Investigations
1st investigations to order
CD4 cell count
Test
The CD4 count is a highly sensitive and specific marker that determines the susceptibility to particular opportunistic infections.[77] Tuberculosis can occur throughout the course of HIV disease but the risk increases as the CD4 count decreases.[1][47][78]Individuals with a CD4 count below 250 cells/microlitre living in or visiting areas in which Coccidioides species are endemic are at increased risk for coccidioidomycosis.[42] Pneumocystis jirovecii pneumonia and candidiasis are more likely when the CD4 count is below 200 cells/microlitre. Individuals with a CD4 count below 150 cells/microlitre who live in or have travelled to areas endemic for Histoplasma capsulatum are at increased risk for developing progressive disseminated histoplasmosis.[1] When the CD4 count is below 100 cells/microlitre, people are at a higher risk for toxoplasmosis.[1] Cytomegalovirus end-organ disease and Mycobacterium avium complex are more likely with CD4 count below 50 cells/microlitre.[1] More than three-quarters of cryptococcal infections associated with AIDS develop when the CD4 count falls below 50 cells/microlitre.[54]
Result
variable
sputum stain and culture
Test
In diagnosis of tuberculosis (TB) three sputum specimens should be obtained for acid-fast bacilli (AFB) smear and cultured.[82]
Examination of induced sputum is recommended as the initial screening test for Pneumocystis jirovecii pneumonia (PCP). Specimens should be evaluated with particular staining methods (e.g., Gomori-methenamine silver, Wright-Giemsa, Diff-Quick, or immunofluorescence). Since the negative predictive value of this test is relatively low, a negative test should prompt a referral for fibreoptic bronchoscopy with BAL, which significantly increases the diagnostic yield to >90%.[108]
Sputum culture is a definitive test for coccidioidomycosis infection. Isolation of Coccidioides organisms from a patient’s sputum or other clinical specimen definitively establishes the diagnosis of coccidioidomycosis and should not be considered to represent colonisation. Organisms grow well within 5-7 days of incubation on most mycological and bacteriological media. When growth is noted, appropriate biocontainment measures are imperative, as cultures are highly infectious and laboratory personnel are at risk of infection.
Result
AFB stain and mycobacterial culture are positive in TB and MAC; staining is positive in PCP; positive in coccidioidomycosis infection
blood cultures
Test
Two blood cultures are usually sufficient for the detection of disseminated Mycobacterium avium complex (MAC). One blood culture identifies 91% of people with MAC bacteraemia, while a second blood culture increases the yield to 98%.[177] However, up to 6 weeks of culture may be required, which limits the clinical utility.
Three-quarters of people with HIV-associated cryptococcosis have positive blood cultures for Cryptococcus neoformans.[140] Blood cultures may be helpful if disseminated disease is suspected in the absence of meningitis.
Mycobacterial blood cultures may be positive in disseminated TB.[97]
Blood cultures may be particularly useful for diagnosing disseminated histoplasmosis, especially in severely immunosuppressed individuals. Culture is a definitive diagnostic method for Histoplasma capsulatum.[1]
Result
may be positive in MAC cryptococcus infection, disseminated TB, and disseminated histoplasmosis
adenosine deaminase
Test
Measuring adenosine deaminase in pleural fluid is an easy and inexpensive test that may help establish the diagnosis of pleural tuberculosis when significantly elevated.[92] Adenosine deaminase sensitivity was 94% and specificity 95% in people living with HIV, regardless of CD4 counts, when the cut-off value was 30 U/L.[93]
Result
elevated in TB pleuritis
Toxoplasma gondii serology
Test
Presence of anti-T gondii IgM antibodies does not necessarily indicate a recently acquired infection as they can remain elevated for more than 1 year. Recent T gondii infection is likely when serial specimens obtained at least 3 weeks apart and tested in parallel reveal at least a 4-fold increase in IgG titres.[178] A negative T gondii IgG makes toxoplasmic encephalitis significantly less likely but does not rule it out.[1]
Result
anti-T gondii IgM suggests recent infection and anti-T gondii IgG suggests past infection
Coccidioides serology
Test
Serological tests are most frequently used to diagnose primary pulmonary coccidioidomycosis and are highly specific for infection.[151] If serological testing is negative in suspected infection, repeat serological testing is recommended since it can take several weeks to develop an adequate antibody response.
Coccidioides serology should be performed in individuals with compatible clinical presentations if they have resided in or visited endemic regions.
Enzyme immunoassay (EIA) is widely available and detects specific IgM and IgG antibodies against Coccidioides. Positive EIA results should be confirmed with immunodiffusion and complement fixation.[1]
Result
positive IgM and/or IgG
cryptococcal polysaccharide antigen
Test
Very specific and highly sensitive (>90%). High serum titers (≥1:160) indicates a higher risk for CNS disease in asymptomatic individuals.[179] High initial cerebrospinal fluid (CSF) titres (≥1:1024) are a marker of poor prognosis and correspond to a high organism burden. CSF antigen titres typically fall with successful treatment.[160]
Result
from serum or CSF
Histoplasma capsulatum antigen
Test
Antigen detection tests for H capsulatum are the preferred diagnostic method for disseminated histoplasmosis. These assays demonstrate high sensitivity and specificity, particularly in disseminated cases. They allow for rapid diagnosis, reducing the time to treatment initiation.
Urine or blood samples are the preferred detection methods. Antigen testing may also be performed on cerebrospinal fluid and bronchoalveolar lavage fluid in cases involving the central nervous system or lungs.[1]
Result
Positive for H capsulatum
Histoplasma capsulatum culture
Test
Culture is a definitive diagnostic method for H capsulatum, but requires several weeks for results due to the slow growth of the organism. H capsulatum can be cultured from blood, bone marrow or respiratory secretions. Blood cultures may be particularly useful for diagnosing disseminated histoplasmosis, especially in severely immunosuppressed individuals.[1]
Result
Positive for H capsulatum
FBC
Test
Anaemia is common in people living with HIV with opportunistic infections. In disseminated Mycobacterium avium complex, the anaemia is an important negative predictor for survival, is usually profound, and is often accompanied by leukopenia and thrombocytopenia.[180][181]
Result
anaemia often accompanied by leukopenia and thrombocytopenia
LFT
LDH
Test
Often elevated in disseminated Mycobacterium avium complex infection, and frequently elevated in Pneumocystis jirovecii pneumonia (PCP).
However, elevated LDH should be interpreted with caution, because it is also elevated in people with various other lung diseases, such as pulmonary tuberculosis and other bacterial pneumonias, as well as a variety of non-infectious conditions.[109][182]
Result
elevated (but non-specific)
ABG
Test
The arterial oxygen pressures (PaO₂) and P(alveolar-arterial [A-a])O₂ gradient correlate with prognosis. A P(A-a)O₂ of greater than 35 mmHg or a PaO₂ of less than 70 mmHg are associated with poorer outcome, and provide an indication for adjunctive corticosteroid therapy.[106]
Result
increased alveolar-arterial oxygen gradient, P(A-a)O₂; PaO₂ values vary widely depending on disease severity
CXR
Test
Should be performed in any person living with HIV with pulmonary symptoms.
Findings in people living with HIV with tuberculosis (TB) and a CD4 above 200 cells/microlitre: upper lobe infiltrates and cavitation; a CD4 below 200 cells/microlitre: mediastinal adenopathy is common and probably reflects an ineffective immune response.[2][154]
The prevalence of normal CXRs in individuals with confirmed Pneumocystis jirovecii pneumonia (PCP) is about 10%.[157] Various atypical radiographic manifestations have been reported, including nodular densities and cavitary lesions. Pneumothorax can occur, and its management is often difficult.[184] If the CXR appears normal but PCP is suspected, a high-resolution CT should be ordered.
Imaging studies are typically useful during the initial evaluation of coccidioidomycosis, but radiographic findings are non-specific.
CXR may be used in subsequent monitoring for improvement.[185]
Result
variable; TB: upper lobe infiltrates and cavitation or mediastinal adenopathy; PCP: bilateral diffuse infiltrates beginning in the perihilar regions; cytomegalovirus: interstitial infiltrate; histoplasmosis: diffuse bilateral reticulonodular infiltrates
head CT
Test
Should be ordered in all individuals with altered mental status.
In toxoplasmic encephalitis, lesions tend to involve the basal ganglia and hemispheric corticomedullary junction.[186]
In cryptococcal meningitis, CT findings may include single or multiple nodules (cryptococcomas), cerebral oedema, or hydrocephalus.[160]
Result
toxoplasma encephalitis: multiple, bilateral, hypodense, ring-enhancing lesions; cryptococcal disease: normal or may show meningeal enhancement; tuberculosis meningitis: tuberculomas, post-contrast basal enhancement, hydrocephalus, and infarcts
Investigations to consider
tuberculosis (TB) nucleic acid amplification
Test
Several rapid nucleic acid amplification tests (NAATs) are available for the diagnosis of TB, and some are also able to detect resistance to some TB drugs. Although NAATs were originally designed and approved for respiratory specimens, they may also be requested on specimens from other sites where involvement of TB is suspected (e.g., cerebrospinal fluid, lymph node aspirate, lymph node biopsy, pleural fluid, peritoneal fluid, pericardial fluid, synovial fluid, or urine).[83] In the US, use of NAATs for extrapulmonary specimens is not approved by the US Food and Drug Administration and therefore use in this setting is off-label.
Result
positive in TB infection
lateral flow urine lipoarabinomannan (LF-LAM) assay
Test
Lateral flow tests that detect lipoarabinomannan (LAM) antigen in urine are potential point-of-care tests for TB. One Cochrane review found the lateral flow urine lipoarabinomannan (LF-LAM) assay to have a sensitivity of 42% in diagnosing TB in HIV-positive individuals with TB symptoms, and 35% in HIV-positive individuals not assessed for TB symptoms.[90] The World Health Organization (WHO) recommends that LF-LAM can be used to assist in the diagnosis of active TB in HIV-positive individuals.[83] This approach is supported by another Cochrane review, which found reductions in mortality and an increase in treatment initiation with use of LF-LAM in inpatient and outpatient settings.[91]
Culture would still be required for drug susceptibility testing (DST).
Result
positive
bronchoalveolar lavage (BAL)
oropharyngeal scrapings (KOH preparation) and culture
Test
Potassium hydroxide 10% (KOH) slide preparation can confirm the diagnosis of candidiasis. Pseudohyphae and budding yeast are characteristic findings.
Cultures alone are not diagnostic because colonisation is common and are usually not necessary unless the lesions fail to resolve on standard antifungal treatment.[190]
Result
presence of Candida species
cerebrospinal fluid (CSF) analysis
Test
Due to the risk of brain herniation if mass effect is present, caution should be exercised when considering lumbar puncture.[178]
Toxoplasmosis infection: Giemsa staining can show tachyzoites.
Cryptococcal meningitis: India ink examination is positive in 70% to 90% of people living with HIV. CSF culture requires 48-72 hours. The opening pressure in the CSF is elevated in up to 75% of individuals; CSF pressures above 250 mm of H₂O may require large-volume CSF drainage.[160][172] CSF cryptococcal antigen is also important to include, as it is a very sensitive and specific test and can be rapidly performed.
Result
toxoplasma encephalitis: mild pleocytosis of mononuclear predominance and elevated protein; cryptococcal meningitis: WBC elevated with lymphocytosis, elevated protein, and low glucose
polymerase chain reaction (PCR): cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL) fluid, and vitreous and aqueous humor specimen for Toxoplasma gondii
Test
Can be useful in the diagnosis of toxoplasmic encephalitis. PCR in CSF has a sensitivity that varies from 12% to 70% and a specificity of approximately 100% in individuals with toxoplasmic encephalitis. A positive PCR in brain tissue does not necessarily indicate active infection because tissue cysts persist in the brain long after acute infection.[191]
Result
positive
bone marrow aspirate and culture
Test
May be helpful in the diagnosis of disseminated Mycobacterium avium complex and tuberculosis, although acid-fast bacilli (AFB) blood cultures are the test of choice. Diagnostic sensitivity of bone marrow cultures may be no greater than that of blood cultures in these individuals, but the histopathological examination of bone marrow specimens results in the relatively rapid identification of infections in some individuals who are culture negative.[192] In early disease, the sensitivity of bone marrow culture may actually be higher than that of AFB blood culture.
Result
granulomas; organism seen on acid-fast stain
lymph node aspirate or biopsy
Test
Lymph node fine-needle aspiration or biopsy allows a specific diagnosis in 100% of individuals with Mycobacterium avium complex focal lymphadenitis.[193]
Result
granulomas; organism seen on acid-fast stain
tissue biopsy
Test
Should be considered when other less invasive tests have been non-diagnostic and the pulmonary process is progressive.
In individuals with suspected tuberculosis (TB), trans-bronchial biopsy or biopsy of an extrapulmonary site (e.g., bone marrow, vertebral column) may be required. On pathological examination tuberculous granulomas are detected in 60% to 100% of cases, depending on the person's immune status and the site of sampling.[96]
The specimen obtained should also be examined for acid-fast bacilli (AFB) and cultured for mycobacteria.
Individuals with clinical illness suggestive of tissue-invasive cytomegalovirus (CMV) disease should have tissue specimen (liver, intestinal mucosa, lung) obtained by biopsy for the detection of CMV.[194]
Positive histopathology gives a definitive diagnosis of coccidioidomycosis.
Periodic acid-Schiff or Gomori methenamine silver staining may be used to identify Histoplasma capsulatum. Histopathology is particularly useful for rapid presumptive diagnosis in acute clinical scenarios of histoplasmosis.[27]
Result
TB: granulomas, visualisation of AFB; CMV: demonstration of CMV-specific cytoplasmic and intra-nuclear inclusions in liver, intestinal mucosa, or lung specimens; coccidioidomycosis: identification of spherules on microscopy; histoplasmosis: H capsulatum appear as relatively small (2-4 microns), ovoid, intracellular yeast
brain biopsy
Test
Can provide a definitive diagnosis of toxoplasmic encephalitis.
Excisional brain biopsy findings range from granulomatous reaction with gliosis and microglial nodules to necrotising encephalitis. The presence of tachyzoites or cysts surrounded by inflammation is considered diagnostic.[159]
Result
toxoplasmic encephalitis: presence of tachyzoites or cysts surrounded by inflammation
abdominal CT
Test
Consider in individuals with abdominal pain or if disseminated Mycobacterium avium complex (MAC) is suspected.
The CT scan in abdominal tuberculosis (TB) usually shows ascites and omental thickening.[154][195] Hepatomegaly, splenomegaly, and small wall thickening may also be noted.[105]
Result
abdominal TB: visceral lesions and intra-abdominal lymphadenopathy with necrosis; disseminated MAC: enlarged intra-abdominal mesenteric and/or retro-peritoneal lymph nodes
high-resolution CT (HRCT) of the chest
Test
When chest radiographic findings are normal, in someone with possible Pneumocystis jirovecii pneumonia (PCP), HRCT, which is more sensitive than chest x-ray, may reveal more faint ground-glass opacities or other subtle lesions. The sensitivity, specificity, positive predictive value, and negative predictive value of HRCT for the diagnosis of PCP are 100%, 83.3%, 90.5%, and 100%, respectively. HRCT is a reliable method for differentiating PCP from other infectious processes in HIV-positive individuals and a good method to rule out PCP.[115][184][196]
Findings include bilateral ground-glass opacities, patchy bilateral consolidation, and multiple nodules or mass-like areas of consolidation.[197]
Ground-glass opacities can be homogeneous (24% of episodes), spare the lung periphery (41%), or display a mosaic pattern (29%). Other findings can include: reticulation, tree-in-bud appearance, consolidation, or cystic lesions.[197]
Result
PCP: ground-glass opacities; cytomegalovirus: diffuse interstitial and parenchymal changes
polymerase chain reaction (PCR) for Pneumocystis jirovecii pneumonia (PCP)
beta-D-glucan
Test
Beta-D-glucan, a cell wall component of most fungi, may be useful for non-invasive diagnosis of Pneumocystis jirovecii pneumonia (PCP). Although elevated beta-D-glucan is not specific to PCP, it should raise suspicion if raised in a person with risk factors and a compatible clinical presentation.[112]
Result
Elevated (but non-specific for PCP)
brain MRI
Test
More sensitive than CT scan for toxoplasmic encephalitis and the preferred imaging technique, especially in individuals without focal neurological abnormalities. Individuals with only one lesion or no lesions apparent on CT scan should undergo MRI to determine whether more than one lesion is present.[121]
MRI scan is more sensitive for detecting multiple enhancing nodules within the brain parenchyma, meninges, basal ganglia, and midbrain.[160]
Result
toxoplasmic encephalitis: multiple or solitary focal brain lesions; cryptococcal disease: multiple enhancing nodules
thallium single photon emission tomography and PET
Test
Useful for distinction between central nervous system (CNS) lymphoma and infectious processes in people living with HIV with focal brain lesions.[123]
Result
increased uptake is an indicator of malignancy (CNS lymphoma)
upper gastrointestinal endoscopy and colonoscopy
Test
On biopsy, cytomegalovirus (CMV) intra-nuclear inclusions are identified in the densely inflamed granulation tissue of the ulcer base.[198]
In oesophageal candidiasis, pathological findings include: desquamated superficial hyperplastic hyper-keratotic squamous epithelium and inflammatory cells, with infiltration by fungal elements.[199]
Result
CMV colitis: lesions vary from punctate and superficial erosions to deep ulcerations, with granular and friable intervening mucosa
coccidioidal antigen testing
Test
Coccidioidal antigen testing can be performed on serum, urine, and cerebrospinal fluid.
Result
positive for coccidioidal antigen
polymerase chain reaction (PCR) for Coccidioides
Test
Real-time PCR can be used for detection of Coccidioides although availability is generally limited.
Result
positive for coccidioidal DNA
polymerase chain reaction (PCR) for Histoplasma
Test
PCR-based molecular diagnostic methods demonstrate high sensitivity and specificity for detecting Histoplasma DNA. These methods can serve as adjunctive tests, particularly when conventional diagnostics yield inconclusive results.
However, availability and standardisation of molecular assays remain limited in clinical practice.
Result
Positive for Histoplasma DNA
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