

1.Rosen MJ. Pneumonia in patients with HIV infection. Medical Clinics of North America 1994;78:1067-1078.  Back to cited text no. 1    
2.Rewari BB (Ed.) Spectrum of oppurtunistic infections in AIDS. Chapter 11. In: Specialists training and reference module. State Pram (Delhi) National AIDS Control Organisation, New Delhi:111-120.  Back to cited text no. 2    
3.Rewari BB (Ed.) Virology and Immunopathology of HIV/AIDS, Chapter5. In: Specialist's training and reference module, State Pram (Delhi) National AIDS Control Organisation (New Delhi):34-48.  Back to cited text no. 3    
4.Walker PA, White DA. Pulmonary disease. Medical clinics of North America, 1996;80:1337-1362.  Back to cited text no. 4    
5.Joshi PL, Mishra SN. Opportunistic infections in HIV/AIDS Patients : An over view, Chapter 1. In : Manual on laboratory diagnosis of common opportunistic infections associated with HIV/AIDS. Baweja UK, Sokhey J (Eds.) Govt. of India. National Institute of communicable diseases, (New Delhi):3-4.  Back to cited text no. 5    
6.Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC Jr. The role of Microbiology laboratory in the diagnosis of infectious disease: Guidelines to practice and management, Chapter 2. In: Color atlas and text book of diagnostic microbiology, 5th edition. Lippincott, Philadelphia, New York (Pubs.) 1997:69.  Back to cited text no. 6    
7.Garcia LS, Procop GW, Roberi GD, Thompson RB, Jr. Over view of conventional methods for bacterial identification, Chapter13. In : Bailey and Scott's diagnostic microbiology,10th edition. Forbes BA, Sahm DF, Weissfeld AS (Eds.) (Mosby).1998:167-181.  Back to cited text no. 7    
8.Mikro Lachema test Kit- Entero Rapid 24. Kit insert.  Back to cited text no. 8    
9.Frederick S. Nolte and Beverly Metchock Mycobacterium, Chapter31. In : Manual of clinical microbiology, 6th edition. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (ASM press, Washington D.C.) 1995:400.  Back to cited text no. 9    
10.Siddiqi SH. The BACTEC 460 TB System, Product and Procedure Manual. Becton Dickinson, USA.  Back to cited text no. 10    
11.Procop GW, Robert GD. Laboratory methods in basic Mycology, Chapter 65. In: Bailey and Scott's Diagnostic microbiology, 10th edition. Betty A. Forbes Daniel F. Sahm, Alice S, Weissfied (Mosby) 1998: 871.  Back to cited text no. 11    
12.Levine SJ. Pneumocystis carinii pulmonary complications of HIV Infection Clinics in Chest Medicine 1996;17:665-695.  Back to cited text no. 12    
13.Bhalla P. Non mycobacterial and bacterial infections in HIV/AIDS, Chapter15. In : Manual on laboratory diagnosis of common opportunistic infections associated with HIV/AIDS. Baveja UK, Sokhey J (Eds). National Institute of Communicable Diseases, New Delhi. 100-118.  Back to cited text no. 13    
14.Tchamran M. Bacterial lung disease from common bacteria during HIV infection in African adults hospitalized in Abidjan. Bull Soc Pathol Exot1997;90:370-372.  Back to cited text no. 14    
15.Dropulic LK, Leslie JM, Eldred LJ, Zenilman J, Sears CL. Clinical manifestations and risk factors of Pseudomonas areuginosa infection in patients with AIDS. J Infectious Diseases 1995;171:930-937.  Back to cited text no. 15    
16.Sehgal SC, Shaimy IAI. Detection of Moraxella cattarrhalis in sputa of adult patients and its significance as a lower respiratory tract pathogen. Indian J Med Microbiol 1994;12:165-170.  Back to cited text no. 16    
17.Khan ZU, Qasem JA, Mustaja AS, Chugh TD. Nocardia an emerging pathogen. IAMM-1999; invited presentations, Abstract 1-15: xxi.  Back to cited text no. 17    
18.Lee CC, Loo LW, Lam MS. Case reports of Nocardia in patients with HIV. Ann Acad Medicine 2000;29:119-126.  Back to cited text no. 18    
19.Uttamchandani RB, Daikos GL, Reyes RR, Fischl MA, et al. Nocardiosis in 30 Patients with advanced Human immunodeficiency virus infection: Clinical features and outcome. Clinical Infectious diseases 1994;18:343-353.  Back to cited text no. 19    
20.Mohanthy KC, Sundrani RM, Nair S. HIV Infection in patients with respiratory diseases. Indian J Tuber1993;40:5-12.  Back to cited text no. 20    
21.Mohanthy KC, Nair S, Sahasrabudhe T. Changing trend of HIV infection in patients with Respiratory disease in Bombay since 1988. Indian J Tuber 1994;41:147-150.  Back to cited text no. 21    
22.Batungwanayo J, Taelman H, Bogaerts J, Allen S, et al. Pulmonary cryptococcosis associated with HIV-1 in Rwanda: a Retrospective study of 37 cases. AIDS 1994;8:1271-1276.  Back to cited text no. 22    
23.Meyohas MC, Roux P,Meynard JL, Frottier J. Aspergillosis in AIDS. Pathol Biol (Paris) 1994;42:647-651.  Back to cited text no. 23    
24.Shivananda PG. Pulmonary aspergillosis and its serological studies. ICMR Bulletin 1992;22:107-108.  Back to cited text no. 24    
25.Lakshmi G. Pulmonary aspergillosis in Chennai. Abstract 20, National Symposium on Mycosis 1999, May.  Back to cited text no. 25    
26.Pankajalakshmi VV, Taralakshmi VV. Some Emerging respiratory fungal infections. Abstract 14, National Symposium on Mycosis,1997.  Back to cited text no. 26    
27.Singh YN, Singh S, Rattan A, et al. Pneumocystis carinii infection in patients of AIDS in India. JAPI 1993;41:41-41.  Back to cited text no. 27    
28.White DA, Zaman MK. Medical Management of AIDS patients : Pulmonary Disease. Med Clinics North America 1992;76:19-44.  Back to cited text no. 28    
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