Background: Human Immunodeficiency Virus (HIV)-reactive patients are more prone to infections. The morbidity and mortality in HIV-reactive patients is due to opportunistic infections. Most of the infections seen in Acquired Immunodeficiency Syndrome are endemic to that geographical region. Hence, this study was undertaken to document the occurrence of pulmonary bacterial and fungal infections in HIV patients. Materials and Methods: Expectorated and induced sputum samples were collected from 100 HIV-reactive patients and processed for bacterial and fungal pathogens including Pneumocystis carinii. Results: Of 100 samples, 66 were culture positive. Among the isolates, Mycobacterium tuberculosis constituted the highest number, 55 (83.3%), followed by other bacterial infections, 11 (16.6%), and fungi, 2 (3.03%). Tuberculosis patients had a CD4 count of less than 250 cells/μl with a mean count of 186 cells/μl and those with bacterial infections had a CD4 count of more than 300 cells/μl. The study showed that males were infected with HIV more than females and most of them belonged to the adult age group in the prime of their working life. Weight loss followed by fever and cough were the most common symptoms. Conclusion: M. tuberculosis is the most common opportunistic pathogen followed by bacterial pathogens infecting the lung in HIV. Low CD4 count is a dangerous signal of decreased immune status and higher chances of opportunistic infections and high mortality.
Keywords: Bacteria, CD4 Count, fungi, HIV, Mycobacterium tuberculosis
|How to cite this article:
Shreevidya K, Dias M. Pulmonary bacterial and fungal infections in human immunodeficiency virus patients: A study from India. Ann Trop Med Public Health 2012;5:80-4
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Shreevidya K, Dias M. Pulmonary bacterial and fungal infections in human immunodeficiency virus patients: A study from India. Ann Trop Med Public Health [serial online] 2012 [cited 2021 Mar 6];5:80-4. Available from: https://www.atmph.org/text.asp?2012/5/2/80/95954
Acquired Immunodeficiency Syndrome (AIDS) caused by Human Immunodeficiency Virus (HIV). HIV infection is a global pandemic, with cases reported from virtually every country.  HIV has posed a major challenge to public health in the present time. AIDS was first recognized in the United States in 1981 with reports of unexplained opportunistic infections, including Pneumocystis jirovecii pneumonia (PCP) and Kaposi’s sarcoma, among homosexual men in New York and San Francisco. 
HIV made a delayed entry into India, but its spread has been very rapid and at present, it is in an advanced stage of the epidemic.  Though HIV is the causative agent of AIDS, most morbidity and mortality in AIDS patients results from opportunistic infections. About 80% of these patients are seen to die as a result of such an infection rather than from HIV. , The spectrum of HIV-related respiratory diseases has evolved since initial years of the epidemic. 
Of the global total population who are living with HIV infection, 95% live in developing countries. Changes in epidemiology have influenced the spectrum of pulmonary diseases in HIV-infected population. The increasing proportion of patients with AIDS, who are intravenous drug users and members of racial or ethnic minorities, correlate with increasing cases of bacterial pneumonia and tuberculosis (TB). 
The present study was undertaken to record the occurrence of various bacterial and fungal agents causing lower respiratory infections in HIV-reactive patients. No viral pathogens were included due to lack of diagnostic facilities.
|Materials and Methods|
Present study was conducted in the department of Microbiology, at Fr. Muller Medical College hospital Mangalore during December 2008 to May 2010.
A total number of 100 patients were included in the study, of which 66 patients’ sputum showed significant growth. The HIV-infected patients were all diagnosed as HIV reactive as per the NACO guidelines. 
Early morning expectorated and induced sputum samples were collected from all patients in separate sterile, wide mouthed, disposable, plastic containers. Expectorated sputum was used to detect the bacterial and fungal pathogens and induced sputum was used for detection of trophozoites and cysts of P. carinii. Quality of the expectorated sputum was assessed both by macroscopic and microscopic examinations. Specimens which were clear, thin, and watery with no purulent material were rejected. Microscopically, Bartlett’s scoring method was used to assess the quality of the sputum.
Smears were prepared and subjected to Gram’s staining, Ziehl-Neelsen staining [20% H 2 SO 4 and1% H 2 SO 4 ], and Toluidine O stain. KOH mount was done for fungi.
The specimens were cultured on 5% Sheep Blood agar, MacConkey agar, heated Blood agar, Lowenstein Jensen Media, and Sabouraud’s dextrose agar. The plates were incubated at 37°C for 18 to 24 hours in humid air plus 5 to 10% CO 2 . Sabouraud’s dextrose agar slopes were incubated in duplicates at 28°C and another at 37°C for 4 weeks and observed for growth at intervals. Lowenstein Jensen media was incubated at 37°C for 4 weeks and observed for growth. Identification of the organisms was conducted according to standard microbiological procedures. ,
Antibiotic sensitivity testing was done by Kirby Bauer disc diffusion methods according to CLSI guidelines.  Antibiotic discs were selected as per CLSI (Clinical and Laboratory Standards Institute).
Candida in the sputum was regarded as pathogen only after obtaining the same strain in repeated samples in pure growth, observing numerous polymorphonuclear leucocytes on Gram’s stain of the sputum samples along with pseudohyphae. All Candida obtained in Sabouraud’s dextrose agar was then processed for identification of species. Germ tube test was done. All Candida were inoculated on corn meal agar and incubated at 25°C to demonstrate chlamydospore formation and to look for typical morphology. Sugar assimilation test was done on Yeast Nitrogen Base agar for further speciation, using the following sugars: glucose, maltose, sucrose, lactose, trehalose, raffinose, and galactose.
Nocardia was suspected when Gram-positive, thin, branching-filaments were observed on Gram’s staining of sputum smears. Ziehl-Neelsen staining with 1% H 2 SO 4 was done, which showed acid fast bacilli with branching filaments. The specimen was then inoculated on Lowenstein Jensen medium and Sabouraud’s dextrose agar without antibiotics. The colonies obtained were white, glabrous, chalky, and wrinkled.
The CD4 count of the patients included in the study was done. The method used was Flow cytometry method.
Results were statistically analyzed by using SPSS version13 software for MS-Windows.
A total of 100 HIV-reactive patients were admitted during the study period, of which 66 patients showed growth. In the present study, most of the HIV-reactive patients were in the age group of 31 to 40 years of age and the second most common age group was 41 to 50 years of age followed by 21 to 30 years. Pediatric patients were not included in the study.
In our study, we noticed male preponderance. Among the 66 patients of HIV, 53 were males and 13 were females [Table 1].
|Table 1: Organisms isolated from HIV-reactive group|
Among the respiratory pathogens, Mycobacterium tuberculosis constituted the maximum number, 55 (83.3%), followed by other bacterial infections, 11 (16.6%), and fungi, 2 (3.03%). None of the culture isolates belonged to Mycobacterium avium-complex. The other bacterial isolates were Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Methicillin-Resistant Staphylococcus aureus, Acinetobacter baumannii, and Nocardia asteroides. Candida albicans and Candida tropicalis were the fungal isolates.
[Table 2] shows that the patients with TB had a lower range of CD4 count and those patients without TB but with other bacterial infections had a CD4 count of above 200 cells/μl.
|Table 2: Distribution of CD4 count in HIV reactive
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According to the NACO 2006 estimates, national adult HIV prevalence in India is approximately 0.36%, amounting to between 2 and 3.1 million people. If an average figure is taken, this comes to 2.5 million people living with HIV and AIDS. 
NACO 2006  report says that more men are HIV positive than women. Nationally, the prevalence rate for adult females is 0.29%, while for males it is 0.43%. Prevalence is also high in the 15 to 49 age group (88.7% of all infections), indicating that AIDS still threatens the cream of society, those in the prime of their working life. In our study, we noticed that males were leading the HIV-reactive status than females. Among the 66 patients of HIV, 53 were males and 13 were females. We observed the high incidence in the middle age group of society. Also, most of the HIV-positive patients with TB also belonged to the age group of 31 to 40 years of age. This is the dynamic age of maximum outdoor involvement and sexually active age. Thus, this study correlates with the above report in parameters of age and gender distribution of HIV.
In this study, most of the cases were newly diagnosed HIV-reactive cases. The most common mode of acquisition of HIV infection was the heterosexual route, 48 (72.72%). A small number of geriatric patients had acquired the infection through blood transfusion, 3 (4.54%). This study did not include any pediatric patients.
Sixty-four (96.9%) of HIV-reactive patients had >10% of presumed or measured weight loss. And, 2 (3.03%) had <10% weight loss. Severe weight loss is a common presenting feature of HIV-infected patients presenting with TB. Fever was the most common symptom in HIV-positive patients, accounting for 90.9% of the cases, followed by cough and breathlessness.
The occurrence of respiratory infections in HIV-reactive patients is well documented. In our study, among the respiratory pathogens, M. tuberculosis constituted the maximum number, 55 (83.3%), followed by other bacterial infections, 11 (16.6%) and fungi, 2 (3.03%). None of the culture isolates belonged to M. avium-complex. Most of the infections seen in AIDS are endemic to that geographical region. M. tuberculosis is endemic in India and thus, is the commonest opportunistic infection in India. 
In a study conducted at YRG Care, Chennai, TB was the most common opportunistic infection, with pulmonary TB affecting 35% of the HIV-positive group and extrapulmonary TB in 11% of them.  The risk of developing TB after an infectious contact is 5 to 10% per year among the HIV-infected individuals when compared with 5 to 10% during their lifetime in HIV-negative individuals. 
In a study conducted by Shailaja et al.  44.3% were bacteria, 42.9% were mycobacteria, and 12.8% were fungi. Bacterial pathogens were K. pneumoniae, S. pneumoniae, S. aureus, P. aeruginosa, Moraxella catarrhalis, Escherichia coli, and N. asteroides.
In our study, the other bacterial isolates were K. pneumoniae, P. aeruginosa, S. pneumoniae, Methicillin-Resistant S. aureus, A. baumannii, and N. asteroides. C. albicans and C. tropicalis were the fungal isolates.
Polymicrobial etiology was noticed in eight (12.12%) of the patients indicating the severity of infection in these patients.
P. aeruginosa pneumonia is often community acquired and is associated with high mortality. Dropulic et al. in their 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. 
In our study, there were two isolates of P. aeruginosa causing pneumonia. Among them, one was a sensitive strain which showed sensitivity to piperacillin, ceftazidime, and higher line of drugs. But the other strain was highly resistant. It showed resistance to all the baseline drugs and even to carbapenems and Monobactams. It was sensitive only to cefoperazone-sulbactam. Infection with the resistant strain of Pseudomonas is associated with bad prognosis and this patient expired.
K. pneumoniae isolates (two) and S. pneumoniae (two) were sensitive strains and thus treatment was easier when compared with other bacteria. A. baumannii is known for its inherent resistance to several drugs due to the presence of the large “resistance island” in its genome. The single strain obtained was highly resistant to all the drugs that were tested, except for levofloxacin and colistin.
In the present study, N. asteroides was isolated from two patients. N. asteroides is the species among Nocardia group that is most commonly associated with invasive disease. Among the infections caused by Nocardia, pulmonary infection is the most common, which is acquired following inhalation of fragmented bacterial mycelia.  In AIDS, Nocardiosis usually affects persons with <250 CD4 T cells/ μl.  But, the CD4 count of the two cases with Nocardia in our study was not available, since both the patients did not return for follow-up.
During the last few decades, there has been an increased incidence of Candidiasis due to the emergence of pandemic of AIDS. Candidiasis is undoubtedly the most common fungal infection in HIV-infected individuals. Although C. albicans remains the most common causative agent, an increasing incidence of non-albicans species of Candida has been seen in the last few years.  Introduction of fluconazole and itraconazole has increased the incidence of non-albicans Candida. These species include C. tropicalis, C. krusei, C. glabrata, and C. parapsilosis. We isolated C. albicans and C. tropicalis. The pathogenicity was proved by repeated isolation in pure culture, plenty of polymorphs with pseudohyphae, and improvement in their health after treatment with antifungals.
PCP was not isolated in this study. This was also the observation made by Shailaja et al.  It is the most common AIDS-defining illness in the developed world. Unlike in the West, the prevalence of PCP is low or negligible in India. In India, very low rates (5 to 7%) , of PCP have been reported. Some reasons for this could be the predominance of other pulmonary diseases like TB, or due to the extensive use of cotrimoxazole in the prophylaxis of PCP in HIV-reactive individuals with CD 4 count of less than 200 cells/μl.
The immune status of the patients was assessed using CD4 count. The CD4 count of HIV-positive patients who were diagnosed to have TB was low. The mean CD4 count was 186 cells/μl in this group of patients.
The HIV-positive patients who did not have TB had a mean CD4 count of 361 cells/μl. This finding correlates with the statement of association of TB with low CD4 count. HIV-reactive patients who had a CD4 count of more than 300 cells/ μl were not positive for TB but had other bacterial infections. Those who had TB had a CD4 count of less than 250 cells/μl, with mean count of 186 cells/μl which demonstrated that TB is common in HIV patients when their immune status is low.
Also, mortality was high in those patients with very low CD4 count, thus proving the association of CD4 count with the immune status of the body.
There were six deaths (10.9%) among the HIV patients who had TB. All these patients had a CD4 count of less than 50 cells/μl and also the smear grading of TB was 3+ in them. This finding triggers the thought of association of low CD4 count and higher smear grading of TB. This can say the luxuriant growth of TB bacilli in a patient with low CD4 count.
Patients with CD4 counts less that 200 cells/μl are 19 times more likely to die than those with CD4 counts greater than 350 cells/μl. 
Since this study was on pulmonary infections, we did not include extrapulmonary TB. The patient with resistant strain of P. aeruginosa, who expired, had a CD4 count of 280 cells/μl. Here, the death may be attributed to the pathogenicity of the bacteria, difficulty in treatment due to drug resistance, as well as a low CD4 count.
All the TB patients were started on DOTS (direct observation therapy) treatment and most of them have responded well with smear negative for TB after treatment for 6 months. Few patients did not return for follow-up.
HIV-reactive patients were started on Anti-Retroviral Treatment depending on their CD4 count and opportunistic infections.
With the availability of HAART (Highly effective antiretroviral therapy), treatment can vastly reduce the morbidity and mortality of HIV-reactive patients.
We conclude that M. tuberculosis is the most common opportunistic pathogen infecting the lung in HIV-infected individuals. Also, they are prone for other bacterial and fungal infections. Low CD4 count is a dangerous sign of decreased immune status and higher chances of opportunistic infections and high mortality.
HIV-TB co infection continues to be a serious problem in India because of endemicity to TB in India. Treatment with DOTS drugs and HAART is very helpful in giving better life to these patients.
The authors thank Dr. Kiran Kumar N, ART Medical officer, Wenlock Government hospital and Ms. Sucharita Suresh Dept of Hospital administration, FMMC.
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Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]